Download 141120 Koong Luke Dissertation

yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Document related concepts
no text concepts found
Luke Yun-Kong Koong
The Dissertation Committee for Luke Yun-Kong Koong Certifies that this is the
approved version of the following dissertation:
Cheryl S Watson, PhD, Mentor
Darren Boehning, PhD, Chair
Gracie Vargas, PhD
Randall M Goldblum, MD
Nancy Ing, DVM, PhD
Dean, Graduate School
Luke Yun-Kong Koong, BS
Presented to the Faculty of the Graduate School of
The University of Texas Medical Branch
in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy
The University of Texas Medical Branch
December, 2014
This dissertation is dedicated to my family, whom has supported me with love and
encouragement throughout my life; my friends, who push me to greater heights; and most
importantly God, who continues to bless me every day with life and joy.
I would like to acknowledge my mentor, Cheryl S Watson, who has guided me
through my graduate work. She has shown me how to be a true scientist and a responsible
steward of our environment. I am forever grateful for the avenues she has opened up for
my life and career. Additionally, I would like to thank my committee members for all of
their constructive ideas throughout my project, as well as encouragement along the way.
Another key figure in my graduate career is Jennifer Jeng of the Watson laboratory, who
helped teach me many of the assays used in this study, and who was also available for
advice and suggestions. The National Institutes of Environmental Health Sciences
(NIEHS) Environmental Toxicology Training Grant helped me acquire numerous skills
toward my career, as well as monetary support, and for that I am truly grateful. Finally, I
want to acknowledge the GSBS and the support they provided me in finishing my degree.
This work was supported by the NIEHS T32ES007254 Environmental
Toxicology Training Grant.
Publication No._____________
Luke Yun-Kong Koong, PhD
The University of Texas Medical Branch, 2014
Supervisor: Cheryl S Watson
Prostate cancer is the most common non-cutaneous cancer among men, and
diethylstilbestrol (DES) is an estrogen that has been used clinically to combat advanced
tumors. Estrogens can indirectly decrease androgen production by central negative
feedback inhibition, but may also have direct tumor killing mechanisms by a less well
understood mechanism. To elucidate these mechanisms and provide understanding for
potential future therapies, we identified cellular pathways and rapid signaling events that
act via estrogen receptors (ERs) and contribute to estradiol (E2) or DES-mediated cell
killing/growth arrest. E2 was much more effective than DES at reducing cell numbers of
both LAPC-4 early-stage androgen-dependent and PC-3 late-stage androgen-independent
prostate cancer cells. Both E2 and DES rapidly (within minutes) activated mitogenactivated protein kinases, generated reactive oxygen species (ROS), induced apoptosis
and necroptosis, and regulated the activation and levels of cell cycle proteins. Regulation
of cyclin D1 played a major role in blocking cell proliferation, but extracellular signal-
regulated kinase (ERK) causing ROS generation, phosphorylation of p38, apoptosis, and
phosphorylation of p16INK4A also contributed. Our rapid effects suggested the
participation of membrane ERs (mERs). ERα and β mediated E2’s ability to increase
ROS through sustained ERK activation, and increased p-cyclin D1 levels in LAPC-4
cells. However, ERβ and GPR30 mediated these responses in PC-3 cells. Apoptosis was
initiated in LAPC-4 and PC-3 cells by E2 only, and not by DES. Necroptosis was not
altered by estrogens in either cell line. We showed for the first time the presence of mERs
in both early and late prostate cancer cells. Then several xenoestrogens (XEs) were
evaluated for their ability to increase or decrease prostate cancer cell numbers.
Coumesterol and genistein unexpectedly stimulated prostate cancer cell growth, while
resveratrol minimally increased cell numbers in both early and late-stage cells. Bisphenol
A (BPA) slightly, though significantly, increased cell numbers in LAPC-4 cells only.
Again, control of cyclin D1 protein levels was a key mediator of the growth change
effects. The findings of these studies should be relevant to the clinical treatment of
prostate tumors, including development of ER subtype-specific agents to control
proliferation and cell death signaling pathways. Our findings regarding XEs should help
establish guidelines for prostate cancer patients regarding consumption of safe dietary
concentrations of phytoestrogens and acceptable exposure levels to BPA.
List of Tables .............................................................................................................ix
List of Figures ............................................................................................................x
List of Abbreviations .................................................................................................xiii
Chapter 1: Introduction ..............................................................................................15
Prostate tumors and methods of treatment ........................................................15
Prostate function and anatomy..........................................................................17
Prostate cancer treatment regimens ..................................................................22
Development of androgen-independent tumors ................................................24
Estrogens and estrogen receptor physiology ....................................................27
Roles of membrane estrogen receptors in nongenomic responses ...................31
Estrogen receptor control of cell proliferation or death ....................................33
Estrogen receptors in the prostate and prostatic disease ...................................34
Xenoestrogens and effects on living systems ...................................................35
Aims of studies in this dissertation ...................................................................37
Aim 1 .......................................................................................................37
Aim 2 .......................................................................................................38
Chapter 2: Direct estradiol and diethylstilbestrol actions on early- vs. late-stage
prostate cancer cells ..........................................................................................39
Abstract .............................................................................................................39
Introduction .......................................................................................................40
Materials and Methods......................................................................................43
Discussion .........................................................................................................58
Chapter 3: Rapid, nongenomic signaling effects of several xenoestrogens involved in
early- vs. late-stage prostate cancer cell proliferation ......................................64
Abstract .............................................................................................................64
Introduction .......................................................................................................65
Materials and Methods......................................................................................68
Results and Discussion .....................................................................................72
Chapter 4: Conclusions and Future Directions ..........................................................87
Major Conclusions from Our Studies ...............................................................87
In vivo model ....................................................................................................90
Receptor targeted agonists (Acadia ERβ agonists) ...........................................94
Other potential studies and directions ...............................................................96
References ..................................................................................................................99
Vita 154
List of Tables
Table 2.1. Summary of mechanisms contributing to estrogen-induced decline in
numbers of LAPC-4 or PC-3 prostate cancer cells..........................60
List of Figures
Figure 1.1. Functional domains of AR. ..................................................................19
Figure 1.2. Canonical AR signaling pathway. .......................................................21
Figure 1.3. Summary of roles for membrane and intracellular ERs. .................28
Figure 1.4. ERα and ERβ splice variants. ..............................................................30
Figure 1.5. Comparison of the structures of E2, DES, BPA, and three
phytoestrogens. ....................................................................................36
Figure 2.1. Cell viability after 3 days of E2 or DES treatment. ...........................46
Figure 2.2 Phospho-ERK (pERK) levels in LAPC-4 and PC-3 cells after E2 or
DES treatments. ..................................................................................48
Figure 2.3. ROS measured in LAPC-4 vs. PC-3 cells treated with 0.1 nM E2 or 1
µM DES................................................................................................49
Figure 2.4. Phospho-JNK (pJNK) levels in LAPC-4 and PC-3 after E2 or DES
treatments. ...........................................................................................51
Figure 2.5. Caspase 3 activity levels after E2 or DES treatments. .......................52
Figure 2.6. Necroptosis after E2 or DES treatments. ............................................52
Figure 2.7. Phospho-p16 (p-p16) levels after E2 or DES treatments. ..................54
Figure 2.8. Activated p38 (p-p38) levels after treatment with 0.1 nM E2 and 1 µM
Figure 2.9. Phosphoryated cyclin D1 (p-cyclin D1) vs. total cyclin levels and their
ratios after treatment with 0.1 nM E2 and 1 µM DES. ....................56
Figure 2.10. ER subtype-selective antagonists inhibit E2- or DES-induced ROS
and p-cyclin D1 responses. .................................................................57
Figure 2.11. Direct mechanisms of E2/DES action on prostate cancer cell
Figure 3.1. Cell number after 3 days of XE treatment. ........................................72
Figure 3.2. ER subtype (α, β, and GPR30) levels (total vs. membrane) in LAPC-4
and PC-3 prostate cancer cells. ..........................................................74
Figure 3.3. Phospho-ERK (pERK) levels in LAPC-4 and PC-3 cells after XE
treatments. ...........................................................................................76
Figure 3.4. ROS levels after treatment with 10-10M E2, 10-6M H2O2, 10-9M BPA,
10-7M coumestrol, 10-7M genistein, and 10-8M resveratrol, ± ER
subtype-selective antagonists. ............................................................77
Figure 3.5. Cyclin D1 phosphorylation and degradation by XEs, and inhibition
by ER-selective antagonists. ...............................................................79
Table 3.1 Summary of XE responses for mechanisms that affect the number of
viable LAPC-4 vs. PC-3 cells. ............................................................84
Figure 4.1. Various components of the prostate tumor microenvironment. ......91
Figure 4.2 Primary steps in establishing a xenograft prostate cancer model in
SCID mice. ...........................................................................................92
Figure 4.3 Number of viable prostate cancer cells following Acadia ERβ agonists.
Figure 4.4. Improper epigenetic regulation of normal prostate cells may lead to
an increased chance for prostate cancer. ..........................................97
List of Abbreviations
Androgen Receptor
Androgen Response Element
Bisphenol A
Cyclic Adenosine Monophosphate
Ethinyl Estradiol
Epidermal Growth Factor
Estrogen Receptor
Extracellular Signal-Regulated Kinase
Estrogen Response Element
Follicle Stimulating Hormone
Insulin-like Growth Factor
c-Jun N-terminal Kinase
Luteinizing Hormone
Luteinizing-Hormone-Releasing Hormone
Membrane Estrogen Receptor
Mitogen Activated Protein Kinase
Phosphoinositide 3-kinase
Prostate Specific Antigen
Reactive Oxygen Species
Severe Combined Immunodeficiency
Signal Transducers and Activators of Transcription
Vascular Endothelial Growth Factor
Chapter 1: Introduction
Prostate tumors and methods of treatment
Androgens have been known to stimulate the growth of the prostate gland and
tumors derived from them since the late 18th century, but the first written report of
prostate cancer did not appear until 1853, when John Adams was able to study the
histology of a prostate tumor [reviewed in 1]. Early treatments employed surgical
castration as a means to decrease androgen levels and reduce prostate gland size 2. The
use of estrogens to treat prostate cancers was first used in the 1940s by Charles Huggins
and Clarence Hodges who administered the pharmaceutical estrogen diethylstilbestrol
(DES) and estradiol benzoate to patients as a form of androgen ablation via central
negative feedback control 3, slowing metastatic prostate cancer cell growth and inhibiting
release of acid phosphatase. A study in the 1960s by the Veteran’s Administration
Cooperative Urological Research Group then found that DES administration was as
effective as orchiectomy in treating prostate tumors 4. While these estrogens indirectly
inhibit prostate cancer cell growth, the rapid, direct mechanisms of estrogen action
on prostate tumor cells have not been considered.
In recent years, prostate cancer has become the most diagnosed non-cutaneous
cancer in men, with close to 200,000 cases diagnosed each year and about 30,000 deaths
annually in the United States
5, 6
. The incidence rate varies across ethnicities, but has
shown a steady overall decline since 1999
7, 8
. The occurrence of prostate tumors
increases with age, and patient age correlates to the ability to histologically detect
cancerous cells in prostate gland biopsies. Most tumors are commonly found in males in
their 60-70’s 9. Precancerous lesions are the precursors for eventual prostate tumors and
can be found in the prostates of 33% of healthy men, but the development of lesions into
actual cancerous cells is significantly less (1 in 9 patients) 9. Additionally, the incidence
of prostate cancer in the US (1 in 7) is higher than in Asian countries (1 in 10,000)
5, 10
This large discrepancy has led many to examine the differences in diets between the US
and East Asia and how they might affect prostate cancer
, which has largely focused
on the soy-based isoflavones. There is also evidence however that the large difference
seen between the two populations is simply due to better reporting and screening
practices in the US 16.
The susceptibility of developing prostate cancer does not appear to be genetic
, with the exception of a late-developing chromosomal translocation causing the fusion
of the transmembrane protease serine 2 (TMPRSS2) gene to the coding region of the
erythroblast transformation-specific (ETS) family of transcription factors, leading to
androgen-independent growth. This translocation can be found in 40-70% of prostate
cancer patients according to several studies, but these lesions may not be continually
selected as the tumor progresses
. However, tumors with this translocation have
increased metastatic potential and can be more aggressive, leading to higher grade
19, 22
. Common ETS family proteins involved in this gene fusion include ETS-
related gene, ETS translocation variant 1, and protein C-ets-1
gene fusions are rarely detected in benign prostate tissue. Interestingly, this major
genetic mutation shows a reliance on estrogens and estrogen receptors (ERs) for
genomic regulation resulting in prostate cancer cell proliferation, specifically via
ERβ 19. ERβ binds to an upstream site of the TMPRSS2 promoter, directly regulating the
transcription of the TMPRSS2-ETS fusion products. (Because our project instead detects
inhibition of prostate cancer cell proliferation by estrogens, this genetic mutation is not a
candidate responsible mechanism in our nongenomic studies.) Another common genetic
subtype of prostate cancers contains a mutation in the speckle-type POZ protein (SPOP)
gene preventing down-regulation of ARs and promoting cell growth, and is found in 15%
of patients with prostate tumors
24, 25
. Other more rare genetic alterations that have been
found are losses in NKX3.1
and PTEN 27, and gene amplification of the AR (regions
Xq11-Xq13) 28.
Prostate cancer cell lines that are the focus of our study have also been reported to
have some genetic modifications. The hypotetraploid LAPC-4 androgen-dependent
prostate cancer cells possess a deletion of chromosome 12p12
, while chromosomes of
Group A, No.5, No.15, and the entire Y chromosome are absent in androgen-independent
PC-3 cells
. Neither of these cell lines possess the TMPRSS2-ERG gene fusion
Another major gene product, tumor-suppressor p53, is mutated in LAPC-4 cells and not
found in PC-3 cells 32. We did not use the commonly used androgen-dependent cell line
LnCAP because it has a mutated AR, unlike our choice of LAPC-4 cells that have wildtype ARs; PC-3 cells do not possess ARs, contributing to the difference in androgen
requirement for tumor cell growth 32. PC-3 cells are typical of the most advanced cancers
due to their hormone insensitivity, level of dedifferentiation, and chromosomal loss.
Prostate function and anatomy
The prostate is a male reproductive gland that provides seminal fluid that protects
and sustains sperm. During ejaculation, the prostate releases this fluid into the urethra, as
well as prevents urine from mixing with semen
. The prostate is typically divided into
three major zones: the peripheral zone, where about 75% of tumors originate; the central
zone, which rarely has tumors; and the transition zone, where benign prostatic
hyperplasia occurs 34-37. The human prostate can also be divided into four lobes: anterior,
posterior, lateral, and median
.The prostate gland, and tumors that develop from it,
rely on androgens for sustained growth, in particular 5α-dihydrotestosterone (DHT)
The indirect ability of DES and other estrogens to inhibit the growth of prostate cancer
cells has mainly been attributed in the past to a decline in androgen production through
negative feedback control via the hypothalamic-pituitary-testicular axis 42. Androgens are
synthesized in the testes and adrenal glands from cholesterol
. The hypothalamus
releases gonadotropin-releasing hormone (or luteinizing-hormone-releasing hormone,
LHRH), which stimulates the release of luteinizing hormone (LH) and folliclestimulating hormone (FSH) from the pituitary gland. LH and FSH are responsible for
stimulating the production of testosterone (T) and also a small amount of E2 in the Leydig
cells of the testes. T can be further converted into DHT by 5α-reductase, or into E2 by the
enzyme aromatase, which is found and functional in several tissues throughout the body,
including the prostate gland 44-46.
Early-stage, androgen dependent LAPC-4 and late-stage, androgen-independent
PC-3 prostate cancer cells express aromatase, but PC-3 cells have decreased expression
. DHT is essential to normal growth of the prostate, as well as defining male
secondary sex characteristics
. There are epidemiological studies to suggest that
mutations in the 5α-reductase enzyme increases the risk of prostate cancer, particularly in
men of African descent 49. The polymorphism at codon 89 increases enzyme activity and
increases prostatic levels of DHT
. Estrogens, such as E2, are traditionally viewed as
female steroid hormones, but have several important functions in mediating the growth
and differentiation of the prostate. Its specific and broader roles are highlighted later in
this review.
Figure 1.1. Functional domains of AR.
The four major functional domains: N-terminal transactivation domain (NTD); DNA binding
domain (DBD); hinge and ligand binding domain (LBD) (Figure from Gao et al., 2005).
Androgens exert their hormonal actions through androgen receptors (AR), which
are part of the steroid nuclear-receptor superfamily 51, 52. The human AR gene is located
at position 12 on the long arm of chromosome X
form of AR
54, 55
. There is currently only one known
, unlike ER, which has two functional genes within this family of
proteins (although a type of G protein-coupled receptor can also mediate estrogenic
responses, see below), as well as several splice variants 56-58. AR has four major domains:
the N-terminal transactivation domain (NTD); the DNA binding domain; hinge, and the
C-terminal ligand binding domain (Figure 1.1)
. ARs function similar to other steroid
hormones, inducing transcription upon ligand activation of DNA binding. However, ARs
have also recently been associated with nongenomic signaling events
. Genomically
inactive AR is found localized in the cytoplasm, bound to a heat shock protein complex,
including Hsp90, p23, and FKBP 63. Upon ligand binding (typically with androgens), AR
is released from the complex and changes conformation, dimerizes, and becomes
activated through phosphorylation
52, 64
. The AR homodimer then translocates to the
nucleus and binds to androgen-response elements of target genes, leading to cellular
growth, differentiation, and the synthesis and release of prostate specific antigen (PSA)
from the prostate epithelium. PSA is an enzyme found in semen, responsible for
cleaving semenogelin I and II in the seminal coagulum
. This protein is upregulated in
prostate cancer, and has consequently been used as a biomarker for prostate cancer
(Figure 1.2), though the usefulness of this biomarker is currently being
. In advanced prostate tumors, AR function is apparently no longer needed to
sustain cell proliferation, and is often lost entirely
. This transition to hormone
independence is typical of many types of steroid growth-driven tumors
. However,
estrogens, which are not the tumor-sustaining hormone of the prostate, have been
shown to have tumor-shrinking effects on these advanced tumors 3, and we have
hypothesized that these effects are directly mediated by ERs via nongenomic
signaling mechanisms.
Figure 1.2. Canonical AR signaling pathway.
Androgens enter prostate cells through the membrane, then bind to inactive ARs. Binding induces
a conformational change in AR. ARs will then be phosphorylated and dimerize, translocating to
the cell nucleus. Transcription begins after binding to AREs and co-activators (ARA70 and
general transcription activators (GTA)) are recruited. Biological responses include increased PSA
levels, cell growth, and cell survival (Figure from Feldman & Feldman 2001).
Prostate cancer treatment regimens
If a prostate tumor is detected early enough, localized prostatectomy and radiation
can be effective treatments. One of the first forms of therapy developed for prostate
tumors other than tumor resection was orchiectomy, to deprive the tumor of the
androgens that early-stage tumors often require to proliferate. Recent pharmaceutical
advances have presented chemical castration as a viable option over surgical
prostatectomy and castration 1.
Treatment with estrogens, specifically DES, was one of the first regimens
discovered to chemically castrate patients 1. High-doses of DES used to be a typical
treatment for late-stage cancers, but this treatment is no longer available in the United
States. Ethinyl estradiol (EE) given at a dose of 0.05mg/day decreased plasma T serum
concentrations below castration levels
. However, when administered alone, estrogens
such as DES (lowest clinical dosage 1mg/day) and EE can induce thromboembolic
events. Most DES and EE treatments now are given in conjunction with antithromboembolic medication as a result
77, 78
. Serum levels of DES resulting from
treatment administration can reach as high as 10µM
where they work as effective
inhibitors of luteinizing hormone secretion from the pituitary. This causes a subsequent
decline in synthesis and secretion of testosterone from the testes by up to 95%
80, 81
Clinical use of DES in the United States has declined however as a result of
cardiovascular threats and the efficacy of other treatments, such as LHRH agonists,
without the need for additional medication 82, 83.
Research in the 1970-80’s highlighted the use of synthetic LHRH agonists as a
means to decrease serum testosterone levels. In particular LHRH agonists downregulated the LHRH receptors present in the pituitary gland, as well as the subsequent
levels of androgens in the body 84. Some common LHRH analogs currently in use for this
treatment include leuprolide (Lupron®), goserelin (Zoladex®), triptorelin (Trelstar®),
and histrelin (Vantas®)
. Both surgical and chemical castration result in 70-80%
reductions in tumor growth for patients with androgen-responsive cancers (early-stage
. LHRH receptor antagonists such as degarelix (Firmagon®) have also been
recently discovered that work equally as well, with the added benefit of no testosterone
flare (an initial increase in testosterone levels during the first 1-3 weeks of treatment with
LHRH agonist) when first used 86, 87.
Potent anti-androgen therapies were developed between 1960-70
, and have
been shown to reduce tumor size similar to treatments with DES and LHRH agonists
Anti-androgenic compounds are now rarely given as the sole means of treatment, and are
most often used in combination with chemical/surgical castration. Flutamide (Eulexin ®),
bicalutamide (Casodex ®), and nilutamide (Nilandron ®) are currently the most
commonly used anti-androgens
. Anti-androgens can be classified as steroidal or
nonsteroidal. Steroidal compounds can also act on progesterone receptors (as many
steroids can bind an alternative receptor at high concentrations), and often cause
impotence and decreased libido. Nonsteroidal compounds work only on ARs, while
maintaining libido and sexual potency 88.
In recent decades, radiation therapy has become another of frontline therapy 1.
Radiation therapy is most effective when tumor cells are still localized to the prostate
gland, and is often administered in conjunction with other therapies, such as localized
prostatectomy and anti-androgens
. However, X-ray radiation therapy can have
significant negative side effects such as rectal toxicitiy and bleeding, erectile dysfunction,
disruption of proper gastrointestinal function, and urinary incontinence
. Proton beam
therapy has also garnered interest due to the ability to localize high radiation doses on
tumorous cells, but has not received full use yet due to concerns over cost, toxicity, and
long-term effectiveness 93.
Late-stage, usually metastatic and androgen-independent cancers are more
difficult to treat. Chemotherapy is one of the treatments of last resort at this stage.
Docetaxel (Taxotere ®), Paclitaxel (Taxol ®), and Estramustine (Emcyt ®) are some
common chemotherapeutics; use of these compounds has shown up to 75% declines in
serum PSA 94. Radiation however is not feasible once metastasis has occurred, but use of
chemotherapeutics is not specific to the tumor site. Side-effects of chemotherapeutics
range in severity, from hair-loss, rashes, gynecomastia, soreness of the mouth, to
decreased white blood cell count. Estramustine use can elicit severe cardiovascular sideeffects, such as heart attacks, edema, and blood clots
. Additionally, prostate cancer
cells can eventually develop a resistance to chemotherapeutics like docetaxel 96.
Development of androgen-independent tumors
The number of androgen-independent (compared to androgen–dependent) cells in
early tumors has been estimated to 1 in 1,000,000 97, so even the most effective androgen
ablation treatments will leave surviving cells. Androgen-independence often occurs when
tumors return after initial treatment, and these returning tumors are typically more
aggressive. Surviving cells from the initial treatment often develop mechanisms to evade
detection by cellular processes that result in their death. They include: dysregulation of
steroid receptors that sustain growth, like AR; loss of regulatory proteins like glutathione
S-transferase π; and alterations in proliferative cell cycle controls. All are typical
progressive hallmarks of prostate cancer “evolution” to androgen-independent tumors in
. Initial therapies targeting early-stage cancer cells will no longer provide
therapeutic benefit once these cellular changes have occurred.
There are several mechanisms behind the development of advanced androgenindependent cancers
. The first mechanism is not actual androgen-independence, but
rather the development of hypersensitivity to lower concentrations of serum androgens
. Just following castration the majority (~70%) of the tumor cells will die due to lower
androgen levels, but a small subset of androgen-dependent prostate cancer cells continue
to grow
because they utilize low concentrations of androgens more effectively. It is
important to note that this subset of cells is still androgen-dependent, as they die with
complete removal of androgens. Lingering serum androgens in minute, detectable levels
are still produced by the adrenal glands, even after chemical/surgical orchiectomy
Additionally, these tumor cells may also show an increase in AR levels. It is reported that
close to 30% of prostate tumors have increased duplications of AR gene exons, such as
exon 3 in 22Rv1 cells, as well as subsequent increases in AR production and expression
28, 103-105
. These higher levels of AR may increase the sensitivity of the tumor to minute
amounts of androgens. Interestingly, this phenomenon also occurs in breast cancers,
suggesting that this is a common exploit for endocrine tumors 106.
Another mechanism of androgen-independent growth is the ability of ARs to bind
other ligands, which then mimic regular androgen/AR activity. Mutations in the AR
ligand binding domain allow for higher levels of promiscuous binding of other
hormones, such as estrogens, or small molecules resembling androgens
107, 108.
commonly used prostate cancer cell line LnCAP harbors an alanine to threonine
substitution at residue 877, and allows estrogens, and other steroid hormones to bind
readily 107. The MDA prostate cancer cell line also has a promiscuous mutated AR, which
can bind glucocorticoids, but deceases the ability of androgens to bind
109, 110
. Aberrant
binding of non-androgenic ligands then leads to inappropriate AR activity and signaling.
Other mechanisms supporting androgen-independent growth include the ability of
proliferative cellular pathways to bypass hormonal control and activate without any
androgenenic ligand or AR present. These pathways are stimulated by or utilize growth
factors and kinases, which then stimulate AR activity
111, 112
. Insulin-like growth factor
(IGF) stimulates cell proliferation by activating the phosphoinositide 3-kinase (PI3K)
signaling pathway and inhibiting of FOXO1’s suppressive activity on AR-DNA binding,
increasing AR transactivation
. Epidermal growth factor (EGF) increases signal
transducers and activators of transcription 3-AR (STAT3-AR) complex formation and
AR activity, in conjunction with IL6 114. Vascular endothelial growth factor C (VEGF-C)
is up-regulated in tissues with low androgen concentrations, such as with androgenindependent prostate cancer cells, and increases AR activity by stimulating the expression
of BAG-1L, an AR co-activator
. Improper AR activity in the absence of androgens
may be mediated by the NTD of the receptor - amino acids 294-556 were found to be
required for androgen independent localization to the nucleus 116.
Nongenomic signaling through ARs has been relatively unstudied to date, but has
been shown to mediate growth-stimulatory effects. These signals can emanate from
caveolin-1 through Akt and ERK, preventing apoptosis
. Nongenomic signaling
through ARs can also rapidly activate L-type calcium channels through an inhibitory Gprotein, stimulating PKC activity and increasing gene transcription
. In the nervous
system, androgens can rapidly activate the ERK signaling pathway, serving as a
neuroprotective against against β-amyloid 119 and serum deprivation 120.
Another way to increase the number of cells in a tumor is to block the process of
apoptosis. The anti-apoptotic bcl-2 gene in androgen-responsive cells is suppressed by
androgens, but is overexpressed in androgen-independent tumors, allowing for
uncontrolled growth
121, 122
. Endothelin-1, which is produced from cells of the prostate
epithelium, downregulates pro-apoptotic proteins, through phosphorylation of Akt and
. On the other hand, some androgen-independent prostate cancer cells have also
developed a resistance to TRAIL-induced apoptosis through constitutively active Akt
. This immunity to apoptosis then allows the surviving androgen-
independent cells to grow unchecked.
Evading destruction by the body’s immune system is another key hallmark of
cancer 125. Cells that are androgen-dependent will die when androgen-ablation therapy is
given, while the subset of cells that are androgen-independent will survive or “lurk”,
evading immune cells that would otherwise kill them
. The cells employ mechanisms
such as recruiting immune cells that inhibit effector T-cells
maturation of dendritic cells via increased VEGF secretion
127, 128
and preventing
. These surviving “lurker”
cells then slowly repopulate the tumor 130.
Estrogens and estrogen receptor physiology
As described above, estrogens such as DES have shown some beneficial indirect
effects by controlling androgen production that supports prostate tumor growth.
However, the potential direct effects of estrogens on prostate cancer cells are not
well understood
In particular, the nongenomic effects of estrogens, as mediated
by mERs, is entirely unknown in this type of tumor cell.
Estrogens are traditionally viewed as a female hormone, and are responsible for
the regulation of the female reproductive cycle and proper development of secondary
sexual characteristics. Estrogens also have many regulatory functions in males though,
such as proper storage of mature sperm cells
, cardiovascular health
, differentiation of the reproductive
, and potentially mental health
. Estrogen
deficiency in males can lead to impaired reproductive fecundity, decreased libido,
uncontrolled bone growth, and brittle bones 138.
