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(1) Specific Aims.
Activation of the estrogen receptor has been shown to be important for the development
and growth of many breast cancers. Accordingly, anti-estrogens such as tamoxifen are important
therapeutic agents in the treatment and chemoprevention of breast cancers. However, other
compounds such as phytoestrogens, anandamide and retinoid X receptor (RXR) agonists have
also demonstrated effectiveness against breast cancer in cell lines and in animal models, but it
has been difficult to put all of these compounds together in a common pathway. In vitro,
phytoestrogens, estrogen antagonists such as tamoxifen, anandamide and RXR agonists all share
the ability to activate a heterodimer of RXR and the steroid and xenobiotic receptor (SXR),
which in turn activates a cytochrome-P450-mediated steroid and xenobiotic destruction pathway.
Some breast cancer cell lines have been shown to express SXR leading to our hypothesis that the
protective effect of these diverse compounds is through activation of SXR:RXR. In order to
investigate the mechanism behind the observed effectiveness of these anti-breast cancer
compounds, we will address the following specific aims:
1. What is the expression pattern of SXR in normal mammary cells and breast cancer
cell lines?
2. Are the protective effects of phytoestrogens, fatty amides (e.g., anandamide), antiesrogens and RXR agonists due to activation of SXR:RXR in breast cancer cells by
these compounds?
3. Is the in vivo target of the SXR:RXR heterodimers the promoter of genes, such as
cytochrome P450 enzymes, involved in the destruction of steroids and xenobiotics or
is the activation of other pathways involved?
(2) Background and Significance.
Nuclear receptor ligands as breast cancer therapeutics
Retinoids, derivatives of vitamin A, play important roles in proliferation and
differentiation of a number of cell types (Sporn and Roberts, 1991). Retinoids exert their action
by binding to a family of nuclear receptor proteins termed retinoic acid receptors or RARs.
Liganded RARs primarily exert their effects by binding to the promoters of genes containing
retinoic acid response elements and thereby modulating target gene expression. A second class
of nuclear receptors related to RARs has been identified called the retinoid X receptors or RXRs.
Heterodimerization with RXR is required for RAR activity. Heterodimerization with RXR is also
required for a number of other receptor-mediated signaling pathways (e.g. thyroid hormone,
Vitamin D3, oxysterol, etc.)(Mangelsdorf et al., 1990; reviewed in Mangelsdorf and Evans,
1995). RXR can also form homodimers that are activated by 9-cis retinoic acid (9cRA). 9cRA,
is effective both in the chemoprevention and treatment of N-nitroso-N-methylrurea-induced rat
mammary carcinoma (Moon, 1994; Anzano et al., 1994; 1996), however, the mechanism by
which it exerts these effects is currently unknown. Synthetic RXR ligands, such as targretin are
also effective in prevention of induced rat mammary carcinomas (Gottardis et al., 1996).
Whether RXR homodimers or RXR heterodimers with other receptors are the targets remains to
be determined, however, it is likely that RXR plays an important role in the effectiveness of 9-cis
retinoic acid and synthetic RXR ligands in breast cancer treatment and prevention.
Breast cancer and the estrogen receptor
Breast cancer is the second leading cause of death among women. Both epidemiological
and cell biology studies have implicated the steroid hormone estrogen and the estrogen receptor
in breast cancer development and tumor growth. Estrogen promotes breast cancer by stimulating
increased proliferation of tissues expressing estrogen receptor. Further cell proliferation in turn
promotes cancer development by increasing the chances that a cell containing a potentially
cancer-causing mutation will multiply (Service, 1998). Estrogen metabolites themselves have
also been implicated in initiating the damage that can lead to mutations in cultured cells and
cancer in lab animals. Therefore, two potential avenues for breast cancer treatment or prevention
lie in interfering with the action of estrogen on the estrogen receptor, or by triggering
mechanisms to rid cells of excess estrogen and its metabolites.
Anti-estrogens such as tamoxifen have proven efficacy in the treatment of breast cancer.
Tamoxifen binds to the estrogen receptor and blocks estrogen itself from binding. At the same
time, tamoxifen fails to induce the cell proliferation that estrogen typically causes upon binding
to its receptor. Although tamoxifen is a highly effective therapeutic for breast cancer, toxic side
effects limit its use, and resistance to its anti-breast cancer effects usually occur within five years
of use (Jordan, 1993). In addition, anti-estrogens are not effective in the treatment of nonestrogen dependent breast cancers. However, combination of tamoxifen therapy with retinoid X
receptor (RXR) selective ligands, such as targretin can cause complete regression of existing
mammary carcinomas in the rat mammary carcinoma model (Ratko et al., 1989; Anzano et al.,
1994; Bischoff et al., 1998). The combination of targretin with tamoxifen was more efficacious
than either compound alone, and this combination holds promise as a new method for anti-breast
cancer therapy.
