Search

The majority of prostate cancer (PCa) patient receiving androgen ablation therapy eventually develop castration-resistant prostate cancer (CRPC). We previously reported that androgen treatment suppresses Skp2 and c-Myc through androgen receptor (AR) and induced G1 cell cycle arrest in androgen-independent LNCaP 104-R2 cells, a late stage CRPC cell line model. However, the mechanism of androgenic regulation of Skp2 in CRPC cells was not fully understood. In this study, we investigated the androgenic regulation of Skp2 in two AR-positive CRPC cell line models, the LNCaP 104-R1 and PC-3AR Cells. The former one is an early stage androgen-independent LNCaP cells, while the later one is PC-3 cells re-expressing either wild type AR or mutant LNCaP AR. Proliferation of LNCaP 104-R1 and PC-3AR cells is not dependent on but is suppressed by androgen. We observed in this study that androgen treatment reduced protein expression of Cdk2, Cdk7, Cyclin A, cyclin H, Skp2, c-Myc, and E2F-1; lessened phosphorylation of Thr14, Tyr15, and Thr160 on Cdk2; decreased activity of Cdk2; induced protein level of p27(Kip1); and caused G1 cell cycle arrest in LNCaP 104-R1 cells and PC-3AR cells. Overexpression of Skp2 protein in LNCaP 104-R1 or PC-3AR cells partially blocked accumulation of p27(Kip1) and increased Cdk2 activity under androgen treatment, which partially blocked the androgenic suppressive effects on proliferation and cell cycle. Analyzing on-line gene array data of 214 normal and PCa samples indicated that gene expression of Skp2, Cdk2, and cyclin A positively correlates to each other, while Cdk7 negatively correlates to these genes. These observations suggested that androgen suppresses the proliferation of CRPC cells partially through inhibition of Cyclin A, Cdk2, and Skp2.

Introduction Triple negative breast cancer (TNBC) is a heterogeneous collection of biologically diverse cancers, which contributes to variable clinical outcomes. Previously, we identified a TNBC subtype that has a luminal phenotype and expresses the androgen receptor (AR+). TNBC cells derived from these luminal AR + tumors have high frequency phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations. The purpose of this study was to determine if targeting phosphoinositide 3-kinase (PI3K) alone or in combination with an AR antagonist is effective in AR + TNBC. Methods We determined the frequency of activating PIK3CA mutations in AR + and AR- TNBC clinical cases. Using AR + TNBC cell line and xenograft models we evaluated the effectiveness of PI3K inhibitors, used alone or in combination with an AR antagonist, on tumor cell growth and viability. Results PIK3CA kinase mutations were highly clonal, more frequent in AR + vs. AR- TNBC (40% vs. 4%), and often associated with concurrent amplification of the PIK3CA locus. PI3K/mTOR inhibitors had an additive growth inhibitory effect when combined with genetic or pharmacological AR targeting in AR + TNBC cells. We also analyzed the combination of bicalutamide +/- the pan-PI3K inhibitor GDC-0941 or the dual PI3K/mTOR inhibitor GDC-0980 in xenograft tumor studies and observed additive effects. Conclusions While approximately one third of TNBC patients respond to neoadjuvant/adjuvant chemotherapy, recent studies have shown that patients with AR + TNBC are far less likely to benefit from the current standard of care chemotherapy regimens and novel targeted approaches need to be investigated. In this study, we show that activating PIK3CA mutations are enriched in AR + TNBC; and, we show that the growth and viability of AR + TNBC cell line models is significantly reduced after treatment with PI3K inhibitors used in combination with an AR antagonist. These results provide rationale for pre-selection of TNBC patients with a biomarker (AR expression) to investigate the use of AR antagonists in combination with PI3K/mTOR inhibitors. Electronic supplementary material The online version of this article (doi:10.1186/s13058-014-0406-x) contains supplementary material, which is available to authorized users.

Triple negative breast cancers are a heterogeneous group of tumors characterized by poor patient survival and lack of targeted therapeutics. Androgen receptor has been associated with triple negative breast cancer pathogenesis, but its role in the different subtypes has not been clearly defined. We examined androgen receptor protein expression by immunohistochemical analysis in 678 breast cancers, including 396 triple negative cancers. Fifty matched lymph node metastases were also examined. Association of expression status with clinical (race, survival) and pathological (basal, non-basal subtype, stage, grade) features was also evaluated. In 160 triple negative breast cancers, mRNA microarray expression profiling was performed, and differences according to androgen receptor status were analyzed. In triple negative cancers the percentage of androgen receptor positive cases was lower (24.8% vs 81.6% of non-triple negative cases), especially in African American women (16.7% vs 25.5% of cancers of white women). No significant difference in androgen receptor expression was observed in primary tumors vs matched metastatic lesions. Positive androgen receptor immunoreactivity was inversely correlated with tumor grade (p

Prostate cancer cells may lose androgen-sensitivity after androgen ablation therapy, becoming highly invasive and metastatic. The biological mechanisms responsible for higher tumurogenicity of androgen-independent prostate carcinomas are not entirely known. We demonstrate that androgen receptor regulation of adhesion and invasion of prostate cancer cells through modulation of α6β4 integrin expression may be one of the molecular mechanisms responsible of this phenomenon. We found that protein and gene expressions of α6 and β4 subunits were strongly reduced in the androgen-sensitive cell line LNCaP respect to the androgen-independent PC3 and that transfection of PC3 cells with a full-length androgen receptor expression vector resulted in a decreased expression of α6β4 integrin, reduced adhesion on laminin, and suppressed Matrigel invasion. Growth in soft agar was also suppressed in androgen receptor-positive PC3 clones. Treatment of androgen receptor positive clones with the synthetic androgen R1881 further...

Aim—To investigate the correlation between immunohistochemical and biochemical steroid receptor analyses by measurement of oestrogen, progesterone, and androgen receptor status in ovarian neoplasia. Methods—Tissue samples were obtained from 27 ovarian neoplasms, including two borderline tumours. Immunohistochemical staining of the tissue slides was scored semiquantitatively, incorporating the intensity and percentage of positive staining (histoscore). Tumours with a histoscore of 10 or more were considered steroid receptor positive. The epithelial and stromal fractions of the tumours were analysed separately. To study the uniformity of receptor expression throughout a tumour, up to four samples were analysed. Results—Immunohistochemical histoscores of the oestrogen receptor in the epithelial fractions were significantly correlated with the biochemical oestrogen receptor values (r = 0.408). Androgen receptor status in the epithelial fraction was correlated with that in the stromal fraction (r = 0.741), while androgen receptor histoscores in the epithelial fraction correlated with the biochemical assay values (r = 0.463). On biochemical analysis, 17 of the 27 ovarian tumours were oestrogen receptor positive and seven were progesterone receptor positive. On immunohistochemical analysis, eight tumours were oestrogen receptor positive and two were progesterone receptor positive. Biochemical analysis showed that 14 of the 26 tumours were slightly androgen receptor positive (10–50 fmol/mg protein), while all the others were negative. On immunohistochemical analysis, seven of the 26 tumours were androgen receptor positive. When two or more specimens from one tumour were analysed, marked differences in steroid status were found, especially in progesterone receptor and androgen receptor expression. Some parts of a tumour were steroid receptor positive, while other parts were negative owing to heterogeneity of expression. Conclusions—Immunohistochemical and biochemical analysis of steroid receptors in ovarian tumours correlated weakly or not at all. Heterogeneity of expression within a tumour and the presence of progesterone and androgen receptors in the stromal fraction partly accounted for this observation. Biochemical and immunohistochemical androgen receptor status was much lower than in previous reports. Key Words: steroid receptors • ovarian neoplasia • immunohistochemical analysis • biochemical analysis

