Androgen Prostate Cancer Research Articles

Androgen-Regulated Expression of Arginase 1, Arginase 2 and Interleukin-8 in Human Prostate Cancer

BackgroundProstate cancer (PCa) is the most frequently diagnosed cancer in North American men. Androgen-deprivation therapy (ADT) accentuates the infiltration of immune cells within the prostate. However, the immunosuppressive pathways regulated by androgens in PCa are not well characterized. Arginase 2 (ARG2) expression by PCa cells leads to a reduced activation of tumor-specific T cells. Our hypothesis was that androgens could regulate the expression of ARG2 by PCa cells.Methodology/Principal FindingsIn this report, we demonstrate that both ARG1 and ARG2 are expressed by hormone-sensitive (HS) and hormone-refractory (HR) PCa cell lines, with the LNCaP cells having the highest arginase activity. In prostate tissue samples, ARG2 was more expressed in normal and non-malignant prostatic tissues compared to tumor tissues. Following androgen stimulation of LNCaP cells with 10 nM R1881, both ARG1 and ARG2 were overexpressed. The regulation of arginase expression following androgen stimulation was dependent on the androgen receptor (AR), as a siRNA treatment targeting the AR inhibited both ARG1 and ARG2 overexpression. This observation was correlated in vivo in patients by immunohistochemistry. Patients treated by ADT prior to surgery had lower ARG2 expression in both non-malignant and malignant tissues. Furthermore, ARG1 and ARG2 were enzymatically active and their decreased expression by siRNA resulted in reduced overall arginase activity and l-arginine metabolism. The decreased ARG1 and ARG2 expression also translated with diminished LNCaP cells cell growth and increased PBMC activation following exposure to LNCaP cells conditioned media. Finally, we found that interleukin-8 (IL-8) was also upregulated following androgen stimulation and that it directly increased the expression of ARG1 and ARG2 in the absence of androgens.Conclusion/SignificanceOur data provides the first detailed in vitro and in vivo account of an androgen-regulated immunosuppressive pathway in human PCa through the expression of ARG1, ARG2 and IL-8.

Atmospheric pressure photoionization tandem mass spectrometry of androgens in prostate cancer.

Androgen deprivation therapy is the most common treatment option for advanced prostate cancer. Almost all prostate cancers recur during androgen deprivation therapy, and new evidence suggests that androgen receptor activation persists despite castrate levels of circulating androgens. Quantitation of tissue levels of androgens is critical to understanding the mechanism of recurrence of prostate cancer during androgen deprivation therapy. A liquid chromatography atmospheric pressure photoionization tandem mass spectrometric method was developed for quantitation of tissue levels of androgens. Quantitation of the saturated keto-steroids dihydrotestosterone and 5-alpha-androstanedione required detection of a novel parent ion, [M + 15](+). The nature of this parent ion was explored, and the method was applied to prostate tissue and cell culture with comparison to results achieved using electrospray ionization.

Redefining hormone resistance in prostate cancer.

Prostate cancer relies on signaling through the androgen receptor (AR) for maintenance and progression; and androgen-deprivation therapy remains a cornerstone of treatment for advanced prostate cancer. An effective clinical classification of prostate cancer should account for the extent of the disease as well as the mechanisms that are driving the growth of the tumor. The previous terms hormone-sensitive and hormone-refractory described response to treatment. It has become clear that these terms do not reflect the mechanism of disease relapse; however over the last decade there has been a better understanding of androgen-receptor mediated signaling effects and incomplete suppression of androgens in prostate cancer. The Prostate Cancer Clinical Trials Working Group 2 (PCWG2) now recommends addressing the spectrum of clinical states based on castration status as this ligand-centered terminology can more accurately describe the patients' disease, and ultimately provides a useful framework for patient management and drug development. Optimized use of androgen-deprivation therapy, low molecular weight inhibitors of adrenal androgen biosynthesis, and new AR antagonists are promising new therapeutics that can further define the meaning of castrate state. As hormone resistance is redefined to include patients that are refractory to treatments that ablate adrenal and in situ tumoral androgens, a meaningful new clinical state in patients will be forged. We propose a model for incorporating these patients into the current PCWG2 conceptualization of the disease.

