Androgen Receptor Stimulation Research Articles

HSD3B1, prostate cancer mortality and modifiable outcomes.

Androgen receptor stimulation by testosterone and dihydrotestosterone is crucial for prostate cancer progression. Despite the initial effectiveness of androgen deprivation therapy (ADT), castration-resistant prostate cancer eventually develops in most men. A common germline missense-encoding polymorphism in HSD3B1 increases extra-gonadal androgen biosynthesis from adrenal precursors owing to increased availability of the encoded enzyme 3β-hydroxysteroid dehydrogenase 1 (3βHSD1) - hence, it is called the adrenal-permissive enzyme. This mechanism explains the more rapid progression to castration-resistant prostate cancer in men who inherit this allele than in men without it via sustained androgen receptor activation despite ADT. Multiple clinical studies, including data derived from prospective phase III studies, have linked adrenal-permissive allele inheritance to inferior clinical responses to ADT and increased mortality, but reversal is possible with upfront adrenal androgen blockade. The adrenal-permissive allele exhibits divergent frequencies across various groups worldwide, which could contribute to differences in clinical outcomes among these populations. Large-scale data from the Million Veteran Program have shown homozygous HSD3B1 adrenal-permissive allele inheritance to be an independent biomarker of prostate cancer-specific mortality. Together, these observations support the integration of HSD3B1 into germline testing and clinical trials as it might help to identify groups at increased likelihood of benefiting from early, intensified, AR-targeting interventions. Lastly, 3βHSD1 is a promising target for pharmacological inhibition, which enables new strategies for systemic prostate cancer therapy.

7609 Androgen Receptor Positive DCIS in a Transmasculine Individual on Testosterone Therapy

Abstract Disclosure: J. Kelly: None. B. Cohen: None. Introduction: The rate of breast cancer in transgender men is lower than in cisgender women, perhaps due to lower estrogen levels in the setting of suppression from exogenous testosterone, lack of clear guidelines on screening after top surgery, and increase in mastectomies. However, the impact of testosterone therapy on existing breast cancer is not known. Concerns regarding use of testosterone in patients with breast cancer include the potential for conversion of testosterone to estradiol, as well as direct androgen receptor (AR) stimulation. However, for some patients, hormone therapy is lifesaving and stopping gender affirming hormone therapy (GAHT) will negatively impact patient well-being. This creates a challenging clinical scenario when transgender patients on testosterone are diagnosed with breast cancer.Case Presentation: We present a case of a 29-year-old self-identified transmasculine nonbinary individual (assigned female at birth) who underwent chest reconstruction surgery after several months on daily transdermal testosterone therapy. A 4 cm DCIS ER+/AR+ with possible micro invasion was incidentally noted on pathology. They had no family history of breast cancer. The patient underwent chest MRI which was negative for persistent disease. The treating oncologist did not recommend Tamoxifen. The patient chose to discontinue testosterone therapy due to uncertainty regarding future breast cancer risk. However, due to worsening depression and fatigue, the patient chose to resume GAHT after receiving counseling of possible risk, though likely low, of cancer recurrence. Given that they had undergone bilateral mastectomy which, unlike chest reconstruction surgery where some breast tissue is left for contouring, all breast tissue was removed and given that chest MRI was negative for disease, cancer recurrence was felt to be low. Furthermore, though AR positivity was noted, the clinical implications are unclear and it is not known whether testosterone would stimulate tumor growth or potentially have antagonistic effects on ER signaling. Additionally, any stimulatory effects of AR signaling may be balanced by androgen suppression of gonadotroph production and reduction in estrogen production; this is more clearly associated with stimulatory effects on cancer growth. And finally, the potential risks of therapy must be weighed against the benefits provided to the patient’s well-being and mental health. Ultimately, the patient resumed testosterone therapy and has been doing well.Teaching Point: This case illustrates the uncertainties regarding the management of gender affirming testosterone therapy in patients with DCIS or breast cancer. It also highlights the need for a patient centered approach to decisions regarding changes to GAHT, to appropriately weigh the risks and benefits of continuing therapy. Presentation: 6/2/2024