Triple negative breast cancer (TNBC) is a heterogeneous collection of biologically diverse cancers, which contributes to variable clinical outcomes. Previously, we identified a TNBC subtype that has a luminal phenotype and expresses androgen receptor (AR+). TNBC cells derived from these luminal AR+ tumors have high frequency PIK3CA mutations. The purpose of this study was to determine if targeting PI3K alone or in combination with an AR antagonist is effective in AR+ TNBC. Methods: We determined the frequency of activating PIK3CA mutations in AR+ and AR- TNBC clinical cases. Using AR+ TNBC cell lines and xenograft models we evaluated the effectiveness of PI3K inhibitors, used alone or in combination with an AR antagonist, on tumor cell growth and viability. Results: PIK3CA kinase mutations were highly clonal, more frequent in AR+ vs. AR- TNBC (40% vs. 4%), and often associated with concurrent amplification of the PIK3CA locus. PI3K/mTOR inhibitors had an additive growth inhibitory effect when combined with genetic or pharmacological AR targeting in AR+ TNBC cells. We also analyzed the combination of bicalutamide +/- the pan-PI3K inhibitor GDC-0941 or the dual PI3K/mTOR inhibitor GDC-0980 in xenograft tumor studies and observed additive effects. Conclusions: While approximately one third of TNBC patients respond to neoadjuvant/adjuvant chemotherapy, recent studies have shown that patients with androgen receptor positive (AR+) TNBC are far less likely to benefit from the current standard of care chemotherapy regimens and novel targeted approaches need to be investigated. In this study, we show that activating PIK3CA mutations are enriched in AR+ TNBC; and, the growth and viability of AR+ TNBC cell line models is significantly reduced after treatment with PI3K inhibitors used in combination with an AR antagonist. These results provide rationale for pre-selection of TNBC patients with a biomarker (AR expression) to investigate the use of AR antagonists in combination with PI3K/mTOR inhibitors. Citation Format: Brian D Lehmann, Joshua A Bauer, Johanna M Schafer, Christopher S Pendleton, Luojia Tang, Kimberly C Johnson, Xi Chen, Justin M Balko, Henry Gomez, Carlos L Arteaga, Gordon B Mills, Melinda E Sanders, Jennifer A Pietenpol. PIK3CA mutations in androgen receptor-positive triple negative breast cancer confer sensitivity to the combination of PI3K and androgen receptor inhibitors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-03.

Androgens regulate breast cancer cell proliferation via androgen receptor (AR)-mediated mechanisms. To investigate further the androgen-responsiveness of human breast tumours, we examined the immunohistochemical expression of the AR and two androgen-regulated proteins, prostate-specific antigen (PSA) and gross cystic disease fluid protein-15 (GCDFP-15), in 72 primary breast tumours. AR immunoreactivity was present in the nuclei of breast tumour cells and was correlated with oestrogen receptor (ER; P < 0.05) and progesterone receptor (PR; P < 0.01) status. PSA and GCDFP-15 immunoreactivity was present in the cytoplasm of tumour cells but not the adjacent stromal cells. AR-positive cells were present in 85% (61/72) of breast tumours, and 98% (43/44) of PSA-positive and 92% (44/48) of GCDFP-15-positive tumours were also positive for AR. Positive immunoreactivity for both PSA and GCDFP-15 in breast tumours was highly dependent on AR status (odds ratios of 24.0 and 4.5 respectively), but unrelated to age, ER and PR status and axillary lymph node involvement. PSA immunoreactivity was more frequently observed in moderate and well-differentiated tumours and was significantly (P < 0.001) associated with GCDFP-15 immunoreactivity. In conclusion, PSA and GCDFP-15 immunoreactivity was dependent on the presence of AR, but not ER or PR in primary breast tumours. Images Figure 1

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 diseas...

Full expression of testosterone (T) actions in the brain requires both direct binding to androgen receptors (AR) and in situ aromatization to estradiol (E2). To determine the cellular basis of constitutively high aromatase and AR binding activities in teleost fish brain, and the neuroanatomic location and spatial relations of cells of each type, an immunocytochemical mapping study of goldfish (Carassius auratus) brain was carried out using antibodies to human placental aromatase and human/rat AR peptide and the avidin-biotin-peroxidase technique. Both antibodies specifically labeled cells that were neuronal in appearance and were most numerous in reproductive control centers: medial and ventral telencephalon (TEL) and preoptic and hypothalamic periventricular nuclei. Additional populations of aromatase- and AR-labeled cells were present in the olfactory bulbs, central telencephalon, and stratum periventriculare of the optic tectum. Anti-aromatase, but not anti-AR, labeled fiber tracts and fibrous layers in visual and auditory pathways, and perikarya and processes of premotor neurons known to integrate sensory input (reticulospinal neurons, Mauthner cells). Anti-AR selectively labeled lateral TEL regions, the nucleus ventromedialis thalami, and discrete cell clusters in the medial tegmental nucleus. Aromatase-immunoreactivity (-ir) was primarily cytoplasmic, whereas AR-ir was primarily nuclear, but relative intensity of nuclear vs cytoplasmic labeling with each antibody differed by brain region. Aromatase- and AR-ir cells were not obviously more numerous in goldfish brain than previously seen in birds and mammals, suggesting that enhanced expression occurs on a per cell basis. We conclude that T exerts its actions coordinately via direct and indirect pathways in most brain regions but independently via AR- or aromatase-mediated mechanisms in selected areas. These studies point to a wide role for androgen in modulating primary sensory signals as well as in classical reproductive processes.

BACKGROUND Patients with prostate carcinoma generally respond to androgen withdrawal therapy, but subsequent progression to androgen-independence is frequently observed. Since androgen receptors play a key role in androgen action, the ratio of androgen receptor-containing cells in cancerous tissues was determined by immunohistochemical staining of prostate biopsy specimens for comparison with the outcome. METHODS Sixty-two patients with untreated Stage D2 prostate carcinoma who received endocrine therapy between 1986 and 1992 were included in the present study. Biopsy tissue was stained with anti-human androgen receptor antibody, and the ratio of positively stained cells was estimated by counting 700 to 1000 cancer cells from each patient. Histologic grade, extent of bone metastases, clinical response to endocrine therapy, and outcome, were compared with androgen receptor content. RESULTS Cancers with a low Gleason score had a significantly higher androgen receptor content than those with a high Gleason score. Androgen receptor content was not significantly correlated with extent of disease or tumor marker response at three months. Patients with 48% or more androgen receptor positive cells had a statistically significant better outcome, in terms of both progression free and cause-specific survival, than patients with less than 48% androgen receptor content. Multivariate analysis demonstrated that androgen receptor content, extent of disease, and tumor marker response at three months were significant predictors of outcome. CONCLUSIONS Androgen receptor content measured immunohistochemically is a useful prognostic indicator for patients with Stage D2 prostate carcinoma treated with endocrine therapy. Cancer 1996;77:934-40.

