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Copyright
by
Luke Yun-Kong Koong
2014
The Dissertation Committee for Luke Yun-Kong Koong Certifies that this is the
approved version of the following dissertation:
THE DIRECT EFFECTS OF ESTRADIOL AND SEVERAL
XENOESTROGENS ON CELL NUMBERS OF EARLY- VS. LATESTAGE PROSTATE CANCER CELLS
Committee:
Cheryl S Watson, PhD, Mentor
Darren Boehning, PhD, Chair
Gracie Vargas, PhD
Randall M Goldblum, MD
Nancy Ing, DVM, PhD
_______________________________
Dean, Graduate School
THE DIRECT EFFECTS OF ESTRADIOL AND SEVERAL
XENOESTROGENS ON CELL NUMBERS OF EARLY- VS. LATESTAGE PROSTATE CANCER CELLS
by
Luke Yun-Kong Koong, BS
Dissertation
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
Dedication
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.
Acknowledgements
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.
iv
THE DIRECT EFFECTS OF ESTRADIOL AND SEVERAL
XENOESTROGENS ON CELL NUMBERS OF EARLY- VS. LATESTAGE PROSTATE CANCER CELLS
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-
v
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.
vi
TABLE OF CONTENTS
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
Results...............................................................................................................46
Discussion .........................................................................................................58
Conclusions.......................................................................................................63
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
vii
Results and Discussion .....................................................................................72
Conclusions.......................................................................................................84
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
viii
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
ix
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
DES.......................................................................................................54
x
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
survival.................................................................................................59
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
xi
Figure 4.3 Number of viable prostate cancer cells following Acadia ERβ agonists.
..............................................................................................................95
Figure 4.4. Improper epigenetic regulation of normal prostate cells may lead to
an increased chance for prostate cancer. ..........................................97
xii
List of Abbreviations
AR
Androgen Receptor
ARE
Androgen Response Element
BPA
Bisphenol A
cAMP
Cyclic Adenosine Monophosphate
DES
Diethylstilbestrol
DHT
Dihydrotestosterone
E2
Estradiol
EE
Ethinyl Estradiol
EGF
Epidermal Growth Factor
ER
Estrogen Receptor
ERK
Extracellular Signal-Regulated Kinase
ERE
Estrogen Response Element
FHS
Follicle Stimulating Hormone
IGF
Insulin-like Growth Factor
JNK
c-Jun N-terminal Kinase
LH
Luteinizing Hormone
LHRH
Luteinizing-Hormone-Releasing Hormone
mER
Membrane Estrogen Receptor
MAPK
Mitogen Activated Protein Kinase
PI3K
Phosphoinositide 3-kinase
PSA
Prostate Specific Antigen
xiii
ROS
Reactive Oxygen Species
SCID
Severe Combined Immunodeficiency
STAT
Signal Transducers and Activators of Transcription
T
Testosterone
VEGF
Vascular Endothelial Growth Factor
XE
Xenoestrogen
xiv
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
11-15
, 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
18
17,
, 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
19-21
. However, tumors with this translocation have
increased metastatic potential and can be more aggressive, leading to higher grade
cancers
19, 22
. Common ETS family proteins involved in this gene fusion include ETS-
related gene, ETS translocation variant 1, and protein C-ets-1
23
. The TMPRSS2-ETS
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
16
found are losses in NKX3.1
26
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
29
, while chromosomes of
Group A, No.5, No.15, and the entire Y chromosome are absent in androgen-independent
PC-3 cells
30
. Neither of these cell lines possess the TMPRSS2-ERG gene fusion
31
.
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
33
. 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
38-40
.The prostate gland, and tumors that develop from it,
rely on androgens for sustained growth, in particular 5α-dihydrotestosterone (DHT)
41
.
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
17
synthesized in the testes and adrenal glands from cholesterol
43
. 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
levels
47
. DHT is essential to normal growth of the prostate, as well as defining male
secondary sex characteristics
48
. 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
50
. 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.
18
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
53
. 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)
51
. 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
59-62
. 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
19
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)
65-67
from the prostate epithelium. PSA is an enzyme found in semen, responsible for
cleaving semenogelin I and II in the seminal coagulum
68
. This protein is upregulated in
prostate cancer, and has consequently been used as a biomarker for prostate cancer
screening
debated
72
69-71
(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
41
. This transition to hormone
independence is typical of many types of steroid growth-driven tumors
73-75
. 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.
