DHT Stimulation Research Articles

P10.17.B Androgen receptor signalling has independent prognostic value and is associated with resistance to temozolomide and tumour growth in glioblastoma

Abstract Background The spectrum of sex differences in glioblastoma (GBM) is a rising topic. GBM is more frequent (sex ratio M:F=1.6) and seem to have worse prognosis in males (M) compared to females. Androgen receptor (AR) is expressed in GBM but its role in GBM biology and clinics is not yet fully understood. Material and Methods We have selected from our tumour tissue bank: (i) GBM samples with clinical, pathological and molecular annotations and (ii) GBM-patient derived cell lines -GBM-PDCL- with transcriptomic profiling and available tissue from parental tumours.AR expression was investigated using multiplex IHC Opal stained-FFPE sections. Sensitivity of PDCL to hormonotherapy (dihydrotestosterone or enzalutamide EZT) alone or combined with temozolomide (TMZ) was tested using WST1 viability assay. Response to drugs were correlated to AR expression (high: log2 [FPKM] >2 or low: log2 [FPKM] <2). PDCLs dihydrotestosterone secretion was examined by ELISA in both supernatants and cell lysates. Results In our cohort of 1188 IDH muted GBM patients, the median follow-up was 27.7 (25.8-31.3). The sex ratio M:F was 1.68 and the median age at diagnosis was 57 years [18-69]. Compared to males, females have better prognosis with a median overall survival of 26.1 months vs 20.8 months (p<0.0001). In multivariate analysis, male gender was an independent prognostic factor with a HR of 1.43 CI95%(1.21-1.69) (p<0.0001) adjusted on age, MGMT status and Karnofsky indice . Although interaction between age and sex was not significant (p = 0.854), HR of gender varied through age category 1.53 CI95%( 1.01-2.33 ), 1.51 CI95%( 0.888-2.55 ) and 1.38 CI95%( 0.857-2.21) for males <45 years, between 46-55 years and > 55 years respectively. In GBM tumours, AR staining was positive in 5- 30% tumour cells. After 72h exposure to 10nM dihydrotestosterone, ARhigh GBM-PDCLs exhibit AR nuclear translocation and c-myc expression upregulation, suggesting functional activation of the receptor. AR pharmacological blockade increased temozolomide sensitivity in ARhigh GBM-PDCLs regardless of the patient’s sex. Moreover, ARhigh PDCLs secreted DHT, suggesting autocrine and paracrine AR stimulation in tumour microenvironment. Conclusion Our study highlights the independent prognostic significance of sex in GBM patients, where males exhibit a worse prognosis. In GBM-PDCL, we show that androgen receptor is (i) functional in vitro and (ii) associated with resistance to temozolomide. Further studies are needed to investigate the role of AR in tumour tissue microenvironment.

Abstract 2650: Targeting the DNA-binding domain of the androgen receptor in castration-resistant prostate cancer

Abstract Background: The androgen receptor (AR), which plays a major role in prostate cancer (PCa), consists of a C-terminal ligand-binding domain (LBD), a hinge domain, a DNA-binding domain (DBD), and an N-terminal domain (NTD). Current AR antagonists and AR signaling inhibitors (ASIs), used in androgen deprivation therapy (ADT) and to prevent disease progression, often fail due to production of alternatively spliced AR variants (AR-Vs) that lack the LBD but retain the DBD, which is essential to AR transcriptional activity. This project examines a series of novel compounds designed to inhibit AR-DNA interaction and study how these compounds affect castration-resistant PCa (CRPC). Methods: Compounds potentially targeting the DBD were used to predict direction and position of compounds when docked to the AR. MTT assays, flow cytometry, immunoblots, qRT-PCR, luciferase assay, and immunofluorescence were used to test compounds on AR and PCa. Drug Affinity Responsive Target Stability (DARTS) tested potential binding of the compounds to the AR. Studies in intact mice were used to determine toxicity of the compounds and the maximum tolerated dose. Results: Of 48 compounds synthesized, C15 had the highest and most consistent efficacy overall. C15 decreased metabolic activity in 22Rv1 and CWR-R1 cells that express AR-Vs and in C4 and C4-2 that do not express AR-Vs. Further, C15 had a reduced effect in AR-null PC3 cells and minimal effect in normal human dermal fibroblast (NHDF) cells. In C4-2 and 22Rv1, C15 significantly reduced PSA mRNA and AR transcriptional activity, and in C4-2, C15 significantly reduced TMPRSS2 and Nrdp1 mRNA levels. C08 and C15 reduced pronase-induced AR proteolysis only when the AR DBD is present. Furthermore, C15 reduces AR binding at the PSA enhancer, but DHT stimulation may weaken this binding inhibition and reduction in AR transcriptional activity. However, combining C15 with an AR inhibitor targeting the LBD (enzalutamide, darolutamide, apalutamide) restores the full inhibitory potential and even reduces metabolic activity and AR transcriptional activity further than either drug alone in both C4-2 and 22Rv1s. No toxicity in intact mice was observed with C15 with the highest dose tested being 50 mg/kg. Conclusion: Our preliminary data demonstrate that C15 directly binds to the DBD of the AR and potently inhibits CRPC cells, including those that express AR-Vs missing the LBD. Although increased DHT levels may weaken C15’s inhibition of the AR DBD, addition of a LBD-targeting AR inhibitor improves the inhibitory potential of both drugs. Based on these results, we conclude that C15 may hold great clinical potential as it successfully inhibits CRPC by targeting the AR DBD with minimal toxicity, and it can improve the effects of standard-of-care LBD-targeting AR inhibitors by reducing AR-V-induced resistance. Citation Format: Thomas M. Steele, Elisabeth A. Messner, Maria Malvina Tsamouri, Salma Siddiqui, Brian Bennion, Ruiwu Liu, Paramita Ghosh. Targeting the DNA-binding domain of the androgen receptor in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2650.