The significance of androgen receptor (AR) expression in triple-negative breast cancer (TNBC) is unclear, and published studies so far have been inconclusive.A tissue microarray was constructed using tissue obtained from 119 patients with primary TNBC and stained for AR expression. Other tissue types obtained included recurrent TNBC, normal breast tissue, adjacent ductal carcinoma-in situ (DCIS), lymph node (LN) and distant metastases. Positive AR expression was defined as ≥10% nuclear staining.Epithelial tissue was present and evaluable in 94 TNBC patients with a total of 177 tissue cores. AR expression in TNBC was 22 of 94 (23%). AR expression was higher in normal breast tissue (88%) and adjacent DCIS (73% overall). All LN metastases from AR-positive TNBC patients were also AR positive; in addition, no AR-negative TNBC patient had AR-positive LNs. AR expression was associated with older patient age (63 vs. 57 years, respectively, p = 0.051) and LN metastases (p = 0.033). Locoregional recurrence and overall/disease-specific survival were similar between AR-positive and AR-negative patients, although AR-positive patients had more advanced disease. On multivariate analysis, the presence of LN metastases was associated with poorer recurrence-free survival in AR-positive patients (hazard ratio, 4.34) (p = 0.031).The AR is expressed in normal breast tissue, and expression decreases with advancement to DCIS and invasive cancer. AR-positive TNBC was more common in older patients and had a higher propensity for LN metastases. AR-positive TNBC may represent a breast cancer subtype with unique features that may be amenable to treatment with alternative targeted therapies.

Distribution and regulation of androgen receptor expression during fetal and neonatal virilization of the rat fetus was assessed by immunohistochemistry. In mesonephric duct derivatives the androgen receptor expression became evident first in the efferent ductules and epididymis (on fetal day 14), subsequently in the vas deferens and finally in the seminal vesicle. Mesenchymal cells of the urogenital tubercle were positive for androgen receptors from fetal day 14 onwards. In the mesenchymal cells of the prostate anlagen, androgen receptor positive cells were found first on fetal day 16. Administration of 5α-dihydrotestosterone to pregnant rats from day 11 to day 20 of gestation caused a stabilization of the wolffian duct in female fetuses. The androgen receptor expression pattern became similar as found in male fetuses, and showed an increase in density and in frequency of androgen receptor positive cells. Administration of the androgen antagonist flutamide during the same interval caused a reduction in density and frequency of androgen receptor positive cells in male fetuses. These findings indicate that androgens enhance the expression of androgen receptors in the developing rat genital tract by induction of androgen receptor positive cells, and by increasing the frequency. The developmental pattern of androgen receptor expression in the rat mesonephric duct system reflects the androgen-responsiveness of the ducts, and is consistent with induction of the androgen receptor along the ducts by testosterone reaching these structures in an exocrine fashion.

Prostate cancer is the most common cancer and the second leading cause of cancer related death among men in the United States. Current therapy for advanced prostate cancer is mainly based on androgen deprivation, though in many cases tumors become androgen-independent and resistant to available treatments. Thus, accessing novel therapies for prostate cancer and resistant tumors remains an important goal in the field. ONC201 is a first-in-class small molecule anti-cancer drug, shown to selectively induce cell death in most cancer cells tested in contrast to matched normal cells. In this study we investigated the single-agent and combination efficacy of ONC201 on a panel of prostate cancer cell lines with varied androgen receptor (AR) and receptor tyrosine kinases (RTKs) expression status. Our preliminary data suggests that the pro-apoptotic effects of ONC201 correlate with expression of androgen receptor and other receptor tyrosine kinases (RTKs). Specifically, high AR and low RTK (c-MET, EGFR, HER2 and IGFR) expression demonstrated higher sensitivity to ONC201 as compared to low AR and high RTK expression as evaluated by cell viability using the Cell-Titer Glo assay. We further modeled these findings in ONC201-sensitive 22Rv1 and ONC201-resistant DU145 cell lines. ONC201 induced robust apoptosis (cleaved PARP) in ONC201-sensitive 22Rv1 cells as compared to ONC201-resistant DU145 cells In addition, ONC201-sensitive 22RV1 cells showed abrogation of total AR expression 72 hours following treatment with ONC201. Thus, AR and RTK status seem to be emerging efficacy markers for response of ONC201 in prostate cancer cells. We are further investigating the involvement of ONC201 in the signal transduction pathway of AR and other RTKs in prostate cancer. Lastly, our initial screening for treatment of cells with a combination of ONC201 and FDA-approved therapies for prostate cancer showed synergistic potential of ONC201 with everolimus and docetaxel in both AR-dependent and independent cells. As expected, synergism of anti-androgen MDV3100 (enzalutamide) with ONC201 was restricted to AR positive prostate cancer cell lines. Our results indicate that ONC201 has therapeutic potential both as a single agent and in combination therapy for prostate cancer. Our long-term goal is to integrate our pre-clinical studies and translate the findings to single agent/combination trials of ONC201 for prostate cancer patients. Citation Format: Avital Lev, Amriti R. Lulla, David T. Dicker, Wafik S. El-Deiry. ONC201 induces cell death in androgen receptor positive prostate cancer cells and shows synergistic effect with anti-prostate cancer drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1825.

TPS5602Background: Approximately 75% of women with epithelial ovarian cancer (OC) present with advanced disease. The majority of these women will ultimately recur and require life-long treatment fo...

e17559Background: SDC is a rare and highly aggressive disease. AR+ metastatic SDC can be successfully treated with a complete anti-androgen blockade (CAB) approach . In case of progression a second...

Background: The androgen receptor (AR) is the most highly expressed steroid receptor in breast cancer (BC) with expression seen in up to 95% of estrogen receptor positive (ER+) and up to 50% of ER negative disease. Historically, steroidal androgens exhibited virilizing side effects, thus limiting clinical use. In TNBC, the expression of AR and androgen synthesizing enzymes is associated with lower proliferation, lower tumor grade, better overall survival, and more favorable clinical outcomes as compared to those patients with TNBC not expressing AR. Data from two trials targeting AR in TNBC indicates low level but encouraging clinical activity. A non-steroidal, tissue-selective, AR modulator (SARM), such as GTx-024, offers a targeted approach of AR activation in AR+ TNBC without virilization or estrogenic effects. Trial Design: Open label, multicenter, multinational, Phase 2 study for the treatment of advanced or metastatic TNBC. Subjects will receive GTx-024, 18 mg orally (PO) daily, continued until evidence of disease progression or toxicity. Subjects whose tumors demonstrate Clinical Benefit (CB) will be treated on study drug until progression. Eligibility Criteria: Inclusion: Female, ≥18 years old, confirmed AR+ (≥10% staining) TNBC (confirmed by medical history), up to 1-2 prior chemotherapy regimens, available archived tumor tissue, measurable or bone-only disease, ECOG 0 or 1, with prior toxicities from chemotherapy resolved. Exclusion: Life expectancy Specific Aims: The primary aim is to measure the proportion of AR+ subjects with CB at 16 weeks; defined as subjects with a complete response (CR), partial response (PR), or stable disease (SD); per modified RECIST 1.1. Secondary endpoints include: objective response rate, progression free survival, and time to progression. Statistical Methods: Simon's two-stage (optimal) design will be used to assess primary efficacy, requiring up to 41 evaluable subjects; i.e., subjects with centrally confirmed AR+ who receive at least one dose of study drug. The first stage will be assessed among the first 21 evaluable subjects. If at least 2/21 achieve CB per modified RECIST 1.1 at 16 weeks, then the trial will proceed to the second stage of recruitment of up to a total of 41 subjects in the evaluable subset of the Full Analysis Set. Otherwise, the trial will be discontinued for lack of efficacy. The trial will test for an unacceptably low CBR of ≤5% versus a CBR more consistent with ≥20%. Target Accrual: Up to 55 subjects will be enrolled. Trial Information: www.gtxinc.com Citation Format: Rugo H, Overmoyer B, Schwartzberg L, Palmieri C, Taylor R, Hancock M, Small S, Johnston MA. A phase 2 open label, multi-center, multinational study investigating the efficacy and safety of GTx-024 on advanced, androgen receptor-positive triple negative breast cancer (AR+ TNBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT2-01-07.