Phytoestrogens and cannabinoids in breast cancer prevention
There is strong epidemiological evidence that diet has a role in the development of breast
cancer. In particular, a diet high in fats appears to predispose a woman to breast cancer. In
contrast, there is mounting evidence that phytoestrogens, which are obtained from diets low in
fats and high in whole grains, seeds, fruits and vegetables, have protective effects against the
development of breast cancer. For example, women from Asian populations that consume large
amounts of phytoestrogens derived from a diet high in soy foods have a substantially lower
frequency of breast tumors than women in Western populations that consume much lower
quantities of phytoestrogens (Aldercreutz et al., 1995).
In animal and cell culture studies, phytoestrogens present in soy-containing foods are
tumor inhibiting (Messina et al., 1994). Phytoestrogens are thought to exert their anti-tumor
effects by interfering with the estrogen receptor, and by acting as estrogen antagonists.
However, phytoestrogens such as coumestrol and genistein , bind to the estrogen receptor with
low affinity compared to native estrogen (Mathieson and Kitts, 1980). In addition, it has been
demonstrated that genistein inhibits growth of breast cancer cells by mechanisms independent
from the estrogen receptor (Peterson and Barnes, 1991). In addition, the anti-breast cancer effect
caused by certain phytoestrogens (such as isoflavonoids and lignans) is apparent using estrogendependent and estrogen-independent breast cancer cell lines (Wang and Kurzer, 1997).
Therefore, phytoestrogen binding directly to the estrogen receptor may account for some of the
observed effects, but additional pathways involving these compounds may elicit the majority of
their protective effects against breast cancer.
Recently, the endogenous cannabinoid, anandamide has also proven effective in
inhibition of human breast cancer cell proliferation. Anandamide binds to the same cell surface
receptor as compounds derived from Cannabis sativa such as delta 9-tetrahydrocannabinol or
THC. Interestingly, the breast cancer inhibitory effects of anandamide and other cannabinoid
compounds are independent of the estrogen receptor (Ruh et al., 1997; De Petracellis et al.,
1998). It is not yet clear if cannabinoids exert their effects through suppression of prolactin
action, or through a mechanism independent of both estrogen and prolactin receptors. However,
anandamide is a strong activator of the nuclear receptor SXR (our unpublished results), which
could implicate a nuclear receptor-related pathway in its efficacy.
The nuclear receptor SXR
SXR is a novel nuclear hormone receptor that is activated by diverse steroids and
xenobiotic compounds (Blumberg et al., 1998). SXR is highly expressed in liver and at more
moderate levels in the intestine where it appears to regulate catabolic cytochrome P450 genes
and subsequently cause degradation of steroids and xenobiotics. Preliminary experiments have
also demonstrated expression of SXR in human osteosarcoma and breast cancer cell lines (our
unpublished results).
SXR requires heterodimerization with RXR for DNA binding and target gene activation.
The SXR:RXR heterodimer is permissive in that either partner may be freely activated by its
ligand. In vitro, SXR:RXR has been shown to activate transcription of the cytochrome P450 gene
CYP3A4. Expression of the CYP3A4 gene is induced by virtually all known SXR activators and
these activators also serve as substrates for the induced enzyme (Blumberg et al., 1998, Lehmann
et al., 1998, Bertilsson et al., 1998). Therefore, in vivo targets of the SXR:RXR heterodimer are
CYP3A4 and likely other cytochrome P-450-mediated steroid and xenobiotic destruction
pathways.
SXR can be activated by a wide range of steroid and xenobiotic compounds including
estrogen and the estrogen antagonist tamoxifen, as well as by partially metabolized steroids,
phytoestrogens such as genistein (Blumberg et al., 1998) and fatty amides including anandamide
(our unpublished results). Interestingly, certain breast cancer cell lines such as MCF-7 have
shown expression of SXR (our unpublished results). Expression of SXR in breast cancer cells is
of particular interest with respect to the anti-breast cancer effects of compounds that activate
SXR. Furthermore, since the SXR:RXR heterodimer is permissive for RXR activation, RXR
ligands such as 9 cis-retinoic acid and targretin , previously shown to be effective against breast
cancers, may be acting through SXR. The effectiveness of tamoxifen and RXR agonists against
breast cancer, both individually and in combination, taken together with the observed anti-breast
cancer effects of phytoestrogens and anandamide, is intriguing with respect to the ability of each
of these compounds to activate the SXR:RXR heterodimer. Our central hypothesis is that
SXR:RXR is an important target for these diverse compounds in the prevention and treatment of
breast cancer.