Dihydrotestosterone activates AP-1 in LNCaP prostate cancer cells.

A cross-talk between androgen/androgen receptor signaling and the AP-1 transcription factor has been proposed. In this study, we asked whether activation of AP-1 modifies androgen-responsive gene transcription, and whether androgens effect AP-1-regulated gene transcription. We show that activation of AP-1 via expression of a constitutively active mutant of mitogen-activated/extracellular signal responsive kinase kinase (MEK) kinase-1 did not increase the activity of the androgen-responsive probasin promoter. Likewise, expression of a constitutively active mutant of the transcription factor c-Jun, which is a major constitutent of AP-1, did not increase the activity of the probasin promoter. In contrast, 5α-dihydrotestosterone (DHT) activated both the probasin promoter and the AP-1-regulated collagenase promoter in LNCaP prostate cancer cells. The AP-1 binding site within the collagenase promoter was identified as DHT-responsive element. In line with this, DHT increased the activities of the c-Jun promoter and the tumor necrosis factor alpha promoter, which both contain AP-1 binding sites. The signal transduction pathway coupling DHT stimulation with AP-1 activation required c-Jun, MAP kinases and androgen receptors, but was independent of transient receptor potential melastatin-8 (TRPM8) channels, proposed to function as ionotropic testosterone receptors. Expression of the GTPase activating protein RGS2 attenuated DHT-induced activation of AP-1, indicating that the DHT-induced signaling cascade involves G proteins.

DHX15 is up-regulated in castration-resistant prostate cancer and required for androgen receptor sensitivity to low DHT concentrations.

DHX15 is a member of the DEAH-box (DHX) RNA helicase family. Our previous study identified it as an AR coactivator which contributes to prostate cancer progression. We investigated DHX15 expression in castration resistant prostate cancer specimens and the influence of DHX15 on the responsiveness of prostate cancer cells to DHT stimulation. We also explored the role DHX15 played in enzalutamide resistance and the interacting domain in DHX15 with AR. DHX15 expression level in human CRPC specimens and prostate cancer specimens was detected by tissue microarray (TMA) immunostaining analysis. Colony formation assay was performed to determine the proliferation of cells treated with enzalutamide or DHT. siRNAs were used to knockdown DHX15. The interactions between DHX15 and AR were detected using co-immunoprecipitation assay. The expression level of DHX15 was upregulated in human CRPC specimens compared with hormone naïve prostate cancer specimens. DHX15 knockdown reduced AR sensitivity to low DHT concentrations in C4-2 cells. Inactivation of DHX15 sensitizes the enzalutamide treatment in C4-2 cells. Deletion mutagenesis indicated that DHX1 5 interacts with AR through its N terminal domain. These findings suggest that DHX15 contributes to prostate cancer progression. DHX15 is required for androgen receptor sensitivity to low DHT concentrations and contributes to enzalutamide resistance in C4-2 cells. Targeting DHX15 may improve the ADT treatment.