MiR-331-3p Regulates ERBB-2 Expression and Androgen Receptor Signaling in Prostate Cancer

MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression and are aberrantly expressed in human cancer. The ERBB-2 tyrosine kinase receptor is frequently overexpressed in prostate cancer and is associated with disease progression and poor survival. We have identified two specific miR-331-3p target sites within the ERBB-2 mRNA 3'-untranslated region and show that miR-331-3p expression is decreased in prostate cancer tissue relative to normal adjacent prostate tissue. Transfection of multiple prostate cancer cell lines with miR-331-3p reduced ERBB-2 mRNA and protein expression and blocked downstream phosphatidylinositol 3-kinase/AKT signaling. Furthermore, miR-331-3p transfection blocked the androgen receptor signaling pathway in prostate cancer cells, reducing activity of an androgen-stimulated prostate-specific antigen promoter and blocking prostate-specific antigen expression. Our findings provide insight into the regulation of ERBB-2 expression in cancer and suggest that miR-331-3p has the capacity to regulate signaling pathways critical to the development and progression of prostate cancer cells.

Suppressive Roles of Calreticulin in Prostate Cancer Growth and Metastasis

Calreticulin is an essential, multifunctional Ca(2+)-binding protein that participates in the regulation of intracellular Ca(2+) homeostasis, cell adhesion, and chaperoning. Calreticulin is abundantly expressed and regulated by androgens in prostate epithelial cells. Given the importance of both calreticulin in multiple essential cellular activities and androgens in prostate cancer, we investigated the possibility of a role for calreticulin in prostate cancer progression. Immunohistochemistry revealed the down-regulation of calreticulin in a subset of human prostate cancer specimens. Prostate cancer cells overexpressing exogenous calreticulin produced fewer colonies in both monolayer culture and soft agar. Furthermore, calreticulin overexpression also inhibited tumor growth in the orthotopic PC3 xenograft tumor model and macroscopic lung metastasis in the rat Dunning AT3.1 prostate tumor model. To address the potential mechanism of calreticulin suppression of prostate cancer, we generated calreticulin mutants with different functional domains deleted. The calreticulin mutants containing the P-domain, which binds to other endoplasmic reticulum chaperone proteins, were sufficient for the suppression of PC3 growth in colony formation assays. Overall, our data support the hypothesis that calreticulin inhibits growth and/or metastasis of prostate cancer cells and that this suppression requires the P-domain.

NF‐κB2/p52 enhances androgen‐independent growth of human LNCaP cells via protection from apoptotic cell death and cell cycle arrest induced by androgen‐deprivation

Androgen-deprivation therapy only causes a temporary regression of prostate cancer, as all tumors will eventually progress to refractory to hormonal therapy after 1-3 years of treatment. The underlying mechanisms of prostate cancer androgen refractory progression are incompletely understood. In this study, we employed in vitro as well as in vivo models to examine the role of NF-kappaB2/p52 in prostate cancer growth and androgen independent progression. The effects of NF-kappaB2/p52 on cell growth, androgen responsiveness, cell cycle and apoptosis were examined in androgen sensitive LNCaP cells. The effect of NF-kappaB2/p52 on tumor growth was examined in intact and castrated male mice. Overexpression of NF-kappaB2/p52 enhances androgen-sensitive LNCaP human prostate cancer cell growth and clonogenic ability in androgen-deprived condition in vitro. NF-kappaB2/p52 induced androgen-independent growth is through protecting LNCaP cells from apoptotic cell death and cell cycle arrest induced by androgen-deprivation. In addition, NF-kappaB2/p52 stimulates Cyclin D1 expression and knock down of Cyclin D1 expression by siRNA abolished NF-kappaB2/p52-induced cell growth in vitro. Adenoviral mediated NF-kappaB2/p52 expression in LNCaP cells enhances tumor growth in intact male nude mice and induces tumor growth in castrated male nude mice, suggesting that overexpression of NF-kappaB2/p52 induces androgen-independent growth of androgen-sensitive LNCaP cells. Overexpression of NF-kappaB2/p52 protects androgen sensitive LNCaP cells from apoptotic cell death and cell cycle arrest induced by androgen-deprivation. NF-kappaB2/p52 activation induces androgen-independent growth in vitro and in vivo.