There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK) that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets.

INTRODUCTION AND OBJECTIVES: Calcium (Ca ), a ubiquitous secondary messenger, is involved in several cellular processes such as apoptosis, proliferation and carcinogenesis. It has also been shown that Ca signaling is involved in androgen-induced PSA secretion. Recently published epidemiologic data demonstrated that patients on calcium channel blockers had significantly lower incidences of prostate cancer. Therefore, we examined the role of various voltagegated calcium channels in androgen signaling and prostate cancer development. METHODS: Multiple prostate cancer cell lines including LNCaP, LAPC-4, and C4-2 (all androgen receptor positive) were treated with calcium channel blockers nifedipine, mibefradil, and tetrandine. The medications were added to the cell culture media at a dose range of 5-50 micromolar for various time points between 2 and 24 hours. Cell proliferaion was measured using an MTS assay. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine expression of several Ca channel genes in different cell lines. Knockdown of Ca channel expression using an siRNA was also utilized to determine effects on cell lines. RESULTS: Blocking L-type channels with nifedipine and tetrandrine significantly suppressed cell proliferation and androgen receptormediated gene expression in prostate cancer cells. Higher doses of tetrandrine also induced a dramatic cytotoxic effect. In contrast, a T-type specific channel blocker mibefradil induced androgen receptor transactivation without androgen addition and also drastically enhanced androgen receptor transactivation in a reporter gene assay. RT-PCR demonstrated that only L-type CACNA1D (cav1.3) and T-type CACNA1H (cav3.2) alpha-subunit genes are highly expressed in androgen receptor-positive prostate cancer cell lines, while L-type CACNA1F (cav1.4) is only weakly expressed in androgen receptorpositive cell lines but highly expressed in androgen receptor-negative cell lines. Other subunits are not expressed in any prostate cancer cells. Furthermore, consistently, knocking down CACNA1D (cav1.3) expression by the siRNA approach abrogated AR transactivation while knocking down CACNA1H (cav3.2) expression enhanced AR transactivation. CONCLUSIONS: These results suggest that the L-type calcium channel subunit (cav1.3) has the potential as a therapeutic target for prostate cancer intervention, although further clinical testing is warranted.

Androgen receptor has a critical role in prostate cancer development and progression. Cell death via autophagy may also contribute to prostate cancer progression. We determined the role and regulatory effects of androgen receptor on the autophagy process of prostate cancer cells.Using a series of morphological approaches, such as transmission electron microscopy, monodansylcadaverine (Sigma®) and GFP-LC3 fluorescence microscopy assay, and Western blot we monitored the autophagic process in 3 pairs of prostate cancer cell lines to study the relationship to androgen receptor signals.Androgen receptor knockdown in androgen receptor positive cells, such as LNCaP or CWRrv1 human prostate cancer cells, led to increased autophagy. Adding functional androgen receptor to androgen receptor negative cells, such as PC3 human prostate cancer cells, resulted in decreased autophagy. This suggests that androgen receptor could have a negative role in regulating autophagy. Mechanism dissection indicated that androgen receptor might repress autophagy via modulation of p62 expression. A therapeutic approach of targeting androgen receptor to increase autophagy using the androgen receptor degradation enhancer ASC-J9® suppressed prostate cancer growth.Findings provide evidence that the androgen receptor might promote prostate cancer cell growth via autophagy down-regulation. Targeting the androgen receptor via ASC-J9 might lead to tumor suppression via the induction of autophagy. This may represent a new, potential therapeutic approach and mechanism for prostate cancer.

Objective To establish a mouse xenograft model of metastatic prostate cancer (PCa) and investigate the relationship between metastasis and circulating tumor cells. Methods Flow cytometry (FACS) was used to detect suitable PCa cells and markers for detecting circulating tumor cells in vivo. We orthotopically injected androgen receptor-positive and androgen-independent C4-2B PCa cells into 12 severe combined immunodeficiency (SCID) mouse prostates, including 1 vehicle control. We measured the serum prostate-specific antigen levels biweekly after tumor inoculation. Circulating tumor cells (CTCs) were measured qualitatively by fluorescent microscopy immediately after the mice were sacrificed. The mouse prostates and lungs were examined for tumor formation using immunohistochemistry because we found no apparent metastasis, except in the lung. Results FACS analyses in vitro identified the marker, prostate-specific membrane antigen, and C4-2B cells to be appropriate for additional in vivo study. We confirmed that the serum prostate-specific antigen increase was dependent on time and prostate tumor weight in mice. Of the 11 mice, 6 could be used as the mouse PCa xenograft model. Fluorescent microscopy detected CTCs in the peripheral blood in 5 of the 6 mice constituting the PCa model. Human prostate-specific antigen expression was detected by immunohistochemistry in the prostates of all the mice and in the lung of 2 of the 6 mice, suggesting 2 mice with lung metastasis. Conclusion We have shown the potential establishment of a mouse lung metastatic xenograft model of androgen receptor-positive and androgen-independent C4-2B PCa tumor. However, the present model requires improvement to be a more reproducible, accurate and complete experimental model. Additional study is necessary to verify the relationship between metastasis and CTCs.

Breast cancer is a heterogeneous disease and several distinct subtypes exist based on differential gene expression patterns. Molecular apocrine tumours were recently identified as an additional subgroup, characterised as oestrogen receptor negative and androgen receptor positive (ER− AR+), but with an expression profile resembling ER+ luminal breast cancer. One possible explanation for the apparent incongruity is that ER gene expression programmes could be recapitulated by AR. Using a cell line model of ER− AR+ molecular apocrine tumours (termed MDA-MB-453 cells), we map global AR binding events and find a binding profile that is similar to ER binding in breast cancer cells. We find that AR binding is a near-perfect subset of FoxA1 binding regions, a level of concordance never previously seen with a nuclear receptor. AR functionality is dependent on FoxA1, since silencing of FoxA1 inhibits AR binding, expression of the majority of the molecular apocrine gene signature and growth cell growth. These findings show that AR binds and regulates ER cis-regulatory elements in molecular apocrine tumours, resulting in a transcriptional programme reminiscent of ER-mediated transcription in luminal breast cancers.