Anabolic-androgenic steroids in sport

Introduction and objective Anabolic-androgenic steroids (AAS) are a group of doping substances. They cause muscle hypertrophy and an increase in strength. Moreover, they may lead to dangerous side effects. Knowledge of the side effects of AAS use is necessary because in clinical practice we often encounter patients who decide to supplement with steroids. The aim of the study is to present information on the epidemiology, mechanism of action, methods of detecting AAS, the impact of the substance on the male and female body, and a description of organ side effects, highlighting: the cardiovascular system, the nervous system and the psyche. Review methods Literature search was conducted in the PubMed database with inclusion criteria ‘free full texts’ and publications date between 2015 – 2024. A total of 7272 results were found; 30 publications were ultimately included in the review. Abbreviated description of the state of knowledge Overusing AAS is very popular among people training at the gym. They have an anabolic effect on muscle tissue. They contribute to infertility in men and masculinization in women. Stimulation of androgen receptors above physiological doses of AAS leads to vascular changes that, in extreme cases, may lead to sudden cardiac death. Moreover, they accelerate brain aging. An increase in aggression and risky sexual behavior has been observed among people using doping. The diagnostic material used in anti-doping tests is urine and blood serum. Summary AAS abuse is a serious public health problem. Men who want to improve their figure and decide to use doping are particularly at risk. Due to the easy availability of steroids, society should be made aware of the negative effects of steroid use.

The influence of ghrelin agonist ipamorelin acetate on the hypothalamic-pituitary-testicular axis in a cichlid fish, Oreochromis mossambicus

Ghrelin, a peptide found in the brain and gut, is predicted to play a significant role in the control of various physiological systems in fish. The objective of this study was to examine the impact of ipamorelin acetate (IPA), a ghrelin agonist, on the reproductive axis of the tilapia Oreochromis mossambicus. The administration of either 5 or 30 µg of IPA for 21 days led to a significant and dose-dependent rise in food intake concomitant with a significant increase in the numbers of primary spermatocytes, secondary spermatocytes, and early spermatids compared to the control group. There was a significant rise in the number of late spermatids, as well as the areas of the lobule and lumen, in fish treated with 30 µg of IPA, compared to the control group. Moreover, there was no significant difference in the percentage of gonadotropin-releasing hormone (GnRH)-immunoreactive fibres in the hypothalamus and anterior pituitary gland across different groups. However, a significant elevation in the expression of androgen receptor protein was observed in fish treated with 30 µg of IPA. Furthermore, the concentrations of luteinizing hormone (LH) and 11-ketotestosterone (11-KT) in the serum of fish treated with either 5 or 30 µg of IPA were significantly elevated in comparison to the control group. Collectively, these findings suggest that the administration of ghrelin enhances the development of germ cells during the meiosis-I phase and that this effect might be mediated via the stimulation of 11-KT and androgen receptors at the testicular level and LH at the pituitary level in the tilapia.

Antifungal activity and hair growth stimulation of purple sweet potato leaf fraction (Ipomoea batatas (L.) Lamk) and its molecular mechanism through androgen receptor inhibition

Alopecia presented a global challenge, spurring the search for new treatments. This study evaluated Ipomoea batatas leaf extracts for their ability to stimulate hair growth and inhibit Malassezia furfur. Secondary metabolites were identified and assessed for their potential to inhibit androgen receptors (AR) via LC-MS/MS and in silico analysis. The hair tonic formulation was optimized using a D-optimal mixture design to improve physicochemical properties. The plant’s extracts and fractions exhibited strong antifungal activity against M. furfur and significant hair growth stimulation compared to minoxidil. In silico analysis identified pyropheophorbide A, methyl-Pyropheophorbide A, hyperoside, and quercetin with superior affinity and stability in interacting with AR. The optimized formulation included 96% ethanol, propylene glycol, and Tween 80 to enhance hair tonic properties. I. batatas leaves showed promising potential in treating alopecia through hair growth stimulation, antifungal activity, and potential inhibition of AR. These findings opened avenues for further research and development in alopecia therapeutics.

Analysis of the responsiveness to antiandrogens in multiple breast cancer cell lines.