SNORA42 enhances prostate cancer cell viability, migration and EMT and is correlated with prostate cancer poor prognosis.

Prostate cancer (PCa) is one of the most common invasive cancers and the second leading cause of cancer-related death in male worldwide, reflecting the needs of diagnostic and prognostic biomarkers for PCa. Emerging evidence has revealed small nucleolar RNAs (snoRNAs) playing a significant role in tumorigenesis and cancer progression. However, there are few reports about snoRNAs in PCa. Here, we found SNORA42 rather than its host gene (KIAA0907) was up-regulated in PCa cell lines. Meanwhile, an obvious up-regulation of SNORA42 was observed in cancer tissues compared to their adjacent normal tissues. SNORA42 could be induced by DHT stimulation. Over-expression of SNORA42 increased prostate cancer cell proliferation and inhibited apoptosis. Importantly, SNORA42 increased prostate cancer cell migration and invasion. Higher SNORA42 expression level was found to be correlated with shorter survival in metastatic PCa tissues by Kaplan-Meier survival analysis, but this effect was not found in primary PCa tissues. In conclusion, over-expression of SNORA42 could have an oncogenic effect on the progression of PCa. SNORA42 might serve as a prognostic biomarker in PCa.

Abstract 2476: Discovery of an androgen-responsive long noncoding RNA that associates with progression of ERG-overexpressing prostate cancers

Abstract Androgen receptor (AR) signaling pathway has an important role in the growth and development of normal prostate, but also in tumorigenesis and progression of prostate cancer (PC). Although the mechanisms of AR signaling have been widely studied and utilized for treatment in advanced PC, the exact role of AR in development of primary PC is unclear. Former studies have found that AR cistrome is reprogrammed during tumorigenesis to bind novel genomic loci by master regulators, including the ETS family transcription factor ERG. While many AR-induced target genes are known, the effect of AR signaling on regulation of long noncoding RNAs (lncRNAs) is poorly understood, especially in the context of PC progression. Previously, we discovered multiple novel PC-associated transcripts (PCATs) to be aberrantly expressed in PC. Here, we evaluated the expression of 39 Tampere PCATs (TPCATs) in 87 radical prostatectomy specimens using high-throughput real-time PCR, and studied their association with time to PSA progression after prostatectomy. Six TPCATs were significantly associated with time to PSA progression, and four of them also associated with extracapsular extension. In addition, we assessed the expression of TPCATs in the TCGA prostate adenocarcinoma cohort, and found many to be correlated with ERG expression. Moreover, publicly available AR ChIP-seq data from PC tumors indicated that several ERG-associated TPCATs had AR-binding sites on their promoters, some of which overlapped with ERG binding sites. Most notably, we found one progression-associated TPCAT that was regulated by AR in an androgen-sensitive manner according to AR siRNA knockdown and DHT stimulation experiments in vitro. The same TPCAT was also highly associated with overexpression of ERG, and further validated to be a highly PC-specific lncRNA that was abundantly expressed in primary PCs. Taken together, these findings give more insight into the role of AR cistrome in the regulation of lncRNAs in primary PC, and introduce a potential novel prognostic marker to be used in early detection of aggressive PC. Citation Format: Annika Kohvakka, Anastasia Shcherban, Kati K. Kivinummi, Matti Annala, Alfonso Urbanucci, Matti Nykter, Tapio Visakorpi. Discovery of an androgen-responsive long noncoding RNA that associates with progression of ERG-overexpressing prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2476.

Elevated androgen levels induce hyperinsulinemia through increase in Ins1 transcription in pancreatic beta cells in female rats.