Androgens and prevention of prostate cancer

This review provides a description of recent insights into the role of androgens in prostate cancer prevention. Many studies have elucidated a variety of molecular mechanisms involved in the initiation and progression of prostate cancer with many directly or indirectly related to the androgen signaling pathway. Both well known and novel agents for targeting the androgen pathway are under investigation, though very few are in clinical trials. After a review of recent papers describing these mechanisms, their results and implications were summarized. Finasteride remains the only agent proven to reduce the risk of prostate cancer, though there are currently two other ongoing phase III trials with vitamin E, selenium, and dutasteride. An enhanced understanding of complex interactions with the androgen pathways is leading to the exploration of additional promising approaches to mitigating the risk of prostate cancer.

Androgens and the molecular epidemiology of prostate cancer

Despite clinical and experimental evidence that show androgens are important in prostate carcinogenesis, epidemiologic studies of serum androgens have been inconclusive. In this review, we summarize the current state of the literature and provide insights and direction for epidemiologic research on androgens and prostate cancer. To date, data on serum androgens in prostate cancer remain inconclusive. Large studies on variants in some androgen-metabolizing genes [SRD5A2, CYP17A1, and hydroxysteroid dehydrogenase (HSD)17B1] do not show a convincing links to prostate cancer, though there are insufficient data to draw conclusions on other genes related to androgen metabolism, including UDP-glycosyltransferases (UGT), sulfotransferases (SULT), CYP3A, and estrogen-related genes. There is some evidence, although controversial, suggesting that select variants may confer risk to certain subtypes of prostate cancer. The most notable finding in 2007 is the highly reproducible link between the chromosome 8q24 risk region and prostate cancer susceptibility. Besides the link between the 8q24 region and prostate cancer risk, population studies do not convincingly show that polymorphisms in androgen metabolism genes are associated with prostate cancer risk. Large epidemiologic studies with comprehensive gene coverage and reliable exposure data are needed to clarify further the role of androgens and their related genes in prostate cancer.

Androgen regulated immunosuppression in prostate cancer through arginase expression

5150 Background: The significant immunological boost following androgen depletion therapy (ADT) illustrates the immunosuppressive potential of androgens in prostate cancer. As the expression of arginase II (ArgII) appears to be one possible mode of immunosuppression in prostate cancer, it is of interest to determine whether the expression of ArgII in prostate cancer cells is regulated by androgens. Our objectives are to evaluate the in vitro and in vivo expression of ArgII in prostate cancer cell lines and primary tumors. Methods: The androgen-regulated expression of ArgII was evaluated in vivo on a tissue micro-array containing cores from a cohort of 40 control patients (radical prostatectomy only) and 36 ADTx patients (ADT prior to radical prostatectomy). Two independent observers evaluated data from the immunohistochemistry staining against ArgII. The in vitro expression was evaluated in LNCaP cells stimulated for 72 hours with 10nM of R1881. PBMCs from healthy donors were stimulated by 0.250ng/ml of OKT3 in the presence of conditioned media from LNCaP expressing a siRNA against ArgII. PBMCs activation was monitored by ELISA against IFNγ. Results: By immunohistochemistry, in vivo expression of ArgII in ADTx patients was 2x lower in normal adjacent tissues (p6x more ArgII than control LNCaP (p2.5x in the presence of R1881. These results suggest that the presence of androgens favors the expression of ArgII both in vitro and in vivo. PBMCs activated by OTK3 for 24 hours in the presence of conditioned media of LNCaP + SiArgII demonstrated that the absence of ArgII was associated with increased IFNγ secretion: 1,681pg/ml with LNCaP + SiCtrl vs 537pg/ml with LNCaP + SiArgII (p<0.05, Mann-U, n=4). Conclusions: Our results demonstrate that the pro-inflammatory boost following ADT is associated with a lower expression of ArgII. Moreover, our in vitro data suggest an androgen regulation of ArgII expression. Finally, conditioned media from LNCaP transfected with a siRNA against ArgII had improved immunostimulatory potential leading to an increased activation of PBMCs. Our data supports the notion that, in the presence of androgens, prostate cancer cells are directly involved in the development of an immunosuppressive tumor environment through the up-regulation of ArgII. No significant financial relationships to disclose.