Androgen receptor (AR) signals play important roles in bladder carcinogenesis and tumor progression. Activation of the epidermal growth factor receptor (EGFR) family, including EGFR and ERBB2, leads to bladder cancer cell growth and correlates with poor patients' prognosis. However, cross talk between AR and EGFR/ERBB2 pathways in bladder cancer remains poorly understood. In AR-positive bladder cancer UMUC3 and TCC-SUP cells, dihydrotestosterone (DHT) increased the expression of EGFR and ERBB2 both in mRNA and in protein levels, and an anti-androgen hydroxyflutamide antagonized the effect of DHT. The necessity of AR was confirmed by silencing the receptor, using short hairpin RNA (shRNA), in UMUC3 cells, as well as by expressing the receptor in AR-negative 5637 cells. Of note were much higher basal levels of EGFR and ERBB2 in UMUC3-control-shRNA than in UMUC3-AR-shRNA and those of EGFR in 5637-AR than in 5637-V. DHT additionally upregulated the levels of phosphorylation of EGFR (pEGFR) and its downstream proteins AKT (pAKT) and ERK1/2 (pERK), induced by EGF treatment, in AR-positive cells. Immunohistochemistry on cystectomy specimens showed strong associations between expressions of AR and EGFR (P=0.0136), pEGFR (P=0.0041), ERBB2 (P=0.0331), or pERK (P=0.0274), but not of pAKT (P=0.5555). The Kaplan–Meier and log-rank tests further revealed that positivity of AR (P=0.0005), EGFR (P=0.2425), pEGFR (P=0.1579), ERBB2 (P=0.2997), or pERK (P=0.1270) and negativity of pAKT (P=0.0483) were associated with tumor progression. Our results indicate that AR activation upregulates the expression of EGFR and ERBB2 in bladder cancer cells. AR signals may thus contribute to the progression of bladder cancer via regulation of the EGFR/ERBB2 pathways.

A 14-step biomimetic synthetic route to glyceollin I (1.5% overall yield) was developed and deployed to produce the natural enantiomeric form in soy, its unnatural stereoisomer, and a racemic mixture. Enantiomeric excess was assessed by asymmetric NMR shift reagents and chiral HPLC. Antiproliferative effects were measured in human breast, ovarian, and prostate cancer cell lines, with all three chiral forms exhibiting growth inhibition (GI) in the low to mid μM range for all cells. The natural enantiomer, and in some cases the racemate, gave significantly greater GI than the unnatural stereoisomer for estrogen receptor positive (ER(+)) versus ER(-) breast/ovarian cell lines as well as for androgen receptor positive (AR(+)) versus AR(-) prostate cancer cells. Surprisingly, differences between ER(+) and ER(-) cell lines were not altered by media estrogen conditions. These results suggest the antiproliferative mechanism of glyceollin I stereoisomers may be more complicated than strictly ER interactions.

Item does not contain fulltext

The relationship between calcium channel blockers and prostate cancer has been an area of increased interest to investigators. Calcium channel blockers have been shown to influence cell proliferation, differentiation, and apoptosis. Clinically, the association between calcium channel blockers and the development of prostate cancer has been controversial. However, on a basic science level, there is evidence that calcium channel blockers induce cytotoxicity in androgen receptor positive cell lines and may offer an innovative strategy for the treatment of castration-resistant prostate cancer.

The low incidence of prostate cancer in Asians has been attributed to chemopreventative properties of certain chemicals found in their diet. This study characterized the androgenic and chemopreventative properties of the Jamaican bush tea “Bizzy,” using androgen receptor positive and negative cell lines. Exposure of prostate cells to Biz-2 resulted in a growth inhibition (GI50) of 15 ppm in LNCaP cells and 3.6 ppm in DU145 cells. Biz-2 elicited a 2-fold increase in the mRNA of the anti-apoptotic gene Bcl2, with a 10-fold increase in that of the proapoptotic gene Bax. We observed a 2.4- to 7.5-fold change in apoptotic cells in both cell lines. Biz-2 at 10 ppm elicited a time- and dose-dependent stimulation of both the protein and mRNA levels of several androgen-regulated genes. Biz-2 caused a 36% decrease in PSA secretion and a significant increase in PSA mRNA. The relative binding affinity (IC50) of Biz-2 for AR was 2- to 5-fold lower than that of the synthetic androgen R1881. Biz-2 was found to be a specific ligand for the AR in that the natural ligand, DHT, and the anti-androgen, flutamide, displaced Biz-2 bound to AR and inhibited Biz-2-induced transcription and PSA secretion. This study provided evidence that Biz-2 extract possesses the ability to modulate prostate cancer cell biology in an AR-dependent manner.

Steroid receptors and tamoxifen binding sites (TBS) were assayed in the soluble fraction of 121 primary breast cancers. Scatchard analysis of TBS in high speed supernatant (100 000 g ) showed one population of binding sites; however, biphasic plots were obtained in low speed supernatants (40 000 g ). Isoelectric focussing of supernatants preincubated with radioactive tamoxifen identified two classes of TBS (pI 4.1–4.6) which have different binding affinities and bind neither oestradiol nor diethylstilbestrol. Association between TBS and steroid receptors was: TBS positive/progesterone receptor positive 32.6%, TBS positive/glucocorticoid receptor positive 52.7%, TBS positive/oestrogen receptor positive 60% and TBS positive/androgen receptor positive 72.2%. We conclude that heterogeneous TBS are present in low speed fractions and can be easily separated from the oestrogen receptor by isoelectric focussing. The association between TBS and steroid receptor status could be of clinical value in the management of primary breast cancer.

SUMMARY: The objective of this work was identify the presence of interstitial cells of Cajal, muscle cells, nerves and androgen receptor positive cells in adult human testicle, using immunohistochemical detection for c-kit/CD-117, actin smooth muscle specific (ASMS), neurofilament (N) and androgen receptor (AR), respectively. The samples were obtained from patients (n= 10) with diagnosis of prostate cancer, with surgery of orchiectomy. Subsequently were processed by histology and for immunohistochemistry using specific antibodies. It showed the presence of cells c-kit/CD-117, with diverse degrees of positivity, distributed mainly in the interstitial peritubular area of the human testicle. The peritubular myoides cells were positive to the presence of the actin smooth muscle and androgen receptor. The neurofilaments elements (+) only were observed in the vascular tunic. The specific immunohistochemistry describe the presence of the interstitial cells of Cajal in human testicular interstitium, opening a new perspective for the functional interpretation of the testicular cellularity and tubular motility. Possibly associated functionally to peribubulars cells of smooth muscle to regulate the mobility of the seminiferous tubules, whose integration and function would be androgen dependent. The cells that express the c-kit receptor, were found exclusively in the interstitial compartment. This cellular type in addition of the muscular cells of peritubules and the absence of nervous fibers to the interior of the testicle, could be responsible for the regulation of tubular mobility, as it happens in the gastrointestinal apparatus.