20
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).
21
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
76
. 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
79
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
22
treatment include leuprolide (Lupron®), goserelin (Zoladex®), triptorelin (Trelstar®),
and histrelin (Vantas®)
85
. Both surgical and chemical castration result in 70-80%
reductions in tumor growth for patients with androgen-responsive cancers (early-stage
tumors)
42
. 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
88
, and have
been shown to reduce tumor size similar to treatments with DES and LHRH agonists
89
.
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
90
. 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
91
. 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
92
. 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.
23
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
95
. 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
particular
98-101
. 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
41
. The first mechanism is not actual androgen-independence, but
rather the development of hypersensitivity to lower concentrations of serum androgens
24
102
. 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
41
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
42
.
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.
The
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,
25
increasing AR transactivation
113
. 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
115
. 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
117
. Nongenomic signaling
through ARs can also rapidly activate L-type calcium channels through an inhibitory Gprotein, stimulating PKC activity and increasing gene transcription
118
. 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
PI3K
123
. On the other hand, some androgen-independent prostate cancer cells have also
developed a resistance to TRAIL-induced apoptosis through constitutively active Akt
phosphorylation
124
. 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”,
26
evading immune cells that would otherwise kill them
126
. The cells employ mechanisms
such as recruiting immune cells that inhibit effector T-cells
maturation of dendritic cells via increased VEGF secretion
129
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
131.
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
organs
133-135
, cardiovascular health
136
132
, differentiation of the reproductive
, and potentially mental health
137
. Estrogen
deficiency in males can lead to impaired reproductive fecundity, decreased libido,
uncontrolled bone growth, and brittle bones 138.
27
ERα or ERβ
GPR30
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
139
. They each have their own splice variants (Figure 1.4)
141-143
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
145
. 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.
28
Truncated forms of ERα have typically shown activity at or near the plasma
membrane
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
149
148
. 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
150
. 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.
29
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
159
. Glycosylation influences the
cellular fate and destination for ERα and ERβ and many other proteins
160-162
.
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
30
membrane
165-167
. Membrane ERs are localized to the membrane via palmitoylation,
tethering them close to the inner surface, mainly in association with caveolae rafts
168
.
Disruption of palmitoylation or the interaction with caveolae has negatively affected cell
proliferation
159
. 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
170-173
.
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
175
139
. 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
initially
177.
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,
apoptosis
178, 184, 185,
170, 181,
178-180,
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
140
. mERα and mERβ do not appear to be different from their
31
nuclear counterparts 189, 190, other than their localization to the plasma membrane through
post-translational modifications
165
. 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
193
. In benign prostatic
hyperplasia, E2 in conjunction with serum hormone binding globulin rapidly increases
(<15 min) cyclic adenosine monophosphate
194
, a signaling molecule that can lead to cell
proliferation and transcriptional regulation of genes
195
. 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
196
, Sp1 transcription factor, or NF-κβ transcription factor,
after stimulation with E2 or another estrogenic ligand
transcription of IGF-I 198, cyclin D1
199
197
. These complexes then regulate
, c-fos 200, and IL-6
201
. Thus, multiple immediate
and delayed cellular outcomes can result from a single ligand binding event.
32
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
proliferation
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
207
.
JNK can also be activated by reactive oxygen species (ROS) after inhibition of MAP
kinase phosphatase activity, leading to eventual apoptosis
208
. Phosphorylated p38
participates in control of cell differentiation 209, proliferation 205, and death 210 in response
to cellular stress
211
, inflammation 212, and UV radiation 213. Estrogens can also use other
signaling cascades such as activation of the PI3K/Akt signaling pathway
214
, or can even
inhibit cell death in some cases by regulating the anti-apoptotic Bcl-2 protein
215
. 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
186,
195
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
217
216
, 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
33
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
products
220
. 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
142.
228,
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
232
, which is different given ERα is often
associated with proliferative effects in other cell types
233
. ERβ is not initially expressed
in the development of the prostate, and ERα is responsible for regulating cell growth.