The Expression of Transcription Factor Activating Transcription Factor 3 in the Human Prostate and its Regulation by Androgen in Prostate Cancer

ATF3 is a member of the basic leucine zipper/cyclic adenosine monophosphate responsive element binding protein family of transcription factors. There is overwhelming evidence that it is a stress inducible factor acting in a signal-type and cell-type dependent manner, and it is involved in cell proliferation and survival. We found that ATF3 was differently expressed in an in vitro prostate cancer tumor progression model and we investigated the possible role of ATF3 in prostate cancer. ATF3 up-regulation in vivo/in vitro and androgen regulation were assessed by immunohistochemistry and immunoblot analysis. Results after forced ATF3 transfection were evaluated by proliferation assay and cell cycle analysis. Immunohistochemistry and immunoblot analysis revealed ATF3 up-regulation in prostate cancer in vitro and in vivo, and stimulation of expression by androgens. Antiandrogen treatment decreased ATF3 expression in androgen sensitive cells but acted as a stimulator in long-term androgen ablated cells representing a model for therapy refractory disease. Expression in tumors increased with higher Gleason scores and highest expression was observed in samples of therapy refractory tumor tissue. Forced ATF3 over expression in a prostate cancer cell line induced cell proliferation and accelerated cell cycle progression from G1 to S-phase. These data provide new insight into the role of ATF3 in prostate cancer development and/or progression. They indicate that ATF3 is an androgen regulated gene that is highly expressed in prostate tumors and stimulating cell proliferation. It represents a possible target for prostate cancer therapy.

Annotation of androgen dependence to human prostate cancer-associated genes by microarray analysis of mouse prostate

In silico methods and array technologies have identified genes differentially expressed in prostate cancer. Biological functions of the identified genes are often unclear. Considering the biological significance of androgens in prostate cancer, we profiled the prostate transcripts of congenital androgen-deficient mice with or without androgen replacement in vivo using murine gene expression array. In parallel genes differentially expressed in human prostate cancer were identified by Digital Differential Display and the Serial Analysis of Gene Expression. Androgen dependence of the identified genes was then determined by the steady-state mRNA levels of the murine orthologs in response to androgen treatment. The annotation is supported by the finding that some of the androgen target genes have been reported previously with independent experiments.

Molecular cloning and characterization of a novel androgen repressible gene expressed in the prostate epithelium

Prostate cancer deaths are due to functional escape of prostate cancer cells from their original androgen-dependent growth. To better understand the origin and evolution of hormone-refractory prostate cancer, it is important to identify and characterize genes expressed in the androgen-deprived prostate. We have verified that the rudimentary prostate of congenital androgen deficient mice ( hpg) is indeed androgen independent. Using suppression subtractive hybridization between mRNA derived from prostates of hypogonadal ( hpg) with or without 14 days of testosterone replacement we have cloned a novel gene from the hpg prostate, termed ADMP (for androgen down regulated gene expressed in mouse prostate), that is down regulated by androgens. ADMP expression is strong in hpg mouse prostate, weak in mature castrated mouse prostate and absent in normal intact or androgen-replaced hpg mouse prostates. While ADMP expression is androgen independent in the hpg prostate, it appears to be androgen-dependent in the kidney and brain of normal intact mouse suggesting tissue specific regulation of ADMP by androgens. Human ADMP mRNA expression is suppressed by androgens in the androgen-sensitive LNCaP cell line. The predicted mouse and human protein of 76 amino acids shares sequence similarity to a putative G-protein coupled receptor indicating its possible role in signal transduction. Human ADMP expression was seen predominantly in the prostate epithelium with weaker expression in the fibroblasts and endothelial cells. Cloning and characterization of ADMP has made it feasible to determine its prospective role in the absence of androgens in prostate cancer.

A20 gene expression is regulated by TNF, Vitamin D and androgen in prostate cancer cells

A20 is a TNF-inducible primary response gene and its product, a zinc finger protein, has antiapoptotic function in several cancer cells. We studied A20 gene expression in the Vitamin D- and TNF-sensitive LNCaP cell line and in the Vitamin D- and TNF-resistant PC-3 cell line. The results of the quantitative real-time RT-PCR analyses demonstrated that the basal level of A20 mRNA production in PC-3 cells was considerably higher than in LNCaP cells that is associated with the resistance of PC-3 cells. TNF induced A20 gene expression in both cell lines, but with different effect. A20 mRNA expression was down-regulated by 10 nM calcitriol within 3–9 h after treatment and up-regulated by androgen reaching maximal values by 6 h after stimulation in LNCaP cells. We conclude that A20 may be involved in the regulation of cell proliferation by TNF, Vitamin D, and androgen in prostate cancer.