ERα or ERβ
Figure 1.3. Summary of roles for membrane and intracellular ERs.
mERs initiate rapid, nongenomic signaling cascades after estrogenic stimulation, leading to such
signaling changes as second messengers (ion flux, lipid signals, cyclic nucleotides) cell cycle
protein posttranslational changes, and kinase and other enzyme activations. Examples of
endpoints are shown such as release of secretory vesicles, efflux, or, trafficking of transporters.
Stimulation of nuclear ERs leads to long-term transcriptional effects. (Figure from Watson 1999).
There are currently three known ERs - α, β, and GPR30
139, 140
. ERα and β are
members of the same (nuclear receptor) gene superfamily and the product of two separate
genes, ESR1 and ESR2
. They each have their own splice variants (Figure 1.4)
that can still be active, such as in the activation of MAPKs by the truncated ERα36 144, or
regulation of proliferative transcription factors by ERβ2
. These truncated forms lack
the transcriptional transactivation capabilities of full-length ERs, but can still mediate
nongenomic responses by coordinating the activities of other signaling molecules.
Truncated forms of ERα have typically shown activity at or near the plasma
146 147
in multiple cell lines. ERα46, but not ERα36, has been shown to bind
the same ligands (including several phytoestrogens and antagonists used in our studies) as
full-length ER, though at much lower binding affinities
E2-induced eNOS activation in endothelial cells
. ERα46 is involved in rapid,
. ERα36 in endometrial cells is
involved in rapid, membrane-initiated ERK and PI3K/Akt pathway signaling in response
to testosterone stimulation
. Truncated forms of ERα have not been reported in
prostate cells yet, but many antibodies used in studies (such as the MC-20 antibody) do
not distinguish between full-length ERα and its truncated forms 151. ERβ1 (wild-type) and
ERβ2 have currently been identified and studied for their roles in proliferation and
metastasis in PC-3 cells and other cell lines, but not in LAPC-4 cells
145, 152, 153
. ERβ
antibody clone 9.88 has not currently been shown to distinguish between wild-type or
ERβ2, but other researchers have developed antibodies specific to truncated forms 154, 155.
ERβ2 has been reported to not bind ligands, but may instead serve to repress
transcriptional activity of full-length ERβ
156, 157
. GPR30 is an entirely different type of
protein -- a seven-transmembrane receptor G-protein coupled receptor so far known to be
involved only in nongenomic actions of estrogens 158.
AF-1 & AF-2 (Activation Function 1 & 2); DBD – DNA-Binding Domain; LBD – LigandBinding Domain
Figure 1.4. ERα and ERβ splice variants.
Domains of the ERs and their splice variants. From left to right: the N-terminal A/B domain that
encodes AF-1; DNA binding domain; the hinge domain; and the C-terminal E/F domain that
encodes the ligand binding domain and AF-2. Many of the splice variants are still functional ERs,
such as ERα36, ERβ2, and ERβ5. Also listed are sites of post-translational modification. Red
triangles indicate the epitopes that are bound by the ER antibodies used in this study (Figure
modified from Thomas & Gustafsson 2011).
Various post-translational modifications can control ER function 159 (Figure 1.4).
Acetylation is mediated by E2 and cAMP responsive element protein, which can help to
stimulate or inhibit the transcriptional activity of ERα
. Glycosylation influences the
cellular fate and destination for ERα and ERβ and many other proteins
Nitrosylation of ERα impairs or inhibits the receptor from binding to EREs
163, 164
Myristoylation and palmitoylation can also direct ERs or their variants to the cell
. Membrane ERs are localized to the membrane via palmitoylation,
tethering them close to the inner surface, mainly in association with caveolae rafts
Disruption of palmitoylation or the interaction with caveolae has negatively affected cell
. It is currently still unknown if mERs are able to span the entire plasma
membrane, due to difficulties studying the subcellular location 169, but this is unlikely due
to the exposure of many epitopes all along the protein on the outside of the cells
These post-translational modifications may also offer a rapid form of ER regulation
without the need to increase or decrease receptor levels.
ERs are expressed in the prostate gland, and contribute to proper function
and development 174. ERs are found both on the membrane and intracellularly in several
cell types, but this has not yet been fully characterized in prostate cancer cells. The
canonical nuclear pathway of action involves estrogenic ligands passing through the lipid
bilayer of the cell and binding to intracellular receptors
from heat shock protein complexes
. The receptors are released
, then dimerize and bind to estrogen response
elements (ERE) to initiate transcription while partnering with coregulator proteins
139, 176
However, membrane initiated responses do not invoke genomic actions, at least
They rapidly activate signaling pathways that cause second messenger
enzymatic synthesis (cyclic nucleotides, phospholipids) or movement (ion flux
resulting in functional changes such as peptide hormone release
location of transporters or channels
182, 183,
178, 184, 185,
170, 181,
activity or
or cell proliferation
186, 187.
Roles of membrane estrogen receptors in nongenomic responses
ERα and ERβ are localized to the plasma membrane through palmitoylation,
while the location of GPR30 has been debated between the endoplasmic reticulum 188 and
the plasma membrane
. mERα and mERβ do not appear to be different from their
nuclear counterparts 189, 190, other than their localization to the plasma membrane through
post-translational modifications
. Palmitoylation at Cys-447 is necessary for ERα
membrane localization and to induce nongenomic signals via the MAPKs – alteration of
this site can lead to cellular and tissue disfunction (loss of signaling pathways, infertility,
disrupted vasculature) 191, 192.
Nongenomic signals as elicited by estrogens can have a variety of functions. Our
laboratory has shown a host of these signaling events and outcomes using several assays
and cell lines. Cell survival and death are usually tied to transcriptional events that take
hours to days to manifest. However, estrogenic compounds working through mERs
initiate the signaling pathways leading to these outcomes in a matter of seconds to
minutes. These signals may also accumulate and have sustained effects, or modify (often
phosphorylate) downstream molecules involved in transcription
. In benign prostatic
hyperplasia, E2 in conjunction with serum hormone binding globulin rapidly increases
(<15 min) cyclic adenosine monophosphate
, a signaling molecule that can lead to cell
proliferation and transcriptional regulation of genes
. While nuclear and membrane
ERs are quite similar, they can initiate different cellular outcomes in response to the same
ligand because they partner with different other proteins.
The genomic and nongenomic signaling pathways are not always independent of
each other. Transcriptional events initiated by nuclear receptors can by influenced by
signaling pathways that are activated by membrane receptors. For instance, the Watson
laboratory previously reported that the transcription factors Elk and ATF-2 are rapidly
phosphorylated following stimulation by E2 or xenoestrogens (XEs) 193. ERs also regulate
transcription without binding to EREs in DNA. Instead, nuclear ERs can form complexes
with proteins such as AP-1
, Sp1 transcription factor, or NF-κβ transcription factor,
after stimulation with E2 or another estrogenic ligand
transcription of IGF-I 198, cyclin D1
. These complexes then regulate
, c-fos 200, and IL-6
. Thus, multiple immediate
and delayed cellular outcomes can result from a single ligand binding event.
Estrogen receptor control of cell proliferation or death
Estrogens and ERs regulate cell fates in a variety of cell types. E2 can
phosphorylate and activate mitogen-activated protein kinases (MAPKs) in tumor cells
originating from the pituitary, neurons, heart, and breast 181, 202-204. MAPKs include ERK,
c-Jun N-terminal kinase (JNK), and p38. ERK activities are often associated with cell
205, 206
, JNK activities are often associated with cell death and apoptosis.
Phosphorylated JNK promotes apoptosis by activating the intrinsic caspase cleavage
pathway of cell death and the release of cytochrome c from mitochondria. JNK can also
further promote cell death by deactivating suppressors of TNF-α driven apoptosis
JNK can also be activated by reactive oxygen species (ROS) after inhibition of MAP
kinase phosphatase activity, leading to eventual apoptosis
. Phosphorylated p38
participates in control of cell differentiation 209, proliferation 205, and death 210 in response
to cellular stress
, inflammation 212, and UV radiation 213. Estrogens can also use other
signaling cascades such as activation of the PI3K/Akt signaling pathway
, or can even
inhibit cell death in some cases by regulating the anti-apoptotic Bcl-2 protein
. Our
laboratory and others have also shown that cyclic nucleotides can be increased in
response to estrogens, through adenylyl cyclase and protein kinase A, in a rapid,
nongenomic manner
stimulating gene transcription and regulating cell
proliferation. GPR30 can inhibit cell proliferation, such as with breast cancer cells
stimulate proliferation, as with thyroid cancer cells
, or
. Therefore, there are many
different signaling interactions initiated at the membrane in which ERs of different types
can be involved.
Estrogens and ERs have also been shown to be important regulators of cell
number in a host of organs. In breast tissue, estrogens via ERs stimulate cell
proliferation at various life stages (puberty, pregnancy, menopause) by increasing
production of several growth factors and altering expression of a host of genes
(amphiregulin, VEGF, loss of Slit2, frizzled-related genes FRP1/FRZB)
218, 219
. Bone
mass (including cells and their products) is regulated by estrogens, which drive the
proliferation of osteoblasts and or death osteoclasts through anti- and pro-apoptotic gene
. The ER subtypes in ovarian cells may be a determinant of sensitivity to
agents that cause ovarian cancer to develop, as well respond to treatments
221, 222
. The
prostate requires estrogens for proper development, including cell proliferation, while ER
status may also influence further tumor development and metastatic potential
223, 224
Estrogens in the colon are necessary for cell growth and division, and may play a
protective role against tumor development, specifically through ERβ
225, 226
. Normal lung
function is maintained by estrogens and their control of cell number, such as formation of
alveoli during development and repair in adulthood 227.
Estrogen receptors in the prostate and prostatic disease
The effect of estrogens on prostate cancer are thought to be mediated by ERβ, the
dominant form of the receptor in the tissue, but this conclusion must await further
examination of all receptors that are present. ERβ is regularly expressed at high levels
in the healthy prostate
compared to ERα
and is mainly found in the epithelial cells vs. the stroma,
During the progression of prostate cancer, the levels of ERα
increase throughout the gland, while ERβ levels decline (yet remain functional), even in
advanced cases of the disease 19, 229, 230. The expression of splice variants ERβ2 and ERβ5
increases metastatic potential
223, 231
. Additionally, exposing neonatal rats to E2 and DES
severely stunted the regular growth of the prostate gland. Interestingly, ERα was shown
to be responsible for this inhibitory effect
, which is different given ERα is often
associated with proliferative effects in other cell types
. ERβ is not initially expressed
in the development of the prostate, and ERα is responsible for regulating cell growth.
Androgen signaling decreases ERα levels, removing the regulation of ERβ expression,
allowing ERβ to rise, and prostate maturation and differentiation to occur
. Selective
estrogen receptor modulators for ERα did not work as a beneficial treatment for advanced
235, 236
prostate cancer patients,
cancer cells in vitro
, though they did inhibit proliferation of PC-3 prostate
, perhaps suggesting an additional compensatory mechanism
present in the actual tumor environment. GPR30 can inhibit prostate cancer cell growth
after treatment with the agonist G-1, through sustained activation of ERK, up-regulation
of the cyclin-dependent kinase p21, and cell cycle arrest in G2 238. It is apparent though
that the overall role of the separate ER subtypes in the prostate may be reversed, and also
depend on male life-stage.
Xenoestrogens and effects on living systems
ERs often bind other estrogenic compounds besides physiological estrogens.
Phytoestrogens, such as coumestrol, genistein, and resveratrol, are estrogens found in
plants. Pharmaceutical estrogens include the aforementioned DES, used clinically, while
EE is used in contraceptives. Synthetic estrogens are man-made compounds found in
many every-day products and are pervasive in the environment. Examples are bisphenol
A (BPA) in plastic products, nonylphenol in paints and oil dispersants, and
dichlorodiphenyltrichloroethane. These estrogen mimetics/XEs can function similarly to
physiological estrogens and activate the same cellular pathways (often inappropriately)
239, 240
, but they are not identical in their actions. Phytoestrogens have been touted as
potential preventatives or therapeutics for diseases
, while environmental estrogens are
pervasive and are associate with negative effects on the health of wildlife, humans, and
the ecosystem
. A further review of XEs is in Chapter 3, highlighting the overall
ability of XE, at dietary- or environmentally-relevant concentrations, to stimulate cell
growth by utilizing signaling pathways differently from E2 and DES, as well as the varied
ER requirements to elicit such cellular responses.
Figure 1.5. Comparison of the structures of E2, DES, BPA, and three phytoestrogens.
Our laboratory has shown that E2 induces rapid, non-genomic signaling as well as
cell death in MCF-7 breast cancer cells expressing high levels of mERα
186, 204
. Our
laboratory has also reported that XEs, agonistic estrogen-like compounds (synthetic
chemicals/phytoestrogens) induce cell proliferation or apoptosis, dopamine transporter
efflux, and prolactin release in MCF-7 breast cancer, PC-12 pheochromocytoma, and
GH3 pituitary cells, respectively 182, 193, 245. The underlying, rapid and direct mechanisms
that contribute to cell proliferation or cell death, in response to estrogens/XEs, have not
been examined in prostate cancer cells. Therefore, we hypothesized that estrogens
directly inhibit prostate cancer cells, rapidly initiating signaling cascades
(MAPKs/caspases) that lead to inhibition of cell proliferation or induction of cell
death. In addition, we hypothesized that XEs also utilized these same pathways,
although not necessarily with the same signaling magnitude or receptor signature of
endogenous estrogens, and that mERs were required in influencing their antiproliferative vs. proliferative effects on prostate cancer cells.
Aims of studies in this dissertation
The work and data for this dissertation can be divided into two parts. The first part
focuses on identifying key cellular pathways that are involved in regulating cell
proliferation or death using E2 or DES, a physiological estrogen vs. a pharmaceutical
mimic. While there are innumerable mechanisms to look at, this work focuses on the
rapid activation or phosphorylation of MAPKs and cell cycle proteins and their
consequences. We also looked at the induction of ROS, apoptosis via caspase 3
activation, and necroptosis (a relatively novel form of programmed cell death). The
second part of this dissertation focuses on the effects of XEs on prostate cancer cells,
with a particular interest in the specific mERs that are present and necessary to elicit or
prevent cell proliferation or death.
Aim 1. To identify and characterize rapid and direct cell mechanisms
involved in E2- or DES-initiated cell proliferation or death of androgen-dependent
LAPC-4 or androgen-independent PC-3 prostate cancer cells.
We will examine several rapid (nongenomic) signaling pathways that have an
influence over cell proliferation or death of prostate cancer cells, as initiated by E 2 or
DES. We anticipate that E2 or DES at physiologically or clinically relevant
concentrations have direct and likely rapid effects on prostate cancer cells that could
provide therapeutic benefits besides central inhibition of androgen production. Multiple
mechanisms, such as MAPK activation, cell cycle protein phosphorylations, and ROS
induction, will be examined to determine if they contribute to the overall balance between
cell proliferation and death of prostate cancer cells. The ERs important for regulating
these pathways will also be elucidated.
Aim 2. To determine if XEs exert anti-proliferative/proliferative effects,
using the same signaling pathways and ERs identified with E2 and DES, in LAPC-4
and PC-3 prostate cancer cells.
We will identify and compare the membrane and intracellular levels of ERs (α, β,
GPR30) that are present in both LAPC-4 and PC-3 cells. Then we will study the XEs
BPA, coumestrol, genistein and resveratrol for their effects on cell viability/proliferation
and cell death, as well as the key cellular pathways identified from Aim 1. The XEs will
be evaluated at environmentally or dietary-relevant concentrations (10-14 M to 10-6 M).
The results of this research should contribute to the current body of knowledge by
showing a direct mechanism through which estrogens and XEs initiate proliferative/antiproliferative effects via mERs in prostate cancer cells. Additionally, the results of this
project should aid other researchers and clinicians in developing much more
specific therapeutic compounds that target the relevant ERs or cell signaling
pathways. At the same time, expounding the proliferative/anti-proliferative effects of
XEs binding to mERs will elucidate the risks or benefits of chronic exposure to
environmental contaminants or dietary phytoestrogens in existing tumors. These results
will also potentially lead to changes in regulating environmental concentrations of
estrogenic compounds or recommendations about dietary intake of phytoestrogens for
cancer patients.
Chapter 2: Direct estradiol and diethylstilbestrol actions on early- vs.
late-stage prostate cancer cells1
Diethylstilbestrol (DES) and other pharmaceutical estrogens have been used at
≥µM concentrations to treat advanced prostate tumors, with successes primarily
attributed to indirect hypothalamic-pituitary-testicular axis control mechanisms.
However, estrogens also directly affect tumor cells, though the mechanisms involved are
not well understood. LAPC-4 (androgen-dependent) and PC-3 (androgen-independent)
cell viability was measured after estradiol (E2) or DES treatment across wide
concentration ranges. We then examined multiple rapid signaling mechanisms at 0.1 nM
E2 and 1µM DES optima including levels of: activation (phosphorylation) for mitogenactivated protein kinases, cell-cycle proteins, and caspase 3, necroptosis, and reactive
oxygen species (ROS). LAPC-4 cells were more responsive than PC-3 cells. Robust and
sustained extracellular-regulated kinase activation with E2, but not DES, correlated with
ROS generation and cell death. c-Jun N-terminal kinase was only activated in E2-treated
PC-3 cells and was not correlated with caspase 3-mediated apoptosis; necroptosis was not
involved. The cell-cycle inhibitor protein p16INK4A was phosphorylated in both cell lines
by both E2 and DES, but to differing extents. In both cell types, both estrogens activated
p38 kinase, which subsequently phosphorylated cyclin D1, tagging it for degradation,
except in DES-treated PC-3 cells. Cyclin D1 status correlated most closely with disrupted
cell cycling as a cause of reduced cell numbers, though other mechanisms also
contributed. As low as 0.1 nM E2 effectively elicited these mechanisms, and its use could
Chapter 2 taken from Koong LY & Watson CS (2014). Direct estradiol and diethylstilbestrol actions on
early- vs. late-stage prostate cancer cells. The Prostate. 74(16):1589-1603.
dramatically improve outcomes for both early- and late-stage prostate cancer patients,
while avoiding the side effects of high-dose DES treatment.
Prostate tumors are usually androgen-dependent in the beginning, so front-line
therapies are initially aimed at reducing the amount of free androgens that sustain these
tumors. Traditional therapies to treat androgen-dependent tumors have included radiation
and surgical removal of tumors, followed by androgen receptor antagonists, or
pharmaceutical castration with luteinizing-hormone-releasing hormone (LHRH) analogs
that block androgen production 246. However, these treatments become ineffective against
tumors that have previously regressed with treatment, and have then escaped these
controls to grow again. These recurrent or advanced prostate tumors often develop
androgen-independence, and as an alternative, synthetic estrogens like diethylstilbestrol
(DES) have been used as treatments
. Estrogen treatment is thought to indirectly
decrease androgen production by negative feedback control on the hypothalamicpituitary-testicular axis
, though with the proven androgen-independence of these
tumors, this may not provide much additional therapeutic benefit. Current clinical
practice employs high doses of DES (≥µM), which can cause many unwanted side-effects
in patients
including erectile dysfunction, decrease in sex drive, weight gain,
gynecomastia, and cardiovascular problems.
Estrogens (or their mimics) could control cellular proliferation or death via a
variety of mechanisms which we examined in these studies. They bind to both
intracellular and membrane receptors that transduce extracellular signals to downstream
186, 247-249
. Downstream mitogen-activated protein kinases (MAPKs) are
responsive to a large number of external stimuli and are nodes of signaling integration for
estrogenic signals, as well as those from other classes of ligands, via their receptors
. There are three primary MAPK subclasses: extracellular signal-regulated kinases
(ERKs); c-Jun N-terminal kinases (JNKs); and p38. ERKs 1 and 2 are well known for
being involved in controlling cell proliferation
by integrating upstream signals to
regulate cell cycle proteins by post-translational modifications
. ERKs also propagate
the signaling cascade to other important cellular response molecules such as c-Myc
, eIF4E
and cyclin D1
. On the other hand JNK is better known for
triggering cellular apoptotic responses, for instance after UV radiation or other types of
DNA damage 208, 257, 258. The MAPK called p38 has some functions similar to JNK, but is
more typically reported to be involved in cytokine, cellular stress, and escalating
inflammatory responses. The p38 MAPK can also control cell number by cyclin D1
phosphorylation at Thr-286 which ubiquitin-tags it for rapid degradation through
, cyclin-dependent kinase (CDK) regulation at major checkpoints
or inducing apoptosis through cellular stress mechanisms
256, 260
. Therefore, there are
multiple mechanistic pathways via which MAPKs are important regulators of cell
A novel role of ERK phosphorylation is the generation of high levels of ROS,
which in turn further sustain ERK activation through the inactivation of dual-specificity
phosphatases. This prolonged activation of ERK will then produce even higher ROS
levels, creating a positive feed-forward mechanism for ramping up this destructive
response quickly. Generation of ROS can lead to cell death via the initiation of apoptosis,
cell senescence mechanisms, or autophagy 262.
As cell cycle proteins drive cell division, they directly cause increased cell
numbers; therefore inhibition of these mechanisms can cause decreases in cell numbers as
cells die and are not replaced. The cell cycle is tightly regulated, being controlled at each
major checkpoint by cyclins and CDKs, as well as inhibitory proteins acting on them.
Cyclin D1 controls the G1/S transition with its partners CDK4/CDK6
. Cyclin D1
protein levels can be changed via p38 phosphorylation (resulting in degradation),
depending on the stage of the cell cycle. CDK4/CDK6 can be regulated by the cell cycle
inhibitor p16INK4A 264, which when phosphorylated binds to CDK4/CDK6, preventing the
formation of the cyclin D1-CDK4/6 holoenzyme required for successful cell cycle
progression. Therefore, signals that activate or inhibit this cascade have profound effects
on cell numbers.
Programmed cell killing can also play a major role in controlling cell numbers.
Several effector caspases feed into executioner caspases, represented in our studies by
caspase 3
, that dismantle DNA. Necroptosis is an alternative, relatively newly
described method of programmed cell death. Cells undergoing this process have
morphologies similar to necrotic cells (plasma membrane integrity loss, increases in cell
and organelle volume), but do not require the activation of caspases
. Though
necroptotic cells are best known to initiate the death process after Fas ligand (FasL)
binding, tumor necrosis factor α (TNFα), or TNF-related apoptosis-inducing ligand
(TRAIL) stimulation
, a growing number of other receptors and stressors are being
reported to be involved in this mechanism
(RIPK1) serves as a scaffold for this process
. Receptor-interacting protein kinase 1
, and the selective inhibitor necrostatin-1
keeps it in an inactive conformation, thus preventing necroptosis 270.
We and others have previously shown that rapidly initiated (nongenomic) steroidinduced signaling events are involved in the modulation of tissue size/cell number
changes (involving both cell proliferation and cell killing) in tumor cell types that contain
membrane estrogen receptors
249, 271, 272
. We now predict that these mechanisms could
also be active in prostate cancer cells, where the expected direct response to estrogens
would be to mediate rapid cellular signaling leading to cell killing, or the slowing of cell
proliferation; direct mechanisms of action of estrogens on prostate tumor cells are still
relatively understudied. Better understanding of such a direct effect could result in
significant improvements in treatment strategies to suppress tumor growth while reducing
harmful side effects due to current high dose DES treatments.
Materials and Methods
Cell Lines and Reagents: We chose cell lines representing the two main types of
human prostate cancers -- androgen-dependent vs. androgen-independent. LAPC-4
androgen-dependent prostate cancer cells 29 were maintained to sub-confluence in phenol
red-free Iscove’s Modified Dulbecco’s Medium (IMDM, MediaTech - Manassas, VA)
with 10% fetal bovine serum (FBS) (Atlanta Biologicals – Lawrenceville, GA), 4 mM Lglutamine (Sigma-Aldrich – St. Louis, MO), and 10 nM dihydrotestosterone (SigmaAldrich). PC-3 androgen-independent prostate cancer cells 30 were maintained by growth
in phenol red-free RPMI 1640 (Sigma-Aldrich) with 10% FBS and 2 mM L-glutamine.
Both cell lines were propagated at 37°C and 5% CO2. E2 and DES (Sigma-Aldrich) were
dissolved in ethanol to a stock concentration of 10 mM (final concentration of EtOH
0.001%) before serial dilution into IMDM or RPMI 1640 at concentrations ranging from
10-14 M to 10-6 M for our studies.
MTT Cell Viability Assay: Cells were plated at 5,000 cells/well in poly-D-lysinecoated 96-well assay plates (BD Biosciences – Bedford, MA; 96-well assay plates:
Corning – Tewksbury, MA), and then allowed to attach overnight. The next day, 100 µl
of medium containing 1% four times charcoal-stripped FBS, plus either vehicle, E2, or
DES was added. After three days, treatment solutions were removed and 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) was
added for 1 hr. Cells were then lysed and the signal read at 590 nm in a Wallac 1420 plate
reader (Perkin Elmer – Waltham, MA) .
Plate Immunoassays: Phosphorylated proteins were assayed by recognition with
antibodies (Abs) specific for these post-translationally modified epitopes:
(Thr202/Tyr204), pJNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), phosphocyclin D1 (Thr286) (all from Cell Signaling – Danvers, MA), or phospho-p16INK4A
(Ser152) (Thermo Scientific – Rockford, IL). Changes in cyclin D1 levels were measured
by using an Ab to the total cyclin D1 (recognizing both modified and unmodified protein;
Cell Signaling Cat. No. 2922). A plate immunoassay developed previously by our
, was adapted for use with the BIOMEK FXP workstation (Beckman
Coulter – Brea, CA) to automate the majority of the liquid handling, thereby reducing
experimental variability and increasing experimental output.
Prostate cancer cells were plated at 10,000 cells/well in 96-well assay plates,
allowed to attach overnight, and weaned from steroids and other small molecules in the
growth media by treatment with 100 µl of media with 1% four times charcoal-stripped
FBS for 48 hr. Cells were then treated with E2 or DES for up to 60 min on the
paraformaldehyde, 1% glutaraldehyde, 0.5% Nonidet P-40, 0.15 M Sucrose). Primary Ab
to the phosphorylated epitopes was then added and incubated with the cells overnight.
The next day, biotinylated anti-mouse/anti-rabbit IgG secondary Ab (Vector Labs –
Burlingame, CA) was added for 1 hr. Next, cells were incubated with avidin-biotinylated
conjugated alkaline phosphatase (ABC-AP, Vector Labs) for 1 hr, then for 30 min with
para-nitrophenylphosphate substrate (Sigma Aldrich), allowing the yellow color of the
para-nitrophenol product to accumulate. Plates were read at 405 nm in a Wallac 1420
plate reader. Readings were then normalized to cell number, estimated by the crystal
violet dye (Sigma-Aldrich) assay, as described previously 274.
ER Antagonist Assays: To investigate the involvement of different ERs and their
potential role in altering ROS formation or cyclin D1 phosphorylation, the following ER
antagonists were used at their most selective concentrations: for ERα, 10-7 M 1,3-bis(4hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole
dihydrochloride (MPP, Tocris Bioscience – Minneapolis, MN); for ERβ, 10-6 M 4-[2phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl] phenol PHTPP (Tocris);
and for GPR30, 10-6 M G15 (Tocris). Cells were pre-incubated with antagonists for 30
min prior to estrogen treatments.
Caspase 3 Assays: To determine estrogen-induced activations of caspase 3, cells
were seeded into black optical 96-well plates (Corning) at a density of 5,000 cells/well
and allowed to attach overnight. Estrogen treatments were started the next day in media
with 1% four times charcoal-stripped FBS, for times ranging from 2-24 hr (only the 8 hr
optimum time point data are shown in Fig. 2.5). After treatment, plates were centrifuged
at 300 g for 5 min, and treatment-containing media were suctioned off. Cells were then
lysed with 50 µL of lysis buffer (10 mM HEPES; 2 mM EDTA; 0.1% CHAPS; 1 mM
DTT; pH 7.4) and stored at -20°C until assay. Assay buffer (50 µL of 50 mM HEPES;
100 mM NaCl; 0.1% CHAPS; 1 mM EDTA; 10% glycerol; 10 mM DTT; pH 7.4)
containing a 50 µM final concentration of Ac-DEVD-AFC caspase-3 assay substrate
(Enzo Life Sciences – Farmingdale, NY) was added. The cellular enzyme-catalyzed
release of 7-Amino-4-trifluoromethylcoumarin was monitored using a FlexStation 3
microplate reader (Molecular Devices – Sunnyvale, CA) at an excitation wavelength of
400 nm and an emission wavelength of 505 nm. Staurosporine at 1µM was used as a
positive control for inducing caspase activity.
Necroptosis Assays: This mechanism was defined by the use of a selective
necroptosis-inhibitor in the MTT assay (described above). Cells were treated for three
days with EtOH (0.0001%) vehicle or 10-10 M E2 or 10-6 M DES; TNFα (10 ng/ml,
Millipore) plus cyclohexamide (10 µg/ml, Sigma-Aldrich) were used together to provide
a positive control for necroptosis. Necrostatin-1 (20 µM, Millipore – Billerica, MA) was
used to specifically define necroptosis by the inhibition of RIP1 kinase 275.