Significance of the proposed research
Estrogen antagonists such as tamoxifen, RXR selective agonists, fatty amides and
phytoestrogens have therapeutic value in the treatment and prevention of breast cancer.
Although it is certainly possible that each of these compounds exerts its effects independently,
each of them can also activate SXR:RXR. Hence this heterodimer could provide a common
target for all four classes of compounds (and potentially others). This, in turn could provide a
common mechanism and therapeutic target for development of novel anti-breast cancer
therapies. In addition, a basic understanding of the mechanism by which dietary factors such as
phytoestrogens are affording their protective effects against breast cancer should provide critical
information for developing dietary guidelines that will reduce the risk of developing breast
cancer. Results from these studies will contribute to the overall goals of the Women’s Health
Initiative by providing new information on reduction of breast cancer via prevention and
intervention strategies.
(3) Research Design and Methods
1. What is the expression pattern of SXR in normal mammary cells and breast
cancer cell lines?
As an essential prerequisite to studying the effects of SXR agonists on breast cancer cells, we
first wish to determine which of the cell lines that will be used express SXR and at what levels.
We will first isolate RNA from a panel of normal and malignant mammary cell lines and
evaluate SXR expression using Northern blotting and RNAse protection. Northern blots will tell
us which of the five previously identified transcripts (Blumberg et al., 1998) are expressed in
each line. RNAse protection will be used to quantitate SXR expression and this information will
be invaluable in the studies described in section 2.
Our collaborator, Dr. Powel Brown has access to biopsy materials from normal breast
tissue and breast tumors and has agreed to make them available for our studies. Dr. Blumberg’s
laboratory has extensive experience with in situ hybridization so the techniques will be locally
available.
We will use in situ hybridization to determine in which cells SXR is expressed within breast
tissue. Initially, we will use standard sectioned in situ hybridization but will switch to PCR-based
in situ hybridization if detection is problematic. Determining which cell type SXR is expressed
in will be helpful in evaluating whether it has a role in the normal regulation of mammary gland
differentiation.
2.
Are the protective effects of phytoestrogens, anti-estrogens, fatty amides and RXR
agonists due to activation of SXR:RXR in breast cancer cells by these compounds?
In addition to their ability to act against breast cancer, phytoestrogens such as genistein, antiestrogens such as tamoxifen, fatty amides such as anandamide and RXR agonists such as 9cRA
are each able to activate the SXR:RXR heterodimer in vitro (Blumberg et al., 1998; our
unpublished results). This suggests that activation of SXR is a common pathway through which
these compounds exert their activity and we aim to explore this hypothesis in detail. In order to
evaluate the activation of SXR:RXR in breast cancer cells, we will ask the following questions:
A) Does the potency and efficacy of individual compounds in SXR activation parallel
their activity against breast cancer? The following compounds will be tested using amounts
comparable to those from previously published trials for anti-breast cancer activity in a panel of
breast cancer cell lines: the phytoestrogens coumestrol, genistein and daidzein, anti-estrogens
tamoxifen and raloxifene, anandamide and the RXR agonist targretin (LGD1069). Each of these
compounds was previously shown to activate SXR in transfected cells (Blumberg et al., 1998)
and we now propose to extend those results to establish efficacy and rank order of potency for
SXR activation. In addition to standard tissue culture cell lines, we will transfect a panel of
mammary cell lines and breast cancer cell lines with SXR and reporter constructs to establish
that SXR can be active in these cells. All compounds will be tested using a concentrations series
to establish dose-responsive.
All of the compounds will subsequently be tested in various breast cancer cell lines obtained
from our collaborator, Dr. Powell Brown. Cells will be treated with the various compounds in
culture for three, six or nine days followed by viability assays and anti-proliferation assays.
FACS analysis, as well as 3H-thymidine incorporation measurements for DNA content, will be
performed to determine whether treated cells continue to proliferate similar to the methods used
by De Petrocellis et al. (1998). Equipment for FACS analysis is available in the Developmental
Biology Center at UC Irvine. A slow down in the cell cycle and therefore a reduction in DNA
content is indicative of cell differentiation and the anti-proliferative and anti-cancer effects of
different treatments. In control experiments, we will transfect SXR-dependent reporters such as
tk(CYP3A4)3-luc (Blumberg et al., 1998) into the same cells to test whether SXR is being
activated. This will enable us to distinguish SXR-dependent from SXR-independent effects of
each class of compounds. Data from cell proliferation experiments will be expressed as a
percentage of cell proliferation in untreated cells, and will be compared using the unpaired
Student’s t test (level of significance P<0.05).