Review article Testosterone therapy in the ageing male: what about the prostate?

The concerns about testosterone therapy in ageing men with late-onset hypogonadism mainly address the risk of prostatic disease, i.e. either benign prostatic hyperplasia (BPH) or prostate cancer (PCa). Both conditions are highly dependent on androgen action and recent clinical data on the cancer-preventive effect of the 5alpha-reductase inhibitor finasteride have supported the possible role of androgens in PCa. However, the clinical data especially on the long-term effects of exogenous androgen substitution in regard to prostate safety are nonconclusive in many respects. As sufficient clinical studies on these risks will not be available in the near future, the approach of testosterone therapy towards prostate complications should be kept on a safe but practical basis. This review includes some recommendations in regard to testosterone therapy and prostate monitoring in patients with BPH and bladder outlet obstruction, with previous history of curative treatment for PCa or with prostatic intraepithelial neoplasia.

Regulation of matrix metalloproteinase 13 expression by androgen in prostate cancer

The matrix metalloproteinases (MMPs) are members of a family of endopeptidases that are able to degrade extra-cellular matrix. MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), play a key role in the migration of normal and malignant cell. Interaction of MMPs and TIMPs has been involved in the process of tumor invasion and metastasis. Using cDNA microarray as a screening tool to find androgen regulated gene in prostate cancer, we have found that expression of MMP-13 is regulated by androgen in prostate cancer derived cell line LNCaP. This regulation was further confirmed and quantified by real-time RT-PCR. In addition, the upregulation of MMP-13 mRNA by androgen could be abolished by the androgen antagonist Casodex but not the protein inhibitor cycloheximide. Western blot and immunohistochemistry of MMP-13 confirmed the androgen regulation at the protein level. We have furthermore shown that MMP-13 expression is presented in human prostate cancer obtained from aspiration biopsy. In summary, MMP-13 is androgen regulated and detectable in prostate cancer. Further study of MMP-13 in prostate cancer may help us to understand the progression of the cancer and can lead to new therapeutic options.

1462: Prostate Cancer Androgen Independence During Salvage Hormone Therapy After Radiation Failure

1462: Prostate Cancer Androgen Independence During Salvage Hormone Therapy After Radiation Failure

In vivo regulation of hTERT expression and telomerase activity by androgen. Guo C, Armbruster BN, Price DT, Counter CM, Department of Pharmacology, Duke University Medical Center, Durham, NC 27710: J Urol 2003;170(2 Pt 1):615–618

The most effective therapy for metastatic prostate cancer is androgen deprivation. Genes activated directly or possibly even indirectly by this steroid hormone represent potential targets for anticancer therapy. One such gene may be hTERT, which encodes the catalytic subunit of telomerase. In prostate cancer cells telomerase is activated, permitting sustained proliferation. Therefore, we tested whether hTERT gene expression is modulated in prostate cancer cells in vitro and in vivo by androgens. Transcriptional activation of hTERT during androgen stimulation was assayed by luciferase assays using the hTERT promoter fused to the luciferase gene and by reverse transcriptase-polymerase chain reaction to detect endogenous hTERT mRNA in LNCaP cells. hTERT mRNA levels and telomerase activity were also measured in CWR22 prostate cancer cells implanted in mice that were subsequently castrated and left untreated or administered androgen. We report that the endogenous hTERT promoter is activated during the administration of androgen to androgen sensitive LNCaP prostate cancer cells. However, this effect was indirect since an hTERT promoter construct was not activated by androgens and transcription of the endogenous gene was not stimulated early enough in cultured cells to be considered a direct target of this steroid hormone. Importantly in an in vivo model of human prostate cancer androgen deprivation led to a decrease in hTERT expression, followed by a decrease in telomerase activity, which was reversed by a single administration of androgen. The hTERT gene is regulated in human prostate cancer cells in vivo by androgens.

In Vivo Regulation of hTERT Expression and Telomerase Activity by Androgen.