ROS Assays: Cells were plated at 10,000 cells/well in a 96-well plate, allowed to
attach overnight, and then treated with 100 µl of media containing 1% four times
Dichlorodihydrofluorescein diacetate (DCDHF) (Enzo Life Sciences) for 1 hr. Then the
production of ROS was measured in cells after E2 or DES treatment for 15 min.
Hydrogen peroxide (1µM, Fisher Scientific – Pittsburg, PA) and ethanol (0.0001%) were
used as positive and negative controls, respectively. For both E2 and DES treatments, the
concentrations spanned 10-14 to 10-6 M. Dichlorofluorescein production, formed as a
result of ROS/DCDHF interaction, was measured at an excitation of 485 nm, and an
emission of 538 nm in a SpectraMax M3 Multi-Mode Microplate Reader (Molecular
Devices). For studies with MEK inhibitor U0126 (Promega – Madison, WI), cells were
co-incubated with 10-7M inhibitor during the last 30 min of the DCDHF incubation.
Statistics: One-way analysis of variance was conducted for each experiment. A
Holm-Sidak post hoc test was used to measure the significance of each treatment vs. the
vehicle control. Significance was set at P<0.05.
Figure 2.1. Cell viability after 3 days of E2 or DES treatment.
LAPC-4 and PC-3 prostate cancer cells were treated with either estrogen, and cell viability was
measured by the MTT assay. In all figures throughout the manuscript white symbols denote
LAPC-4 cells and black symbols PC-3 cells; triangles represent E2 treatments, and circles
represent DES treatments. *denotes significance from vehicle (V) controls at P<0.05, and shaded
gray bars represent the response to vehicle ± SEM.
Cell Viability: E2 treatment (represented by triangles in this and all subsequent
line graphs) for three days effectively decreased the number of viable cells by 20-30%
below the vehicle treatment level at all concentrations tested (10-14 to 10-6 M) in LAPC-4
androgen-dependent (early-stage) prostate cancer cells (represented by white symbols
and bars in all figures) (Fig. 2.1A) and at 10-10 to 10-8 M concentrations in PC-3
androgen-independent (late-stage) prostate cancer cells (represented by black symbols
and bars in all figures). DES treatment (represented by circles in all line graphs)
decreased cell viability by as much as 20% in LAPC-4 cells only at 10-14 to 10-12 M and
10-6 M, while the cell viability of PC-3 cells was not significantly decreased by DES at
any concentration (Fig. 2.1B). For comparison (positive control), when these cells were
grown in complete serum-containing media, LAPC-4 cell numbers increased by 27%,
while PC-3 increased by 19% (not shown).
Phospho-ERK Driven ROS Accumulation and Phospho-JNK Driven Caspase
Activation: In both LAPC-4 and PC-3 cells, most concentrations of E2 and DES elicited
similar rapid ERK and JNK activations and deactivations in a typical oscillating time
pattern 181, 204, 276 (Figs. 2.2 & 2.4). However, there were some instances where values for
ERK activations at some concentrations did differ (for E2 at 1 and 10 min, and for DES at
1 and 15min); we chose the non-variable 5 min time point for subsequent measurements
at single time points. To better visualize the composite temporal pattern, the insets in
these figures show the averages of these changes for all concentrations. Because of these
largely similar responses (most not significantly different from one another due to
concentration), we chose conditions for all further assays to mimic physiologically (10-10
M E2)- and clinically (10-6 M DES)-relevant concentrations that were also active in
reducing cell viability (see Fig. 2.1). The response for those chosen concentrations is
shown in bold lines for each in Figs. 2.2 and 2.4. At these selected concentrations E2treated LAPC-4 cells showed a rapid and significant increase in phospho-ERK levels
(16%) after 5 min (Fig. 2.2A), and a maximal response (26%) after 15 min of treatment.
After falling to control levels, activation was seen again at 60 min, as we have often
observed in other cell lines 181, 204, 273. A similar but less robust response was seen in PC-3
cells [ERK activated by 8% after 10 min of E2 treatment, with another rise to 18% at 60
min (Fig. 2.2B)]. In both cell lines, DES at most concentrations moderately, though
significantly, decreased phospho-ERK levels rapidly (Fig. 2.2C & D). In LAPC-4 cells,
the ≤20% decrease in phospho-ERK levels was mostly maintained throughout the time
course. The activations of ERK in our studies were not consistent with the traditional role
for ERK in causing cell proliferation, as our cells numbers instead declined.
Figure 2.2 Phospho-ERK (pERK) levels in LAPC-4 and PC-3 cells after E2 or DES
Cells were treated with E2 or DES at different concentrations (different color of symbols and
lines). pERK was measured by plate immunoassay for up to 60 min. Insets show the average of
all [E2] (A & B) or all [DES] (C & D) treatments. * denotes significance from vehicle response
(shown as 0 time) at P<0.05. The shaded gray horizontal bars represent the response to vehicle ±
Therefore, we next investigated the role of sustained ERK in the generation of
ROS. The production of ROS and associated sustained activation of ERK can lead to cell
death, via a positive feed forward mechanism
. To test this possibility we treated
LAPC-4 and PC-3 cells with E2, which increased ROS across all concentrations after 15
min (the optimum in the time course, not shown) in both cell lines (Figs. 2.3A & C).
Addition of MEK inhibitor U0126 inhibited ROS increases by ~50% in both cells lines,
indicating ERK1/2 is involved in this E2-mediated pathway, but not exclusively
responsible for ROS generation. However, DES did not significantly alter the levels of
ROS at any concentration in either cell line (2.3B & D), consistent with the more robust
cell-killing effect by E2.
Figure 2.3. ROS measured in LAPC-4
vs. PC-3 cells treated with 0.1 nM E2
or 1 µM DES.
ROS levels were measured after 15 min
of each estrogen treatment (A-D). Black
stars represent response to 1 µM H2O2,
a positive control for ROS generation.
White symbols denote LAPC-4 cells,
black symbols for PC-3 cells; triangles
represent E2, and circles DES
treatments. * denotes significance from
vehicle control at P<0.05. The shaded
gray horizontal bars represent the
response to vehicle ± SEM.
E2 treatment evoked different JNK responses from androgen-dependent compared
to androgen-independent cell lines (Fig. 2.4). In LAPC-4 cells, JNK was slightly
deactivated after 1 min, and was intermittently deactivated thereafter (Fig. 2.4A).
However, in PC-3 cells, JNK was activated (20%) at 1-10 min of E2 treatment, (Fig.
2.4B) followed by a return to control levels and a 10% decrease at 30 min, rising again to
baseline at 1 hr. In contrast, DES decreased JNK activation in both cell lines by 5-10% as
early as 5 min, an effect that mostly persisted through the remainder of the time course
(Fig. 2.4C & D). This is mostly inconsistent with the typical role of JNK in decreasing
cell viability compared to the cell line-specific viability responses we saw (Fig. 2.1).
Therefore, we next examined caspase activation to determine if apoptosis contributed to
any of the cell number declines observed in our viability assays (using staurosporine as a
positive control)
. A time-course study at 2-24 hrs showed that the highest levels of
caspase 3 activity were seen after eight hrs in both cell lines (entire time course not
shown); this time was therefore chosen for the comparative studies. Caspase 3 was
significantly activated in LAPC-4 cells by both E2 and DES, but not in PC-3 cells (Fig.
2.5). Though not entirely consistent with the cell-killing effects we saw in our viability
assays, nor with the traditional role of JNK in activating caspases, these data could
contribute some mechanistic explanations for cell-killing to the balance of multiple
mechanisms affecting cell number.
Figure 2.4. Phospho-JNK (pJNK) levels in LAPC-4 and PC-3 after E2 or DES treatments.
Cells were treated with different concentrations of E2 or DES (each line) and then pJNK
was measured by plate immunoassay for up to 60 min. Insets show the average response
values of all [E2] (A & B) or all [DES] (C & D) treatments. * denotes significance from
the vehicle response (time 0) at P<0.05. The shaded gray horizontal bars represent the
response to vehicle ± SEM.
Figure 2.5. Caspase 3 activity levels after E2 or
DES treatments.
LAPC-4 and PC-3 cells were treated with 0.1 nM E2
or 1 µM DES and caspase 3 activity measured after 8
hr (which is the response optimum; time course not
shown). White bars denote LAPC-4 cells, and black
bars PC-3 cells. * denotes significance from vehicle
(V) at P<0.05. The shaded gray horizontal bars
represent the response to vehicle ± SEM.
Staurosporine (Stauro) at 1µM was the positive
control for caspase 3 activation.
Necroptosis: We next examined the possibility
that this alternative mechanism of cell death
contributed to these reductions in cell viability caused by estrogens. This form of
programmed cell death was not observed in either cell line after either estrogen treatment
(Fig. 2.6). The combination of TNFα and cyclohexamide served as a positive control for
necroptosis. Addition of the necroptosis inhibitor necrostatin-1 blocked the cell-killing
effects of the positive control, but did not alter the cell number decreases caused by the
estrogens. Therefore, necroptosis did not appear to be involved in the killing of these
cells by estrogens.
Figure 2.6.
Necroptosis after
E2 or DES
LAPC-4 and PC-3
cells were treated
with 0.1 nM E2 or 1
µM DES and cell
measured via MTT
assays after 3 days.
White bars denote
LAPC-4 cells; black
bars are PC-3 cells.
* denotes significance from vehicle (V) at P<0.05. The shaded gray horizontal bars represent the
response to vehicle ± SEM. TNFα plus cyclohexamide (TNF/Cyclo) were positive controls for
inducing necroptosis in these cells which is defined by necrostatin (NecS) reversal of the
response. The cell viability in complete growth medium is shown for comparison at this 3 day
time point (Complete).
Phospho-p16INK4A: Next we turned to modulation of the cell cycle through
phosphorylation of the CDK inhibitor, p16INK4A. We hypothesized that activation of
p16INK4A would interfere with CDK‘s partnering with cyclin D1, and thereby hinder
progression through the cell cycle, leading to reduced cell numbers. In LAPC-4 cells, the
levels of phospho-p16INK4A rapidly increased ~45% in1-5 min after both E2 and DES
treatment (Fig. 2.7), and then fell to 20-25% increase for the duration of the time course
(Figs.2.7A & B). In PC-3 cells, E2 initially activated p16 less robustly, but increasing to
about 37% by 15 min and sustaining this level throughout the time course (Fig. 2.7C).
However, DES activation of p16 in PC-3 cells though significant, was severely blunted
(Fig. 2.7D). Therefore, these data could help explain in part the data of Fig. 1 where
DES-treated PC-3 cell numbers did not decline.
Phospho-p38: Cyclin D1 can be directly phosphorylated by p38 at a site that
directs its degradation
, so we next examined whether p38 was activated by estrogens
in our model systems. In both LAPC-4 cells and PC-3 cells, both E2 and DES phosphoactivated p38 rapidly (within 1 min) with similar patterns (Fig. 2.8). The most robust
response was In LAPC-4 cells, where DES activated p38 by almost 100% after 5 min of
treatment (Fig. 2.8B). All other increases were in the ~50% ranges (Figs. 2.8A, C, and
D). Interestingly, these activations were sustained for at least 60 min under all conditions,
and could therefore be available to direct phospho-activation of cyclin D1.
Figure 2.7. Phosphop16 (p-p16) levels
after E2 or DES
LAPC-4 and PC-3
cells were treated
with 0.1 nM E2 or 1
µM DES and p-p16
was measured for up
to 60 min by plate
immunoassays. White
LAPC-4 cells, and
black symbols PC-3
represent E2, and
denotes significance
from vehicle (at time 0) at P<0.05. The shaded gray horizontal bars represent the response to
vehicle ± SEM.
Figure 2.8. Activated
p38 (p-p38) levels
after treatment with
0.1 nM E2 and 1 µM
LAPC-4 and PC-3
cells were treated
with E2 or DES and pp38 was measured for
up to 60 min via plate
immunoassays. White
LAPC-4 cells, black
PC-3 cells. Triangles
represent E2, and
denotes significance
from vehicle (time 0)
at P<0.05. The shaded
gray horizontal bars represent the response to vehicle ± SEM.
Phospho-cyclin D1 and Total Cyclin D1: As p38-mediated phosphorylation of cyclin D1
leads to its degradation, this could in turn slow or halt progress through the cell cycle
, causing cell numbers to decline. Both estrogen treatments of LAPC-4 cells caused a
rapid rise in cyclin D1 phosphorylation (Fig. 2.9A and B), as did E2 treatment of PC-3
cells (Fig. 2.9C), though the latter was not as well sustained. However, DES treatment of
PC-3 cells instead caused a dephosphorylation of cyclin D1 (Fig. 2.9D). Although this
decrease in phosphorylation fluctuated, it lasted at least 30 min. If these cyclin
phosphorylations affect the levels of cyclin proteins as expected, they could explain our
estrogen-driven changes in cell numbers. Therefore, we next examined total cyclin D1
levels to determine if phosphorylation did indeed correlate with degradation shortly
thereafter. To assess this, we treated LAPC-4 and PC-3 cells with E2 or DES over a time
course of 5-950 min (16 hrs.). Decreases in total cyclin D1 were observed in each
scenario where cyclin D1 was phosphorylated (Fig. 2.9E, 2.9F, 2.9G). E2 caused a
significant decrease in total cyclin D1 within an hr in both LAPC-4 and PC-3 cells, and
DES acted similarly in LAPC-4 cells. Maximum reductions at 16 hrs in these three cases
were ~25%. However, there was no significant decrease in total cyclin D1 levels in PC-3
cells after DES treatment (Fig. 2.9H), which correlated with its lack of phosphorylation
and its inability to reduce the numbers of viable cells in this case. The ratio of p-cyclin
D1 to total cyclin D1 shows a very rapid change in cyclin D1 kinase “activity” which in
this case is a marking of the protein for degradation, except in DES-treated PC-3 cells
(Fig. 2.9I-2.9L). This last view of the data best correlates with the pattern of estrogeninduced decrease in cell number.
Figure 2.9.
cyclin D1 (pcyclin D1) vs.
total cyclin
levels and their
ratios after
treatment with
0.1 nM E2 and 1
LAPC-4 (early)
and PC-3 (late)
cells were treated
with E2 or DES.
The p-cyclin D1
were measured
for up to 60 min
and the total
cyclin D1 levels
measured up to
16 hrs via plate
The ratio of pcyclin D1 to total
cyclin D1 was
overlapping time
points at 5, 15,
and 60 min (I-L).
White symbols
denote LAPC-4
cells and black
represent E2 and
circles DES. *
significance from
vehicle (time 0)
at P<0.05. The
horizontal bars
represent the response to vehicle ± SEM.
ER-selective Antagonists: Next we examined the involvement of different estrogen
receptor subtypes (Fig. 2.10), focusing on the mechanisms that had given the most robust
of our explanations for estrogen-induced declines in cell numbers -- ROS generation and
cyclin D1 phosphorylation/degradation. For this we blocked each receptor subtype with a
selective antagonist at a very selective concentration, and then measured the estrogeninduced responses at time points selected as optimal for each mechanism. We found that
the ROS production caused by 15 min of E2 treatment in androgen-dependent LAPC-4
cells was antagonized by both ERα- and ERβ-selective inhibitors (Fig. 2.10A), while the
GPR30 antagonist had no effect. Androgen-independent PC-3 cells also had increased
ROS levels after E2 treatment, but in this case antagonizing only ERβ and GPR30
decreased the amount of ROS produced (Fig. 2.10B). DES was not active in generating
ROS, and so was not tested for receptor participation.
Figure 2.10. ER
antagonists inhibit E2or DES-induced ROS
and p-cyclin D1
LAPC-4 and PC-3 cells
were pretreated with
antagonists for each of
the three ER subforms:
α (MPP), β (PHTPP),
and GPR30 (G15) and
then treated at the
response time optima
with 0.1 nM E2 or 1 µM
measured after 15 min
(A & B). P-cyclin D1
levels were measured after 1 min for E2-treated PC-3 cells and 15 min for all others (C & D).
White bars denote LAPC-4 cells, and black bars PC-3 cells. * denotes significance from vehicle
(V) response at P<0.05. # denotes significance from the E2 response. The shaded gray horizontal
bars represent the response to vehicle ± SEM.
The roles of the three known estrogen receptors in altering p-cyclin D1 levels
after 15 min of E2 treatment were dictated both by the cell type in which the tests were
done and the estrogen mediating the response. That is, in LAPC-4 cells, E2 again
operated via ERα and ERβ to increase cyclin D1 phosphorylation (Fig. 2.10C), while in
PC-3 cells, a 1 min E2 treatment acted via ERβ and GPR30 to increase p-cyclin D1 levels
(Fig. 2.10D).
DES was also active in cyclin D1 activation, but only in LAPC-4 cells, where it
required ERβ and GPR30. Strikingly, a 15 min DES treatment in PC-3 cells reduced
cyclin D1 phosphorylation in a way that was independent of any of these three ERs (Fig.
2.10D), suggesting that at this high concentration this synthetic estrogen works via nonreceptor-mediated mechanisms in ways that did not reduce cell number.
Our results indicate that when several mechanisms of estrogen action are
considered using cell lines that represent different stages of human prostate cancer, DES
at a concentration achieved by typical clinical treatments (1 mg, three times daily leading
to serum concentrations measured at ≥1 µM)
does not kill or otherwise reduce the
numbers of cells that represent androgen-independent late-stage tumors (PC-3 cells).
Ironically, these late-stage tumors are the ones usually treated with DES. While cellkilling effects have been observed at concentrations 10-100 fold higher than those used in
our studies
, such high concentrations can cause many unwanted side-effects in
patients, and are unlikely to act via receptors. However, in our studies, E2 was much more
potent in decreasing the numbers of both androgen-dependent and -independent prostate
cancer cells in a concentration range (optimal at 0.1 nM) that may be far better tolerated
by patients. The mechanisms that we examined for participation in estrogen-induced cell
number decline in prostate cancer cell number are depicted in Figure 2.11 and their
effects summarized in Table 2.1.
Figure 2.11. Direct mechanisms of E2/DES action on prostate cancer cell survival.
E2/DES initiate rapid signals at mERs and through the activation or deactivation of MAPKs.
These signals can also lead to apoptosis, ROS increases, or phosphorylation of cell-cycle proteins
that delay the cycle. Each of these mechanisms can contribute to the estrogenic control of cell
MAPKs are important signal integrators of external stimuli leading to cell
proliferation or death. The most commonly cited mechanisms explain cell number
decreases by ERK inactivation (halting proliferation) or JNK activation (inducing
apoptosis via caspases). Instead, we demonstrated that E2 rapidly increased pERK levels
in both early- and late-stage prostate cancer cell lines. The activation of JNK did not in
any case correlate with the expected effect on caspase activity, so it did not explain any
of our cell viability results. Therefore, we had to entertain other explanations and
pathways for the therapeutic (tumor cell-killing) actions of these estrogens via MAPKs.
Table 2.1. Summary of mechanisms contributing to estrogen-induced decline in numbers of
LAPC-4 or PC-3 prostate cancer cells.
E2/DES activate differing pathways (listed) in LAPC-4 vs. PC-3 cells, which can lead to cell
death (apoptosis/necroptosis, ROS increases) or lack of cell proliferation (↓ pERK,
phosphorylation of cell cycle proteins). Each of the mechanisms listed in red or green font were
able to contribute to the estrogenic control of cell numbers in our studies. Mechanisms in the gray
font indicates a pathway that was tested but does not contribute. These number of mechanisms
contributing to each outcome were summed. The red text mechanisms and summary numbers
indicate participation of mechanisms that will kill cells or stop their growth; the green text
mechanisms and summary numbers indicate mechanisms that will cause cells to proliferate or
survive. We counted changes in ERK and ROS as separate mechanisms. Only a 0.5 score is
awarded to the p-p16 result for DES-treated PC-3 cells because it was a very low response
compared to the others. For non-color print versions: These are the mechanisms that decrease
cell number: ↑↑ERK→ROS, ↓ERK, ↑JNK, ↑ caspase 3, ↑p-p-16, ↑p-cyclin→↓total cyclin. They
are summed by the top number in each panel. Mechanisms that increase cell number: ↓JNK, ↓pcyclin→↑total cyclin -- are summed by the bottom number in each panel.
Sustained ERK activation (often throughout 60 min) associated with ROS
generation-mediated cell killing
demonstrated in our studies did correlate with E2-
induced declines in cell numbers. In this type of response ROS activates even more ERK,
likely participating in a positive feed-forward loop 262. The increased ROS production we
observe may not be due solely to sustained pERK, as use of MEK inhibitor U0126 only
abrogated the response by about 50%. Other possible sources of E2-generated ROS
include E2-induced DNA damage or intercalation of E2 into the plasma membrane, where
it can undergo redox cycling
280, 281
. Activated JNK levels did not correlate with nor
explain decreased cell numbers via apoptosis. Apoptosis, apparently not controlled by
JNK, played a role only in the cell model of early-stage prostate cancer (LAPC-4) where
this type of killing was caused by both estrogens. Late-stage cancer cells represented by
the PC-3 cell line were unaffected by either of these mechanisms. Neither cell type
underwent estrogen-induced necroptosis. Again, such a lack of response to estrogens in
cells representing late-stage prostate tumors was surprising, given that this is the type of
patient who is most often treated with the estrogen DES 282.
We showed that blocking the actions of proteins that drive the cell cycle was also
involved in these estrogen-induced declines in cell numbers. The major cell-cycle protein
CDK inhibitor p16INK4A and the cyclin D1-phosphorylating kinase p38 were both induced
by estrogens in all cases, regardless of whether these treatments actually reduced cell
numbers; so while these proteins may participate (be permissive), they do not alone or
primarily control a critical step in determining the cell viability outcome. However cyclin
D1 phosphorylation mediated by p38
, leading to its rapid degradation, was directly
correlated with estrogen-induced cell number declines in all cases. Cyclin D1 availability
and activity are major deciding factors for cell-cycle progression, and a diminished
ability to move through this checkpoint readily decreases cell numbers. E2 performed
much better in this pathway endpoint, providing a treatment advantage for both early- and
late-stage cancer cells. It is unknown why activated p38 cannot mediate DES-induced
phosphorylation of cyclin D1 in PC-3 cells, and this perhaps points to the involvement of
another unknown factor.
Changes in cell viability and caspase activation were measured over a course of
several hours to days, and therefore the involvement of both genomic and nongenomic
signals is likely. However, for our signal transduction assays, the mechanisms we
examined were activated rapidly (within minutes) after estrogen treatments, and likely
were mediated by a membrane form of these estrogen receptors, as we have shown in
other cancer cell types (GH3 pituitary, MCF-7 breast, PC-12 pheochromocytoma)
phosphorylation/activation of Elk-1 and ATF-2 transcription factors as early as 10-15
. This illustrates how signaling initiated early via nongenomic mechanisms can
progress to downstream genomic mechanisms. Our previous studies demonstrated rapid
activation via mERs for the three MAPK activations demonstrated here. In addition, our
previous work on glucocorticoid-induced killing of T lymphoma cells showed
dependence on the presence of a membrane form of the glucocorticoid receptor
. Our
demonstration here of novel non-genomic actions of estrogens on prostate cancer cells
should open new avenues of thinking about therapeutic approaches using estrogens of
many types. Estrogen receptors play a major role in normal physiological regulation and
development of many types of cells including the prostate, but also in cell survival and
cancer development
. The primary ER of the prostate gland is ERβ, with lower levels
of ERα and GPR30. However, each is a possible target for cancer therapy 290. Our results
using ER subtype-selective antagonists suggest that estrogens mediate specific
mechanisms (increased phosphorylation of cyclin D1 resulting in its degradation, ROS
generation via ERK activation) via some but not all ERs -- and not always the same ones
-- to affect cell viability. E2 required ERβ to operate the most important contributory
mechanisms related to cell number declines (increasing ROS and p-cyclin in both cell
types), but ERα and GPR30 participated variably, especially depending upon early- vs.
late-stage tumor cell type. However, the high concentration DES effects in late-stage PC3 cells required no receptor involvement at all, were in the opposite direction from those
mechanisms associated with the decline in cell numbers, and were therefore in agreement
with the inability of DES to reduce cell numbers. It has previously been observed that at
such high concentrations DES can bypass estrogen receptor-mediated mechanisms, and
instead alter such chemical properties as fluidity, lipophilicity, and polarity of the lipid
291, 292
, so perhaps DES is operating on these cells via these other less productive
The degree to which these treatments are more effective because they engage
more of the mechanisms that can reduce tumor cell numbers are summarized in Table 1;
we listed the active mechanisms in color, and then added up how many mechanisms were
active in each case toward increasing (green) or decreasing (red) cell number. Only by
examining all of these responses together were we able to comprehensively consider
which pathways contributed to the therapeutic cell-killing effects of estrogens on prostate
cancer cells of different stages. These studies are an important example of how
systematic examination of multiple mechanisms can elucidate the extent to which a
therapeutic agent will be effective on tumor cell stages with distinct characteristics. We
showed that estrogens have a rapid and direct effect on prostate tumor cells, and that
multiple, but not all cell-killing mechanisms contribute to the therapeutic response. E2
was much more potent and efficacious than DES, suggesting that E2 could be a better
form of treatment for men with all stages of prostate cancer. This represents a particularly
important opportunity for treatment of advanced prostate cancers where treatment options
are limited. However, these results also suggest that very early-stage developing tumors
in high-risk cancer-susceptible men could benefit from low-dose E2 treatment, the natural
levels of which may decline in men during aging 293, 294.
Chapter 3: Rapid, nongenomic signaling effects of several xenoestrogens
involved in early- vs. late-stage prostate cancer cell proliferation
Xenoestrogens (XEs) are exogenous mimics capable of binding to estrogen
receptors (ERs), competing with/disrupting the actions of physiological estrogens, and
promoting tumor growth in the prostate and other endocrine tissues. Humans are exposed
to numerous XEs including environmental contaminants such as plastics monomer
bisphenol A (BPA), and dietary phytoestrogens such as coumestrol and genistein from
soy, and resveratrol, highest in red grapes. There is growing interest in the ability of
phytoestrogens to prevent or treat tumors. We previously reported that multiple cellular
mechanisms influence the number of prostate cancer cells after estradiol or
diethylstilbestrol treatment. We now examine the effect of these XEs on signaling
mechanisms that alter the number of LAPC-4 (androgen-dependent) and PC-3 (androgenindependent) cells at environment- and diet-relevant concentrations. Coumesterol and
genistein both increased the number of LAPC-4 and PC-3 cells dramatically. Rapid
alterations of phospho- and total-cyclin D1 levels most closely correlated with the XEinduced changes in cell numbers. Sustained activation (phosphorylation) of the
extracellular signal-regulated kinases 1 and 2 as a prelude to generation of reactive
oxygen species also partially contributed to the XE’s effects on cell numbers. Early-stage
cells expressed higher levels of all three ERs (including those in membranes) than did
late-stage cells; ER subtypes were variably involved in the signaling responses. Taken
together, these results show that each XE can elicit its own signature constellation of
signaling responses, highlighting the importance of managing exposures to both
environmental and dietary XEs for existing prostate tumors. These mechanisms may offer
new cellular targets for therapy.
Prostate cancers are well-known for their initial androgen responsiveness, which
diminishes with the progression of disease stage. While the corresponding decrease in
androgen receptor (AR) levels that accompanies this decline in responses has been well
, little is known about the relationship of tumor progression with the
estrogen receptor (ER) types that might be involved, such as those that are thought to
mediate the therapeutic effects of the pharmaceutical estrogen diethylstilbestrol (DES).
There are many types of xenoestrogens (XEs) – exogenous estrogen-like compounds that
bind to ER ligand binding pockets
58, 296, 297
. In normal or cancer cells, they imitate,
compete with, or disrupt the actions of physiological estrogens
298, 299
. Some XEs are
known to promote tumor development in many tissues by stimulating inappropriate
endocrine responses via ERs, promoting angiogenesis, increasing DNA adducts, or
altering the epigenome 300-304. Actions of XEs via ERs have also been shown to cause the
proliferation of established endocrine tumors or tumor cell lines of many types, including
those from brain, breast, kidney, lung, pancreas, prostate, and testis 287, 305-311.
Alternatively, some XEs, especially dietary compounds, have been credited with
preventing tumors in some of these tissues
, highlighting a broad range of XE
response profiles. Genistein is a phytoestrogen found in soy products, fava beans, and
some coffee bean preparations 316 that can cause cell cycle arrest and growth inhibition at
concentrations within the ranges achieved by the diets of some cultures (10-8 M to 10-6
M), via the down regulation of cyclin B
317, 318
. Coumestrol (found in red clover, alfalfa
sprouts, and also some soy products) can kill breast and colon cancer cells by producing
reactive oxygen species (ROS)
. Resveratrol, found in grapes, can decrease cell
numbers by increasing intracellular calcium levels, disrupting G1/S progression, and
stimulating apoptosis 320-324.