We will also ask if the pathway of phytoestrogen activation of SXR can be dissected from
other cellular pathways using related compounds, such as genistein and daidzein. Genistein and
daidzein are structurally similar isoflavones, however, genistein is known to have effects on a
tyrosine phosphorylation cascade whereas daidzein does not (Akiyama et al., 1987). Other
related isoflavones will be tested for effectiveness. This will aid in supporting a model where
SXR activation is the pathway involved in phytoestrogen effectiveness.
B) Can transfection of breast cancer cells with constitutively active SXR mimic
treatment of the same cells with the compounds tested in A)? We have already constructed a
constitutively active SXR by fusing the SXR coding sequence to the strong activator VP16 as
has been described for RARs (Blumberg et al., 1997). This construct will be transfected into each
of the cell lines tested in A), and its effects on viability and proliferation compared with those
elicited by treatment with the test compounds will be evaluated.
C) Can transfection of breast cancer cells with dominant negative SXR constructs
blunt or block the effectiveness of the anti-breast cancer compounds being tested? A
dominant negative SXR has been constructed by deleting the AF-2 activation domain, similar to
that described for the RARs (Blumberg et al., 1997). This dominant negative inhibits activation
of SXR in a dose-dependent manner and will be transfected into each of the cell lines tested in
A). Each cell line will then be treated with phytoestrogens, anandamide, tamoxifen or targretin.
The effectiveness of each compound will be measured using the proliferation assays.
Analysis of the constitutively active and dominant negative mutant SXRs should correlate
with the results obtained with the activators in part A) if the SXR signaling pathway is involved
in regulating the growth of breast cancer cells.
D) What is the role of SXR in estrogen versus non-estrogen dependent breast
cancers? SXR may be the common link in effectiveness for diverse compounds like
phytoestrogens, anandamide, anti-estrogens and RXR agonists against breast cancer. However,
it is important to determine if SXR is an active target in all breast cancers, or only certain
subsets, particularly non-estrogen-dependent breast cancers. Non-estrogen dependent breast
cancers are difficult to treat because of the lack of effective targets for anti-breast cancer
therapeutics. If SXR expression correlates with the efficacy of the anti-breast cancer compounds
tested in non-estrogen dependent lines, SXR will provide an urgently needed new therapeutic
target for the treatment of non-estrogen dependent breast cancers.
The phytoestrogens genistein and coumestrol, the fatty amide anandamide, the estrogen
antagonist tamoxifen and RXR-selective retinoids such as targretin will be tested at
concentrations that bracket their reported effective concentrations against proliferation of breast
cancer cells in vitro. Anti-proliferative effects of these compounds will be tested on the
estrogen-dependent cell lines such as MCF-7, T47D, ZR-75 and BT-474 and non-estrogen
dependent lines such SK-BR3, MDA-MB-231 and MDA-MB-435. We will then evaluate
whether differences in effectiveness of these compounds on the various cell lines correlates with
the differential expression of SXR measured in aim 1). Our model holds that the expression of
SXR will correlate with the anti-breast cancer effectiveness of its activators. Any differences
between the expression of SXR and the activity of an individual compound would be informative
in determining which pathway mediates the effect.
3.
Is the in vivo target of the SXR:RXR heterodimer the promoter of genes, such as the
cytochrome P-450 enzymes, involved in the destruction of steroids and xenobiotics or is the
activation of other pathways involved?
SXR is highly expressed in liver, a site of metabolism for many compounds including steroid
hormones such as estrogen. In the liver, steroid hormones are degraded through the cytochrome
P450 pathway. The SXR:RXR heterodimer had a demonstrated specificity for certain types of
direct repeat DNA elements, some of which were present in the promoters for the steroid
hydroxlyases, P450 oxidoreductase and UDP-glucuronosyltransferase, and SXR:RXR was
shown to bind to the promoter of the steroid hydroxylase gene CYP3A4 in vitro (Blumberg et al.,
1998). In addition, expression of CYP3A4 is induced by many SXR activators including
estrogen metabolites (Blumberg et al., 1998; Lehmann et al., 1998). Therefore, potential in vivo
targets of SXR:RXR are the cytochrome components of the steroid destruction pathway. Timely
removal of estrogen and estrogen metabolites from cells can have anti-cancer effects in both
normal and breast cancer cells by reducing the potentiation of growth induced by estrogen and
by reducing the DNA damaging effects caused by estrogen metabolites.