Antiandrogens were originally developed as therapeutic agents for prostate cancer but are also expected to be effective for breast cancer. However, the role of androgen signaling in breast cancer has long been controversial due to the limited number of experimental models. Our study aimed to comprehensively investigate the efficacy of antiandrogens on breast cancer. In the present study, a total of 18 breast cancer cell lines were treated with the agonist or antagonists of the androgen receptor (AR). Among the 18 cell lines tested, only T-47D cells proliferated in an androgen-dependent manner, while the other cell lines were almost irresponsive to AR stimulation. On the other hand, treatment with AR antagonists at relatively high doses suppressed the proliferation of not only T-47D cells but also some other cell lines including AR-low/negative cells. In addition, expression of the full-length AR and constitutively active AR splice variants, AR-V7 and ARV567es, was not correlated with sensitivity to AR antagonists. These data suggest that the antiproliferative effect of AR antagonists is AR-independent in some cases. Consistently, proliferation of AR-knockout BT-549 cells was inhibited by AR antagonists. Identification of biomarkers would be necessary to determine which breast cancer patients will benefit from these drugs.

Regulation of androgen receptor stability by the β1 Pix/STUB1 complex.

The androgen receptor (AR) is essential in the development and differentiation of testes and male genitalia. AR expression is tightly regulated at the translational and posttranslational levels. AR posttranscriptional regulation is a major determinant of AR availability since AR is a direct target of E3 ubiquitin ligase STUB1. Our work indicated that the Rac/Cdc42 guanosine triphosphatase guanine nucleotide exchange factor, β1 Pix, enhanced AR levels after AR stimulation in HEK293 and HeLa cells. AR stimulation decreased AR ubiquitination which is accompanied by increased β1 Pix binding to AR. Ectopic expression of β1 Pix decreased AR ubiquitination in Tm4 and HEK293 cells. We demonstrated that the formation of a multimolecular complex comprised of AR/β1 Pix/STUB1 responded in a time-dependent manner to AR stimulation. β1 Pix binding dissociated STUB1 from AR and thus prevented STUB1 from catalyzing receptor ubiquitination. β1 Pix enhanced AR transcriptional activity and increased AR target gene expression. Irrespective of treatment, immunofluorescence analysis showed a strong nuclear colocalization of endogenous AR and endogenous βPix in Tm4 cells. However, using Tm4 cell fractionation, AR stimulation decreased βPix/AR association in the cytosolic fraction and increased binding of AR to βPix in the nuclear fraction. To support the role of β1 Pix in androgen regulation, we found that individuals lacking this gene have a significant increase in genitourinary malformations associated with androgen dysfunction. Our data indicate that β1 Pix is an important modulator of AR stability and ligand-dependent AR transcriptional activity. We propose that β1 Pix could serve as a promising therapeutic target to modulate AR signaling.

Abstract 3080: Characterization of steroid receptor mediated activation of GLI as driving force of breast cancer growth

Abstract Background: Hedgehog (Hh) signaling pathway plays a fundamental role in the early stages of development by regulating morphogenesis. GLI transcription factors are drivers of this signaling pathway by regulating the expression of growth related genes. Hyperactivation of GLI proteins have been associated with several cancers including medulloblastoma, glioblastoma, ovarian, prostate and breast cancers. Our study in breast cancer (BCa) suggests transcriptional activation of GLI3 in estrogen receptor alpha (ERα) positive cells upon estradiol stimulation. Loss of ERα greatly decreases GLI3 protein stability and stimulation with estradiol significantly increases GLI3 stability in ER-positive BCa cells. We have discovered that GLI3 forms nuclear complexes with ERα upon estradiol stimulation and that the loss of GLI3 reduces BCa cells growth. Therefore, we hypothesize that ERα-GLI3 complex orchestrates BCa transcriptome required for cell growth and development. Our study focuses on characterizing ERα mediated activation of GLI3 in BCa cells and the possible compensation by other steroid receptors such as the androgen receptor (AR) and the glucocorticoid receptor (GR) in ER-negative BCa cells. Method: We examined ERα- GLI transcriptional activity by RNA sequencing upon stimulation with estradiol and inhibition of GLI activity by GANT61 in ER-positive MCF7 cells. We also identified ERα domains that are essential for GLI3 binding by immunoprecipitation and proximity ligation assay (PLA). To identify ERα-GLI3 interactome, we have optimized Rapid Immunoprecipitation Mass Spectrometry of Endogenous protein (RIME). We also examined the role of GLI3 in ER-negative BCa cell growth and characterized the role of AR and GR in activating GLI3 by luciferase reporter assay.Results: Inhibition of GLI DNA binding by GANT61 reduced the expression of GLI regulated (CDC20, CDK1, UBE2C) and modified expression of ERα regulated (FOXM1, SPC24, KIF20A, BIRC5) genes, suggesting cooperative role of ERα-GLI3 in mediating growth and metastasis. The optimized RIME assay suggests immunoprecipitation of ERα with GLI3 in BCa cells upon estradiol induction. Immunoprecipitation and PLA studies suggest that ERα-N terminal, DNA binding and C-terminal domains interact with GLI3. We also showed that knockdown of GLI3 reduces growth in ER negative BCa cells and that AR or GR stimulation by dihydrotestosterone and dexamethasone respectively, are important for GLI3 transcriptional activity. Conclusion: Collectively, our results suggest a new role of steroid receptors in regulating GLI oncogenic transcriptional activity in BCa, leading to new therapeutic possibilities for patients. Citation Format: Shabnam Massah, Maria Guo, Jane Foo, Ralph Buttyan, Artem Cherkasov, Nada Lallous. Characterization of steroid receptor mediated activation of GLI as driving force of breast cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3080.