Hyperandrogenism is associated with hyperinsulinemia and insulin resistance in adult females. We tested whether androgens dysregulate pancreatic beta cell function to induce hyperinsulinemia through transcriptional regulation of insulin gene (Ins) in the islets. Adult female Wistar rats implanted with dihydrotestosterone (DHT; 7.5-mg, 90-d release) or placebo pellets were examined after 10 weeks. DHT exposure increased plasma DHT levels by 2-fold similar to that in polycystic ovary syndrome in women. DHT exposure induced hyperinsulinemia with increased HOMA-IR index in fasting state and glucose intolerance and exaggerated insulin responses following glucose tolerance test. DHT females had no change in islet number, size and beta cell proliferation/apoptosis but exhibited significant mitochondrial dysfunction (higher ADP/ATP ratio, decreased mtDNA copy number, increased reactive oxygen production and downregulation of mitochondrial biogenesis) and enhanced glucose-stimulated insulin secretion. Ins expression was increased in DHT islets. In vitro incubation of control islets with DHT dose dependently stimulated Ins transcription. Analysis of Ins1 gene revealed a putative androgen responsive element in the promoter. Chromatin-immunoprecipitation assays showed that androgen receptors bind to this element in response to DHT stimulation. Furthermore, reporter assays showed that the promoter element is highly responsive to androgens. Insulin-stimulated glucose uptake in skeletal muscle was decreased with associated decrease in IRβ expression in DHT females. Our studies identified a novel androgen-mediated mechanism for the control of Ins expression via transcriptional regulation providing a molecular mechanism linking elevated androgens and hyperinsulemia. Decreased IRβ expression in the skeletal muscles may contribute, in part, to glucose intolerance in this model.

Relationship between serum response factor and androgen receptor in prostate cancer.

Serum response factor (SRF) is an important transcription factor in castrate-resistant prostate cancer (CRPC). Since CRPC is associated with androgen receptor (AR) hypersensitivity, we investigated the relationship between SRF and AR. Transcriptional activity was assessed by luciferase assay. Cell proliferation was measured by MTT and flow cytometry. Protein expression in patients was assessed by immunohistochemistry. To investigate AR involvement in SRF response to androgen, AR expression was down-regulated using siRNA. This resulted in the abrogation of SRF induction post-DHT. Moreover, DHT stimulation failed to induce SRF transcriptional activity in AR-negative PC346 DCC cells, which was only restored following AR over-expression. Next, SRF expression was down-regulated by siRNA, resulting in AR increased transcriptional activity in castrate-resistant LNCaP Abl cells but not in the parental LNCaP. This negative feedback loop in the resistant cells was confirmed by immunohistochemistry which showed a negative correlation between AR and SRF expression in CRPC bone metastases and a positive correlation in androgen-naïve prostatectomies. Cell proliferation was next assessed following SRF inhibition, demonstrating that SRF inhibition is more effective than AR inhibition in castrate-resistant cells. Our data support SRF as a promising therapeutic target in combination with current treatments.

Androgens enhance the glycolytic metabolism and lactate export in prostate cancer cells by modulating the expression of GLUT1, GLUT3, PFK, LDH and MCT4 genes.

The present study aims to investigate the role of androgens in controlling the glycolytic metabolism and lactate efflux in prostate cancer (PCa) cells. Androgen-responsive LNCaP cells were treated with 5α-dihydrotestosterone (DHT, 10nM) for 12-48h, and their glycolytic metabolism, lactate production and viability were analyzed. Intracellular and extracellular levels of glucose and lactate were determined spectrophotometrically, and the expression of glucose transporters (GLUT1/GLUT3), phosphofructokinase 1, lactate dehydrogenase (LDH) and monocarboxylate transporter (MCT4) was analyzed by real-time PCR and Western blot. The enzymatic activity of LDH was determined by means of a colorimetric assay. Experiments were reproduced in androgen-non-responsive DU145 and PC3 cells. Androgens stimulated glucose consumption in LNCaP cells by increasing the expression of GLUT3, GLUT1 and PFK, which was underpinned by increased cell viability. Accordingly, lactate production by LNCaP cells was enhanced upon DHT stimulation as evidenced by the increased levels of lactate found in cell culture medium. Although LDH enzymatic activity decreased in LNCaP cells treated with DHT, the expression of MCT4 was significantly increased with androgenic treatment, which sustains the increase on lactate export. Glucose consumption and the expression of GLUTs and PFK remained unchanged in DHT-treated DU145 and PC3 cells. The results obtained establish androgens as modulators of glycolytic metabolism in PCa cells by stimulating glucose consumption, as well as the production and export of lactate, which may represent a crucial issue-driven prostate tumor development. These findings also highlight the importance of PCa therapies targeting AR and metabolism-related proteins.