XEs also include environmental contaminants from the manufacture, use, or
leachates of consumer products (e.g. plastics, chlorinated pesticides, alkylphenol
surfactants). Bisphenol A (BPA) is a component of many plastic products [such as water
bottles, food containers, receipt paper, and the inner coatings of food cans
] and
leaches from these products more readily with heat or acidity. As a result, BPA is a
common environmental and human/animal contaminant
cell proliferation
kinases (MAPKs),
that has been shown to alter
, cell signaling through the activation of mitogen-activated protein
and intracellular calcium levels
330, 331
, prostate cancer cell
migration 332, and increase susceptibility to certain diseases 300, 333.
Epidemiological studies generally support an association between diets high in
phytoestrogens and low cancer incidence
incidence of prostate cancer
12, 334, 335
. African-Americans have a higher
, and dietary differences are being investigated as a
possible factor in tumor development and progression within that population. East Asians
consume high amounts of phytoestrogens, and their incidence of many types of cancer,
including prostate cancer, is much less
. Asian diets contain high levels of soy
ingredients, with the best-known active estrogenic components being daidzein, genistein,
and coumestrol
. Genetics may also play a role in the sensitivity of various cancer-
relevant mechanisms to estrogens, including the ability to metabolize dietary
phytoestrogens to more active compounds
, though this can also be due to the type of
gut microbiome present 338.
Until recently, most studies on XEs were focused on the gene expression
(genomic) consequences of exposures
. Even at high concentrations, XEs generally
elicited only low levels of transcriptional responses, so these compounds were thus
labeled as weak estrogens 339, 342. However, XEs can also initiate non-genomic responses,
so classifying them as weak without taking these more rapid cellular responses into
consideration may be misleading
287, 307, 343-347
. Some endogenous estrogen metabolites
such as estriol were formerly labeled as weak because of their limited ability to activate
specific transcription, but have recently been found to have profound effects on disease
348, 349
. Estriol, like XEs can have quite potent effects on nongenomic
. Because of this belief that XEs were weak, many past studies did not
evaluate estrogens at environmentally relevant low doses [reviewed in
]. Dose
responses to estrogens are typically non-monotonic and therefore must be assessed over a
wide and detailed range of concentrations (reaching down to the femtomolar to
nanomolar range) to predict their ability to act, especially at relevant environmental and
dietary levels 287, 302, 350, 352.
We have previously shown that XEs can rapidly activate cellular signaling
pathways in tumor cells of other tissues (pituitary, breast, adrenal), and when in
combination with them can modify the actions of physiological estrogens
328, 329, 344, 353-
. MAPKs can be rapidly activated or deactivated by XEs, leading to alterations in such
functional end points as proliferation, apoptosis, and prolactin release
287, 356
. Moreover,
these MAPK phosphorylations [such as those for the extracellular signal regulated
kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 kinase] often occur at low
physiological estrogen and XE concentrations 287, 328, 357.
We also recently demonstrated that estradiol (E2) and diethylstilbestrol (DES) can
rapidly stimulate or deactivate ERKs in LAPC-4 and PC-3 prostate cancer cells
when sustained, cause ROS generation, contributing to a decrease in viable cells
, and
262, 358
In addition, estrogen-induced rapid phosphorylation of cyclin D1 led to its subsequent
prompt degradation, which in turn was correlated to the ability of E2 and DES to inhibit
growth of these cells
. We will now investigate if some XEs also alter the viability of
prostate cancer tumor cells via these mechanisms. Elucidating how these XEs function in
early- vs. late-stage prostate tumor cells could lead to selective advice for patients about
diet and exposure to environmental estrogens.
Materials and Methods
Cell lines and hormones: We chose cell lines representing the two main types of
prostate cancers – androgen-dependent vs. androgen-independent. LAPC-4 androgendependent prostate cancer cells (passages 45-50)
were maintained to sub-confluence
in phenol red-free Iscove’s Modified Dulbecco’s Medium (IMDM; MediaTech,
Manassas, VA) with 10% fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville,
GA), 4 mM L-glutamine (Sigma-Aldrich, St. Louis, MO), and 10-9M dihydrotestosterone
(Sigma-Aldrich). PC-3 androgen-independent prostate cancer cells (passages 18-23)
were maintained by growth in phenol red-free RPMI 1640 (Sigma-Aldrich) with 10%
FBS and 2mM L-glutamine. Both cell lines were propagated at 37°C in 5% CO2. BPA,
coumestrol, genistein, and resveratrol (all from Sigma-Aldrich) were dissolved in ethanol
to a stock concentration of 10mM before serial dilution into IMDM or RPMI 1640 at
concentrations ranging from 10-14M to 10-6M (and a final EtOH concentration of
MTT Cell Viability Assay: Cells were plated at 5,000 cells/well in poly-D-lysinecoated (BD Biosciences, Bedford, MA) 96-well assay plates, (Corning, Tewksbury, MA),
and then allowed to attach overnight. The next day, XE treatments were added in 100µL
of medium with 1% 4x charcoal-stripped FBS. Extensive charcoal stripping of serum was
done to remove and thus minimize the effect of any steroid hormones already present;
these conditions were previously optimized to demonstrate effects of steroids and mimics
on cell proliferation for these cell lines. After three days, treatments were removed and 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) was
added for 1 hr. Cells were then lysed and the signal read at 590nm in a Wallac 1420 plate
reader (Perkin Elmer, Waltham, MA).
Plate Immunoassays: Phosphorylated proteins were recognized by antibodies
(Thr202/Tyr204) and phospho-cyclin D1 (Thr286) (both from Cell Signaling, Danvers,
MA). Changes in total cyclin D1 levels were measured by using an Ab to cyclin D1
recognizing both modified and unmodified protein cyclin D1 (Cell Signaling Cat. No.
2922). ER Abs used included ERα (MC-20, Santa Cruz Biotechnology, Dallas, TX); ERβ
(clone 9.88, Sigma-Aldrich); and GPR30 (Cat. No. NLS4271, Novus Biologicals,
Littleton, CO). Membrane and total ER levels were measured by controlling for
permeabilization of the cell membrane. A plate immunoassay developed in our lab 284 and
used in many of our past studies was recently adapted 360 for use with the BIOMEK FXP
workstation (Beckman Coulter, Brea, CA) to automate the majority of the plate assay’s
liquid handling, decreasing experimental variability and increasing experimental output.
Prostate cancer cells were plated at 10,000 cells/well in 96-well assay plates,
allowed to attach overnight, and given 100µL of medium with 1% 4x charcoal-stripped
FBS for 48 hr. Cells were then treated with XEs for up to 60 min on the workstation,
followed by fixation (2% paraformaldehyde, 1% gluteraldehyde) ± permeabilization
(0.15M sucrose, 0.5% Nonidet P-40; to access internal vs. extracellular epitopes). The
primary Ab to the phosphorylated epitopes was then added and incubated with the cells
overnight. The next day, biotinylated anti-mouse/anti-rabbit IgG secondary Ab (Vector
Labs, Burlingame, CA) was added for 1 hr. Next, cells were incubated for 1 hr with
avidin-biotinylated conjugated alkaline phosphatase (ABC-AP, Vector Labs), then for 30
min with para-nitrophenylphosphate substrate (Thermo Scientific, Rockford, IL),
allowing the yellow color of the para-nitrophenyl product to accumulate. Plates were read
at 405nm in a Wallac 1420 plate reader. Readings were then normalized to cell number,
estimated by the crystal violet dye (Sigma Aldrich) assay as described previously 284.
Subtype-selective ER Antagonist Assays: To further determine ER subtype
involvement in altering ROS formation or cyclin D1 phosphorylation, the following ER
antagonists were used at their receptor-selective concentrations: for ERα, 10-7M MPP; for
ERβ, 10-6M PHTPP; and for GPR30, 10-6M G15 (all from Tocris Bioscience,
Minneapolis, MN). All were dissolved in ethanol to a stock concentration of 10mM, then
serially diluted into culture medium. Final ethanol concentrations were 0.0001%, which
was used as vehicle control for all studies. Cells were incubated with antagonists for 30
min before XE treatments.
ROS Assays: Cells were plated at 10,000 cells/well in a 96-well assay plate, then
allowed to attach overnight. Cells were then treated with 100µL of medium containing
2’,7’-Dichlorodihydrofluorescein diacetate
1% 4x charcoal-stripped FBS for 48 hr.
(DCDHF, Enzo Life Sciences, Farmingdale, NY; 15µM) was loaded into cells for 1 hr,
and XE treatments were then administered for 15 min. Hydrogen peroxide (Fisher
Scientific, Pittsburg, PA) and ethanol (0.0001%) were used as positive and negative
controls, respectively. E2 (1nM) was a positive control for previously determined
estrogenic responses
. Dichlorofluorescein production, formed as a result of
ROS/DCDHF interaction, was measured at an excitation of 485 nm, and an emission of
538 nm in a SpectraMax M3 Multi-Mode Microplate Reader (Molecular Devices).
Statistics: All experiments were conducted a minimum of three times. One-way
analysis of variance was conducted for all experiments except ER quantification, which
was analyzed using a Student’s t-test. A Holm-Sidak post hoc test was used to measure
the significance of each treatment versus the vehicle control. Significance was set at
p<0.05, unless otherwise stated.
Results and Discussion
Figure 3.1. Cell number after 3 days of XE treatment.
LAPC-4 and PC-3 prostate cancer cells were treated with XEs and viable cells were measured by
the MTT assay. In all figures throughout the manuscript white symbols denote LAPC-4 cells and
black symbols PC-3 cells. *denotes significance from vehicle (V) controls at p<0.05, and shaded
horizontal bars represent the response to V ± SEM. In this and other graphs, where error bars are
not visible, they were within the size of the symbol. Dietary or environmentally relevant
concentration ranges are shown by the solid horizontal bars below the graphs for each XE. The
insets show cell numbers after three days of E2 treatment, for comparison (and see 358)
XE Effects on the Number of Viable Cells: XEs at environment- or diet-relevant
concentrations caused some increases in the numbers of LAPC-4 and PC-3 prostate
cancer cells, observed here after three days of exposure in media containing 1% charcoalstripped serum (Figs. 3.1A & B). Coumestrol increased viable cell numbers at all but the
lowest concentration assessed (10-14M) in both cell lines (by >200% in LAPC-4 cells,
>400% in PC-3 cells). There was a strikingly different response to genistein between cell
lines representing different tumor stages; genistein did not affect LAPC-4 cells, while it
strongly stimulated the growth of PC-3 cells (by ~4-fold at concentrations from 0.1nM to
1µM, all levels achievable by some diets). BPA caused a small stimulatory effect in
LAPC-4 cells, at concentrations >10-12M (maximal increases of ~40-50%), but had no
effect on PC-3 cells. Resveratrol showed minimal, but significant stimulation compared
to vehicle in both cell lines at most concentrations tested. For comparison to other
estrogens assessed in our previous studies 358 (see insets), even at physiological (10-10-108
M) concentrations E2 instead caused a significant decrease in cell viability for both cell
lines by ~20-30%. The actions of these dietary estrogens in causing prostate cancer cell
growth are perhaps unexpected, given the epidemiological evidence that cultures with
diets rich in genistein and coumestrol show decreased levels of prostate cancer
14, 361
However, some other studies have also shown a prostate cell proliferation effect by these
phytoestrogens 362-364.
Subcellular Location and Levels of ERs: We next asked which types of ERs were
present in LAPC-4 and PC-3 cell lines that could mediate these changes in viable cell
number. We had previously noted a variable dependence of rapid responses on all three
ER subtypes (determined by using selective antagonists) in our studies on E2 vs. DES
treatment of these cells
. Plasma membrane versions of estrogen receptors (mERs) are
thought to mediate rapid signaling involved in cell number changes in other cell types
[reviewed in 186, 353, 365], so we examined the subcellular location of these receptors here.
Figure 3.2. ER subtype (α, β, and GPR30) levels (total vs. membrane) in LAPC-4 and PC-3
prostate cancer cells.
The negative control samples used no primary antibody (Ab) for any of the ER subtypes, as
indicated by the first bar and the shaded bar extending horizontally across the graph (average
±SEM). *denotes significance from controls at p<0.05.
Using our plate immunoassay ± cell permeabilization with detergent (Fig. 3.2),
we observed that late-stage tumor cells (panel B) had much lower expression of ERs than
did early-stage cells (panel A; note the ~6-fold vertical scale difference between panels A
and B), a frequent finding among steroid receptors in endocrine cancers of multiple types
, although this evaluation for mERs in prostate tumor cells is novel. We also saw
that membrane receptor populations were much lower than total (and thus intracellular)
receptor forms in early-stage cells; the levels of mERs α and β were about 20% and 24%
of their total receptor populations, respectively, as we have seen previously for the
proportion of membrane versions of these receptors in other tumor cell types 171, 286, 369.
Although much lower, we detected significant levels of all three ER types in PC-3
cells. In these late-stage tumor cells the membrane receptor population was a much larger
percentage of the total receptor numbers, perhaps in keeping with their more
undifferentiated state, as we have seen with membrane glucocorticoid receptors in human
lymphoma cells compared to normal circulating lymphocytes
. ERβ predominated in
LAPC-4 cells (and to a lesser extent in PC-3 cells), as expected based on the literature
regarding the dominance of this receptor type in normal prostate tissues and the earlystage tumors that arise from them
57, 223
. However, there were also significant levels of
ERα and GPR30, suggesting that they might also play a role in mediating estrogenic
mechanisms. Interestingly, we found that the sizable amount of GPR30 was largely
intracellular in LAPC-4 cells. GPR30 has been identified in other prostate cancer studies,
but the subcellular location was not elucidated
206, 238, 370
. The subcellular location of
GPR30 in other tissues and their cancers has been a point of contention; different groups
have demonstrated this receptor form as either primarily in the plasma membrane or in
the endoplasmic reticulum 371, 372.
Phospho-ERK: Our next goal was to identify pathways and mechanisms
responsible for any changes in numbers of viable cells, and ERK phosphorylation is one
mechanism that has traditionally been associated with cell proliferation
. We selected
an effective and environment- or diet-relevant concentration for each XE studied (10-9M
BPA, 10-7M coumestrol, 10-7M genistein, and 10-8M resveratrol; see Fig. 3.1 for relevant
ranges), and measured their ability to elicit ERK phosphorylation in both cell lines over
60 min (Figs. 3.3A & B). We observed activation for all compounds except genistein, but
found that a sustained (60 min) pERK response did not predict a XE’s positive influence
on cell number, as has been a long-held association 373, 374.
Figure 3.3. Phospho-ERK (pERK) levels in LAPC-4 and PC-3 cells after XE
LAPC-4 and PC-3 cells were treated with 10–9M BPA, 10-7M coumestrol, 10-7M genistein, and
10-8M resveratrol. pERK was measured up to 60 min via the plate immunoassay. *denotes
significance from vehicle (shown at time 0) controls at p<0.05, and horizontal shaded bars
represent the response to vehicle ± SEM.
The most striking result was the difference between cell line-specific responses
after resveratrol treatment, which caused a strong ERK deactivation (40%) in LAPC-4
cells, while it had slightly increased the number of viable cells (Fig. 3.1). In PC-3 cells
resveratrol rapidly activated and sustained pERK (at 60 min), and caused modest cell
proliferation. BPA and coumestrol rapidly stimulated ERK phosphorylation in both cell
lines (Figs. 3.3A & B), all with sustained levels at 60 min, but had no proliferation
effects in late-stage cells. Genistein rapidly though modestly deactivated ERK in both
cell lines, but substantially increased viable PC-3 cell numbers. Only the activation of
ERK by coumestrol in both cell types correlated with its ability to cause these cells to
proliferate. Therefore, these XEs elicited unique patterns of ERK activation/deactivation,
which could contribute to cell survival, cell death, or proliferation
262, 373, 375
, but clearly
more mechanisms needed to be considered.
Figure 3.4. ROS levels after treatment with 10-10M E2, 10-6M H2O2, 10-9M BPA, 10-7M
coumestrol, 10-7M genistein, and 10-8M resveratrol, ± ER subtype-selective
Antagonists (Antag) were 10-7M MMP for ERα; 10-6M PHTPP for ERβ; and 10-6M G15 for
GPR30. ROS levels were measured after 15 min of each XE treatment (the optimal response
time). *denotes significance from vehicle (V) controls at p<0.05, while # denotes significance
from paired XE treatment values (p<0.05). ERα inhibition was significantly different vs.
resveratrol alone in PC-3 cells ($) at p<0.09. The shaded horizontal bars represent the response to
vehicle (V) ± SEM.
ROS generation: Others have recently associated sustained ERK activations with
ROS generation leading to cell killing 262, and we recently extended this to the ability of a
physiological estrogen (E2) to induce cell death in early-stage prostate cancer cells
Therefore, we examined if XEs (at optimal concentrations for such ERK responses) could
be linked to any ROS elevations (measured at the peak time of 15 min, time course not
shown). The positive controls for ROS generation, including both H2O2 and E2, caused
robust ROS generation (Figs. 3.4A and B), and as we saw previously, cell death 358. Most
XE treatments generated significant ROS levels regardless of whether they had caused
sustained ERK activation (Fig. 3.3), though BPA and genistein did so only in one cell
line each. These ROS increases were all modest compared to those caused by E2.
Therefore, ROS elevation due to sustained ERK activation was not considered to be a
primary mechanistic determinant of viable cell number in these studies. Perhaps higher
levels of ROS need to be generated to kill this cell type.
Therefore, these MAPK, and potentially linked ROS responses, did not
individually predict cell proliferation vs. cell killing effects. Possibly a more traditional
route of ROS generation not involving ERKs was involved in these cells. Estrogens have
been shown to damage DNA 281, which can also cause ROS generation 376, 377. The ability
of these XEs to induce ROS was different from that of E2
, further highlighting the
imperfect mimicry of physiological estrogens by XEs. Overall, the pERK and ROS
responses to XE treatments in both cell lines do not appear to be lone driving
mechanisms that elicit changes in cell numbers. Therefore, we have to consider the
combined contribution of these responses to an overall balance of competing mechanisms
(see below).
We observed previously that E2 required different ER subtypes to elicit ROS
responses in LAPC-4 vs. PC-3 cells
. Here XEs also demonstrated unique ER-use
signatures for this response (Fig. 3.4). In LAPC-4 (early-stage) cells, BPA caused these
modest ROS increases independent of any known ERs, and genistein did not raise ROS
levels (Fig. 3.4A). The increases due to coumestrol required ERα and GPR30, while
resveratrol required only ERα. A somewhat different profile was evident in PC-3 (latestage) cells, where coumestrol and resveratrol both increased ROS via ERβ, while
genistein stimulation of ROS did not require any known ERs, and BPA did not cause a
response (Fig. 3.4B). Clearly, the regulation of this pathway via ERs became quite
different as cell types progressed to a less differentiated state with far lower receptor
numbers (Fig. 3.2). The involvement of more than one ER subtype in some XE-generated
responses also suggests the participation of multiple pathways.
Figure 3.5. Cyclin D1 phosphorylation and degradation by XEs, and inhibition by ERselective antagonists.
Cyclin phosphorylation was measured at 1-60 min, and total cyclin D1 levels over 16 hr of XE
treatment. For 5A & 5B, LAPC-4 and PC-3 cells were pretreated with antagonists (Antag) for
each of the three ER subtypes: α (MPP), β (PHTPP), and GPR30 (G15,) and then treated with 109
M BPA, 10-7M coumestrol, 10-7M genistein, 10-8M resveratrol, 10-10M E2 or 10-6M DES. Shaded
horizontal bars represent V ± SEM. * denotes significance compared to vehicle (V) at p<0.05. #
denotes significance from paired XE treatment responses at p<0.05. For 5C & 5D, LAPC-4 and
PC-3 cells were treated with each XE for the times indicated and total cyclin D1 levels were
measured with a plate immunoassay.
Total and Phosphorylated Cyclin D1: Control of cyclin D1 levels
378, 379
is a
mechanism we previously identified as being a significant contributor to E2- or DESevoked changes in prostate cancer cell survival 358. Both estrogens caused rapid cyclin D1
phospho-activation leading to swift degradation of this cell-cycle protein. This response
to E2 and DES is also shown here in Fig. 3.5A & B. Others have also shown that
phytoestrogens can affect other cell-cycle protein levels
317, 318, 322, 323, 380
, which in turn
affect the number of cells. Of the four XEs studied here, the three phytoestrogens
(coumestrol, genistein, and resveratrol) all affected cyclin D1 phosphorylation levels,
though in distinctly different directions, and differently for early- vs. late-stage cells
(Figs. 3.5A & B). In each case where cyclin D1 was phosphorylated, the corresponding
expected rapid decline in total cyclin D1 levels occurred (panels C and D). Interestingly,
we observed significant declines in total cyclin D1 as early as 5 min after genistein or
resveratrol treatment (~10%), but the largest decreases for most compounds were seen
after 4 hr. Coumestrol caused cyclin dephosphorylation, resulting in cyclin D1 level
increases, correlating very well with its ability to increase cell numbers. Resveratrol
signaling significantly phosphorylated cyclin D1 in both cell lines, driving total cyclin D1
levels down, yet while eliciting very small increases in cell proliferation, a less perfect
correlation. Genistein was the only compound that caused opposing effects on these
mechanisms in the two cell lines. In LAPC-4 cells, it increased phosphorylated cyclin D1,
causing its degradation, but that did not correlate with measured changes in viable cell
numbers. However, in PC-3 cells, genistein depressed cyclin phosphorylation, allowing
increases in cyclin levels and correlating with a strikingly robust cell proliferative
response. BPA did not affect cyclin D1 phosphorylation in either cell line, nor did it
change levels of total cyclin D1, in keeping with its minimal effects on cell numbers.
While these correlations generally go in the expected direction, they do not entirely
predict the degree of the functional (cell number-changing) responses. Therefore, we
ultimately considered all of these mechanisms together (see summation in Table 3.1), to
see if other mechanisms in some cases modified this dominant response to cyclin D1
changes (see Conclusions).
Other signaling pathways that we have not examined here may also contribute to
the net change in cyclin D1 phosphorylation and consequent total protein levels.
Glycogen synthase kinase 3β, which is regulated by the phosphoinositide 3kinase/protein kinase B pathway (PI3K/Akt), has also been shown to phosphorylate
cyclin D1 on Thr286, as well as regulate the protein’s subcellular location in mouse
. However, the exact role of GSK-3β/PI3K/Akt in driving phosphorylation
of cyclin D1 has been debated, as Guo et al., found that the activity of those pathways did
not change (during the relevant S-phase), nor decrease cyclin D1 protein levels in mouse
or human fibroblasts
. In addition, inhibition of GSK-3β in MCF-7 breast cancer cells
did not completely disrupt cyclin D1 protein degradation
. Other pathways that can
cause degradation of cyclin D1 include p38, which we previously studied for activation
by DES and E2 358 and did not include here because it was activated for all cell types and
treatments. Cyclin D1 phosphorylation through p-p38 seems to be especially prevalent in
response to the damage to DNA caused by environmental agents which require rapid
cellular responses to prevent propagation of genomic mistakes
384, 385
. The Mirk/Dyrk1b
kinase, active during G0/G1, has also been shown to regulate cyclin D1 protein levels
through phosphorylation at Thr288
. Therefore, multiple pathways can cause
phosphorylation of cyclin D1, but they may influence degradation of cyclin D1 to varying
degrees, and may be tissue-selective.
We next examined which ER subtypes (α, β, or GPR30) might be involved in the
cyclin D1 phosphorylations, again using selective antagonists for each receptor subtype.
For genistein, either the ERβ or GPR30 antagonist reversed the cyclin D1
phosphorylation in LAPC-4 cells, but only the ERβ antagonist decreased p-cyclin D1
levels in PC-3 cells. Coumestrol apparently did not utilize any of the known ER subtypes
in LAPC-4 cells to decrease p-cyclin D1 levels, but ERα was required in PC-3 cells.
Coumestrol’s lack of dependence on any known receptor subtype in LAPC-4 cells is
surprising, given the plentiful expression of all of these receptors in that cell line. It is
possible that the low p-cyclin levels and thus larger errors in the measurement, caused by
coumestrol treatment made antagonist reversals difficult to detect. Resveratrol’s
induction of p-cyclin D1 levels in both cell lines showed a dependence on ERs α and
GPR30 in LAPC-4 cells, and on ERα in PC-3 cells. Therefore, each XE showed a
dependence on a different ER subtype or subtype combination in the two cell lines. These
dependencies are consistent with what has been previously shown about receptor subtype
binding preferences for these XE compounds. For example, resveratrol has a higher
binding preference for ERα than for ERβ
, while coumestrol and genistein are strong
ERβ agonists 297, but are still capable of binding to ERα 299, 387, 388. The predominance of
ERβ in these prostate cell lines may influence their responses to these XEs that affect cell
number. However, mostly genomic pathways have been examined in the past, such as the
ability to activate ER reporter constructs, with differences between cell types for different
. Few comparisons for nongenomic responses are available, though we
previously observed different MAPK activation patterns and mostly positive proliferative
responses to E2 and various XEs in GH3/B6/F10 pituitary cells276, 329, 392 that have high
mERα and low mERβ levels
Also consistent with our results are known XE effects that do not involve these
ERs. An example is the well-known direct inhibitory effect of genistein on tyrosine
. In other instances, small lipophilic compounds like these can intercalate
into cell membranes and as “border lipids” influence the actions of proteins embedded in
them. Lipophilic estrogens can change membrane fluidity
291, 292
, especially when they
are present at relatively high concentrations (as is true for most effective phytoestrogen
concentrations resulting from dietary exposures). Because changes in cell numbers are
best observed after three days, the nuclear-localized receptors forms involved in slower
transcriptional regulation
396, 397
may also be relevant to these effects, which we did not
examine in our studies of these more novel rapid actions.
Another possible contributor to tumor cell behavior in prostate cell lines is the
tumor-suppressor p53 32. It is mutated in LAPC-4 cells, and not present in PC-3 cells, and
therefore unlikely to drive estrogen-mediated prostate tumor viability in our present
studies. We also chose cell models for our studies that do not present the added
complication of mutant ARs (such as in LnCaP cells
) to which estrogens can more
readily bind and elicit effects, especially at high concentrations. LAPC-4 cells have wildtype ARs and PC-3 cells do not express ARs 32.
Table 3.1 Summary of XE responses for mechanisms that affect the number of viable
LAPC-4 vs. PC-3 cells.
Estradiol is shown for comparison, summarizing the data from our previous publication 358.
Mechanisms in red text contribute to decreases in viable cell numbers, while mechanisms in
green text increase the number of viable cells. Gray text indicates mechanisms that did not make
any contribution to changes in cell numbers. These mechanistic contributions are summed in the
red and green numbers in the upper right-hand corner of each box. ∆ = change.
The estrogen-induced mechanism that dominated our effective predictions of cell
growth behavior in these studies was the rapid phosphorylation of cyclin D1, followed
shortly thereafter by its degradation. This mechanism largely predicted the primary
response to each XE in terms of cell number changes (except in the case of resveratrol).
However, no single mechanism entirely predicted the degree of these XE-induced
changes, so we also examined the balance of the effects of other pathways (summarized
in Table 3.1). In this table each of the mechanisms examined in these studies for each
cell type due to each XE is summarized in colored text: red denotes actions causing
decreases in viable cells, while green represents mechanisms that increase cell numbers,
and gray indicates no effect. These mechanistic contributions are summed in the red and
green numbers in the upper right-hand corner of each box. The modest effects of these
XEs are in contrast to the strong cell growth inhibitory/cell killing effects we saw
previously with E2; these previous conclusions
are shown in the first column of the
table for the sake of comparison. E2 engaged a total of seven signaling responses in
LAPC-4 cells and PC-3 cells -- though in this table we list only the 4 that were examined
here that gave the best predictions of therapeutic responses (decreases in cell numbers) to
compare with XEs. Coumestrol is a robust stimulator of cell growth in both early- and
late-stage prostate cancer cells, matching its strong positive effects on cyclin D1 status
and levels. The ability to activate sustained ERK, and through it to increase ROS levels,
did not decrease the cyclin D1-driven outcome. Genistein’s different effects on early- vs.
late-stage cells could also be largely explained by the cyclin D1 changes. Resveratrol had
a very small effect in both cell lines, though the altered cyclin D1 levels and the ROS
generation should have predicted a large decrease in cell viability, which was not
observed. BPA showed the smallest changes in these responses that we examined,
corresponding to only minimal growth stimulation, in only LAPC-4 cells.