The most effective therapy for metastatic prostate cancer is androgen deprivation. Genes activated directly or possibly even indirectly by this steroid hormone represent potential targets for anticancer therapy. One such gene may be hTERT, which encodes the catalytic subunit of telomerase. In prostate cancer cells telomerase is activated, permitting sustained proliferation. Therefore, we tested whether hTERT gene expression is modulated in prostate cancer cells in vitro and in vivo by androgens. Transcriptional activation of hTERT during androgen stimulation was assayed by luciferase assays using the hTERT promoter fused to the luciferase gene and by reverse transcriptase-polymerase chain reaction to detect endogenous hTERT mRNA in LNCaP cells. hTERT mRNA levels and telomerase activity were also measured in CWR22 prostate cancer cells implanted in mice that were subsequently castrated and left untreated or administered androgen. We report that the endogenous hTERT promoter is activated during the administration of androgen to androgen sensitive LNCaP prostate cancer cells. However, this effect was indirect since an hTERT promoter construct was not activated by androgens and transcription of the endogenous gene was not stimulated early enough in cultured cells to be considered a direct target of this steroid hormone. Importantly in an in vivo model of human prostate cancer androgen deprivation led to a decrease in hTERT expression, followed by a decrease in telomerase activity, which was reversed by a single administration of androgen. The hTERT gene is regulated in human prostate cancer cells in vivo by androgens.

IMAGE ANALYSIS OF ANDROGEN RECEPTOR IMMUNOSTAINING IN PROSTATE CANCER ACCURATELY PREDICTS RESPONSE TO HORMONAL THERAPY

Purpose Immunostaining for androgen receptor in prostate tumor specimens has revealed that the majority of primary and advanced stage cancers are positive for this regulatory transcription factor. Consequently, its use as a marker for tumor behavior and therapeutic response has been discounted. However, past reports have noted significant heterogeneity of androgen receptor immunostaining between prostate tumor cells in contrast to staining homogeneity in normal epithelium, which indicates that variability in androgen receptor content may exist within certain tumor specimens. To analyze this phenomenon more thoroughly and to determine whether this variability possesses clinical correlates, androgen receptor immunostaining profiles within androgen receptor positive prostate tumor specimens were categorized using an image analysis based system. Materials and Methods Tumor specimens were obtained before hormone therapy from 44 patients with advanced stage prostate cancer and 4 with early stage disease who later had progression. Response to antiandrogen therapy and survival was monitored. Paraffin embedded tumor sections were processed for immunocytochemistry and stained for androgen receptor. A Quantimet* image analysis system was used to analyze nuclear immunostaining for androgen receptor and Receptogram [dagger] patterns were established for each specimen based on univariate distributions of nuclear receptor content and concentration. *Leica, Inc., Deerfield, Illinois. [dagger] R.J. Sklarew Imaging Associates, Inc., Larchmont, New York. Results Data revealed that 17 of 18 responders to hormone therapy possessed type 1 (15) or type 3 (2) Receptograms, which are characterized by a unimodal peak or multimodal peaks within a narrow concentration range. Of the 17 cases that stabilized following therapy 16 had type 3 Receptograms and 1 was characterized as type 1. In contrast, all 13 patients in whom endocrine treatment failed had either type 2 or 4 Receptograms, which are characterized by a highly skewed or bimodal androgen receptor distribution. Positive and negative predictive values for this assay were 100 and 93%, respectively. In addition, the type 1/3 Receptogram patterns were correlated with longer mean survival. Conclusions Image analysis of prostate cancer androgen receptor immunostaining with a pattern oriented approach for response is capable of accurately predicting response to hormone therapy in patients with advanced stage disease. Application of this analytic scheme may assist the clinician with therapeutic management of advanced prostate cancer.

Interaction of drugs, hormones, and nutrition in the causes of cancer

Hormones may act as promoters in the carcinogenic process, and occasionally their metabolites may act as antihormones or have new physiologic effects. Drugs can interact with the endocrine system in many ways. They can promote secretion of a hormone, alter its rate of removal from plasma, change plasma protein-binding characteristics, or modify routes of metabolism. Estrogens have a preparative effect on the uterine endometrium. There are biologic, clinical and epidemiologic reasons for believing that estrogen administration to postmenopausal women increases the risk for endometrial cancer. Although there are similar biologic reasons to associate prolonged estrogenic stimulation with breast cancerr, evidence for such an association is weak. Oral contraceptive use has been associated with a variety of hepatocellular tumors. Although estrogens, per se, can effect several hepatic functions, it seems likely that the 17 alpha-alkyl and 17 alpha-ethinyl functions of the progestins and estrogens are involved in this process. The role of estrogen use during pregnancy in the causation of vaginal cancer in female offspring and the role of androgens in prostate cancer have been discussed.