34
Androgen signaling decreases ERα levels, removing the regulation of ERβ expression,
allowing ERβ to rise, and prostate maturation and differentiation to occur
234
. 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
237
, 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
dichlorodiphenyldichloroethylene,
a
breakdown
product
of
the
pesticide
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
241
, while environmental estrogens are
pervasive and are associate with negative effects on the health of wildlife, humans, and
the ecosystem
242-244
. A further review of XEs is in Chapter 3, highlighting the overall
ability of XE, at dietary- or environmentally-relevant concentrations, to stimulate cell
35
growth by utilizing signaling pathways differently from E2 and DES, as well as the varied
ER requirements to elicit such cellular responses.
E2
BPA
Coumestrol
Genistein
DES
Resveratrol
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,
36
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
37
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.
38
Chapter 2: Direct estradiol and diethylstilbestrol actions on early- vs.
late-stage prostate cancer cells1
Abstract
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
1
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.
39
dramatically improve outcomes for both early- and late-stage prostate cancer patients,
while avoiding the side effects of high-dose DES treatment.
Introduction
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
77
. Estrogen treatment is thought to indirectly
decrease androgen production by negative feedback control on the hypothalamicpituitary-testicular axis
42
, 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
77
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
effectors
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
40
250,
251
. 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
251
by integrating upstream signals to
regulate cell cycle proteins by post-translational modifications
252
. ERKs also propagate
the signaling cascade to other important cellular response molecules such as c-Myc
Elk-1
254
, eIF4E
255
and cyclin D1
256
253
,
. 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
proteasomes
259
, cyclin-dependent kinase (CDK) regulation at major checkpoints
or inducing apoptosis through cellular stress mechanisms
261
256, 260
,
. Therefore, there are
multiple mechanistic pathways via which MAPKs are important regulators of cell
numbers.
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
263
. Cyclin D1
protein levels can be changed via p38 phosphorylation (resulting in degradation),
41
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
265
, 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
266
. 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
267
, a growing number of other receptors and stressors are being
268
reported to be involved in this mechanism
(RIPK1) serves as a scaffold for this process
269
. 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.
42
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:
pERK1/2
(Thr202/Tyr204), pJNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), phosphocyclin D1 (Thr286) (all from Cell Signaling – Danvers, MA), or phospho-p16INK4A
43
(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
laboratory
273
, 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
workstation,
followed
by
simultaneous
fixation
and
permeabilization
(2%
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);
44
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
charcoal-stripped
FBS
for
48
hr.
Cells
were
loaded
with
15µM
2’,7’-
Dichlorodihydrofluorescein diacetate (DCDHF) (Enzo Life Sciences) for 1 hr. Then the
45
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.
Results
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.
46
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.
47
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
treatments.
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 ±
SEM.
48
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
262
. 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.
49
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)
277
. 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.
50
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.
51
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
treatments.
LAPC-4 and PC-3
cells were treated
with 0.1 nM E2 or 1
µM DES and cell
viability
was
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
52
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
278
, 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.
53
Figure 2.7. Phosphop16 (p-p16) levels
after E2 or DES
treatments.
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
symbols
denote
LAPC-4 cells, and
black symbols PC-3
cells.
Triangles
represent E2, and
circles
DES.
*
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
DES.
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
symbols
denote
LAPC-4 cells, black
PC-3 cells. Triangles
represent E2, and
circles
DES.
*
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
54
259,
278
, 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.
55
Figure 2.9.
Phosphoryated
cyclin D1 (pcyclin D1) vs.
total cyclin
levels and their
ratios after
treatment with
0.1 nM E2 and 1
µM DES.
LAPC-4 (early)
and PC-3 (late)
cells were treated
with E2 or DES.
The p-cyclin D1
levels
(A-D)
were measured
for up to 60 min
and the total
cyclin D1 levels
(E-H)
were
measured up to
16 hrs via plate
immunoassays.
The ratio of pcyclin D1 to total
cyclin D1 was
calculated
for
overlapping time
points at 5, 15,
and 60 min (I-L).
White symbols
denote LAPC-4
cells and black
PC-3
cells.
Triangles
represent E2 and
circles DES. *
denotes
significance from
vehicle (time 0)
at P<0.05. The
shaded
gray
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
56
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
subtype-selective
antagonists inhibit E2or DES-induced ROS
and p-cyclin D1
responses.
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
DES.