Abstract 4599: Negative feedback loop between serum response factor and androgen receptor in castrate-resistant prostate cancer

Abstract Despite initial response to androgen ablation, patients with advanced disease relapse to develop castrate-resistant prostate cancer (CRPC) which is difficult to treat. We previously identified Serum Response Factor (SRF) as an important transcription factor in an in vitro model of CRPC. Since CRPC is associated with androgen receptor (AR) hypersensitivity and previous studies linked SRF with AR, we investigated the relationship between SRF and AR. DHT stimulation increased SRF transcriptional activity in both parental LNCaP cells and their castration-resistant Abl subline. To investigate AR involvement in this process, AR expression was down-regulated using siRNA, resulting in the abrogation of SRF induction post-DHT. Moreover, DHT stimulation failed to induce SRF transcriptional activity in the AR-negative PC346 DCC cells. However, when AR was over-expressed, SRF induction post-DHT was restored. To explore whether this was reciprocal, SRF was down-regulated by siRNA and AR transcriptional activity and protein expression were assessed. These experiments showed AR increased activity and expression following SRF knock-down, suggesting a negative feedback loop, which was confirmed by immunohistochemistry (IHC) in CRPC bone metastases (n=42), showing a negative association between SRF and AR positivity. This negative feedback was also supported by a computational model based on our experimental data. We next assessed cell proliferation following SRF inhibition and demonstrated that SRF inhibition by siRNA or CCG-1423 was as effective as AR inhibition in decreasing cell proliferation. Moreover concomitant inhibition of AR and SRF showed a synergistic effect. To confirm SRF relevance in clinical samples, we performed IHC staining for SRF on 94 primary tumours and 21 castration-resistant patients who received a transurethral resection of the prostate. This analysis showed that SRF nuclear staining was associated with castration-resistance with 95% of castration-resistant patients showing positive SRF staining versus only 50% of primary tumours. In addition SRF expression in bone metastases in a cohort of 38 patients who died of CRPC, was an independent predictor of survival from (a) prostate cancer diagnosis and (b) diagnosis with CRPC. Overall our data highlight the importance of SRF in CRPC and support SRF as a promising therapeutic target alone or in combination with current treatments. Citation Format: Maria Prencipe, Gillian O'Hurley, Amanda O'Neill, Dara Lundon, Lan K. Nguyen, Susie Boyce, Colm Morrissey, Helmut Klocker, Elaine W. Kay, William Gallagher, William Watson. Negative feedback loop between serum response factor and androgen receptor in castrate-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4599. doi:10.1158/1538-7445.AM2014-4599

Abstract P2-09-05: The potent anti-androgen enzalutamide demonstrates broad anti-tumour activity across all androgen receptor-positive triple-negative breast cancer subtypes

Abstract Background: Up to 30% of triple-negative breast cancers (TNBC) show expression of the androgen receptor (AR) and may therefore be candidates for AR-targeted therapy. Molecular profiling of TNBC has identified the luminal AR (LAR) subset, which shares many aspects of ER-positive luminal cancers but is resistant to ER-targeted therapy. LAR cancers highly express the AR, are heavily enriched in hormone-regulated pathways and demonstrate tumour growth in response to androgen stimulation that can be inhibited by the mixed AR agonist/antagonist bicalutamide. The objective of this study was to investigate the role of AR signalling in TNBC in more detail and to evaluate the potential of the second generation anti-androgen Enzalutamide in this setting relative to bicalutamide. Methods: A panel of 13 TNBC breast cancer cell lines was selected, representing different molecular subtypes of TNBC (including 4 LAR cell lines). Baseline AR expression was determined using qPCR (Taqman) and Western-Blot (Antibody, Santa Cruz Biotechnology). The effects of enzalutamide and bicalumatide on cell proliferation and survival were determined using MTT assays with and without DHT stimulation. Comprehensive molecular profiling (including Illumina Human HT-12 v4 whole genome mRNA and Illumina Human 450K methylation microarrays) was applied before and after enzalutamide treatment to define the molecular signature associated with sensitivity to enzalutamide therapy. All statistical calculations and IC50 doses were calculated using Prism v6.0 (GraphPad). Results: A wide range of AR expression was confirmed across the cell line panel defining 3 groups with complete lack of AR expression, low/moderate AR expressions levels, or very high expression in the LAR subtype (with up to 100-fold greater AR expression compared to other TNBC subtypes). Enzalutamide demonstrated broad anti-tumour activity across all AR+ TNBC subtypes (but not in AR-negative subtypes), with IC50 values being similar in LAR and non-LAR cell lines. Treatment with enzalutamide resulted in more effective growth inhibition and a broader range of activity compared to bicalutamide. Data on molecular profiles of enzalutamide sensitivity will be presented at the meeting. Conclusion: AR inhibition is a promising therapeutic strategy for both LAR and non-LAR AR-expressing TNBC. The potent anti-androgen enzalutamide shows broad activity across all AR+ TNBC subtypes and warrants further clinical evaluation. A phase 2 study of enzalutamide in AR+ TNBC has been initiated. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-05.