Once again, mechanistic responses (phosphorylations, cyclin level changes, and
ROS generation) to these varied estrogens were documented to be very rapid, supporting
the notion that important tumor-altering effects can occur, or at least be initiated, via
nongenomic signaling mechanisms. This was supported by our demonstration that
membrane versions of these receptors are present in these tumor cells. Our present and
287, 328, 329, 331, 343, 344, 353, 356, 399
studies continue to support conclusions about the
ability of XEs to imperfectly mimic physiological and pharmaceutical estrogens, as well
as their unique patterns of mechanism engagement and ER requirements 286, 287, 328, 329, 343,
344, 353, 356, 399, 400
. Because of these differences, it is important to consider the potential
effects of each XE individually at its typical culturally- or environmentally-relevant
levels, to determine what exposure advice these mechanistic studies may point to. Given
that some of these compounds can have profound stimulatory effects on prostate cancer
cell numbers (particularly the soy-related phytoestrogens coumestrol and genistein, and
especially in late-stage tumor cells), it may be prudent to advise such patients against
consuming foods that contain these phytoestrogens. On a lower priority level, resveratrol
and BPA exposures may warrant similar warnings (Table 3.1). Because BPA and
genistein had different effects on the number of viable cancer cells, depending on prostate
cancer stage, patients having recurring or long-term tumors may need different exposure
advice. Because cell growth-promoting mechanisms receive stimulation via different ER
subtypes depending upon the compound, it may be prudent to recommend blocking of
these effects via all of these receptors. Alternatively, testing of a patient’s individual
tumor receptor profiles may allow for tailoring of therapies with antagonists for
individual receptor subtypes.
Our initial hypothesis was that some of these alternative dietary estrogens might
fulfill the hoped for anti-tumor signaling and cell growth effects. It appears that this is not
the case, and the best estrogen to mediate tumor cell killing effects is E2, profiled in more
detail for these mechanisms (and others) in our previous report
. Taken together, these
findings should have profound implications for dietary recommendations for prostate
cancer patients, as well for as the development of ER-specific treatments to shrink tumors
or slow tumor progression.
Chapter 4: Conclusions and Future Directions
Showed for the first time membrane estrogen receptors (α, β, GPR30) in earlyand late-stage prostate cancer cell lines
Identified several direct, non-genomic cellular pathways that are rapidly activated
by estrogens, both physiological and exogenous, which contribute to the overall
control of prostate cancer cell numbers
Delineated the differences in signaling and proliferative responses between earlyand late-stage prostate cancer cells to estrogenic therapies, dietary estrogens, and
environmental contaminants
Characterized the membrane estrogen receptors involved in propagating nongenomic signals after stimulation with estrogens
Identified potential cellular signaling targets and estrogenic compounds for future
study and prostate cancer therapies
The interconnected web of rapid, estrogen-induced cell signaling is capable of
producing a myriad of responses in prostate cancer cells. We have also seen that the
numerous mechanisms and pathways activated in our two cell lines must be taken into
consideration as a whole, not as individual causes of actions. There were no single
pathways that completely dominated the fate of the prostate cancer cells, but there were
several signals that strongly indicated whether a cell would survive, proliferate, or die.
This was true regardless of the stage of the prostate cancer cells (LAPC-4 vs PC-3),
though the ER controls governing later stage cancers exhibit less influence than their
early counterparts. Most importantly, we have shown novel mechanisms for direct
controls over prostate cancer cells, independent of central control alterations in serum
hormone levels. These findings should be useful in the identification and development of
effective therapies for prostate tumors, as well as the recognition of potential risks to
patients with existing carcinomas.
Many past studies have also observed opposing effects of E2 and DES, mainly on
fetal development
, but these opposing effects could also still be applicable to disease
progression. Several assay results from chapter 2 showed opposing effects due to E2 or
DES treatment. This may be due to E2 and DES having different binding affinities toward
ERs - DES and other similar synthetic estrogens show greater binding affinities (2-3
times higher) for ERs than E2
299, 402
. In addition, DES showed a very slow dissociation
from ERs, similar to E2 403. The different strength and stability of DES’s interactions with
ERs may therefore convey a different message to the cellular machinery and signaling
pathways, compared to E2.
Rapid effects initiated from the membrane ERs have the ability to influence
longer, genomic actions of the cell (although not all nongenomic effects have a nuclear
endpoint). This division of cellular responses affords multiple layers of control to the cell
in response to external stimuli. Duration, strength, and localization of signals can help
determine the type of cellular response elicited, such as with the activation of ERK and
. The length of these activations can then influence the genes that are
induced in responses
. In addition, many transcriptionally active proteins are activated
and regulated through signals from mERs, thus providing a junction between nongenomic
and genomic signals
. It is likely that these same categories of events are occurring in
our two cell lines. Our experiments do not directly investigate the long-term effects of
nongenomic signals, so further experiments would be necessary. A possible target to
study would be Elk-1, which we have observed to be rapidly activated in GH3 cells
An effective assay to test this hypothesis would be to develop a transcription factorspecific blocker, such as those for STAT3 and STAT5
405, 406
, disrupt the activity of a
rapidly activated transcription factor, such as Elk-1, and then measure the outcome on
The data from our xenoestrogen studies provided insight into possible dietary
considerations for prostate cancer patients, but some results between assays appeared to
conflict. For example, resveratrol slightly increased prostate cancer cell numbers in both
cell lines, yet results from the p-cyclin and total cyclin assays would have suggested that
this XE had the ability to kill or prevent the proliferation of cells. It is clear therefore that
the scope of our experiments is limited, and does not fully examine all the rapid signaling
events initiated by XE stimulation that can affect cell number. As we have mentioned
before, other pathways that we did not examine are likely to be contributing to the overall
ability of resveratrol to slightly increase cell numbers, and that our current assay results
alone do not explain how the XE works. Additional pathways, such as through Akt/PKC,
may offer additional insight.
The decline in male estrogen levels with age has been well documented lately 293,
, and this decrease in hormone may account for the increased chance for prostate
cancer development. Additionally, the development of the prostate in fetuses is heavily
influenced by estrogens
. Our results from chapter two indicate that E2 is capable of
controlling prostate cancer cell number at physiological concentrations. However, the
lowest concentration of E2 (10-16M) had no effect on cell numbers, suggesting a lower
limit to possible sub-physiological E2 levels. Further studies to investigate this regulatory
role of E2 are warranted.
One of the largest drawbacks to our results is that the findings are currently
limited to in vitro cell line models. Future studies would have to be extended to an in vivo
model, to determine if other regulatory factors in an animal will influence estrogens’
actions on prostate tumors. Cell cultures are limited to a single type of cell, and do not
represent the complexity of cell types in the prostate or other forms of regulation present
in the body. Our findings are also currently only applicable to already developed prostate
cancer cells. There may be differences in responses and signaling pathway stimulation
regarding initial development of prostate tumors (carcinogenesis). Targeted drug therapy
is an emerging interest in the clinical and pharmaceutical industry, and identification of
cellular targets, such as ERs or signaling nodes, may provide an additional alternative to
radical surgery or chemotherapy. Additionally, it will be interesting to study the
interaction between phytoestrogens and environmental contaminants with physiological
estrogens in inhibiting or enhancing tumor growth. Some of these potential future studies
are discussed as follows.
In vivo model
The results of this study have helped develop a clearer picture for direct
estrogenic effects on prostate cancer cells. However, these findings must currently be
limited to in vitro systems of prostate cancer. The tumor microenvironment of the
originating or metastatic site contains many supporting cells that are necessary for the
development and maintenance of cancerous cells
(Figure 4.1). Therefore,
expanding upon these findings in an in vivo model is necessary. We have shown that
estrogens have a direct, nongenomic effect on prostate cancer cells, and it is likely they
can also affect neighboring non-cancerous cells of the tumor microenvironment. In fact,
the interplay between tumor cells and microenvironment has been observed to be
necessary for many tumor functions, particularly in metastasis and migration
411, 412
Skeletal metastasis in prostate cancer patients accounts for 90% of reported deaths,
suggesting that the microenvironment of the skeletal system is amenable to metastatic
prostate cancer cells
413, 414
. Typical components of the tumor microenvironment include
endothelial cells, fibroblasts secreting extracellular matrix proteins, muscle cells, immune
cells, and of course tumor cells 415. The cellular responses of these non-cancerous cells in
the tumor microenvironment may induce paracrine signaling that can increase or decrease
the growth of tumor cells.
Figure 4.1. Various components of the prostate tumor microenvironment.
Numerous types of cells support a prostate tumor cell and its ability to proliferate and eventually
metastasize. (1) Support cells that develop from prostate epithelial stem cells. (2) Connexins may
play a role in the further development of prostate tumors (Figure from Czyz et al., 2012).
Estrogenic effects on individual components of the tumor microenvironment have
already been shown. E2 modulates the levels of nitric-oxide synthase and nitric oxide in
endothelial cells, which is vital in angiogenesis
416, 417
, as well as endothelial cell
. Fibroblast migration and proliferation can be controlled by E2 as well, in
benign and tumor cells
419, 420
. ERα in the fibroblasts of the prostate cancer
microenvironment also regulate the ability of prostate cancer cells to invade the
surrounding tissue, as well as the amount of extracellular matrix molecules produced
The smooth muscle cells of the prostate stroma proliferate in response to E 2 and show an
increase in cell cycle proteins like cyclin D1
, which can be mediated by ERα
Immune cells, such as macrophages, are also associated with the prostate tumor stroma,
and proinflammatory or anti-inflammatory cytokine production is usually decreased by
E2 228, 424. Therefore, understanding the interplay of these cells and molecules should be
the next step toward elucidating rapid, direct estrogenic responses, and in particular,
through mERs.
Figure 4.2 Primary steps in
establishing a xenograft
prostate cancer model in
SCID mice.
The major steps in developing a
prostate cancer xenograft model
in SCID mice, as developed by
Lawrence and colleagues. The
addition of stromal cells and
location of implanted cells
closely mimics normal tumor
Lawrence et al., 2013).
Our laboratory has previously done similar in vivo experiments in female F344
Sprague-Dawley rats, measuring the induction of pituitary tumors after exposure to
phytoestrogens over an eight week period
. The use of severe combined
immunodeficiency (SCID) mice with prostate cancer xenografts and silastic implants
containing E2/DES/XEs is of particular interest. Mice do not develop prostate cancer
naturally, and thus provide a solid platform to study prostate cancer, without concerns
about background
. Prostate cancer models using SCID mice and estrogen treatments
have already been developed, but those studies focus on the systematic alteration of
hormone levels and metastatic potential, as opposed to the localized signals initiated by
tumor cell ERs 427, 428.
The method developed by Lawrence and colleagues provides an optimal platform
upon which to study estrogenic responses on tumor cells within a tumor
microenvironment and with supporting cells (Figure 4.2). In short, an in vivo model for
our study would entail xenografts of LAPC-4 or PC-3 prostate cancer cells implanted
subcutaneously or orthotopically
429, 430
into male SCID mice. Alternatively, cells could
be injected via the tail vein, though that model would be more appropriate for studying
metastasis 431. Tumor cells introduced to the mouse in this protocol maintain their regular
characteristics and show the ability to proliferate normally. Additionally, prostate tumor
cells are supplemented with the appropriate stromal cells, further mimicking
physiological conditions and signaling
. This model is particularly strong due to the
limitation of studies to localized tumor cell interactions with the microenvironment. This
however means that studying the ability of cells to metastasize in this model is not viable.
Also, due to a lack of or severe depression of the immune system function in SCID mice,
studying the interaction of tumor cells with macrophages and immune cells is not
. LAPC-4 prostate cancer cells growth would be supplemented with DHT
during the seeding period, as they are androgen-dependent cells. Additionally, LAPC-4
cells can become androgen-independent if deprived of androgens 29.
Following initial seeding and implantation of prostate cancer cells, the mice are
left untreated for 6-8 weeks, allowing growth of the xenograft and supporting cells. Once
the xenografts have been given the chance to establish, measurements in tumor size
would be made before subcutaneous silastic implants, containing cholesterol, E2, and
DES, would be inserted. Mice would be treated for an additional three weeks, after which
tissue would be harvested and analyzed for changes in tumor size, ER levels, and cell
cycle proteins levels, such as cyclin D1 and p16 INK4A.
Live tissue can also be assayed using our laboratory designed plate immunoassay,
as long as assay conditions are properly optimized, as we have developed for rat brain
slices, human blood cells, and rat hemi-pituitary gland (unpublished protocols). The
tissue and cells obtained from the mouse would still harbor many components of the
normal tumor microenvironment. Prostate tumor tissue obtained in this manner could be
assayed for rapid phosphorylation of MAPKs and cell cycle proteins. An alternative to
mimic tumor and paracrine signaling would be to collect media from an estrogen-treated
subset of cells (tumor associated fibroblasts for example) and then treat tumor cells with
this conditioned media, in conjunction with an estrogen 434.
Receptor targeted agonists (Acadia ERβ agonists)
Direct targeting of specific ER subtypes has become an area of interest in
estrogen-mediated/affected cancers. In prostate cancer, ERβ has drawn a lot of attention
due to its predominance in the prostate gland and the tumors arising from it, as well as the
potential inhibitory role in prostate cancer cell growth 176, 435. To this effect, we evaluated
five ERβ specific agonists from Acadia Pharmaceuticals (San Diego, CA) and measured
prostate cancer cell viability. LAPC-4 and PC-3 prostate cancer cells were treated with
each ERβ agonists for three days, then a MTT cell viability assay was conducted.
Agonizing ERβ in PC-3 cells did not have any cell killing effects, similar to what
we have seen when the same cells are treated with DES (Figure 4.3). In fact, there were
several concentrations where all agonists except 74131 increased viability, suggesting
that agonizing ERβ in PC-3 cells is not a viable therapeutic. On the other hand, LAPC-4
cells showed different responses to the five agonists. Agonist 269623 may be of
particular interest due to the 15-20% decrease in viable cells (10-13-10-9M), while 270957
at 10-6M may also warrant further study (E2 elicited a 25-30% decline in viable cells in
LAPC-4 cells). At lower concentrations, 270957 stimulated growth, and the three other
remaining agonists also showed the ability to increase cell viability. Further studies into
the key signaling pathways and cell proliferation mechanisms are warranted to identify if
these compounds interact with prostate cancer cells similarly to E2 or DES.
Figure 4.3 Number of viable prostate cancer cells following Acadia ERβ agonists.
MTT assay results after 3 day treatment with agonists in LAPC-4/PC-3 cells.
Given the ERβ rich environment of the prostate and prostatic tumors
, it is
surprising that only two of the agonists were able to reduce cell numbers, and that those
actions were limited to early-stage prostate cancer cells. As discussed in Chapter 3, this
increased ability to kill LAPC-4 cells may be due to the significant differences in ER
levels. Higher expressions levels of ERβ may then provide estrogens and estrogen
mimetics a better opportunity to induce cell killing effects
. Estrogenic stimulation of
supporting tumor cells may also play a role in the effectiveness of ERβ agonists, as
discussed previously.
Based on these preliminary results, the clinical application of using selective ERβ
agonists could provide therapeutic options for prostate cancer patients. Targeting the
ERβ-rich cells of the prostate may prove a more direct and effective measure, as opposed
to the standard therapy of LHRH agonists and antagonists, anti-androgens, and surgery,
which can have profound effects on a patient’s hormone levels. Further investigations are
necessary however, particularly with an in vivo model, to study the potential side-effects.
ERβ is found in various other tissues of the body, including serving major regulatory
roles in the nervous system and heart 437, 438.
Other potential studies and directions
Our studies focused on the effects of estrogens on established prostate cancer cell
lines. While our results reveals the mechanisms involved in killing or promoting growth
of tumor cells, it does not explain the progression of prostate cells from a nonmalignant
state to a malignant state. There are many studies focused on the prevention of prostate
tumors, particularly through the use of phytoestrogens like genistein
439, 440
. Studies on
prostatic stem cells could also further elucidate how prostatic tumors form. Epigentic
modifications to these pioneer cells has become an emerging field of study (Figure 4.4).
Figure 4.4. Improper epigenetic regulation of normal prostate cells may lead to an increased
chance for prostate cancer.
Improper regulation of DNA methylation may cause the suppression of important regulatory
genes (Figure from Cooper & Foster, 2008).
Increased environmental exposure to estrogen-like compounds, such as BPA may be
increasing the susceptibility of prostate diseases or deformities via inappropriate
methylation patterns 300, 441, 442. There are numerous genes that show hypermethylation in
prostate cancers, including INK4A and APC, both of which are known tumor suppressors
. Methylation of the glutathione S-transferase-pi gene is found in over 90% of prostate
cancers, but not in normal prostate cells
. Hypermethylation of the E-cadherin gene
may even promote the further development of cancers to a metastatic state
Interestingly, ESR1 and ESR2 are hypermethylated, ,preventing ER expression, and
strengthening the regulatory role for estrogens and ERs in the prostate and associated
diseases 443.
The estrogenic signals exerted on prostate cancer cells are not restricted to only
one compound, as in the current paradigm for our in vitro model. As already discussed
above, many signals are propagated to prostate cancer cells from the tumor
microenvironment and the endocrine system. Specifically, there are other estrogens and
mimetics circulating throughout the body, and they often affect the same targets, such as
ERs. Therefore, a more physiological and comprehensive study would be to elucidate the
effects of estrogenic mixtures on prostate cancer cell survival, both in vitro and in vivo.
Endocrine responses exerted by other physiological hormones or estrogens (physiological
or XE) on the tumor and entire body could interact or interfere with the signals initiated
at membrane and nuclear ERs of prostate cancer cells. These combinations of estrogens
and their responses may not always cause additive or subtractive responses, and may in
fact be synergistic, as we have previously seen in our laboratory 239, 276, 328.
In conclusion, the application of data from this dissertation can be expanded upon
by investigating tumor microenvironment and systems that closely mimic physiological
conditions, particularly in live animals. Further knowledge on the important estrogenregulated cell signals and mechanisms may increase and improve the arsenal of
therapeutics available for patients and clinicians to choose from. Improving and creating
more specific therapies to target prostate tumor cells, such as ERs or their downstream
targets, may also help reduce unwanted side-effects from more traditional approaches
such as radical surgery (prostatectomy and orchiectomy) and broad target
chemotherapeutics. Furthermore, developing an understanding of how prostate tumors
develop, as well as the other cellular messages they require will provide a platform for
preventative, rather than reactionary measures.
Denmeade SR, Isaacs JT. A history of prostate cancer treatment. Nat Rev Cancer
2002; 2:389-96.
Lytton B. Prostate cancer: a brief history and the discovery of hormonal ablation
treatment. J Urol 2001; 165:1859-62.
Huggins C, Hodges CV. Studies on Prostatic Cancer: I. The Effect of Castration,
of Estrogen and of Androgen Injection on Serum Phosphatases in Metastatic Carcinoma
of the Prostate. Cancer Res 1941; 1:293-7.
Byar DP. Treatment of prostatic cancer: studies by the Veterans Administration
cooperative urological research group. Bull N Y Acad Med 1972; 48:751-66.
Society AC. Cancer Facts & Figures 2014. In: Society AC, ed., 2014:1-72.
Denmeade SR, Isaacs JT. Development of prostate cancer treatment: the good
news. Prostate 2004; 58:211-24.
Saman DM, Lemieux AM, Nawal Lutfiyya M, Lipsky MS. A review of the
current epidemiology and treatment options for prostate cancer. Dis Mon 2014; 60:150-4.
Group UCSW. United States Cancer Statistics: 1999–2010 Incidence and
Mortality Web-based Report. Department of Health and Human Services, Centers for
Disease Control and Prevention, and National Cancer Institute, 2013.
Abate-Shen C, Shen MM. Molecular genetics of prostate cancer. Genes Dev
2000; 14:2410-34.
Pu YS, Chiang HS, Lin CC, Huang CY, Huang KH, Chen J. Changing trends of
prostate cancer in Asia. Aging Male 2004; 7:120-32.
Carter BS, Carter HB, Isaacs JT. Epidemiologic evidence regarding predisposing
factors to prostate cancer. Prostate 1990; 16:187-97.
Adlercreutz H. Phytoestrogens: epidemiology and a possible role in cancer
protection. Environ Health Perspect 1995; 103 Suppl 7:103-12.
Andres S, Abraham K, Appel KE, Lampen A. Risks and benefits of dietary
isoflavones for cancer. Crit Rev Toxicol 2011; 41:463-506.
Hedelin M, Balter KA, Chang ET, Bellocco R, Klint A, Johansson JE, et al.
Dietary intake of phytoestrogens, estrogen receptor-beta polymorphisms and the risk of
prostate cancer. Prostate 2006; 66:1512-20.
Kunisue T, Tanabe S, Isobe T, Aldous KM, Kannan K. Profiles of phytoestrogens
in human urine from several Asian countries. J Agric Food Chem 2010; 58:9838-46.
Gomez SL, Noone AM, Lichtensztajn DY, Scoppa S, Gibson JT, Liu L, et al.
Cancer incidence trends among Asian American populations in the United States, 19902008. J Natl Cancer Inst 2013; 105:1096-110.
Carter BS, Beaty TH, Steinberg GD, Childs B, Walsh PC. Mendelian inheritance
of familial prostate cancer. Proc Natl Acad Sci U S A 1992; 89:3367-71.
Carter BS, Bova GS, Beaty TH, Steinberg GD, Childs B, Isaacs WB, et al.
Hereditary prostate cancer: epidemiologic and clinical features. J Urol 1993; 150:797802.
Setlur SR, Mertz KD, Hoshida Y, Demichelis F, Lupien M, Perner S, et al.
Estrogen-dependent signaling in a molecularly distinct subclass of aggressive prostate
cancer. J Natl Cancer Inst 2008; 100:815-25.
Tomlins SA, Bjartell A, Chinnaiyan AM, Jenster G, Nam RK, Rubin MA, et al.
ETS gene fusions in prostate cancer: from discovery to daily clinical practice. Eur Urol
2009; 56:275-86.
Demichelis F, Fall K, Perner S, Andren O, Schmidt F, Setlur SR, et al.
TMPRSS2:ERG gene fusion associated with lethal prostate cancer in a watchful waiting
cohort. Oncogene 2007; 26:4596-9.
Tomlins SA, Mehra R, Rhodes DR, Smith LR, Roulston D, Helgeson BE, et al.
TMPRSS2:ETV4 gene fusions define a third molecular subtype of prostate cancer.
Cancer Res 2006; 66:3396-400.
Yu J, Mani RS, Cao Q, Brenner CJ, Cao X, Wang X, et al. An integrated network
of androgen receptor, polycomb, and TMPRSS2-ERG gene fusions in prostate cancer
progression. Cancer Cell 2010; 17:443-54.
Barbieri CE, Baca SC, Lawrence MS, Demichelis F, Blattner M, Theurillat JP, et
al. Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in
prostate cancer. Nat Genet 2012; 44:685-9.
An J, Wang C, Deng Y, Yu L, Huang H. Destruction of full-length androgen
receptor by wild-type SPOP, but not prostate-cancer-associated mutants. Cell Rep 2014;
He WW, Sciavolino PJ, Wing J, Augustus M, Hudson P, Meissner PS, et al. A
novel human prostate-specific, androgen-regulated homeobox gene (NKX3.1) that maps
to 8p21, a region frequently deleted in prostate cancer. Genomics 1997; 43:69-77.
Cairns P, Okami K, Halachmi S, Halachmi N, Esteller M, Herman JG, et al.
Frequent inactivation of PTEN/MMAC1 in primary prostate cancer. Cancer Res 1997;
Visakorpi T, Hyytinen E, Koivisto P, Tanner M, Keinanen R, Palmberg C, et al.
In vivo amplification of the androgen receptor gene and progression of human prostate
cancer. Nat Genet 1995; 9:401-6.
Klein KA, Reiter RE, Redula J, Moradi H, Zhu XL, Brothman AR, et al.
Progression of metastatic human prostate cancer to androgen independence in
immunodeficient SCID mice. Nat Med 1997; 3:402-8.
Kaighn ME, Narayan KS, Ohnuki Y, Lechner JF, Jones LW. Establishment and
characterization of a human prostatic carcinoma cell line (PC-3). Investigative urology
1979; 17:16-23.
Saramaki OR, Harjula AE, Martikainen PM, Vessella RL, Tammela TL,
Visakorpi T. TMPRSS2:ERG fusion identifies a subgroup of prostate cancers with a
favorable prognosis. Clin Cancer Res 2008; 14:3395-400.
van Bokhoven A, Varella-Garcia M, Korch C, Johannes WU, Smith EE, Miller
HL, et al. Molecular characterization of human prostate carcinoma cell lines. The
Prostate 2003; 57:205-25.
Purvis K, Attramadal A, Rui H. Secretory function of the prostate gland. Scand J
Urol Nephrol Suppl 1988; 107:46-51.
Cohen RJ, Shannon BA, Phillips M, Moorin RE, Wheeler TM, Garrett KL.
Central zone carcinoma of the prostate gland: a distinct tumor type with poor prognostic
features. J Urol 2008; 179:1762-7; discussion 7.
Lee CH, Akin-Olugbade O, Kirschenbaum A. Overview of prostate anatomy,
histology, and pathology. Endocrinol Metab Clin North Am 2011; 40:565-75, viii-ix.
McNeal JE, Redwine EA, Freiha FS, Stamey TA. Zonal distribution of prostatic
adenocarcinoma. Correlation with histologic pattern and direction of spread. Am J Surg
Pathol 1988; 12:897-906.
McNeal JE. Cancer volume and site of origin of adenocarcinoma in the prostate:
relationship to local and distant spread. Hum Pathol 1992; 23:258-66.
Myers RP. Structure of the adult prostate from a clinician's standpoint. Clin Anat
2000; 13:214-5.
McNeal JE. Normal anatomy of the prostate and changes in benign prostatic
hypertrophy and carcinoma. Semin Ultrasound CT MR 1988; 9:329-34.
McNeal JE. Normal histology of the prostate. Am J Surg Pathol 1988; 12:619-33.
Feldman BJ, Feldman D. The development of androgen-independent prostate
cancer. Nat Rev Cancer 2001; 1:34-45.
Denmeade SR, Isaacs JT. Androgen Deprivation Strategies in the Treatment of
Advanced Prostate Cancer. In: Bast RC, Kufe DW, Pollock RE, Weichselbaum RR,
Holland JF, Frei E, eds. Holland-Frei Cancer Medicine. Hamilton, ON: BC Decker, 2000.
Sharifi N, Auchus RJ. Steroid biosynthesis and prostate cancer. Steroids 2012;
Randall VA. Role of 5 alpha-reductase in health and disease. Baillieres Clin
Endocrinol Metab 1994; 8:405-31.
Meinhardt U, Mullis PE. The essential role of the aromatase/p450arom. Semin
Reprod Med 2002; 20:277-84.
Hsing AW, Reichardt JK, Stanczyk FZ. Hormones and prostate cancer: current
perspectives and future directions. Prostate 2002; 52:213-35.
Zhao H, Kim Y, Wang P, Lapointe J, Tibshirani R, Pollack JR, et al. Genome-
wide characterization of gene expression variations and DNA copy number changes in
prostate cancer cell lines. Prostate 2005; 63:187-97.
Amory JK, Anawalt BD, Matsumoto AM, Page ST, Bremner WJ, Wang C, et al.
The effect of 5alpha-reductase inhibition with dutasteride and finasteride on bone mineral
density, serum lipoproteins, hemoglobin, prostate specific antigen and sexual function in
healthy young men. J Urol 2008; 179:2333-8.
Makridakis NM, Buchanan G, Tilley W, Reichardt JK. Androgen metabolic genes
in prostate cancer predisposition and progression. Front Biosci 2005; 10:2892-903.
Makridakis N, Ross RK, Pike MC, Chang L, Stanczyk FZ, Kolonel LN, et al. A
prevalent missense substitution that modulates activity of prostatic steroid 5alphareductase. Cancer Res 1997; 57:1020-2.
Gao W, Bohl CE, Dalton JT. Chemistry and structural biology of androgen
receptor. Chem Rev 2005; 105:3352-70.
Brinkmann AO, Blok LJ, de Ruiter PE, Doesburg P, Steketee K, Berrevoets CA,
et al. Mechanisms of androgen receptor activation and function. J Steroid Biochem Mol
Biol 1999; 69:307-13.
Migeon BR, Brown TR, Axelman J, Migeon CJ. Studies of the locus for androgen
receptor: localization on the human X chromosome and evidence for homology with the
Tfm locus in the mouse. Proc Natl Acad Sci U S A 1981; 78:6339-43.
Tilley WD, Marcelli M, Wilson JD, McPhaul MJ. Characterization and
expression of a cDNA encoding the human androgen receptor. Proc Natl Acad Sci U S A
1989; 86:327-31.
Trapman J, Klaassen P, Kuiper GG, van der Korput JA, Faber PW, van Rooij HC,
et al. Cloning, structure and expression of a cDNA encoding the human androgen
receptor. Biochem Biophys Res Commun 1988; 153:241-8.