We proposed to investigate the role of our newly designed PET imaging probe (FMDHT) in the early detection of ovarian cancer using PET. Our probe is specifically designed to target androgen receptors. Since ovarian cancers also express these receptors, we anticipated that FMDHT may be able to detect these receptors at an early stage when the tumor isles aggressive and possibly managed easier. To accomplish that, we proposed and performed assessment of FMDHT accumulation in ovarian cancer xenografts (OVCAR) using female mice. Although, FMDHT accumulated in these tumors, the receptor concentration was low in these tumors, thus eluding the copious accumulation of this probe. In addition, we assessed the accumulation of FMDHT in female monkeys, especially in the ovaries. Our initial hypothesis was that since the androgen receptor concentration is low in normal ovaries, FMDHT would accumulate sparingly in normal ovaries of non-human primates. We then hypothesize that the androgen receptor concentration would increase (much higher) during the malignancy, we would observe and increased probe signal in presence of ovarian cancer. As expected, PFDHT accumulation in the ovaries was low and could not differentiate normal ovaries from other normal tissues. This was an important and encouraging property of this imaging probe. In near future, we would like to extend this observation to cancer model in large animal species to assess the ability of FMDHT in detecting androgen receptor positive ovarian cancer.

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are responsible for the pre-receptor reduction/oxidation of steroids at the 17-position into active/inactive hormones, and the 15 known enzymes vary in their substrate specificity, localisation, and directional activity. 17beta-HSD Type 3 (17beta-HSD3) has been seen to be over-expressed in prostate cancer, and catalyses the reduction of androstenedione (Adione) to testosterone (T), which stimulates prostate tumour growth. Specific inhibitors of 17beta-HSD3 may have a role in the treatment of hormone-dependent prostate cancer and benign prostate hyperplasia, and also have potential as male anti-fertility agents. A 293-EBNA-based cell line with stable expression of transfected human 17beta-HSD3 was created and used to develop a whole cell radiometric TLC-based assay to assess the 17beta-HSD3 inhibitory potency of a series of compounds. STX2171 and STX2624 (IC(50) values in the 200-450nM range) were two of several active inhibitors identified. In similar TLC-based assays these compounds were found to be inactive against 17beta-HSD1 and 17beta-HSD2, indicating selectivity. A novel proof of concept model was developed to study the efficacy of the compounds in vitro using the androgen receptor positive hormone-dependent prostate cancer cell line, LNCaPwt, and its derivative, LNCaP[17beta-HSD3], transfected and selected for stable expression of 17beta-HSD3. The proliferation of the parental cell line was most efficiently stimulated by 5alpha-dihydrotestosterone (DHT), but the LNCaP[17beta-HSD3] cells were equally stimulated by Adione, indicating that 17beta-HSD3 efficiently converts Adione to T in this model. Adione-stimulated proliferation of LNCaP[17beta-HSD3] cells was inhibited in the presence of either STX2171 or STX2624. The compounds alone neither stimulated proliferation of the cells nor caused significant cell death, indicating that they are non-androgenic with low cytotoxicity. STX2171 inhibited Adione-stimulated growth of xenografts established from LNCaPwt cells in castrated mice in vivo. In conclusion, a primary screening assay and proof of concept model have been developed to study the efficacy of 17beta-HSD3 inhibitory compounds, which may have a role in the treatment of hormone-dependent cancer. Active compounds are selective for 17beta-HSD3 over 17beta-HSD1 and 17beta-HSD2, non-androgenic with low toxicity, and efficacious in both an in vitro proof of concept model and in an in vivo tumour model.

The aim of this study was to investigate the expression of p21(waf1/cip1), p27(kip1), p63 and androgen receptor proteins in relation to serum prostate specific antigen levels in low and high Gleason score prostate cancers. Biopsies of patients suffering from prostate adenocarcinoma of low (3 + 3 to 3 + 4) and high (5 + 4 to 5 + 5) Gleason scores (13 cases each group) were immunostained for positive regulators of cell cycle control (p21(waf1/cip1) and p27(kip1)), and essential markers of normal prostate gland ontogeny (p63) and growth (androgen receptor) to find differentially expressed markers of malignant progression. Serum prostate specific antigen levels were also monitored at the time of biopsy and following anti-androgen therapy. All cases except one in each group were androgen receptor positive. P63 and p21(waf1/cip1) proteins detected in normal basal cell nuclei were lost in all but one studied tumors respectively. P27(kip1) protein, however, was detected in all low Gleason score prostate cancers, but it was found in only 7/13 high score cases. Prostate specific antigen levels, either pre- or post-treatment, did not show strict correlation with the p27(kip1) results. The low to high grade dedifferentiation of prostate adenocarcinoma is accompanied with the down-regulation of p27(kip1) protein, which may be an important molecular sign of the lost cell cycle control.

Aberrant regulation in the adhesive ability of cancer cells is closely associated with their metastatic activity. In this study, we examine the role of ErbB-2 in regulating the adhesive ability of androgen receptor (AR)-positive human prostate cancer (PCa) cells, the major cell population of PCa. Utilizing different LNCaP and MDA PCa2b cells as model systems, we found that ErbB-2 activity was correlated with PYK2 activity and adhesive ability in those cells. Increased ErbB-2 expression or activity in LNCaP C-33 cells enhanced PYK2 activation and cell adhesion, while the high PYK2 activity and the rapid adhesion of LNCaP C-81 cells were decreased by diminishing ErbB-2 expression or activity. Knockdown studies revealed the predominant role of ErbB-2 in regulating LNCaP C-81 cell adhesion. Coimmunoprecipitation showed that C-81 cells had increased interaction between ErbB-2 and PYK2. Elevated ErbB-2 activity in LNCaP cells correlated with increased ERK/MAPK activity and enhanced adhesive ability, which were abolished by the expression of K457A-PYK2 mutant or the treatment of PD98059, a MEK inhibitor. In summary, our data suggested that ErbB-2, via PYK2-ERK/MAPK, upregulates the adhesive ability of AR-positive human PCa cells.

Prostate tumors are initially dependent on androgens for growth, but the majority of patients treated with anti-androgen therapy progress to androgen-independence characterized by resistance to such treatment. This study investigates a novel role for filamin A (FlnA), a 280 kDa cytoskeletal protein (consisting of an actin-binding domain (ABD) followed by 24 sequential repeats), in androgen-independent (AI) growth. Full-length FlnA is cleaved to 170 kDa (ABD+FlnA1-15) and 110 kDa fragments (FlnA16-24); the latter is further cleaved to a 90 kDa fragment (repeats 16-23) capable of nuclear translocation and androgen receptor (AR) binding. Here, we demonstrate that in androgen-dependent LNCaP prostate cancer cells, the cleaved 90 kDa fragment is localized to the nucleus, whereas in its AI subline C4-2, FlnA failed to cleave and remained cytoplasmic. Transfection of FlnA16-24 cDNA in C4-2 cells restored expression and nuclear localization of 90 kDa FlnA. Unlike LNCaP, C4-2 cells proliferate in androgen-reduced medium and in the presence of the AR-antagonist Casodex. They also exhibit increased Akt phosphorylation compared to LNCaP, which may contribute to their AI phenotype. Nuclear expression of 90 kDa FlnA in C4-2 cells decreased Akt phosphorylation, prevented proliferation in androgen-reduced medium and restored Casodex sensitivity. This effect was inhibited by constitutive activation of Akt indicating that FlnA restored Casodex sensitivity in C4-2 cells by decreasing Akt phosphorylation. In addition, FlnA-specific siRNA which depleted FlnA levels, but not control siRNA, induced resistance to Casodex in LNCaP cells. Our results demonstrate that expression of nuclear FlnA is necessary for androgen dependence in these cells.