Xeno-oestrogens bisphenol a and diethylstilbestrol selectively activating androgen receptor mediated AREs-TATA reporter system

We cloned the three androgen response elements(AREs, including AREI, AREII, and AREIII) with a core transactivation TATA element of the prostate-specific antigen(PSA) promoter into pGL2 basic vector to create an artificial pGL2/AREs-TATA reporter system, which was applied to evaluating the effects of different xeno-oestrogens[bisphenol A(BPA), 4-nonylphenol(4-NP), dichlorodiphenyl trichloroethane(DDT) or diethylstilbestrol (DES)] on androgen receptor(AR) abnormal activation to regulate PSA expression and cell proliferation. In all the three AREs, AREIII-TATA displayed as a major element responsive to AR-mediated DHT stimulation of PSA promoter. Therefore, pGL2/AREIII-TATA reporter was adopted to analyze the activation capacity of AR activated by four different xeno-oestrogens. The activation of pGL2/AREIII-TATA reporter by each xeno-oestrogen was analyzed in two different cell lines, one was HEK293T(Human Embryonic Kidney 293T) cell line, and the other was AR stably expressed DU145 cell line, which was produced by infecting AR with pLenti-puro-AR into the prostate cancer DU145 cells and that were scanned with puromycin and tested by AR antibody. In both the two cell lines, BPA or DES significantly induced AR-mediated transcriptional activity of AREIII-TATA reporter, whereas DDT or 4-nonylphenol did not. Moreover, AR-mediated cell proliferation in response to each of four xeno-oestrogens was measured in MTT assays in both HEK293T cell or AR stably expressed DU145 cell lines. BPA or DES, as an AR inducer, exhibited an enhanced effect in cell proliferation, rather than the effect of DDT or 4-NP, in both cell lines. Finally, we demonstrated that BPA or DES stimulated PSA expression and enhanced the recruitment of AR onto the PSA promoter, resulting in stronger binding to AREIII sites. Taken together, four xeno-oestrogens were identified to have different activities on AR. BPA and DES are demonstrated to be androgenic effectors in the regulation of PSA activation or cell proliferation.

Targeting Androgen Receptor in Estrogen Receptor-Negative Breast Cancer

Endocrine therapies for breast cancer that target the estrogen receptor (ER) are ineffective in the 25%-30% of cases that are ER negative (ER-). Androgen receptor (AR) is expressed in 60%-70% of breast tumors, independent of ER status. How androgens and AR regulate breast cancer growth remains largely unknown. We find that AR is enriched in ER- breast tumors that overexpress HER2. Through analysis of the AR cistrome and androgen-regulated gene expression in ER-/HER2+ breast cancers we find that AR mediates ligand-dependent activation of Wnt and HER2 signaling pathways through direct transcriptional induction of WNT7B and HER3. Specific targeting of AR, Wnt or HER2 signaling impairs androgen-stimulated tumor cell growth suggesting potential therapeutic approaches for ER-/HER2+ breast cancers.

Abstract 2942: The effect of AR expression level on the chromatin binding of the receptor

Abstract Treatment of castration resistant prostate cancer remains to be a challenge. Androgen receptor (AR) is overexpressed and still active at this stage of the disease. Our goal is to identify AR downstream genes and to study the effect of AR overexpression on the chromatin binding. We established, by stable transfection of wtAR into LNCaP cell, two cell lines that over express moderate (LNCaP-ARmo: 2-3 fold) and high (LNCaP-ARhi: 4-5 fold) receptor amounts of protein. We cultured the cell lines in androgen depleted medium for 4 days and subsequently treated them for 2 hours with vehicle or 1nM and 100nM of 5α-dihydrotestosterone (DHT). Subsequently, we used chromosome immunoprecipitation and deep sequencing (ChIP-seq) to identify AR binding. Expression profiling was done by Illumina array. The data analysis is ongoing. We first studied the well known AR target gene PSA. Our data indicated that in depleted medium there is not apparent difference in the loading of the promoter and enhancer of the PSA regulatory region in different cell lines. Instead, the promoter and the enhancer are more loaded in LNCaP-ARmo/−ARhi than in control cell line upon DHT stimulation. Our preliminary data show that there is a positive correlation between AR-levels and whole genome-wide binding upon stimulation with 1 nM DHT. Whereas, in 100 nM DHT, which is a saturating concentration of the ligand, there is a negative correlation. In the 1 nM DHT, AR seems to bind to different genomic loci in LNCaP-ARhi or ARmo than in control cells. In conclusions, the amount of AR has an effect on the chromatin binding of the receptor. Cells overexpressing AR seems to be more androgen-responsive. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2942.