Chaudhri RA, Olivares-Navarrete R, Cuenca N, Hadadi A, Boyan BD, Schwartz
Z. Membrane estrogen signaling enhances tumorigenesis and metastatic potential of
breast cancer cells via estrogen receptor-alpha36 (ERalpha36). J Biol Chem 2012;
Hartman J, Ström A, Gustafsson J-Å. Current concepts and significance of
estrogen receptor β in prostate cancer. Steroids 2012; 77:1262-6.
Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, et al.
Comparison of the ligand binding specificity and transcript tissue distribution of estrogen
receptors alpha and beta. Endocrinology 1997; 138:863-70.
Lutz LB, Jamnongjit M, Yang WH, Jahani D, Gill A, Hammes SR. Selective
modulation of genomic and nongenomic androgen responses by androgen receptor
ligands. Mol Endocrinol 2003; 17:1106-16.
Kousteni S, Bellido T, Plotkin LI, O'Brien CA, Bodenner DL, Han L, et al.
Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors:
dissociation from transcriptional activity. Cell 2001; 104:719-30.
Kampa M, Papakonstanti EA, Hatzoglou A, Stathopoulos EN, Stournaras C,
Castanas E. The human prostate cancer cell line LNCaP bears functional membrane
testosterone receptors that increase PSA secretion and modify actin cytoskeleton. Faseb j
2002; 16:1429-31.
Unni E, Sun S, Nan B, McPhaul MJ, Cheskis B, Mancini MA, et al. Changes in
androgen receptor nongenotropic signaling correlate with transition of LNCaP cells to
androgen independence. Cancer Res 2004; 64:7156-68.
De Leon JT, Iwai A, Feau C, Garcia Y, Balsiger HA, Storer CL, et al. Targeting
the regulation of androgen receptor signaling by the heat shock protein 90 cochaperone
FKBP52 in prostate cancer cells. Proc Natl Acad Sci U S A 2011; 108:11878-83.
Chen S, Xu Y, Yuan X, Bubley GJ, Balk SP. Androgen receptor phosphorylation
and stabilization in prostate cancer by cyclin-dependent kinase 1. Proc Natl Acad Sci U S
A 2006; 103:15969-74.
Lin DY, Fang HI, Ma AH, Huang YS, Pu YS, Jenster G, et al. Negative
modulation of androgen receptor transcriptional activity by Daxx. Mol Cell Biol 2004;
Sharma NL, Massie CE, Ramos-Montoya A, Zecchini V, Scott HE, Lamb AD, et
al. The androgen receptor induces a distinct transcriptional program in castration-resistant
prostate cancer in man. Cancer Cell 2013; 23:35-47.
Shang Y, Myers M, Brown M. Formation of the androgen receptor transcription
complex. Mol Cell 2002; 9:601-10.
Balk SP, Ko YJ, Bubley GJ. Biology of prostate-specific antigen. J Clin Oncol
2003; 21:383-91.
Ragsdale JW, 3rd, Halstater B, Martinez-Bianchi V. Prostate Cancer Screening.
Prim Care 2014; 41:355-70.
Peter J, Unverzagt C, Hoesel W. Analysis of free prostate-specific antigen (PSA)
after chemical release from the complex with alpha(1)-antichymotrypsin (PSA-ACT).
Clin Chem 2000; 46:474-82.
Stephan C, Ralla B, Jung K. Prostate-specific antigen and other serum and urine
markers in prostate cancer. Biochim Biophys Acta 2014; 1846:99-112.
Prensner JR, Rubin MA, Wei JT, Chinnaiyan AM. Beyond PSA: the next
generation of prostate cancer biomarkers. Sci Transl Med 2012; 4:127rv3.
Clarke R, Brunner N, Katzenellenbogen BS, Thompson EW, Norman MJ, Koppi
C, et al. Progression of human breast cancer cells from hormone-dependent to hormoneindependent growth both in vitro and in vivo. Proc Natl Acad Sci U S A 1989; 86:364953.
Deligdisch L, Holinka CF. Progesterone receptors in two groups of endometrial
carcinoma. Cancer 1986; 57:1385-8.
Yuli C, Shao N, Rao R, Aysola P, Reddy V, Oprea-llies G, et al. BRCA1a has
antitumor activity in TN breast, ovarian and prostate cancers. Oncogene 2007; 26:6031-7.
Langeveld JW, Lycklama a Nijeholt AA, Jonas U. Oestrogen in the treatment of
prostatic carcinoma. What is the safe and effective dose of ethinyloestradiol? Br J Urol
1989; 63:76-9.
Malkowicz SB. The role of diethylstilbestrol in the treatment of prostate cancer.
Urology 2001; 58:108-13.
Bosset PO, Albiges L, Seisen T, de la Motte Rouge T, Phe V, Bitker MO, et al.
Current role of diethylstilbestrol in the management of advanced prostate cancer. BJU Int
2012; 110:E826-9.
Kemp HA, Read GF, Riad-Fahmy D, Pike AW, Gaskell SJ, Queen K, et al.
Measurement of diethylstilbestrol in plasma from patients with cancer of the prostate.
Cancer Res 1981; 41:4693-7.
Danutra V, Harper ME, Griffiths K. The effect of stilboestrol analogues on the
metabolism of steroids by the testis and prostate of the rat in vitro. J Endocrinol 1973;
Auclerc G, Antoine EC, Cajfinger F, Brunet-Pommeyrol A, Agazia C, Khayat D.
Management of advanced prostate cancer. Oncologist 2000; 5:36-44.
Chaturvedi S, Garcia JA. Novel agents in the management of castration resistant
prostate cancer. J Carcinog 2014; 13:5.
Seidenfeld J, Samson DJ, Hasselblad V, Aronson N, Albertsen PC, Bennett CL, et
al. Single-therapy androgen suppression in men with advanced prostate cancer: a
systematic review and meta-analysis. Ann Intern Med 2000; 132:566-77.
Schally AV, Comaru-Schally AM, Plonowski A, Nagy A, Halmos G, Rekasi Z.
Peptide analogs in the therapy of prostate cancer. Prostate 2000; 45:158-66.
D'Amico AV. US Food and Drug Administration approval of drugs for the
treatment of prostate cancer: a new era has begun. J Clin Oncol. United States,
Carter NJ, Keam SJ. Degarelix: a review of its use in patients with prostate
cancer. Drugs 2014; 74:699-712.
Thompson IM. Flare Associated with LHRH-Agonist Therapy. Rev Urol 2001; 3
Suppl 3:S10-4.
Crawford ED. Hormonal therapy in prostate cancer: historical approaches. Rev
Urol 2004; 6 Suppl 7:S3-s11.
Pavone-Macaluso M, de Voogt HJ, Viggiano G, Barasolo E, Lardennois B, de
Pauw M, et al. Comparison of diethylstilbestrol, cyproterone acetate and
medroxyprogesterone acetate in the treatment of advanced prostatic cancer: final analysis
of a randomized phase III trial of the European Organization for Research on Treatment
of Cancer Urological Group. J Urol 1986; 136:624-31.
Gillatt D. Antiandrogen treatments in locally advanced prostate cancer: are they
all the same? J Cancer Res Clin Oncol 2006; 132 Suppl 1:S17-26.
Pilepich MV, Krall JM, al-Sarraf M, John MJ, Doggett RL, Sause WT, et al.
Androgen deprivation with radiation therapy compared with radiation therapy alone for
locally advanced prostatic carcinoma: a randomized comparative trial of the Radiation
Therapy Oncology Group. Urology 1995; 45:616-23.
Budaus L, Bolla M, Bossi A, Cozzarini C, Crook J, Widmark A, et al. Functional
outcomes and complications following radiation therapy for prostate cancer: a critical
analysis of the literature. Eur Urol 2012; 61:112-27.
Wisenbaugh ES, Andrews PE, Ferrigni RG, Schild SE, Keole SR, Wong WW, et
al. Proton beam therapy for localized prostate cancer 101: basics, controversies, and facts.
Rev Urol 2014; 16:67-75.
Scher HI, Kelly WM, Zhang ZF, Ouyang P, Sun M, Schwartz M, et al. Post-
therapy serum prostate-specific antigen level and survival in patients with androgenindependent prostate cancer. J Natl Cancer Inst 1999; 91:244-51.
Kitamura T. Necessity of re-evaluation of estramustine phosphate sodium (EMP)
as a treatment option for first-line monotherapy in advanced prostate cancer. Int J Urol
2001; 8:33-6.
Hwang C. Overcoming docetaxel resistance in prostate cancer: a perspective
review. Ther Adv Med Oncol 2012; 4:329-40.
Craft N, Chhor C, Tran C, Belldegrun A, DeKernion J, Witte ON, et al. Evidence
for clonal outgrowth of androgen-independent prostate cancer cells from androgendependent tumors through a two-step process. Cancer Res 1999; 59:5030-6.
Lonergan PE, Tindall DJ. Androgen receptor signaling in prostate cancer
development and progression. J Carcinog 2011; 10:20.
Lee WH, Morton RA, Epstein JI, Brooks JD, Campbell PA, Bova GS, et al.
Cytidine methylation of regulatory sequences near the pi-class glutathione S-transferase
gene accompanies human prostatic carcinogenesis. Proc Natl Acad Sci U S A 1994;
Comstock CE, Augello MA, Schiewer MJ, Karch J, Burd CJ, Ertel A, et al.
Cyclin D1 is a selective modifier of androgen-dependent signaling and androgen receptor
function. J Biol Chem 2011; 286:8117-27.
Chang GT, Blok LJ, Steenbeek M, Veldscholte J, van Weerden WM, van
Steenbrugge GJ, et al. Differentially expressed genes in androgen-dependent and independent prostate carcinomas. Cancer Res 1997; 57:4075-81.
Gregory CW, Johnson RT, Jr., Mohler JL, French FS, Wilson EM. Androgen
receptor stabilization in recurrent prostate cancer is associated with hypersensitivity to
low androgen. Cancer Res 2001; 61:2892-8.
Koivisto P, Kononen J, Palmberg C, Tammela T, Hyytinen E, Isola J, et al.
Androgen receptor gene amplification: a possible molecular mechanism for androgen
deprivation therapy failure in prostate cancer. Cancer Res 1997; 57:314-9.
Brand LJ, Dehm SM. Androgen receptor gene rearrangements: new perspectives
on prostate cancer progression. Curr Drug Targets 2013; 14:441-9.
Dehm SM, Tindall DJ. Alternatively spliced androgen receptor variants. Endocr
Relat Cancer 2011; 18:R183-96.
Pink JJ, Jordan VC. Models of estrogen receptor regulation by estrogens and
antiestrogens in breast cancer cell lines. Cancer Res 1996; 56:2321-30.
Veldscholte J, Ris-Stalpers C, Kuiper GG, Jenster G, Berrevoets C, Claassen E, et
al. A mutation in the ligand binding domain of the androgen receptor of human LNCaP
cells affects steroid binding characteristics and response to anti-androgens. Biochem
Biophys Res Commun 1990; 173:534-40.
Berrevoets CA, Veldscholte J, Mulder E. Effects of antiandrogens on
transformation and transcription activation of wild-type and mutated (LNCaP) androgen
receptors. J Steroid Biochem Mol Biol 1993; 46:731-6.
Zhao XY, Boyle B, Krishnan AV, Navone NM, Peehl DM, Feldman D. Two
mutations identified in the androgen receptor of the new human prostate cancer cell line
MDA PCa 2a. J Urol 1999; 162:2192-9.
Zhao XY, Malloy PJ, Krishnan AV, Swami S, Navone NM, Peehl DM, et al.
Glucocorticoids can promote androgen-independent growth of prostate cancer cells
through a mutated androgen receptor. Nat Med 2000; 6:703-6.
Culig Z, Hobisch A, Cronauer MV, Radmayr C, Hittmair A, Zhang J, et al.
Regulation of prostatic growth and function by peptide growth factors. Prostate 1996;
Craft N, Shostak Y, Carey M, Sawyers CL. A mechanism for hormone-
independent prostate cancer through modulation of androgen receptor signaling by the
HER-2/neu tyrosine kinase. Nat Med 1999; 5:280-5.
Fan W, Yanase T, Morinaga H, Okabe T, Nomura M, Daitoku H, et al. Insulin-
like growth factor 1/insulin signaling activates androgen signaling through direct
interactions of Foxo1 with androgen receptor. J Biol Chem 2007; 282:7329-38.
Aaronson DS, Muller M, Neves SR, Chung WC, Jayaram G, Iyengar R, et al. An
androgen-IL-6-Stat3 autocrine loop re-routes EGF signal in prostate cancer cells. Mol
Cell Endocrinol 2007; 270:50-6.
Rinaldo F, Li J, Wang E, Muders M, Datta K. RalA regulates vascular endothelial
growth factor-C (VEGF-C) synthesis in prostate cancer cells during androgen ablation.
Oncogene 2007; 26:1731-8.
Dar JA, Masoodi KZ, Eisermann K, Isharwal S, Ai J, Pascal LE, et al. The N-
terminal domain of the androgen receptor drives its nuclear localization in castrationresistant prostate cancer cells. J Steroid Biochem Mol Biol 2014; 143:473-80.
Li L, Ren CH, Tahir SA, Ren C, Thompson TC. Caveolin-1 maintains activated
Akt in prostate cancer cells through scaffolding domain binding site interactions with and
inhibition of serine/threonine protein phosphatases PP1 and PP2A. Mol Cell Biol 2003;
Foradori CD, Weiser MJ, Handa RJ. Non-genomic actions of androgens. Front
Neuroendocrinol 2008; 29:169-81.
Ramsden M, Nyborg AC, Murphy MP, Chang L, Stanczyk FZ, Golde TE, et al.
Androgens modulate beta-amyloid levels in male rat brain. J Neurochem 2003; 87:10525.
Hammond J, Le Q, Goodyer C, Gelfand M, Trifiro M, LeBlanc A. Testosterone-
mediated neuroprotection through the androgen receptor in human primary neurons. J
Neurochem 2001; 77:1319-26.
Furuya Y, Krajewski S, Epstein JI, Reed JC, Isaacs JT. Expression of bcl-2 and
the progression of human and rodent prostatic cancers. Clin Cancer Res 1996; 2:389-98.
McDonnell TJ, Troncoso P, Brisbay SM, Logothetis C, Chung LW, Hsieh JT, et
al. Expression of the protooncogene bcl-2 in the prostate and its association with
emergence of androgen-independent prostate cancer. Cancer Res 1992; 52:6940-4.
Nelson JB, Udan MS, Guruli G, Pflug BR. Endothelin-1 inhibits apoptosis in
prostate cancer. Neoplasia 2005; 7:631-7.
Chen X, Thakkar H, Tyan F, Gim S, Robinson H, Lee C, et al. Constitutively
active Akt is an important regulator of TRAIL sensitivity in prostate cancer. Oncogene
2001; 20:6073-83.
Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100:57-70.
Thakur A, Vaishampayan U, Lum LG. Immunotherapy and immune evasion in
prostate cancer. Cancers (Basel) 2013; 5:569-90.
Tien AH, Xu L, Helgason CD. Altered immunity accompanies disease
progression in a mouse model of prostate dysplasia. Cancer Res 2005; 65:2947-55.
Miller AM, Lundberg K, Ozenci V, Banham AH, Hellstrom M, Egevad L, et al.
CD4+CD25high T cells are enriched in the tumor and peripheral blood of prostate cancer
patients. J Immunol 2006; 177:7398-405.
Gabrilovich DI, Chen HL, Girgis KR, Cunningham HT, Meny GM, Nadaf S, et
al. Production of vascular endothelial growth factor by human tumors inhibits the
functional maturation of dendritic cells. Nat Med 1996; 2:1096-103.
Isaacs JT. The biology of hormone refractory prostate cancer. Why does it
develop? Urol Clin North Am 1999; 26:263-73.
Aggarwal R, Weinberg V, Small EJ, Oh W, Rushakoff R, Ryan CJ. The
mechanism of action of estrogen in castration-resistant prostate cancer: clues from
hormone levels. Clin Genitourin Cancer 2009; 7:E71-6.
Hess RA, Bunick D, Lee KH, Bahr J, Taylor JA, Korach KS, et al. A role for
oestrogens in the male reproductive system. Nature 1997; 390:509-12.
Cunha GR, Wang YZ, Hayward SW, Risbridger GP. Estrogenic effects on
prostatic differentiation and carcinogenesis. Reprod Fertil Dev 2001; 13:285-96.
Akingbemi BT. Estrogen regulation of testicular function. Reprod Biol
Endocrinol 2005; 3:51.
Hong JH, Song C, Shin Y, Kim H, Cho SP, Kim WJ, et al. Estrogen induction of
smooth muscle differentiation of human prostatic stromal cells is mediated by
transforming growth factor-beta. J Urol 2004; 171:1965-9.
Klaiber EL, Broverman DM, Haffajee CI, Hochman JS, Sacks GM, Dalen JE.
Serum estrogen levels in men with acute myocardial infarction. Am J Med 1982; 73:87281.
Heller J, Dogan I, Schulz JB, Reetz K. Evidence for gender differences in
cognition, emotion and quality of life in Parkinson's disease? Aging Dis 2014; 5:63-75.
Murata Y, Robertson KM, Jones ME, Simpson ER. Effect of estrogen deficiency
in the male: the ArKO mouse model. Mol Cell Endocrinol 2002; 193:7-12.
Deroo BJ, Korach KS. Estrogen receptors and human disease. J Clin Invest 2006;
Thomas P, Pang Y, Filardo EJ, Dong J. Identity of an estrogen membrane
receptor coupled to a G protein in human breast cancer cells. Endocrinology 2005;
Bollig A, Miksicek RJ. An estrogen receptor-alpha splicing variant mediates both
positive and negative effects on gene transcription. Mol Endocrinol 2000; 14:634-49.
Thomas C, Gustafsson JA. The different roles of ER subtypes in cancer biology
and therapy. Nat Rev Cancer 2011; 11:597-608.
Lee LM, Cao J, Deng H, Chen P, Gatalica Z, Wang ZY. ER-alpha36, a novel
variant of ER-alpha, is expressed in ER-positive and -negative human breast carcinomas.
Anticancer Res 2008; 28:479-83.
Zhang X, Deng H, Wang ZY. Estrogen activation of the mitogen-activated protein
kinase is mediated by ER-alpha36 in ER-positive breast cancer cells. J Steroid Biochem
Mol Biol 2014; 143:434-43.
Dey P, Jonsson P, Hartman J, Williams C, Strom A, Gustafsson JA. Estrogen
receptors beta1 and beta2 have opposing roles in regulating proliferation and bone
metastasis genes in the prostate cancer cell line PC3. Mol Endocrinol 2012; 26:19912003.
Chaudhri RA, Hadadi A, Lobachev KS, Schwartz Z, Boyan BD. Estrogen
receptor-alpha 36 mediates the anti-apoptotic effect of estradiol in triple negative breast
cancer cells via a membrane-associated mechanism. Biochim Biophys Acta 2014.
Li L, Haynes MP, Bender JR. Plasma membrane localization and function of the
estrogen receptor alpha variant (ER46) in human endothelial cells. Proc Natl Acad Sci U
S A 2003; 100:4807-12.
Lin AH, Li RW, Ho EY, Leung GP, Leung SW, Vanhoutte PM, et al. Differential
ligand binding affinities of human estrogen receptor-alpha isoforms. PLoS One 2013;
Figtree GA, McDonald D, Watkins H, Channon KM. Truncated estrogen receptor
alpha 46-kDa isoform in human endothelial cells: relationship to acute activation of nitric
oxide synthase. Circulation 2003; 107:120-6.
Lin SL, Yan LY, Liang XW, Wang ZB, Wang ZY, Qiao J, et al. A novel variant
of ER-alpha, ER-alpha36 mediates testosterone-stimulated ERK and Akt activation in
endometrial cancer Hec1A cells. Reprod Biol Endocrinol 2009; 7:102.
Hattori T, Stawski L, Nakerakanti SS, Trojanowska M. Fli1 is a negative
regulator of estrogen receptor alpha in dermal fibroblasts. J Invest Dermatol 2011;
Lee MT, Ouyang B, Ho SM, Leung YK. Differential expression of estrogen
receptor beta isoforms in prostate cancer through interplay between transcriptional and
translational regulation. Mol Cell Endocrinol 2013; 376:125-35.
Rajapaksa G, Nikolos F, Bado I, Clarke R, Gustafsson JA, Thomas C. ERbeta
decreases breast cancer cell survival by regulating the IRE1/XBP-1 pathway. Oncogene
Rosin G, de Boniface J, Karthik GM, Frisell J, Bergh J, Hartman J. Oestrogen
receptors beta1 and betacx have divergent roles in breast cancer survival and lymph node
metastasis. Br J Cancer 2014; 111:918-26.
Chantzi NI, Palaiologou M, Stylianidou A, Goutas N, Vassilaros S, Kourea HP, et
al. Estrogen receptor beta2 is inversely correlated with Ki-67 in hyperplastic and
noninvasive neoplastic breast lesions. J Cancer Res Clin Oncol 2014; 140:1057-66.
Matthews J, Gustafsson JA. Estrogen signaling: a subtle balance between ER
alpha and ER beta. Mol Interv 2003; 3:281-92.
Cotrim CZ, Fabris V, Doria ML, Lindberg K, Gustafsson JA, Amado F, et al.
Estrogen receptor beta growth-inhibitory effects are repressed through activation of
MAPK and PI3K signalling in mammary epithelial and breast cancer cells. Oncogene
2013; 32:2390-402.
Filardo EJ, Thomas P. Minireview: G protein-coupled estrogen receptor-1,
GPER-1: its mechanism of action and role in female reproductive cancer, renal and
vascular physiology. Endocrinology 2012; 153:2953-62.
Ascenzi P, Bocedi A, Marino M. Structure-function relationship of estrogen
receptor alpha and beta: impact on human health. Mol Aspects Med 2006; 27:299-402.
Medzihradszky KF. Characterization of protein N-glycosylation. Methods
Enzymol 2005; 405:116-38.
Cheng X, Hart GW. Glycosylation of the murine estrogen receptor-alpha. J
Steroid Biochem Mol Biol 2000; 75:147-58.
Cheng X, Hart GW. Alternative O-glycosylation/O-phosphorylation of serine-16
in murine estrogen receptor beta: post-translational regulation of turnover and
transactivation activity. J Biol Chem 2001; 276:10570-5.
Marino M, Ficca R, Ascenzi P, Trentalance A. Nitric oxide inhibits selectively the
17beta-estradiol-induced gene expression without affecting nongenomic events in HeLa
cells. Biochem Biophys Res Commun 2001; 286:529-33.
Garban HJ, Marquez-Garban DC, Pietras RJ, Ignarro LJ. Rapid nitric oxide-
mediated S-nitrosylation of estrogen receptor: regulation of estrogen-dependent gene
transcription. Proc Natl Acad Sci U S A 2005; 102:2632-6.
Acconcia F, Ascenzi P, Bocedi A, Spisni E, Tomasi V, Trentalance A, et al.
Palmitoylation-dependent estrogen receptor alpha membrane localization: regulation by
17beta-estradiol. Mol Biol Cell 2005; 16:231-7.
Resh MD. Fatty acylation of proteins: new insights into membrane targeting of
myristoylated and palmitoylated proteins. Biochim Biophys Acta 1999; 1451:1-16.
Marino M, Ascenzi P. Membrane association of estrogen receptor alpha and beta
influences 17beta-estradiol-mediated cancer cell proliferation. Steroids 2008; 73:853-8.
Levin ER. Cell localization, physiology, and nongenomic actions of estrogen
receptors. J Appl Physiol (1985) 2001; 91:1860-7.
Levin ER. Extranuclear estrogen receptor's roles in physiology: lessons from
mouse models. Am J Physiol Endocrinol Metab 2014; 307:E133-40.
Norfleet AM, Clarke CH, Gametchu B, Watson CS. Antibodies to the estrogen
receptor-alpha modulate rapid prolactin release from rat pituitary tumor cells through
plasma membrane estrogen receptors. Faseb j 2000; 14:157-65.
Watson CS, Campbell CH, Gametchu B. Membrane oestrogen receptors on rat
pituitary tumour cells: immuno-identification and responses to oestradiol and
xenoestrogens. Exp Physiol 1999; 84:1013-22.
Dominguez R, Micevych P. Estradiol rapidly regulates membrane estrogen
receptor alpha levels in hypothalamic neurons. J Neurosci 2010; 30:12589-96.
Monje P, Boland R. Characterization of membrane estrogen binding proteins from
rabbit uterus. Mol Cell Endocrinol 1999; 147:75-84.
Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. Cloning of a
novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci U S A 1996;
Knoblauch R, Garabedian MJ. Role for Hsp90-associated cochaperone p23 in
estrogen receptor signal transduction. Mol Cell Biol 1999; 19:3748-59.
Nilsson S, Gustafsson JA. Estrogen receptors: therapies targeted to receptor
subtypes. Clin Pharmacol Ther 2011; 89:44-55.
Watson CS. Signaling themes shared between peptide and steroid hormones at the
plasma membrane. Sci STKE. United States, 1999:Pe1.
Jeng YJ, Kochukov M, Watson CS. Combinations of physiologic estrogens with
xenoestrogens alter calcium and kinase responses, prolactin release, and membrane
estrogen receptor trafficking in rat pituitary cells. Environ Health 2010; 9:61.
Kuo J, Hamid N, Bondar G, Prossnitz ER, Micevych P. Membrane Estrogen
Receptors Stimulate Intracellular Calcium Release and Progesterone Synthesis in
Hypothalamic Astrocytes. The Journal of Neuroscience 2010; 30:12950-7.
Dominguez R, Dewing P, Kuo J, Micevych P. Membrane-initiated estradiol
signaling in immortalized hypothalamic N-38 neurons. Steroids 2013; 78:607-13.
Watson CS, Jeng YJ, Kochukov MY. Nongenomic actions of estradiol compared
with estrone and estriol in pituitary tumor cell signaling and proliferation. Faseb j 2008;
Alyea RA, Laurence SE, Kim SH, Katzenellenbogen BS, Katzenellenbogen JA,
Watson CS. The roles of membrane estrogen receptor subtypes in modulating dopamine
transporters in PC-12 cells. J Neurochem 2008; 106:1525-33.
Koldzic-Zivanovic N, Seitz PK, Watson CS, Cunningham KA, Thomas ML.
Intracellular signaling involved in estrogen regulation of serotonin reuptake. Mol Cell
Endocrinol 2004; 226:33-42.
Razandi M, Pedram A, Levin ER. Plasma membrane estrogen receptors signal to
antiapoptosis in breast cancer. Mol Endocrinol 2000; 14:1434-47.
Zárate S, Jaita G, Ferraris J, Eijo G, Magri ML, Pisera D, et al. Estrogens Induce
Expression of Membrane-Associated Estrogen Receptor α Isoforms in Lactotropes. PLoS
ONE 2012; 7:e41299.
Zivadinovic D, Gametchu B, Watson CS. Membrane estrogen receptor-alpha
levels in MCF-7 breast cancer cells predict cAMP and proliferation responses. Breast
Cancer Research 2005; 7:R101-R12.
Watters JJ, Campbell JS, Cunningham MJ, Krebs EG, Dorsa DM. Rapid
membrane effects of steroids in neuroblastoma cells: effects of estrogen on mitogen
activated protein kinase signalling cascade and c-fos immediate early gene transcription.
Endocrinology 1997; 138:4030-3.
Prossnitz ER, Arterburn JB, Smith HO, Oprea TI, Sklar LA, Hathaway HJ.
Estrogen signaling through the transmembrane G protein-coupled receptor GPR30. Annu
Rev Physiol 2008; 70:165-90.
Pappas TC, Gametchu B, Watson CS. Membrane estrogen receptors identified by
multiple antibody labeling and impeded-ligand binding. Faseb j 1995; 9:404-10.
Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J, Andersson G, et al.
Mechanisms of estrogen action. Physiol Rev 2001; 81:1535-65.
La Rosa P, Pesiri V, Leclercq G, Marino M, Acconcia F. Palmitoylation regulates
17beta-estradiol-induced estrogen receptor-alpha degradation and transcriptional activity.
Mol Endocrinol 2012; 26:762-74.
Adlanmerini M, Solinhac R, Abot A, Fabre A, Raymond-Letron I, Guihot AL, et
al. Mutation of the palmitoylation site of estrogen receptor alpha in vivo reveals tissue-
specific roles for membrane versus nuclear actions. Proc Natl Acad Sci U S A 2014;
Jeng YJ, Watson CS. Proliferative and anti-proliferative effects of dietary levels
of phytoestrogens in rat pituitary GH3/B6/F10 cells - the involvement of rapidly activated
kinases and caspases. BMC Cancer 2009; 9:334.
Nakhla AM, Khan MS, Romas NP, Rosner W. Estradiol causes the rapid
accumulation of cAMP in human prostate. Proc Natl Acad Sci U S A 1994; 91:5402-5.