Although androgens and the androgen receptor (AR) are involved in tumorigenesis of prostate cancer (PC) in initial phases, less clear is the role played in advanced androgen-independent (AI) stages of the disease. Several recent reports indicated that re-expression of AR in PC-derived cell lines determines a less aggressive phenotype of the cells. We have previously demonstrated that re-expression of AR decreases the invasion ability of PC3 cells in vitro by affecting signalling and internalization processes of epidermal growth factor receptor (EGFR). Here, we show that reduced EGFR internalization is also a characteristic of AR positive PC cell lines LNCaP and 22Rv1. Reduced internalization in PC3-AR cells is associated to a defective interaction between the EGFR and two adaptor proteins which mediate the endocytotic process, Grb2 and c-Cbl. As a consequence of such reduced interaction, ubiquitination of the receptor, which is mainly mediated by c-Cbl, is also altered. In addition, we show that internalized EGFR co-localizes with early endosome antigen-1, a marker of clathrin-mediated endocytosis, in PC3-Neo cells but not in AR positive cell lines. Conversely, EGFR maintains co-localization with caveolin-1 after EGF stimulation in PC3-AR cells. These data suggest that expression of AR affects clathrin-mediated endocytosis pathway of EGFR, which, according to recent findings, plays an essential role in the completeness of signalling of the receptor. Taken together, these data emphasize the role of AR in the regulation of EGFR endocytotic trafficking and active signalling in PC cells. In view of the role of EGFR signalling in invasion of carcinoma cells, our data may explain the lower invasive phenotype observed in AR-positive cell lines.

INTRODUCTION AND OBJECTIVES: The Wnt signaling pathway (canonical and non-canonical) regulates crucial aspects of embryonic development. Previously, we have reported that non-canonical Wnt members promote proliferation of cancer cells by increasing secretion of growth factors and expression of cancer related genes. We have also demonstrated that Wnt5a is predominantly expressed in prostatic stromal cells and plays a key role in stromalepithelial communication and in the development of prostate cancer. The purpose of this study is to reveal the relation between the noncanonical Wnt signaling and AR signaling on prostate. METHODS: Prostate tissue samples were obtained from patients undergoing radical prostatectomy or cystectomy for prostate and bladder cancer respectively. Cells, representing the cancer, stromal from around the cancer, as well as epithelium and stromal from pathologically normal tissues were isolated by laser capture microdissection Stromal cells (WPMY1 and PrSC) were cultured with DHT ligand and protein. The mRNA levels of Wnt5a, AR, and PSA were determined in both sample types. Wnt5a-knockdown in WPMY1 cells (shWnt5a) along with a vector control (shCTL) was performed and effects on growth evaluated by MTT assay. The cells were co-cultured with PC3 cells, and the mRNA expression of AR was evaluated. A DNA fragment upstream of the Wnt5a gene was inserted into pGL4.17 a vector and the luciferase activity representing Wnt5a inductionby DHT was measured. RESULTS: Wnt5a mRNA expression was prediminanly in the stromal cells of the human prostate samples and the expression levels observed were in proportion to PSA mRNA expression levels. DHT increased production of AR as well as PSA and Wnt5a mRNA expression in both WPMY1 and PrSC cells. Wnt5 knockdown decreased the proliferation of WPMY1cells by 50% compared to shCTR. Co-culture of PC3 cells and WPMY1shWnt5a formed fewer and smaller sized colonies than co-culture of PC3 cells and WPMY1shCTR. AR and PSA mRNA expression levels were remarkably lower in WPMY1shWnt5a than in the control. DHT ligand dramatically increased the luciferase activity of Wnt5a promoter in WPMY1 cells. CONCLUSIONS: The AR is expressed not only in prostatic epithelium but also prostatic stromal as measured by their response to DHT. In stromal cells, The studies presented here demonstrated an interplay between AR signaling and Wnt5a that appears to modulate the proliferation of prostate cancer cells providing further support for the importance of Wnt5a in the regulation of prostate cancer.

Background: Steroid hormone receptors such as glucocorticoid receptors, androgen receptors, and oestrogen receptors α (ERα) and β (ERβ) have been identified in synovial cells of patients with rheumatoid arthritis and osteoarthritis. Objectives: To find a quantitative relationship between the number of receptor positive cells and markers of inflammation, and to compare the two groups of patients with rheumatoid arthritis and osteoarthritis. Methods: A total of 36 patients with rheumatoid arthritis (n = 17) and osteoarthritis (n = 19) were included, and receptor positive cells and cellular markers of synovial inflammation were quantified by immunohistochemistry and ELISA (interleukin 6 (IL6) and IL8). Results: Patients with rheumatoid arthritis showed a higher degree of histologically determined inflammation compared with those with osteoarthritis. However, synovial density of gluco-corticoid receptor positive (GR+), androgen receptor positive (AR+), ERα+ and ERβ+ cells were not different among patients with rheumatoid arthritis and osteoarthritis. In patients with osteoarthritis, the density of GR+ cells positively correlated with the density of AR+, ERα+ and ERβ+ cells (p = 0.007), which was not observed in patients with rheumatoid arthritis. This indicates positively coupled steroid hormone receptor expression in patients with osteoarthritis but not in those with rheumatoid arthritis. In patients with rheumatoid arthritis, secretion of synovial IL6 and IL8 positively correlated with the density of ERα+ and ERβ+ cells (not with gluco-corticoid receptor and androgen receptor), which was not found in the synovium of patients with osteoarthritis. This indicates that inflammatory factors might up regulate the expression of oestrogen receptors in patients with rheumatoid arthritis, or vice versa. Conclusions: In patients with osteoarthritis, expression of different steroid receptors is positively coupled, which was not observed in the synovium of patients with rheumatoid arthritis. This uncoupling phenomenon in rheumatoid arthritis might lead to an imbalance of the normal synovial homeostasis.

The molecular and cellular origins of prostate cancer are poorly understood. Recent evidence from our laboratory suggests that prostate cancer may arise from a basal/luminal precursor cell marked by cell surface expression of PSCA. The evidence supporting this hypothesis is that (I) PSCA marks an intermediate cell population that coexpresses basal and luminal cell cytokeratins (2) this cell population is does not express p63 and is androgen receptor positive, all hallmarks of prostate cancer, and (3) PSCA is highly expressed in HGPIN and prostate cancer and in all animal models of prostate cancer. To test this hypothesis and to develop new models of prostate, we propose to determine whether delivery of oncogenes specifically to the PSCA positive cells of mouse prostate is sufficient to cause cancer. To accomplish this, we will develop a transgenic mouse model in which the retroviral receptor gene tva is expressed in the prostate under control of the PSCA promoter. Virus containing one or more oncogenes will be delivered to the prostate and the resulting phenotype characterized.