To test if activation of steroid destruction pathways are relevant to the effectiveness of
SXR:RXR activators against breast cancer, we will address the following questions:
A) Does increased production of CYP3A4 confer a protective effect against breast
cancer proliferation?
At least nine different cytochrome P450s have been detected in the mammary gland of the
female rat including CYP3A (Hellmond et al., 1995). Therefore, it is plausible that SXR is
activating SXR-mediated, cytochrome-P450-dependent steroid destruction pathways in breast
cancer cells to elicit anti-proliferative effects. Since CYP3A4 is thought to be a major SXR
target, in vivo, (Blumberg et al., 1998, Lehmann et al., 1998, Bertilsson et al., 1998) we will test
whether CYP3A4 overexpression can bypass SXR activation and mimic treatment with known
effective compounds. To test this, an overexpression system composed of the CYP3A4 gene
under the control of a strong promoter such as the CMV early promoter will be constructed and
transfected into various breast cancer cell lines. Proliferation assays will be performed as
described previously to determine if overexpression of CYP3A4 can reduce proliferation,
implicating cytochrome-dependent destruction in the anti-breast cancer effects produced by SXR
activators. If CYP3A4 is ineffective, we will try other human P450 cDNAs that have been
published in similar experiments.
B) What are the targets if SXR activation is required for anti-breast cancer effects
but cytochrome P450 induction is not?
An exhaustive search for SXR targets is probably beyond the scope of this proposal but if we
reach this point before completion of the fellowship period then several possibilities may be
considered. The first, and simplest would be a bioinformatic approach, similar to that which
identified CYP genes as potential SXR targets (Blumberg et al., 1998). Public databases will be
searched for candidate SXR-responsive genes using string-searching programs such as
FINDPATTERNS (UWGCG package). Any matches found in the promoters or introns of genes
known to be expressed in breast tissue will be followed up with particular emphasis placed on
genes involved in signal transduction.
Another possibility would be to screen genomic fragments for the presence of SXRdependent transcription units. In this approach, ~2kb fragments of human genomic DNA are
cloned upstream of an appropriate, promoterless reporter gene (such as -galactosidase) and
transfected into cells stably expressing VP16-SXR and RXR. Cells showing -galactosidase
activity are candidates for containing SXR-dependent promoters. The reporter containing the
candidate fragment is then retested using SXR and a potent activator such as rifampicin for
specific response to SXR. Remaining positive clones would be used to isolate the corresponding
cDNA and tested for mammary specific expression. Any SXR-dependent transcription unit
expressed in mammary tissue would be a good candidate for mediating protective or therapeutic
effects of phytoestrogens and other effective compounds.
One further approach would be the CpG island-genomic binding site approach of Watanabe
et al (1998). In this method, recombinant SXR:RXR heterodimers (which we have already
produced in baculovirus (Blumberg et al., 1998) are used to select fragmented CpG islands from
a human genomic CpG island library. This library is enriched in expressed genes and has been
used to isolate novel estrogen-responsive genes from the breast cancer cell line MCF-7
(Watanabe et al., 1998). The genomic fragments are cloned into promoterless reporter constructs
and tested for SXR-dependent regulation. Any positive genomic fragments will also be used to
identify the corresponding cDNA and tissue-specific expression evaluated.
It is more likely that the approaches described in this section (3) will form the basis for
future experiments in my own laboratory. Identification of the promoter targets through which
SXR activators afford their anti-breast cancer effects will be an important area for future research
and could have significant impact on human health.
Conclusion and prospects
The project I have proposed above is feasible during a three year fellowship which will
be conducted in the laboratory most knowledgeable about SXR and its biology. Our model, that
SXR provides a common target for diverse compounds known to be effective against breast
cancer, is a plausible one and would provide an important new target in the fight against breast
cancer if correct. In support of our model, CYP3A4 and other catabolic cytochrome P450 genes
are known to be expressed in normal and malignant mammary cells. However, I am aware that
the project is highly hypothesis-driven and it remains possible that our model is incorrect and
that SXR has no role at all in either mammary development or the treatment of breast cancer. It
seems improbable that these diverse compounds, all of which are effective against breast cancer
and all of which activate the same nuclear receptor, would exert their anti-breast cancer effects
through independent pathways, but this remains a possibility.
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