ROS
were
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
57
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.
Discussion
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)
79
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
279
, 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.
58
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
number.
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.
59
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
262
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
60
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
283
, 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
61
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)
However,
we
have
also
previously
seen
284-286
estrogen-mediated
.
rapid
phosphorylation/activation of Elk-1 and ATF-2 transcription factors as early as 10-15
min
287
. 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
288
. 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
289
. 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
62
instead alter such chemical properties as fluidity, lipophilicity, and polarity of the lipid
bilayer
291, 292
, so perhaps DES is operating on these cells via these other less productive
mechanisms.
Conclusions
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.
63
Chapter 3: Rapid, nongenomic signaling effects of several xenoestrogens
involved in early- vs. late-stage prostate cancer cell proliferation
Abstract
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
64
environmental and dietary XEs for existing prostate tumors. These mechanisms may offer
new cellular targets for therapy.
Introduction
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
documented
295
, 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
312-315
, 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
65
. 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)
319
. 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
325
] 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
327
kinases (MAPKs),
326
that has been shown to alter
, cell signaling through the activation of mitogen-activated protein
327-329
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
336
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
12
. Asian diets contain high levels of soy
ingredients, with the best-known active estrogenic components being daidzein, genistein,
and coumestrol
15
. Genetics may also play a role in the sensitivity of various cancer-
relevant mechanisms to estrogens, including the ability to metabolize dietary
66
phytoestrogens to more active compounds
337
, 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
339-341
. 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
expression
responses
348, 349
350
. 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
351
]. 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
355
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
67
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
358
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
358
. 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)
359
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)
30
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
68
concentrations ranging from 10-14M to 10-6M (and a final EtOH concentration of
0.0001%).
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
(Abs)
specific
for
these
post-translationally
modified
epitopes:
pERK1/2
(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
69
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.
70
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
358
. 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.
71
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
72
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
358
. 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.
73
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
366-368
, 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
74
undifferentiated state, as we have seen with membrane glucocorticoid receptors in human
lymphoma cells compared to normal circulating lymphocytes
288
. 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
262
. 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.
75
Figure 3.3. Phospho-ERK (pERK) levels in LAPC-4 and PC-3 cells after XE
treatments.
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,
76
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.
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
358
.
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
77
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
358
, 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
358
. 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
78
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
79
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
80
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
fibroblasts
381
. 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
382
. In addition, inhibition of GSK-3β in MCF-7 breast cancer cells
did not completely disrupt cyclin D1 protein degradation
383
. 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
386
. 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.
81
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
296
, 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
XEs
389-391
. Few comparisons for nongenomic responses are available, though we
previously observed different MAPK activation patterns and mostly positive proliferative
82
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
kinases
393-395
. 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
398
) 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.
83
Conclusions
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,
84
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
358
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
recent
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
85
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
358
. 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.
86
Chapter 4: Conclusions and Future Directions
MAJOR CONCLUSIONS FROM OUR STUDIES

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
87
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
401
, 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
apoptosis
404
. The length of these activations can then influence the genes that are
induced in responses
114
. In addition, many transcriptionally active proteins are activated
and regulated through signals from mERs, thus providing a junction between nongenomic
and genomic signals
197
. 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
193
.
An effective assay to test this hypothesis would be to develop a transcription factorspecific blocker, such as those for STAT3 and STAT5
88
405, 406
, disrupt the activity of a
rapidly activated transcription factor, such as Elk-1, and then measure the outcome on
viability.
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,
294
, 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
407
. 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
89
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
408-410
(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
90
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
migration
418
416, 417
, as well as endothelial cell
. Fibroblast migration and proliferation can be controlled by E2 as well, in
91
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
421
.
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
422
, which can be mediated by ERα
423
.
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
physiology
(Figure
from
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
425
. 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
92
about background
426
. 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
432
. 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
possible
433
. 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
93
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
94
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
435
, 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
95
436
. 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).
96
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
443
. Methylation of the glutathione S-transferase-pi gene is found in over 90% of prostate
cancers, but not in normal prostate cells
444
. Hypermethylation of the E-cadherin gene
may even promote the further development of cancers to a metastatic state
445
.
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.
97
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.
98
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Vita
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.
Education
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
Publications
154
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),
291-302.
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.
155
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.
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