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 2367: Characterizing the reciprocal regulation of an ABI1-dependent androgen receptor axis in prostate cancer

Abstract Background: Prostate cancer (PCa) affects nearly 50% of males over 60. Therapies for advanced PCa primarily target the androgen receptor (AR), a ligand-activated transcription factor, which is a key driver of PCa tumor growth. Our previous research demonstrated that ABI1 acts as a tumor suppressor in PCa. ABI1 is a scaffold protein for the WAVE Regulatory Complex, positively regulates cell adhesion, and inhibits integrin activation through sequestering key kinases. We have observed a physical interaction between AR and ABI1. In this study, we aim to characterize the ABI1-dependent AR regulation in PCa. Method: We generated an ABI1 KO cell line model in LNCaP cells using CRISPR-Cas9. We then used mutagenesis to develop ABI1 binding mutants (W485N and ∆SH3) and generated ABI1 rescue cell lines in ABI1-KO. We performed qPCR, co-immunoprecipitation, subcellular fractionation, western blotting, in vitro assay, live-cell imaging, and PLA. We purified both AR and ABI1 proteins for in vitro analysis, performed turbidity and liquid-liquid droplet formation assays under DIC. Results: In cellulo binding assays indicate interaction of AR-NTD with the ABI1-SH3 domain. Expression of ABI1-W485N in KO cell line showed decreased binding, while ABI1-∆SH3 showed no observable binding to AR in co-IP assays compared to wildtype control. We then performed subcellular fractionation assays and saw decreased AR nuclear localization compared to ABI1-WT control. Decreased nuclear localization in ABI1-W485N was associated with decreased mRNA expression of hallmark AR target genes, KLK3 and FKBP5. In vitro turbidity assays indicated the conditions in which AR and ABI1 have propensity to phase separate. We then performed a liquid-liquid droplet assay. AR and ABI1 formed liquid-liquid droplets individually. We labeled ABI1 with Alexaflour 488 and saw that ABI1 was recruited to AR droplets indicating that AR and ABI1 can co-phase separate. PLA assays in cellulo and in vivo patient tumor samples showed positive interactions of AR-ABI1 in the nucleus. We then used live-cell observation and saw co-localization of biomolecular condensates for AR and ABI1. Conclusions: Our studies demonstrate that ABI1 plays a role in AR transcriptional pathway. Loss of ABI1 resulted in decreased nuclear AR and subsequent decrease in mRNA expression of AR target genes. Due to the intrinsically disordered structure of AR and ABI1, they were both able to phase separate individually as well as together. AR stimulation increased PLA interactions in nucleus of cells. The co-localization of AR and ABI1 in biomolecular condensates was changed upon androgen deprivation conditions. Future studies will investigate if anti-AR treatments could lead to the dysregulation of ABI1 and promote reactivation of AR pathway through ABI1-AR axis. Findings will allow for novel insights into the mechanisms underlying AR and ABI1 in prostate metastatic progression. Citation Format: Baylee A. Porter-Hansen, Alaji Bah, Alfonso Urbanucci, Konsta Kukkonen, Fan Zhang, Sonia Kung, Ladan Fazli, Martin E. Gleave, Gennady Bratslavsky, Leszek Kotula. Characterizing the reciprocal regulation of an ABI1-dependent androgen receptor axis in 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 2367.