The molecular mechanism of Vav3 oncogene on upregulation of androgen receptor activity in prostate cancer cells

Our previous studies revealed that Vav3 oncogene is overexpressed in human prostate cancer, enhances androgen receptor (AR)-mediated signaling, and may play a role in prostate cancer development and progression. The purpose of this study was to determine the molecular mechanisms responsible for AR activation by Vav3. We found that interaction between N-terminus and C-terminus of AR is essential for its elevated activity stimulated by Vav3. The DH and PH domains of Vav3 are involved in direct interaction with AR. Both cytoplasmic and nuclear levels of AR and Vav3 are elevated and their nuclear localization is further stimulated by DHT in androgen-independent LNCaP-AI cells relative to their parental androgen-dependent LNCaP cells. Vav3 is colocalized with AR, phospho(P)-Akt, and HER2 with a short term stimulation by EGF and DHT. PI3K inhibitor LY294002 blocks colocalization of Vav3 with P-Akt. Consistently, EGF and DHT stimulate Vav3 and AR interaction and enhance PI3K-Akt signaling. Mutation of tyrosines to phenylalanines in the acidic domain or deletion of the SH2 and SH3 domains significantly enhances Vav3 ability for AR activation, while deletion of the DH domain abolishes this activity. Given that an elevated interaction of Vav3 with AR, P-Akt, and HER2 in both cytoplasm and nucleus upon the short-term of DHT stimulation, our data suggest that Vav3 may enhance non-genomic AR activity via PI3K-Akt signaling in addition to AR transcriptional activity and further support a role in androgen-independent growth in prostate cancer.

Nuclear Receptor-Induced Chromosomal Proximity and DNA Breaks Underlie Specific Translocations in Cancer

Chromosomal translocations are a hallmark of leukemia/lymphoma and also appear in solid tumors, but the underlying mechanism remains elusive. By establishing a cellular model that mimics the relative frequency of authentic translocation events without proliferation selection, we report mechanisms of nuclear receptor-dependent tumor translocations. Intronic binding of liganded androgen receptor (AR) first juxtaposes translocation loci by triggering intra- and interchromosomal interactions. AR then promotes site-specific DNA double-stranded breaks (DSBs) at translocation loci by recruiting two types of enzymatic activities induced by genotoxic stress and liganded AR, including activation-induced cytidine deaminase and the LINE-1 repeat-encoded ORF2 endonuclease. These enzymes synergistically generate site-selective DSBs at juxtaposed translocation loci that are ligated by nonhomologous end joining pathway for specific translocations. Our data suggest that the confluence of two parallel pathways initiated by liganded nuclear receptor and genotoxic stress underlies nonrandom tumor translocations, which may function in many types of tumors and pathological processes.

Hair Sciences

1. Nilofer Farjo, MD 1. Manchester, England </br>![][1]</img> In the following article, Dr. Moonkyu Kim and Jung-Chul Kim looked at differences in gene expression between frontal and occipital scalp and beard hair follicles before and after DHT stimulation. We know that DHT has different

Androgens Induce Prostate Cancer Cell Proliferation through Mammalian Target of Rapamycin Activation and Post-transcriptional Increases in Cyclin D Proteins