Aronica SM, Kraus WL, Katzenellenbogen BS. Estrogen action via the cAMP
signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene
transcription. Proc Natl Acad Sci U S A 1994; 91:8517-21.
Jakacka M, Ito M, Weiss J, Chien PY, Gehm BD, Jameson JL. Estrogen receptor
binding to DNA is not required for its activity through the nonclassical AP1 pathway. J
Biol Chem 2001; 276:13615-21.
Bjornstrom L, Sjoberg M. Mechanisms of estrogen receptor signaling:
convergence of genomic and nongenomic actions on target genes. Mol Endocrinol 2005;
Umayahara Y, Kawamori R, Watada H, Imano E, Iwama N, Morishima T, et al.
Estrogen regulation of the insulin-like growth factor I gene transcription involves an AP1 enhancer. J Biol Chem 1994; 269:16433-42.
Liu MM, Albanese C, Anderson CM, Hilty K, Webb P, Uht RM, et al. Opposing
action of estrogen receptors alpha and beta on cyclin D1 gene expression. J Biol Chem
2002; 277:24353-60.
Duan R, Porter W, Safe S. Estrogen-induced c-fos protooncogene expression in
MCF-7 human breast cancer cells: role of estrogen receptor Sp1 complex formation.
Endocrinology 1998; 139:1981-90.
Ray A, Prefontaine KE, Ray P. Down-modulation of interleukin-6 gene
expression by 17 beta-estradiol in the absence of high affinity DNA binding by the
estrogen receptor. J Biol Chem 1994; 269:12940-6.
Improta-Brears T, Whorton AR, Codazzi F, York JD, Meyer T, McDonnell DP.
Estrogen-induced activation of mitogen-activated protein kinase requires mobilization of
intracellular calcium. Proc Natl Acad Sci U S A 1999; 96:4686-91.
Singer CA, Figueroa-Masot XA, Batchelor RH, Dorsa DM. The mitogen-
activated protein kinase pathway mediates estrogen neuroprotection after glutamate
toxicity in primary cortical neurons. J Neurosci 1999; 19:2455-63.
Zivadinovic D, Watson CS. Membrane estrogen receptor-alpha levels predict
estrogen-induced ERK1/2 activation in MCF-7 cells. Breast Cancer Res 2005; 7:R13044.
Zhang W, Liu HT. MAPK signal pathways in the regulation of cell proliferation
in mammalian cells. Cell Res 2002; 12:9-18.
Zhang Z, Duan L, Du X, Ma H, Park I, Lee C, et al. The proliferative effect of
estradiol on human prostate stromal cells is mediated through activation of ERK. Prostate
2008; 68:508-16.
Liu J, Lin A. Role of JNK activation in apoptosis: a double-edged sword. Cell Res
2005; 15:36-42.
Shen HM, Liu ZG. JNK signaling pathway is a key modulator in cell death
mediated by reactive oxygen and nitrogen species. Free Radic Biol Med 2006; 40:928-39.
Cuadrado A, Nebreda AR. Mechanisms and functions of p38 MAPK signalling.
Biochem J 2010; 429:403-17.
Xia Z, Dickens M, Raingeaud J, Davis R, Greenberg M. Opposing effects of ERK
and JNK-p38 MAP kinases on apoptosis. Science 1995; 270:1326 - 31.
Dolado I, Swat A, Ajenjo N, De Vita G, Cuadrado A, Nebreda AR. p38alpha
MAP kinase as a sensor of reactive oxygen species in tumorigenesis. Cancer Cell 2007;
Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, et al. A
protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature
1994; 372:739-46.
Roos WP, Kaina B. DNA damage-induced cell death by apoptosis. Trends Mol
Med 2006; 12:440-50.
Guo RX, Wei LH, Tu Z, Sun PM, Wang JL, Zhao D, et al. 17 beta-estradiol
activates PI3K/Akt signaling pathway by estrogen receptor (ER)-dependent and ERindependent mechanisms in endometrial cancer cells. J Steroid Biochem Mol Biol 2006;
Lewis-Wambi JS, Jordan VC. Estrogen regulation of apoptosis: how can one
hormone stimulate and inhibit? Breast Cancer Res 2009; 11:206.
Ariazi EA, Brailoiu E, Yerrum S, Shupp HA, Slifker MJ, Cunliffe HE, et al. The
G protein-coupled receptor GPR30 inhibits proliferation of estrogen receptor-positive
breast cancer cells. Cancer Res 2010; 70:1184-94.
Vivacqua A, Bonofiglio D, Albanito L, Madeo A, Rago V, Carpino A, et al.
17beta-estradiol, genistein, and 4-hydroxytamoxifen induce the proliferation of thyroid
cancer cells through the g protein-coupled receptor GPR30. Mol Pharmacol 2006;
Russo J, Russo IH. The role of estrogen in the initiation of breast cancer. J Steroid
Biochem Mol Biol 2006; 102:89-96.
LaMarca HL, Rosen JM. Estrogen regulation of mammary gland development
and breast cancer: amphiregulin takes center stage. Breast Cancer Res 2007; 9:304.
Imai Y, Youn MY, Kondoh S, Nakamura T, Kouzmenko A, Matsumoto T, et al.
Estrogens maintain bone mass by regulating expression of genes controlling function and
life span in mature osteoclasts. Ann N Y Acad Sci 2009; 1173 Suppl 1:E31-9.
Harding M, Cowan S, Hole D, Cassidy L, Kitchener H, Davis J, et al. Estrogen
and progesterone receptors in ovarian cancer. Cancer 1990; 65:486-91.
Chan KK, Wei N, Liu SS, Xiao-Yun L, Cheung AN, Ngan HY. Estrogen receptor
subtypes in ovarian cancer: a clinical correlation. Obstet Gynecol 2008; 111:144-51.
Lai JS, Brown LG, True LD, Hawley SJ, Etzioni RB, Higano CS, et al.
Metastases of prostate cancer express estrogen receptor-beta. Urology 2004; 64:814-20.
Bonkhoff H, Fixemer T, Hunsicker I, Remberger K. Estrogen receptor expression
in prostate cancer and premalignant prostatic lesions. Am J Pathol 1999; 155:641-7.
Wong NA, Malcomson RD, Jodrell DI, Groome NP, Harrison DJ, Saunders PT.
ERbeta isoform expression in colorectal carcinoma: an in vivo and in vitro study of
clinicopathological and molecular correlates. J Pathol 2005; 207:53-60.
Martineti V, Picariello L, Tognarini I, Carbonell Sala S, Gozzini A, Azzari C, et
al. ERbeta is a potent inhibitor of cell proliferation in the HCT8 human colon cancer cell
line through regulation of cell cycle components. Endocr Relat Cancer 2005; 12:455-69.
Kazmi N, Marquez-Garban DC, Aivazyan L, Hamilton N, Garon EB, Goodglick
L, et al. The role of estrogen, progesterone and aromatase in human non-small-cell lung
cancer. Lung Cancer Manag 2012; 1:259-72.
Harkonen PL, Makela SI. Role of estrogens in development of prostate cancer. J
Steroid Biochem Mol Biol 2004; 92:297-305.
Fixemer T, Remberger K, Bonkhoff H. Differential expression of the estrogen
receptor beta (ERbeta) in human prostate tissue, premalignant changes, and in primary,
metastatic, and recurrent prostatic adenocarcinoma. Prostate 2003; 54:79-87.
Leav I, Lau KM, Adams JY, McNeal JE, Taplin ME, Wang J, et al. Comparative
studies of the estrogen receptors beta and alpha and the androgen receptor in normal
human prostate glands, dysplasia, and in primary and metastatic carcinoma. Am J Pathol
2001; 159:79-92.
Leung YK, Lam HM, Wu S, Song D, Levin L, Cheng L, et al. Estrogen receptor
beta2 and beta5 are associated with poor prognosis in prostate cancer, and promote
cancer cell migration and invasion. Endocr Relat Cancer 2010; 17:675-89.
Huang L, Pu Y, Alam S, Birch L, Prins GS. Estrogenic regulation of signaling
pathways and homeobox genes during rat prostate development. J Androl 2004; 25:3307.
Strom A, Hartman J, Foster JS, Kietz S, Wimalasena J, Gustafsson JA. Estrogen
receptor beta inhibits 17beta-estradiol-stimulated proliferation of the breast cancer cell
line T47D. Proc Natl Acad Sci U S A 2004; 101:1566-71.
McPherson SJ, Ellem SJ, Risbridger GP. Estrogen-regulated development and
differentiation of the prostate. Differentiation 2008; 76:660-70.
Bergan RC, Reed E, Myers CE, Headlee D, Brawley O, Cho HK, et al. A Phase II
study of high-dose tamoxifen in patients with hormone-refractory prostate cancer. Clin
Cancer Res 1999; 5:2366-73.
Stein S, Zoltick B, Peacock T, Holroyde C, Haller D, Armstead B, et al. Phase II
trial of toremifene in androgen-independent prostate cancer: a Penn cancer clinical trials
group trial. Am J Clin Oncol 2001; 24:283-5.
Rohlff C, Blagosklonny MV, Kyle E, Kesari A, Kim IY, Zelner DJ, et al. Prostate
cancer cell growth inhibition by tamoxifen is associated with inhibition of protein kinase
C and induction of p21(waf1/cip1). Prostate 1998; 37:51-9.
Chan QK, Lam HM, Ng CF, Lee AY, Chan ES, Ng HK, et al. Activation of
GPR30 inhibits the growth of prostate cancer cells through sustained activation of
Erk1/2, c-jun/c-fos-dependent upregulation of p21, and induction of G(2) cell-cycle
arrest. Cell Death Differ 2010; 17:1511-23.
Vinas R, Jeng YJ, Watson CS. Non-genomic effects of xenoestrogen mixtures. Int
J Environ Res Public Health 2012; 9:2694-714.
Fucic A, Gamulin M, Ferencic Z, Katic J, Krayer von Krauss M, Bartonova A, et
al. Environmental exposure to xenoestrogens and oestrogen related cancers: reproductive
system, breast, lung, kidney, pancreas, and brain. Environ Health 2012; 11 Suppl 1:S8.
Patisaul HB, Jefferson W. The pros and cons of phytoestrogens. Front
Neuroendocrinol 2010; 31:400-19.
Ahmed SA. The immune system as a potential target for environmental estrogens
(endocrine disrupters): a new emerging field. Toxicology 2000; 150:191-206.
Soto AM, Chung KL, Sonnenschein C. The pesticides endosulfan, toxaphene, and
dieldrin have estrogenic effects on human estrogen-sensitive cells. Environ Health
Perspect 1994; 102:380-3.
Vidaeff AC, Sever LE. In utero exposure to environmental estrogens and male
reproductive health: a systematic review of biological and epidemiologic evidence.
Reprod Toxicol 2005; 20:5-20.
Watson CS, Bulayeva NN, Wozniak AL, Finnerty CC. Signaling from the
membrane via membrane estrogen receptor-alpha: estrogens, xenoestrogens, and
phytoestrogens. Steroids 2005; 70:364-71.
Cannata DH, Kirschenbaum A, Levine AC. Androgen Deprivation Therapy as
Primary Treatment for Prostate Cancer. Journal of Clinical Endocrinology & Metabolism
2012; 97:360-5.
Cargnello M, Roux PP. Activation and Function of the MAPKs and Their
Substrates, the MAPK-Activated Protein Kinases. Microbiology and Molecular Biology
Reviews 2011; 75:50-83.
Levin ER. Plasma membrane estrogen receptors. Trends in Endocrinology &
Metabolism 2009; 20:477-82.
Pietras RJ, Marquez-Garban DC. Membrane-associated estrogen receptor
signaling pathways in human cancers. Clin Cancer Res 2007; 13:4672-6.
Wagner EF, Nebreda AR. Signal integration by JNK and p38 MAPK pathways in
cancer development. Nat Rev Cancer 2009; 9:537-49.
Roskoski R, Jr. ERK1/2 MAP kinases: structure, function, and regulation.
Pharmacol Res 2012; 66:105-43.
Chang F, Steelman LS, Shelton JG, Lee JT, Navolanic PM, Blalock WL, et al.
Regulation of cell cycle progression and apoptosis by the Ras/Raf/MEK/ERK pathway
(Review). Int J Oncol 2003; 22:469-80.
Pintus G, Tadolini B, Posadino AM, Sanna B, Debidda M, Bennardini F, et al.
Inhibition of the MEK/ERK signaling pathway by the novel antimetastatic agent NAMIA down regulates c-myc gene expression and endothelial cell proliferation. Eur J
Biochem 2002; 269:5861-70.
Gille H, Kortanjann M, Thomae O, Moomaw C, Slaughter C, Cobb MH, et al.
ERK phosphorylation potentiates Elk-1 mediated ternary complex formation and
transactivation. EMBO Journal 1995; 14:951-62.
Meloche S, Pouyssegur J. The ERK1/2 mitogen-activated protein kinase pathway
as a master regulator of the G1- to S-phase transition. Oncogene 2007; 26:3227-39.
Terada Y, Inoshita S, Nakashima O, Kuwahara M, Sasaki S, Marumo F.
Regulation of cyclin D1 expression and cell cycle progression by mitogen-activated
protein kinase cascade. Kidney Int 1999; 56:1258-61.
Dhanasekaran DN, Reddy EP. JNK signaling in apoptosis. Oncogene 2008;
Chen YR, Wang X, Templeton D, Davis RJ, Tan TH. The role of c-Jun N-
terminal kinase (JNK) in apoptosis induced by ultraviolet C and gamma radiation.
Duration of JNK activation may determine cell death and proliferation. J Biol Chem
1996; 271:31929-36.
Alao JP. The regulation of cyclin D1 degradation: roles in cancer development
and the potential for therapeutic invention. Mol Cancer 2007; 6:24.
Thornton TM, Rincon M. Non-classical p38 map kinase functions: cell cycle
checkpoints and survival. Int J Biol Sci 2009; 5:44-51.
Lee MW, Park SC, Yang YG, Yim SO, Chae HS, Bach JH, et al. The
involvement of reactive oxygen species (ROS) and p38 mitogen-activated protein (MAP)
kinase in TRAIL/Apo2L-induced apoptosis. FEBS Lett 2002; 512:313-8.
Cagnol S, Chambard JC. ERK and cell death: Mechanisms of ERK-induced cell
death - apoptosis, autophagy and senescence. The FEBS Journal 2010; 277:2-21.
Pillai MS, Sapna S, Shivakumar K. p38 MAPK regulates G1-S transition in
hypoxic cardiac fibroblasts. Int J Biochem Cell Biol 2011; 43:919-27.
Huschtscha LI, Reddel RR. p16(INK4a) and the control of cellular proliferative
life span. Carcinogenesis 1999; 20:921-6.
Cullen SP, Martin SJ. Caspase activation pathways: some recent progress. Cell
Death Differ 2009; 16:935-8.
Dunai Z, Bauer PI, Mihalik R. Necroptosis: biochemical, physiological and
pathological aspects. Pathol Oncol Res 2011; 17:791-800.
Kaczmarek A, Vandenabeele P, Krysko DV. Necroptosis: the release of damage-
associated molecular patterns and its physiological relevance. Immunity 2013; 38:209-23.
Fulda S. The mechanism of necroptosis in normal and cancer cells. Cancer Biol
Ther 2013; 14:999-1004.
Jouan-Lanhouet S, Arshad MI, Piquet-Pellorce C, Martin-Chouly C, Le Moigne-
Muller G, Van Herreweghe F, et al. TRAIL induces necroptosis involving
RIPK1/RIPK3-dependent PARP-1 activation. Cell Death Differ 2012; 19:2003-14.
Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, et al. Chemical
inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury.
Nat Chem Biol 2005; 1:112-9.
Watson CS, Jeng YJ, Guptarak J. Endocrine disruption via estrogen receptors that
participate in nongenomic signaling pathways. J Steroid Biochem Mol Biol 2011;
Kelly MJ, Levin ER. Rapid actions of plasma membrane estrogen receptors.
Trends Endocrinol Metab 2001; 12:152-6.
Bulayeva NN, Gametchu B, Watson CS. Quantitative measurement of estrogen-
induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways.
Steroids 2004; 69:181-92.
Lottering ML, de Kock M, Viljoen TC, Grobler CJ, Seegers JC. 17beta-Estradiol
metabolites affect some regulators of the MCF-7 cell cycle. Cancer Lett 1996; 110:181-6.
Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, et al. Chemical
inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury.
Nat Chem Biol 2005; 1:112-9.
Vinas R, Watson CS. Bisphenol S disrupts estradiol-induced nongenomic
signaling in a rat pituitary cell line: effects on cell functions. Environ Health Perspect
2013; 121:352-8.
Jeng YJ, Kochukov MY, Watson CS. Membrane estrogen receptor-alpha-
mediated nongenomic actions of phytoestrogens in GH3/B6/F10 pituitary tumor cells. J
Mol Signal 2009; 4:2.
Terada Y, Inoshita S, Nakashima O, Kuwahara M, Sasaki S, Marumo F.
Regulation of cyclin D1 expression and cell cycle progression by mitogen-activated
protein kinase cascade. Kidney Int 1999; 56:1258-61.
Robertson CN, Roberson KM, Padilla GM, O'Brien ET, Cook JM, Kim C-S, et al.
Induction of Apoptosis by Diethylstilbestrol in Hormone-Insensitive Prostate Cancer
Cells. Journal of the National Cancer Institute 1996; 88:908-17.
Simpkins JW, Dykens JA. Mitochondrial mechanisms of estrogen
neuroprotection. Brain Res Rev 2008; 57:421-30.
Cavalieri E, Frenkel K, Liehr JG, Rogan E, Roy D. Estrogens as endogenous
genotoxic agents--DNA adducts and mutations. Journal of the National Cancer Institute
Monographs 2000:75-93.
Clemons J, Glodé LM, Gao D, Flaig TW. Low-dose diethylstilbestrol for the
treatment of advanced prostate cancer. Urologic Oncology: Seminars and Original
Investigations 2013; 31:198-204.
Thornton TM, Rincon M. Non-Classical P38 Map Kinase Functions: Cell Cycle
Checkpoints and Survival. International Journal of Biological Sciences 2009; 5:44-51.
Bulayeva NN, Gametchu B, Watson CS. Quantitative measurement of estrogen-
induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways.
Steroids 2004; 69:181-92.
Zivadinovic D, Watson CS. Membrane estrogen receptor-alpha levels predict
estrogen-induced ERK1/2 activation in MCF-7 cells. Breast Cancer Research 2005;
Alyea R, Laurence S, Kim S, Katzenellenbogen B, Katzenellenbogen J, Watson
C. The roles of membrane estrogen receptor subtypes in modulating dopamine
transporters in PC-12 cells. J Neurochem 2008; 106:1525 - 33.
Jeng Y-J, Watson C. Proliferative and anti-proliferative effects of dietary levels of
phytoestrogens in rat pituitary GH3/B6/F10 cells - the involvement of rapidly activated
kinases and caspases. BMC Cancer 2009; 9:1-17.
Gametchu B, Watson CS. Plasma membrane-associated glucocorticoid hormone
receptor in human leukemic patients: Clinical implications. In: Gametchu B, ed.
Glucocorticoid Receptor Structure and Leukemic Cell Responses. Austin: R.G. Landes
Company, 1995:163-76.
Pearce ST, Jordan VC. The biological role of estrogen receptors α and β in cancer.
Critical Reviews in Oncology/Hematology 2004; 50:3-22.
Bonkhoff H, Berges R. The Evolving Role of Oestrogens and Their Receptors in
the Development and Progression of Prostate Cancer. European Urology 2009; 55:53342.
Golden GA, Mason RP, Tulenko TN, Zubenko GS, Rubin RT. Rapid and
opposite effects of cortisol and estradiol on human erythrocyte Na+,K+-ATPase activity:
relationship to steroid intercalation into the cell membrane. Life Sci 1999; 65:1247-55.
Whiting KP, Restall CJ, Brain PF. Steroid hormone-induced effects on membrane
fluidity and their potential roles in non-genomic mechanisms. Life sciences 2000;
Orwoll E, Lambert LC, Marshall LM, Phipps K, Blank J, Barrett-Connor E, et al.
Testosterone and Estradiol among Older Men. Journal of Clinical Endocrinology &
Metabolism 2006; 91:1336-44.
Finkelstein JS, Lee H, Burnett-Bowie S-AM, Pallais JC, Yu EW, Borges LF, et al.
Gonadal Steroids and Body Composition, Strength, and Sexual Function in Men. New
England Journal of Medicine 2013; 369:1011-22.
Huang CK, Luo J, Lee SO, Chang C. Androgen receptor differential roles in
stem/progenitor cells including prostate, embryonic, stromal, and hematopoietic lineages.
Stem Cells 2014.
Bowers JL, Tyulmenkov VV, Jernigan SC, Klinge CM. Resveratrol acts as a
mixed agonist/antagonist for estrogen receptors alpha and beta. Endocrinology 2000;
McCarty MF. Isoflavones made simple – Genistein’s agonist activity for the beta-
type estrogen receptor mediates their health benefits. Medical Hypotheses 2006; 66:1093114.
Chandsawangbhuwana C, Baker ME. 3D models of human ERα and ERβ
complexed with coumestrol. Steroids 2014; 80:37-43.
Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, et al.
Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta.
Endocrinology 1998; 139:4252-63.
Prins GS, Hu WY, Shi GB, Hu DP, Majumdar S, Li G, et al. Bisphenol A
promotes human prostate stem-progenitor cell self-renewal and increases in vivo
carcinogenesis in human prostate epithelium. Endocrinology 2014; 155:805-17.
Richter C, Birnbaum L, Farabollini F, Newbold R, Rubin B, Talsness C, et al. In
vivo effects of bisphenol A in laboratory rodent studies. Reprod Toxicol 2007; 24:199 224.
Vandenberg L, Colborn T, Hayes T, Heindel J, Jacobs D, Lee D, et al. Hormones
and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose responses.
Endocr Rev 2012; 33:378 - 455.
Bharathi SP, Raj HM, Jain S, Banerjee BD, Ahmed T, Arora VK. Role of
pesticides in the induction of tumor angiogenesis. Anticancer Res 2013; 33:231-40.
De Flora S, Micale RT, La Maestra S, Izzotti A, D'Agostini F, Camoirano A, et
al. Upregulation of clusterin in prostate and DNA damage in spermatozoa from bisphenol
A-treated rats and formation of DNA adducts in cultured human prostatic cells. Toxicol
Sci 2011; 122:45-51.
Giannandrea F, Paoli D, Figà-Talamanca I, Lombardo F, Lenzi A, Gandini L.
Effect of endogenous and exogenous hormones on testicular cancer: the epidemiological
evidence. The International Journal of Developmental Biology 2013; 57:255-63.
Cotter KA, Yershov A, Novillo A, Callard GV. Multiple structurally distinct ERα
mRNA variants in zebrafish are differentially expressed by tissue type, stage of
development and estrogen exposure. General and Comparative Endocrinology 2013;
Wong RLY, Walker CL. Molecular Pathways: Environmental estrogens activate
nongenomic signaling to developmentally reprogram the epigenome. Clinical Cancer
Research 2013; 19:3732-7.
Vilahur N, Molina-Molina JM, Bustamante M, Murcia M, Arrebola JP, Ballester
F, et al. Male specific association between xenoestrogen levels in placenta and
birthweight. Environment International 2013; 51:174-81.
Ibarluzea Jm J, Fernandez MF, Santa-Marina L, Olea-Serrano MF, Rivas AM,
Aurrekoetxea JJ, et al. Breast cancer risk and the combined effect of environmental
estrogens. Cancer Causes Control 2004; 15:591-600.
Wetherill YB, Petre CE, Monk KR, Puga A, Knudsen KE. The xenoestrogen
bisphenol A induces inappropriate androgen receptor activation and mitogenesis in
prostatic adenocarcinoma cells. Mol Cancer Ther 2002; 1:515-24.
Rice S, Whitehead SA. Phytoestrogens and breast cancer--promoters or
protectors? Endocr Relat Cancer 2006; 13:995-1015.
Zhang Y, Li Q, Zhou D, Chen H. Genistein, a soya isoflavone, prevents
azoxymethane-induced up-regulation of WNT/beta-catenin signalling and reduces colon
pre-neoplasia in rats. Br J Nutr 2013; 109:33-42.
Schleipen B, Hertrampf T, Fritzemeier KH, Kluxen FM, Lorenz A, Molzberger
A, et al. ERbeta-specific agonists and genistein inhibit proliferation and induce apoptosis
in the large and small intestine. Carcinogenesis 2011; 32:1675-83.
Chen J, Zeng J, Xin M, Huang W, Chen X. Formononetin induces cell cycle
arrest of human breast cancer cells via IGF1/PI3K/Akt pathways in vitro and in vivo.
Horm Metab Res 2011; 43:681-6.
Shanmugam MK, Kannaiyan R, Sethi G. Targeting cell signaling and apoptotic
pathways by dietary agents: role in the prevention and treatment of cancer. Nutr Cancer
2011; 63:161-73.
Alves RC, Almeida IM, Casal S, Oliveira MB. Isoflavones in coffee: influence of
species, roast degree, and brewing method. J Agric Food Chem 2010; 58:3002-7.
Adjakly M, Ngollo M, Boiteux JP, Bignon YJ, Guy L, Bernard-Gallon D.
Genistein and daidzein: different molecular effects on prostate cancer. Anticancer Res
2013; 33:39-44.
Shenouda NS, Zhou C, Browning JD, Ansell PJ, Sakla MS, Lubahn DB, et al.
Phytoestrogens in common herbs regulate prostate cancer cell growth in vitro. Nutr
Cancer 2004; 49:200-8.
Lee YH, Yuk HJ, Park KH, Bae YS. Coumestrol induces senescence through
protein kinase CKII inhibition-mediated reactive oxygen species production in human
breast cancer and colon cancer cells. Food Chem 2013; 141:381-8.
Bommareddy A, Eggleston W, Prelewicz S, Antal A, Witczak Z, McCune DF, et
al. Chemoprevention of prostate cancer by major dietary phytochemicals. Anticancer Res
2013; 33:4163-74.
Chang HT, Chou CT, Chen IL, Liang WZ, Kuo DH, Huang JK, et al.
Mechanisms of resveratrol-induced changes in [Ca(2+)]i and cell viability in PC3 human
prostate cancer cells. J Recept Signal Transduct Res 2013; 33:298-303.
Wang TT, Schoene NW, Kim YS, Mizuno CS, Rimando AM. Differential effects
of resveratrol and its naturally occurring methylether analogs on cell cycle and apoptosis
in human androgen-responsive LNCaP cancer cells. Mol Nutr Food Res 2010; 54:335-44.
Benitez DA, Pozo-Guisado E, Alvarez-Barrientos A, Fernandez-Salguero PM,
Castellon EA. Mechanisms involved in resveratrol-induced apoptosis and cell cycle arrest
in prostate cancer-derived cell lines. J Androl 2007; 28:282-93.
Scarlatti F, Sala G, Ricci C, Maioli C, Milani F, Minella M, et al. Resveratrol
sensitization of DU145 prostate cancer cells to ionizing radiation is associated to
ceramide increase. Cancer Letters 2007; 253:124-30.
Vandenberg L, Chahoud I, Heindel J, Padmanabhan V, Paumgartten F,
Schoenfelder G. Urinary, circulating, and tissue biomonitoring studies indicate
widespread exposure to bisphenol A. Environmental Health Perspectives 2010;
Vandenberg LN, Hauser R, Marcus M, Olea N, Welshons WV. Human exposure
to bisphenol A (BPA). Reproductive Toxicology 2007; 24:139-77.
Ge LC, Chen ZJ, Liu HY, Zhang KS, Liu H, Huang HB, et al. Involvement of
activating ERK1/2 through G protein coupled receptor 30 and estrogen receptor
alpha/beta in low doses of bisphenol A promoting growth of Sertoli TM4 cells. Toxicol
Lett 2014; 226:81-9.
Vinas R, Watson CS. Mixtures of xenoestrogens disrupt estradiol-induced non-
genomic signaling and downstream functions in pituitary cells. Environ Health 2013;
Bulayeva NN, Watson CS. Xenoestrogen-induced ERK-1 and ERK-2 activation
via multiple membrane-initiated signaling pathways. Environ Health Perspect 2004;
Wang W, Wang J, Wang Q, Wu W, Huan F, Xiao H. Bisphenol A modulates
calcium currents and intracellular calcium concentration in rat dorsal root ganglion
neurons. J Membr Biol 2013; 246:391-7.
Wozniak AL, Bulayeva NN, Watson CS. Xenoestrogens at picomolar to
nanomolar concentrations trigger membrane estrogen receptor-alpha-mediated Ca2+
fluxes and prolactin release in GH3/B6 pituitary tumor cells. Environ Health Perspect
2005; 113:431-9.