DRUG THERAPY FOR ANDROGEN-POSITIVE BREAST CANCER

There are some types of breast cancer (BC) that depend on their molecular characteristics. However, lately researchers provide date regarding the presence of additional receptors of triple negative breast cancer (TNBC). One of them is LA R-subtype that has androgen receptors (AR) on breast cancer cells. The role of AR in the development of this pathology is still controversial. According to some authors, stimulation of AR leads to the proliferation suppression, in the opinion of others – to the activation and potentiation of tumor invasion. There is also a version of the dichotomous effect of androgens that depends on the concentration of this hormone. The various effects of LA R-subtype therapy of BC are also explained by the presence of many other regulator proteins that interact with AR. The first attempts to treat BC with androgen have begun since the 40s of the last century, but in those years it did not have the desired effect and was not used until recently. Currently, the basic mechanisms of the effect of androgens on BC cells on the molecular level are known, and researches try to use androgen therapy, also in combination with aromatase inhibitors to increase the concentration of endogenous testosterone. The effects of selective androgen receptor modulators and anabolic steroids are being studied. However, in the last decade, the largest number of studies focused on the study of anti-androgen therapy. Patients receive AR antagonists, such as enzalutamide, bicalutamide, that are prescribed for prostate cancer. Enzalutamide blocks both androgen- and estrogen-mediated tumor growth, and therefore can be used regardless of the presence of estrogen receptors (ER), in contrast to bicalutamide. The results showed a significant increase in disease free survival up to 16.5 months in patients with hormone positive BC.

Role of SPTSSB-Regulated de Novo Sphingolipid Synthesis in Prostate Cancer Depends on Androgen Receptor Signaling.

SummaryAnti-androgens are a common therapy in prostate cancer (PCa) targeting androgen receptor (AR) signaling. However, these therapies fail due to selection of highly aggressive AR-negative cancer cells that have no therapeutic options available. We demonstrate that elevating endogenous ceramide levels with administration of exogenous ceramide nanoliposomes (CNLs) was efficacious in AR-negative cell lines with limited efficacy in AR-positive cells. This effect is mediated through reduced de novo sphingolipid synthesis in AR-positive cells. We show that anti-androgens elevate de novo generation of sphingolipids via SPTSSB, a rate-limiting mediator of sphingolipid generation. Moreover, pharmacological inhibition of AR increases the efficacy of CNL in AR-positive cells through de novo synthesis, while SPTSSB knockdown limited CNL's efficacy in AR-negative cells. Alluding to clinical relevance, SPTSSB is upregulated in patients with advanced PCa after anti-androgens treatment. These findings emphasize the relevance of AR regulation upon sphingolipid metabolism and the potential of CNL as a PCa therapeutic.

Investigating the Benefit of Combined Androgen Modulation and Hypofractionation in Prostate Cancer.

Hypofractionation is currently considered a valid alternative to conventional radiotherapy for the treatment of patients with organ-confined prostate cancer. Recent data have demonstrated that extreme hypofractionation, which involves the use of a high radiation dose per delivered fraction and concomitant reduction of sessions, is a safe and effective treatment, even though its radiobiological rationale is still lacking. The present work aims to investigate the biological basis sustaining this approach and to evaluate the potential of a hypofractionated regimen in combination with androgen deprivation therapy, one of the major standards of care for prostate cancer. Findings show that androgen receptor (AR) modulation, by use of androgens and antiandrogens, has a significant impact on cell survival, especially in hypoxic conditions (4% O2). Subsequent experiments have revealed that AR activity as a transcription factor is involved in the onset of malignant senescence-associated secretory phenotype (SASP) and activation of DNA repair cascade. In particular, we found that AR stimulation in hypoxic conditions promotes the enhanced transcription of ATM gene, the cornerstone kinase of the DNA damage repair genes. Together, these data provide new potential insights to justify the use of androgen deprivation therapy, in particular with second-generation anti-androgens such as enzalutamide, in combination with radiotherapy.