Androgen receptor (AR) plays a central role in prostate cancer, with most tumors responding to androgen deprivation therapies, but the molecular basis for this androgen dependence has not been determined. Androgen [5alpha-dihydrotestosterone (DHT)] stimulation of LNCaP prostate cancer cells, which have constitutive phosphatidylinositol 3-kinase (PI3K)/Akt pathway activation due to PTEN loss, caused increased expression of cyclin D1, D2, and D3 proteins, retinoblastoma protein hyperphosphorylation, and cell cycle progression. However, cyclin D1 and D2 message levels were unchanged, indicating that the increases in cyclin D proteins were mediated by a post-transcriptional mechanism. This mechanism was identified as mammalian target of rapamycin (mTOR) activation. DHT treatment increased mTOR activity as assessed by phosphorylation of the downstream targets p70 S6 kinase and 4E-BP1, and mTOR inhibition with rapamycin blocked the DHT-stimulated increase in cyclin D proteins. Significantly, DHT stimulation of mTOR was not mediated through activation of the PI3K/Akt or mitogen-activated protein kinase/p90 ribosomal S6 kinase pathways and subsequent tuberous sclerosis complex 2/tuberin inactivation or by suppression of AMP-activated protein kinase. In contrast, mTOR activation by DHT was dependent on AR-stimulated mRNA synthesis. Oligonucleotide microarrays showed that DHT-stimulated rapid increases in multiple genes that regulate nutrient availability, including transporters for amino acids and other organic ions. These results indicate that a critical function of AR in PTEN-deficient prostate cancer cells is to support the pathologic activation of mTOR, possibly by increasing the expression of proteins that enhance nutrient availability and thereby prevent feedback inhibition of mTOR.

Distinct Regulation by Steroids of Messenger RNAs for FSHR and CYP19A1 in Bovine Granulosa Cells

Steroidal regulation of gene expression in follicular cells is not completely defined. Granulosa cells from 5 mm bovine follicles were cultured and treated and steady-state mRNA levels determined for FSHR (follicle-stimulating hormone receptor) and CYP19A1 (aromatase). Cells were treated for 5 days with (0.1-300 ng/ml) 17beta-estradiol (E2), testosterone (T), or 5alpha-dihydrotestosterone (DHT). FSHR mRNA was increased by T and DHT but not E2. In contrast, CYP19A1 mRNA was induced by all doses of E2 but only high doses of T and DHT. Similarly, varying treatment duration (1-5 days) showed that FSHR was increased by T and DHT and CYP19A1 mRNA increased by E2 and T at all times. Synergism between steroid hormones and FSH or forskolin was also evaluated. FSH or E2 did not alter FSHR mRNA and did not enhance DHT stimulation of FSHR mRNA. In contrast, DHT alone had no effect on CYP19A1 mRNA but synergized with FSH plus E2 to increase CYP19A1 mRNA, probably due to induction of FSHR by DHT. Effects of E2 and T on CYP19A1 were blocked by ICI 182,780, indicating mediation by estrogen receptors. However, the specific androgen receptor antagonist bicalutamide did not block E2 or T effects on CYP19A1 but did block T and DHT stimulation of FSHR. Thus, FSHR is specifically regulated through androgen receptor, whereas CYP19A1 is regulated by multiple pathways, including estrogen receptors and cAMP/protein kinase A induced by FSHR activation in granulosa cells. These inter- and intracellular regulatory mechanisms may be critical for normal follicle growth and dominant follicle selection.

Effects of Aroclors and individual PCB congeners on activation of the human androgen receptor in vitro

To investigate possible interactions between the human androgen receptor and PCBs in vitro, we have used a previously characterized human androgen receptor reporter gene assay in which PC-3 LUC AR+ cells respond to 5α-dihydrotestosterone (DHT, 50 pM) with enhanced luciferase activity. The effects of Aroclors, commercial mixtures of PCBs, or polychlorinated terphenyls (PCTs) (0, 0.1, 1.0, and 10.0 μM) on luciferase activity in PC-3 LUC AR+ cells were determined after exposure for 18 h in the presence and absence of DHT (50 pM). In the absence of DHT, none of the Aroclors induced luciferase activity but, in the presence of DHT (50 pM), Aroclors 1016, 1221, 1232, 1242, 1248, 1254, 1260, 5432, and 5442 acted antagonistically at concentrations that did not affect cell viability. Aroclor 5460 was without effect. Similarly, when PCBs found as human milk contaminants were assessed as individual congeners (each at 1 μM, where no cytotoxic effects were observed), none activated luciferase expression in the absence of DHT but PCBs 49, 66, 74, 105, and 118 completely antagonized the stimulation by DHT (50 pM) and PCBs 138, 153, and 156 were less effective antagonists, reducing the DHT stimulation by about 50%. Thus, 30% (by weight) of the PCBs in human milk are androgen antagonists (PCBs 66, 74, 105, and 118) and a further 25% are partial antagonists (PCBs 138, 153, and 156). A proportionally representative mixture of PCBs that contaminate human milk also caused the DHT-mediated activation of luciferase activity in PC-3 LUC AR+ cells to be reduced by more than 50%.