Derouiche S, Warnier M, Mariot P, Gosset P, Mauroy B, Bonnal JL, et al.
Bisphenol A stimulates human prostate cancer cell migration remodelling of calcium
signalling. Springerplus 2013; 2:54.
Midoro-Horiuti T, Tiwari R, Watson CS, Goldblum RM. Maternal bisphenol a
exposure promotes the development of experimental asthma in mouse pups. Environ
Health Perspect 2010; 118:273-7.
Lamartiniere CA. Protection against breast cancer with genistein: a component of
soy. Am J Clin Nutr 2000; 71:1705S-7S; discussion 8S-9S.
Magee PJ, Rowland IR. Phyto-oestrogens, their mechanism of action: current
evidence for a role in breast and prostate cancer. Br J Nutr 2004; 91:513-31.
Abd Elmageed ZY, Moroz K, Srivastav SK, Fang Z, Crawford BE, Moparty K, et
al. High circulating estrogens and selective expression of ERbeta in prostate tumors of
Americans: implications for racial disparity of prostate cancer. Carcinogenesis 2013;
Vergne S, Sauvant P, Lamothe V, Chantre P, Asselineau J, Perez P, et al.
Influence of ethnic origin (Asian v. Caucasian) and background diet on the bioavailability
of dietary isoflavones. Br J Nutr 2009; 102:1642-53.
Renouf M, Hendrich S. Bacteroides uniformis is a putative bacterial species
associated with the degradation of the isoflavone genistein in human feces. J Nutr 2011;
Gaido KW, Leonard LS, Lovell S, Gould JC, Babai D, Portier CJ, et al.
Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid
hormone receptor gene transcription assay. Toxicol Appl Pharmacol 1997; 143:205-12.
Singleton DW, Feng Y, Chen Y, Busch SJ, Lee AV, Puga A, et al. Bisphenol-A
and estradiol exert novel gene regulation in human MCF-7 derived breast cancer cells.
Mol Cell Endocrinol 2004; 221:47-55.
Sheeler CQ, Dudley MW, Khan SA. Environmental estrogens induce
transcriptionally active estrogen receptor dimers in yeast: activity potentiated by the
coactivator RIP140. Environ Health Perspect 2000; 108:97-103.
Gutendorf B, Westendorf J. Comparison of an array of in vitro assays for the
assessment of the estrogenic potential of natural and synthetic estrogens, phytoestrogens
and xenoestrogens. Toxicology 2001; 166:79-89.
Kochukov M, Jeng Y-J, Watson C. Alkylphenol xenoestrogens with varying
carbon chain lengths differentially and potently activate signaling and functional
responses in GH3/B6/F10 somatomammotropes. Env Health Perspect 2009; 117:723 30.
Vinas R, Watson C. Bisphenol s disrupts estradiol-induced nongenomic signaling
in a rat pituitary cell line: effects on cell functions. Environ Health Perspect 2013;
121:352 - 8.
Watson CS, Alyea RA, Jeng YJ, Kochukov MY. Nongenomic actions of low
concentration estrogens and xenoestrogens on multiple tissues. Molecular and Cellular
Endocrinology 2007; 274:1-7.
Zsarnovszky A, Le HH, Wang HS, Belcher SM. Ontogeny of rapid estrogen-
mediated extracellular signal-regulated kinase signaling in the rat cerebellar cortex:
potent nongenomic agonist and endocrine disrupting activity of the xenoestrogen
bisphenol A. Endocrinology 2005; 146:5388-96.
Nadal A, Ropero AB, Laribi O, Maillet M, Fuentes E, Soria B. Nongenomic
actions of estrogens and xenoestrogens by binding at a plasma membrane receptor
unrelated to estrogen receptor alpha and estrogen receptor beta. Proc Natl Acad Sci U S
A 2000; 97:11603-8.
Sicotte NL, Liva SM, Klutch R, Pfeiffer P, Bouvier S, Odesa S, et al. Treatment
of multiple sclerosis with the pregnancy hormone estriol. Ann Neurol 2002; 52:421-8.
Voskuhl R, Wang H, Jackson Wu T, Sicotte N, Bates A, Beaver G, et al. A
combination trial of estriol plus glatimer acetate in relapsing-remitting multiple sclerosis.
American Academy of Neurology 2014 Annual Meeting 2014.
Watson C, Jeng Y-J, Kochukov M. Nongenomic actions of estradiol compared
with estrone and estriol in pituitary tumor cell signaling and proliferation. The FASEB
Journal 2008; 22:3328-36.
vom Saal FS, Welshons WV. Large effects from small exposures. II. The
importance of positive controls in low-dose research on bisphenol A. Environ Res 2006;
Zhong L, Xiang X, Lu W, Zhou P, Wang L. Interference of xenoestrogen o,p′-
DDT on the action of endogenous estrogens at environmentally realistic concentrations.
Bulletin of Environmental Contamination and Toxicology 2013; 90:591-5.
Jeng Y-J, Kochukov M, Watson C. Membrane estrogen receptor-alpha-mediated
nongenomic actions of phytoestrogens in GH3/B6/F10 pituitary tumor cells. Journal of
Molecular Signaling 2009; 4:1-11.
Watson CS, Bulayeva NN, Wozniak AL, Alyea RA. Xenoestrogens are potent
activators of nongenomic estrogenic responses. Steroids 2007; 72:124-34.
Watson CS, Hu G, Paulucci-Holthauzen AA. Rapid actions of xenoestrogens
disrupt normal estrogenic signaling. Steroids 2013.
Jeng Y, Kochukov M, Watson C. Combinations of physiologic estrogens with
xenoestrogens alter calcium and kinase responses, prolactin release, and membrane
estrogen receptor trafficking in rat pituitary cells. Environ Health 2010; 9:61.
Watson CS, Bulayeva NN, Wozniak AL, Finnerty CC. Signaling from the
membrane via membrane estrogen receptor-α: Estrogens, xenoestrogens, and
phytoestrogens. Steroids 2005; 70:364-71.
Koong LY, Watson CS. Direct estradiol and diethylstilbestrol actions on early-
versus late-stage prostate cancer cells d.o.i.10.1002/pros.22875. The Prostate 2014.
Klein KA, Reiter RE, Redula J, Moradi H, Zhu XL, Brothman AR, et al.
Progression of metastatic human prostate cancer to androgen independence in
immunodeficient SCID mice. Nat Med 1997; 3:402-8.
Viñas R, Goldblum RM, Watson CS. Rapid estrogenic signaling activities of the
modified (chlorinated, sulfonated, and glucuronidated) endocrine disruptor bisphenol A.
Endocrine Disruptors 2013; 1:0-9.
Gallagher RP, Kutynec CL. Diet, micronutrients and prostate cancer: a review of
the evidence. Can J Urol 1997; 4:22-7.
Nakamura H, Wang Y, Kurita T, Adomat H, Cunha GR. Genistein increases
epidermal growth factor receptor signaling and promotes tumor progression in advanced
human prostate cancer. PLoS One 2011; 6:e20034.
El Touny LH, Banerjee PP. Identification of a biphasic role for genistein in the
regulation of prostate cancer growth and metastasis. Cancer Res 2009; 69:3695-703.
Wang X, Clubbs EA, Bomser JA. Genistein modulates prostate epithelial cell
proliferation via estrogen- and extracellular signal-regulated kinase-dependent pathways.
J Nutr Biochem 2006; 17:204-10.
Watson C, Gametchu B. Membrane-initiated steroid actions and the proteins that
mediate them. Proc Soc Exp Biol Med 1999; 220:9 - 19.
Hess KR, Pusztai L, Buzdar AU, Hortobagyi GN. Estrogen receptors and distinct
patterns of breast cancer relapse. Breast Cancer Res Treat 2003; 78:105-18.
Bianchini G, Pusztai L, Karn T, Iwamoto T, Rody A, Kelly C, et al. Proliferation
and estrogen signaling can distinguish patients at risk for early versus late relapse among
estrogen receptor positive breast cancers. Breast Cancer Res 2013; 15:R86.
Signoretti S, Loda M. Estrogen receptor beta in prostate cancer: brake pedal or
accelerator? Am J Pathol 2001; 159:13-6.
Campbell CH, Watson CS. A comparison of membrane vs. intracellular estrogen
receptor-alpha in GH(3)/B6 pituitary tumor cells using a quantitative plate immunoassay.
Steroids 2001; 66:727-36.
Comeglio P, Morelli A, Cellai I, Vignozzi L, Sarchielli E, Filippi S, et al.
Opposite effects of tamoxifen on metabolic syndrome-induced bladder and prostate
alterations: a role for GPR30/GPER? Prostate 2014; 74:10-28.
Revankar CM, Mitchell HD, Field AS, Burai R, Corona C, Ramesh C, et al.
Synthetic estrogen derivatives demonstrate the functionality of intracellular GPR30. ACS
Chem Biol 2007; 2:536-44.
Yu X, Filardo EJ, Shaikh ZA. The membrane estrogen receptor GPR30 mediates
cadmium-induced proliferation of breast cancer cells. Toxicology and Applied
Pharmacology 2010; 245:83-90.
Deschenes-Simard X, Kottakis F, Meloche S, Ferbeyre G. ERKs in cancer:
friends or foes? Cancer Res 2014; 74:412-9.
Roskoski Jr R. ERK1/2 MAP kinases: Structure, function, and regulation.
Pharmacological Research 2012; 66:105-43.
Chang F, Steelman LS, Shelton JG, Lee JT, Navolanic PM, Blalock WL, et al.
Regulation of cell cycle progression and apoptosis by Ras/Raf/MEK/ERK pathway.
International Journal of Oncology 2003; 22:469-80.
Yuan L, Dietrich AK, Nardulli AM. 17beta-Estradiol alters oxidative stress
response protein expression and oxidative damage in the uterus. Mol Cell Endocrinol
2014; 382:218-26.
Spencer WA, Vadhanam MV, Jeyabalan J, Gupta RC. Oxidative DNA damage
following microsome/Cu(II)-mediated activation of the estrogens, 17beta-estradiol,
equilenin, and equilin: role of reactive oxygen species. Chem Res Toxicol 2012; 25:30514.
Alao JP. The regulation of cyclin D1 degradation: roles in cancer development
and the potential for therapeutic invention. Molecular Cancer 2007; 6:1-16.
Terada Y, Nakashima O, Inoshita S, Kuwahara M, Sasaki S, Marumo F. Mitogen-
activated protein kinase cascade and transcription factors: the opposite role of MKK3/6p38K and MKK1-MAPK. Nephrology Dialysis Transplantation 1999; 14:45-7.
Zheng J, Li H, Zhu H, Xiao X, Ma Y. Genistein inhibits estradiol- and
environmental endocrine disruptor-induced growth effects on neuroblastoma cells. Oncol
Lett 2013; 5:1583-6.
Diehl JA, Cheng M, Roussel MF, Sherr CJ. Glycogen synthase kinase-3beta
regulates cyclin D1 proteolysis and subcellular localization. Genes & development 1998;
Yang K, Guo Y, Stacey WC, Harwalkar J, Fretthold J, Hitomi M, et al. Glycogen
synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels. BMC cell
biology 2006; 7:33.
Alao JP, Gamble SC, Stavropoulou AV, Pomeranz KM, Lam EW, Coombes RC,
et al. The cyclin D1 proto-oncogene is sequestered in the cytoplasm of mammalian
cancer cell lines. Mol Cancer 2006; 5:7.
Casanovas O, Miro F, Estanyol JM, Itarte E, Agell N, Bachs O. Osmotic stress
regulates the stability of cyclin D1 in a p38SAPK2-dependent manner. J Biol Chem
2000; 275:35091-7.
Mikhailov A, Shinohara M, Rieder CL. Topoisomerase II and histone deacetylase
inhibitors delay the G2/M transition by triggering the p38 MAPK checkpoint pathway. J
Cell Biol 2004; 166:517-26.
Zou Y, Ewton DZ, Deng X, Mercer SE, Friedman E. Mirk/dyrk1B kinase
destabilizes cyclin D1 by phosphorylation at threonine 288. J Biol Chem 2004;
Pettersson K, Delaunay F, Gustafsson JA. Estrogen receptor beta acts as a
dominant regulator of estrogen signaling. Oncogene 2000; 19:4970-8.
van Lipzig MM, ter Laak AM, Jongejan A, Vermeulen NP, Wamelink M, Geerke
D, et al. Prediction of ligand binding affinity and orientation of xenoestrogens to the
estrogen receptor by molecular dynamics simulations and the linear interaction energy
method. J Med Chem 2004; 47:1018-30.
Harris DM, Besselink E, Henning SM, Go VL, Heber D. Phytoestrogens induce
differential estrogen receptor alpha- or Beta-mediated responses in transfected breast
cancer cells. Exp Biol Med (Maywood) 2005; 230:558-68.
Schreihofer DA. Transcriptional regulation by phytoestrogens in neuronal cell
lines. Mol Cell Endocrinol 2005; 231:13-22.
Matthews JB, Twomey K, Zacharewski TR. In vitro and in vivo interactions of
bisphenol A and its metabolite, bisphenol A glucuronide, with estrogen receptors alpha
and beta. Chem Res Toxicol 2001; 14:149-57.
Bulayeva NN, Wozniak AL, Lash LL, Watson CS. Mechanisms of membrane
estrogen receptor-alpha-mediated rapid stimulation of Ca2+ levels and prolactin release
in a pituitary cell line. Am J Physiol Endocrinol Metab 2005; 288:E388-97.
Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, et al.
Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 1987;
Yan GR, Xiao CL, He GW, Yin XF, Chen NP, Cao Y, et al. Global
phosphoproteomic effects of natural tyrosine kinase inhibitor, genistein, on signaling
pathways. Proteomics 2010; 10:976-86.
Daibata M, Mellinghoff I, Takagi S, Humphreys RE, Sairenji T. Effect of
genistein, a tyrosine kinase inhibitor, on latent EBV activation induced by cross-linkage
of membrane IgG in Akata B cells. J Immunol 1991; 147:292-7.
Routledge EJ, White R, Parker MG, Sumpter JP. Differential effects of
xenoestrogens on coactivator recruitment by estrogen receptor (ER) alpha and ERbeta. J
Biol Chem 2000; 275:35986-93.
Leclercq G, Jacquot Y. Interactions of isoflavones and other plant derived
estrogens with estrogen receptors for prevention and treatment of breast cancerconsiderations concerning related efficacy and safety. J Steroid Biochem Mol Biol 2014;
Maggiolini M, Vivacqua A, Carpino A, Bonofiglio D, Fasanella G, Salerno M, et
al. The mutant androgen receptor T877A mediates the proliferative but not the cytotoxic
dose-dependent effects of genistein and quercetin on human LNCaP prostate cancer cells.
Mol Pharmacol 2002; 62:1027-35.
Jeng Y, Watson C. Combinations of physiologic estrogens with xenoestrogens
alter ERK phosphorylation profiles in rat pituitary cells. Environ Health Perspect 2011;
119:104 - 12.
Watson CS, Jeng Y-J, Guptarak J. Endocrine disruption via estrogen receptors
that participate in nongenomic signaling pathways. The Journal of Steroid Biochemistry
and Molecular Biology 2011; 127:44-50.
Davies J, Russell M, Davenport GR. Effects of maternal administration of
diethylstilbestrol and estradiol on the newborn guinea pig. Acta Anat (Basel) 1985;
Blair RM, Fang H, Branham WS, Hass BS, Dial SL, Moland CL, et al. The
estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural
diversity of ligands. Toxicol Sci 2000; 54:138-53.
Rich RL, Hoth LR, Geoghegan KF, Brown TA, LeMotte PK, Simons SP, et al.
Kinetic analysis of estrogen receptor/ligand interactions. Proc Natl Acad Sci U S A 2002;
Chen JR, Plotkin LI, Aguirre JI, Han L, Jilka RL, Kousteni S, et al. Transient
versus sustained phosphorylation and nuclear accumulation of ERKs underlie anti-versus
pro-apoptotic effects of estrogens. Journal of Biological Chemistry 2005; 280:4632-8.
Song H, Wang R, Wang S, Lin J. A low-molecular-weight compound discovered
through virtual database screening inhibits Stat3 function in breast cancer cells. Proc Natl
Acad Sci U S A 2005; 102:4700-5.
Nakonechnaya AO, Jefferson HS, Chen X, Shewchuk BM. Differential effects of
exogenous and autocrine growth hormone on LNCaP prostate cancer cell proliferation
and survival. J Cell Biochem 2013; 114:1322-35.
Taylor JA, Richter CA, Suzuki A, Watanabe H, Iguchi T, Coser KR, et al. Dose-
related estrogen effects on gene expression in fetal mouse prostate mesenchymal cells.
PLoS One 2012; 7:e48311.
Sun Y, Campisi J, Higano C, Beer TM, Porter P, Coleman I, et al. Treatment-
induced damage to the tumor microenvironment promotes prostate cancer therapy
resistance through WNT16B. Nat Med 2012; 18:1359-68.
Morrissey C, Vessella RL. The role of tumor microenvironment in prostate cancer
bone metastasis. J Cell Biochem 2007; 101:873-86.
Czyz J, Szpak K, Madeja Z. The role of connexins in prostate cancer promotion
and progression. Nat Rev Urol 2012; 9:274-82.
Almholt K, Johnsen M. Stromal cell involvement in cancer. Recent Results
Cancer Res 2003; 162:31-42.
Fidler IJ. The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis
revisited. Nat Rev Cancer 2003; 3:453-8.
Singh AS, Figg WD. In vivo models of prostate cancer metastasis to bone. J Urol
2005; 174:820-6.
Bubendorf L, Schopfer A, Wagner U, Sauter G, Moch H, Willi N, et al.
Metastatic patterns of prostate cancer: an autopsy study of 1,589 patients. Hum Pathol
2000; 31:578-83.
Sung SY, Hsieh CL, Wu D, Chung LW, Johnstone PA. Tumor microenvironment
promotes cancer progression, metastasis, and therapeutic resistance. Curr Probl Cancer
2007; 31:36-100.
Cooke JP, Losordo DW. Nitric oxide and angiogenesis. Circulation 2002;
Caulin-Glaser T, Garcia-Cardena G, Sarrel P, Sessa WC, Bender JR. 17 beta-
estradiol regulation of human endothelial cell basal nitric oxide release, independent of
cytosolic Ca2+ mobilization. Circ Res 1997; 81:885-92.
Oviedo PJ, Sobrino A, Laguna-Fernandez A, Novella S, Tarin JJ, Garcia-Perez
MA, et al. Estradiol induces endothelial cell migration and proliferation through estrogen
receptor-enhanced RhoA/ROCK pathway. Mol Cell Endocrinol 2011; 335:96-103.
Stevenson S, Nelson LD, Sharpe DT, Thornton MJ. 17beta-estradiol regulates the
secretion of TGF-beta by cultured human dermal fibroblasts. J Biomater Sci Polym Ed
2008; 19:1097-109.
Liu YM, Choy KW, Lui WT, Pang MW, Wong YF, Yip SK. 17beta-estradiol
suppresses proliferation of fibroblasts derived from cardinal ligaments in patients with or
without pelvic organ prolapse. Hum Reprod 2006; 21:303-8.
Slavin S, Yeh CR, Da J, Yu S, Miyamoto H, Messing EM, et al. Estrogen receptor
alpha in cancer-associated fibroblasts suppresses prostate cancer invasion via modulation
of thrombospondin 2 and matrix metalloproteinase 3. Carcinogenesis 2014.
Luo Y, Waladali W, Li S, Zheng X, Hu L, Zheng H, et al. 17beta-estradiol affects
proliferation and apoptosis of rat prostatic smooth muscle cells by modulating cell cycle
transition and related proteins. Cell Biol Int 2008; 32:899-905.
Zhang J, Hess MW, Thurnher M, Hobisch A, Radmayr C, Cronauer MV, et al.
Human prostatic smooth muscle cells in culture: estradiol enhances expression of smooth
muscle cell-specific markers. Prostate 1997; 30:117-29.
Harkonen PL, Vaananen HK. Monocyte-macrophage system as a target for
estrogen and selective estrogen receptor modulators. Ann N Y Acad Sci 2006; 1089:21827.
Jeng YJ, Kochukov M, Nauduri D, Kaphalia BS, Watson CS. Subchronic
exposure to phytoestrogens alone and in combination with diethylstilbestrol - pituitary
tumor induction in Fischer 344 rats. Nutr Metab (Lond) 2010; 7:40.
Shappell SB, Thomas GV, Roberts RL, Herbert R, Ittmann MM, Rubin MA, et al.
Prostate pathology of genetically engineered mice: definitions and classification. The
consensus report from the Bar Harbor meeting of the Mouse Models of Human Cancer
Consortium Prostate Pathology Committee. Cancer Res 2004; 64:2270-305.
Bastide C, Bagnis C, Mannoni P, Hassoun J, Bladou F. A Nod SCID mouse
model to study human prostate cancer. Prostate Cancer Prostatic Dis 2002; 5:311-5.
Montgomery B, Nelson PS, Vessella R, Kalhorn T, Hess D, Corey E. Estradiol
suppresses tissue androgens and prostate cancer growth in castration resistant prostate
cancer. BMC Cancer 2010; 10:244.
Risbridger GP, Ellem SJ, McPherson SJ. Estrogen action on the prostate gland: a
critical mix of endocrine and paracrine signaling. J Mol Endocrinol 2007; 39:183-8.
Wang Y, Xue H, Cutz JC, Bayani J, Mawji NR, Chen WG, et al. An orthotopic
metastatic prostate cancer model in SCID mice via grafting of a transplantable human
prostate tumor line. Lab Invest 2005; 85:1392-404.
Nemeth JA, Harb JF, Barroso U, Jr., He Z, Grignon DJ, Cher ML. Severe
combined immunodeficient-hu model of human prostate cancer metastasis to human
bone. Cancer Res 1999; 59:1987-93.
Lawrence MG, Taylor RA, Toivanen R, Pedersen J, Norden S, Pook DW, et al. A
preclinical xenograft model of prostate cancer using human tumors. Nat Protoc 2013;
Bosma MJ, Carroll AM. The SCID mouse mutant: definition, characterization,
and potential uses. Annu Rev Immunol 1991; 9:323-50.
Kaminski A, Hahne JC, Haddouti el M, Florin A, Wellmann A, Wernert N.
Tumour-stroma interactions between metastatic prostate cancer cells and fibroblasts. Int J
Mol Med 2006; 18:941-50.
Hartman J, Strom A, Gustafsson JA. Current concepts and significance of
estrogen receptor beta in prostate cancer. Steroids 2012; 77:1262-6.
Dey P, Strom A, Gustafsson JA. Estrogen receptor beta upregulates FOXO3a and
causes induction of apoptosis through PUMA in prostate cancer. Oncogene 2013.
Shughrue PJ, Merchenthaler I. Distribution of estrogen receptor beta
immunoreactivity in the rat central nervous system. J Comp Neurol 2001; 436:64-81.
Murphy E. Estrogen signaling and cardiovascular disease. Circ Res 2011;
Wang J, Eltoum IE, Carpenter M, Lamartiniere CA. Genistein mechanisms and
timing of prostate cancer chemoprevention in lobund-wistar rats. Asian Pac J Cancer
Prev 2009; 10:143-50.
McCormick DL, Johnson WD, Bosland MC, Lubet RA, Steele VE.
Chemoprevention of rat prostate carcinogenesis by soy isoflavones and by Bowman-Birk
inhibitor. Nutr Cancer 2007; 57:184-93.
Hu WY, Shi GB, Hu DP, Nelles JL, Prins GS. Actions of estrogens and endocrine
disrupting chemicals on human prostate stem/progenitor cells and prostate cancer risk.
Mol Cell Endocrinol 2012; 354:63-73.
Nelles JL, Hu WY, Prins GS. Estrogen action and prostate cancer. Expert Rev
Endocrinol Metab 2011; 6:437-51.
Cooper CS, Foster CS. Concepts of epigenetics in prostate cancer development.
Br J Cancer 2009; 100:240-5.
Nakayama M, Bennett CJ, Hicks JL, Epstein JI, Platz EA, Nelson WG, et al.
Hypermethylation of the human glutathione S-transferase-pi gene (GSTP1) CpG island is
present in a subset of proliferative inflammatory atrophy lesions but not in normal or
hyperplastic epithelium of the prostate: a detailed study using laser-capture
microdissection. Am J Pathol 2003; 163:923-33.
Lombaerts M, van Wezel T, Philippo K, Dierssen JW, Zimmerman RM, Oosting
J, et al. E-cadherin transcriptional downregulation by promoter methylation but not
mutation is related to epithelial-to-mesenchymal transition in breast cancer cell lines. Br J
Cancer 2006; 94:661-71.
Luke Yun-Kong Koong was born January 19, 1986 in Starkville, Mississippi to
Kai Siak Koong, Ph.D. and Lai C. Liu, Ph.D. He graduated from McAllen Memorial
High School (McAllen, Texas) in 2004, then attended The University of Texas – Pan
American in Edinburg, Texas. He graduated in 2008 with a B.S. in Biology, minoring in
chemistry and communication-journalism. He entered the graduate program at The
University of Texas Medical Branch in 2008 and joined the laboratory of Cheryl S.
Watson, Ph.D. in 2009. His research focuses on the direct effects of estrogens on the cell
numbers of early- and late-stage prostate cancer cells, the role of membrane estrogen
receptors in prostate cancers and the mechanisms by which xenoestrogens can utilize the
same cellular pathways. This research has been applied to toxicology and environmental
health in regards to prevalence of exposure to exogenous estrogens.
July 2014-Ph.D.-Cell Biology Graduate Program, The University of Texas Medical
Branch, Galveston, TX
Dissertation: The Direct Effects of Estradiol & Several Xenoestrogens on Cell Numbers
of Early- vs. Late Stage Prostate Cancer Cells
May 2008-B.S. Cum Laude, Biology, Minor in Chemistry & Communication-Journalism,
The University of Texas – Pan American, Edinburg, TX
Koong LY & Watson CS (2014). Rapid, nongenomic signaling effects of several
xenoestrogens on early- vs. late-stage prostate cancer cell proliferation (In Submission).
Endocrine Disruptors.
Koong LY & Watson CS (2014). Direct estradiol and diethylstilbestrol actions on earlyvs. late-stage prostate cancer cells. The Prostate. 74(16):1589-603.
Watson CS, Jeng Y-J, Bulayeva NN, Finnerty CC, Koong LY, Zivadinovic D, Alyea RA,
Midoro-Horiuti T, Goldblum RM, Anastasio NC, Cunningham KA, Seitz PK & Smith
TD. (2014). Steroid Receptors, Methods in Molecular Biology. 1204:123-33.
Motamedi S, Shilagard T, Edward K, Koong LY, Qui S, & Vargas G (2011). Gold
nanorods for intravital vascular imaging of preneoplastic oral mucosa. Biomedical Optics
Express. 2(5): 1194-1203.
Koong KS, Koong LY, Liu LC, & Yu M. (2005) An examination of selected drug
availability at online pharmacies. International Journal of Electronic Healthcare. 1(3),
Proceedings and Abstracts
Koong LY & Watson CS. Direct and rapid estrogenic signaling mechanisms that reduce
cell numbers in early- vs. late-stage prostate cancer cells. Endocrine Society. Chicago, IL.
June 2014.
Koong LY & Watson CS. Reduction in cell numbers in early- vs. late-stage prostate
cancer cells via direct and rapid estrogenic signaling mechanisms. Cell Biology Graduate
Program Symposium, UTMB. Galveston, TX. March 2014.
Koong LY & Watson CS. A Direct Signaling Mechanism Involved in Estradiol and
Diethylstilbestrol Effects on Androgen-Dependent and Androgen-Independent Prostate
Cancer Cell Viability. Endocrine Society. Houston, TX. June 2012.
Koong LY & Watson CS. Estrogen-mediated signaling differences related to cell
viability in androgen-dependent and androgen-independent prostate cancer cells. Society
of Toxicology. San Francisco, CA. March 2012.
Koong LY & Watson CS. Rapid, non-genomic signaling in prostate cancer cells via
membrane estrogen receptors. Society of Toxicology. Washington, DC. March 2011.
Motamedi S, Shilagard T, Koong LY, & Vargas G. (2010). Feasibility of using gold
nanorods for optical contrast in two photon microscopy of oral carcinogenesis. Proc.
SPIE Vol. 7576, 75760Z. San Francisco, CA. Jan. 2010.
Jeng Y-J, Guptarak J, Koong LY, & Watson CS. Effects of physiological and
nonphysiological estrogenic compounds on proliferation mechanisms in GH3/B6/F10 rat
pituitary tumor cells. UTMB Cancer Center Day. Galveston, TX. May 2010.
Permanent address:
7424 El Cielo, Galveston, Texas 77551
This dissertation was typed by Luke Yun-Kong Koong.