The degree of mineralization in human thyroid cartilage is gender specific. Until now, laryngeal tissue was tested for sexual hormone receptors by the use of radiolabelled hormones only without exact localization of the receptors. In this study immediately frozen cartilage specimens from seven male and one female patient who underwent laryngectomy were used for immunolocalization of sexual hormone receptors. Additionally, serum sexual hormone levels were measured by means of radioimmunoassay. Alkaline phosphatase was localized enzymohistochemically in another cohort of six male and four female cartilage specimens from laryngectomies and autopsies. Chondrocytes in thyroid cartilage from both sexes reacted with antibodies to the androgen receptor. The low serum testosterone levels, which varied between 1.5 and 3.9 ng/ml, did not correlate with insufficient mineralization of thyroid cartilage in men (r=0.363, P=0.432). Chondrocytes did not react with antibodies to the estrogen receptor alpha and the progesterone receptor in both sexes. Expression of alkaline phosphatase started about the middle of the second decade. Some chondrocytes near the mineralization front were positive for androgen receptor and alkaline phosphatase, other chondrocytes were negative for both. Our results suggest the involvement of androgen receptor positive chondrocytes in thyroid cartilage mineralization, probably by a testosterone-linked stimulation of alkaline phosphatase.

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)–positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A–induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A–induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A–induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21. (Mol Cancer Res 2006;4(2):113–23)

Little is known of the underlying biology of estrogen receptor-negative, progesterone receptor-negative (ER(-)/PR(-)) breast cancer (BC), and few targeted therapies are available. Clinical heterogeneity of ER(-)/PR(-) tumors suggests that molecular subsets exist. We performed genome-wide expression analysis of 99 primary BC samples and eight BC cell lines in an effort to reveal distinct subsets, provide insight into their biology and potentially identify new therapeutic targets. We identified a subset of ER(-)/PR(-) tumors with paradoxical expression of genes known to be either direct targets of ER, responsive to estrogen, or typically expressed in ER(+) BC. Differentially expressed genes included SPDEF, FOXA1, XBP1, CYB5, TFF3, NAT1, APOD, ALCAM and AR (P

Since the human appendix testis is a Mullerian duct derivative it has been theorised that rising oestrogen levels in puberty result in enlargement of this organ and consequent predisposition to torsion. This study sought to establish the presence or absence of both androgen and oestrogen receptors in the human appendix testis. Bilateral appendix testis specimens surgically excised from ten patients undergoing scrotal exploration for acute scrotum were stained immunohistochemically for androgen and oestrogen receptors. These were examined by light microscopy. The human appendix testis was found to express both oestrogen and androgen receptors. Marked regional variation of androgen and oestrogen receptor positivity was demonstrated. The surface epithelium and some stromal cells were androgen receptor positive, whereas oestrogen receptors were confined to ductular invaginations, gland-like structures and some stromal cells. Rising levels of androgens and oestrogens in pubertal boys may account for enlargement and the predisposition of the human appendix testis for torsion since this structure contains receptors for both.

568 Background: The androgen receptor (AR) is the most highly expressed steroid receptor in breast cancer (BC) with 75-95% of estrogen receptor positive (ER+) and 50% of ER negative BCs expressing ...

e17055 Background: AR+ RMSDC can be treated with an approach including anti-androgen drugs. In case of progression a 2nd line with palliative chemotherapy can be proposed. We report 3 cases of a 2n...

TPS1144 Background: Androgen receptor (AR) expression is observed in 10-30% of patients (pts) with triple-negative breast cancer (TNBC). Experiments using AR+ TNBC cell lines have demonstrated enhanced growth in response to androgen stimulation that is inhibited by enzalutamide (ENZA), a potent oral inhibitor of AR signaling (Richer JK. Abstract presented at the AACR Advances in Breast Cancer Research Meeting, San Diego, CA, 2013 October 3-6 and D'Amato NC. Abstract presented at the SABCS, San Antonio, TX, December 4-8, 2012). AR+ TNBC may represent a subtype driven by AR signaling and which may respond to ENZA. Methods: Women with advanced AR+ TNBC (ER and PgR

Background: Adenovirus-mediated overexpression of the apoptosis-inducing protein Bax can induce apoptosis in prostate cancer cell lines. Constitutive overexpression of Bax could result in unwanted apoptosis in every site of accidental Bax accumulation in vivo. Therefore, we developed an adenoviral construct (Av-ARR 2 PB-Bax) in which the probasin promoter, modified to contain two androgen response elements, drives Bax expression. This promoter would be expected to limit expression of Bax to cells expressing the androgen receptor. Methods: A variety of androgen receptor (AR)-positive and -negative cell lines of prostatic or nonprostatic origin were infected with Av-ARR 2 PB-Bax or a control virus, Av-ARR 2 PB-CAT, in which the same promoter drives expression of the chloramphenicol acetyl transferase-reporter gene. Bax expression and apoptosis in vitro were assessed by western blot analysis. Tumor size and apoptosis in vivo were assessed after four weekly injections of Av-ARR 2 PB-Bax or Av-ARR 2 PB-CAT into subcutaneous LNCaP xenografts growing in uncastrated male mice. All statistical tests were two-sided. Results: Bax was overexpressed in an androgen-dependent way in AR-positive cell lines of prostatic origin but not in AR-positive cells of nonprostatic origin or in AR-negative cell lines of either prostatic or nonprostatic origin. The androgen dihydrotestosterone activated apoptosis in LNCaP cells infected with Av-ARR 2 PB-Bax but not in those infected with Av-ARR 2 PB-CAT. Av-ARR 2 PB-Bax-injected LNCaP xenograft tumors decreased in tumor size from 34.1 mm 3 (95% confidence interval [CI] = 25.1 mm 3 to 43.1 mm 3 ) to 24.6 mm 3 (95% CI = -2.5 mm 3 to 51.7 mm 3 ), but the difference was not statistically significant (P = .5). Tumors injected with Av-ARR 2 PB-CAT increased in size, from 28.9 mm 3 (95% CI = 12.7 mm 3 to 45.1 mm 3 ) to 206 mm 3 (95% CI = 122 mm 3 to 290 mm 3 ) (P = .002) and contained statistically significant more apoptotic cells (23.3% [95% CI = 21.1% to 25.6%] versus 9.5% [95% CI = 8.0% to 11.1]) (P

Background We demonstrated that p53-deficiency is sufficient for the establishment of clonal cell lines from the uterus and prostate. In the present study, we improved cloning methods to establish androgen-responsive cell lines. Methods In our previous study, a prostatic cell line was established from the ventral prostate of a p53-deficient mouse and was maintained in a medium containing heat-inactivated fetal calf serum at 10% supplemented with insulin (10 μg/ml), transferrin (10 μg/ml), cholera toxin (10 ng/ml) and selenium (10−8 M). In the present study, 5α-dihydrotestosterone (10−8 M) was added to the medium from the beginning of cloning procedures. Results We succeeded in the establishment of an androgen receptor positive prostatic cell line, designated PEA5. PEA5 cells exhibited a typical epithelial morphology in culture and growth was stimulated by androgens in a dose-dependent manner. In addition, they grew and formed three-dimensional structures in collagen gel, in which growth was also stimulated by androgen. Conclusions Although PEA5 lacks p53 gene, it still retains androgen sensitivity. In collagen gel culture, PEA5 cells can grow and form three-dimensional structures similar to those of the primary cultures reported previously. Furthermore, prostates of p53-deficient mice are shown to be useful sources for obtaining androgen-responsive cells lines. Prostate 46:214–225, 2001. © 2001 Wiley-Liss, Inc.