Identification of retinoid acid induced 16 as a novel androgen receptor target in prostate cancer cells

BackgroundRetinoid acid induced 16 (RAI16) was reported to enhance tumorigenesis in hepatocellular carcinoma (HCC). The androgen receptor (AR) is a nuclear hormone receptor that functions as a critical oncogene in several cancer progressions. However, whether RAI16 is a candidate AR target gene that may involve in prostate cancer progression was unclear. Materials & methodsRAI16 expression was detected in prostate cancer cells with or without the AR agonist R1881 treatment by quantitative RT-PCR and Western blot. Direct AR binding to the RAI16 promoter was tested using AR chromatin immunoprecipitation (ChIP) and luciferase assay. Cell viability and colony formation assays in response to R1881 were analyzed in cells with RAI16 knockdown by specific siRNA. ResultsThe expression of RAI16 was high in LNCaP(AI), LNCaP(AD), C4-2 expressing AR, but low in Du145 and Pc-3 cells without AR expressing. In addition, the expression of RAI16 could be induced by 10 nM R1881 treatment LNCaP(AD) and C4-2 cells, but inhibited by AR specific siRNA treatment. Furthermore, AR binds directly to ARE3 (−2003~-1982bp) of RAI16 promoter region by ChIP and luciferase assay. RAI16 knockdown inhibited the enhancement of cell viability and colony formation of AR stimulation. ConclusionsWe demonstrate for the first time that RAI16 is a direct target gene of AR. RAI16 may involved in cell growth of prostate cancer cells in response to AR signaling.

Abstract 5307: Androgen Receptor promotes localization of E-cadherin to cell-cell junctions through Abi1 signaling

Abstract According to American Cancer Society, prostate cancer is a slow progressing disease that affects nearly 50 percent of males over the age of 60. Every year, 165,000 new cases are diagnosed and 30,000 deaths are reported. Current therapies such as Androgen Depravation Therapy (ADT) and Anti-Androgen treatments such as Enzalutamide increase quality of life and extend survival but do not provide a definitive treatment and are ineffective in highly metastatic prostate cancer. Castrate Resistant Prostate Cancer (CRPC) is an untreatable highly metastatic form of prostate cancer and once diagnosed the survival rate is 1-2 years. Hence, the understanding of molecular mechanisms of CRPC is of critical importance. We previously linked prostate cancer to Abi1 gene. Abl interactor 1 (Abi1) is a scaffolding protein and an integral member of the WAVE Regulatory Complex (WRC). WRC is actin nucleation promoting factor that acts upstream of Arp2/3 to enhance the speed of actin polymerization and is critical for various cellular functions including cell-cell junction formation. Our previous research using a prostate cancer mouse models shows that loss of Abi1 expression leads to down-regulation of cellular adhesion proteins such as E-cadherin. Studies using our CRISPR KO RWPE cell line indicate that loss of Abi1 disrupts cell-cell junction formation via decreased levels of E-cadherin. Analysis of patient data demonstrated loss of Abi1 in a subset of CRPC patients. Castrate resistant tumor growth is androgen independent therefore anti-androgen therapy is ineffective. In search of a link between the androgen signaling and the role of Abi1 in prostate cancer we found that Abi1 gene contains androgen receptor (AR) binding sites. Consistent with the idea that Abi1 is an AR responsive gene, expression levels of Abi1 are increased following AR stimulation with the synthetic androgen, R1881, of the hormone sensitive cell line LNCaP. We also observed increased levels of E-cadherin at the cell-cell junctions. In contrast, cells lacking Abi1 do not form proper cell junctions neither respond to AR stimulation in the same way as wild type cells. We propose that Abi1 is a critical part of androgen signaling by promoting cell-cell junctions and tissue integrity. Supported by NCI R01 CA161018 (LK). Citation Format: Baylee Porter, Disharee Nath, Susan A. Krum, Gennady Bratslavsky, Vladimir A. Kuznetsov, Leszek Kotula. Androgen Receptor promotes localization of E-cadherin to cell-cell junctions through Abi1 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5307.

Androgen receptor promotes oral squamous cell carcinoma cell migration by increasing EGFR phosphorylation.

Objectives: This study is aimed to investigate the role of androgen receptor (AR) in regulating oral squamous cell carcinoma (OSCC) cells migration.Materials and methods: Tumors from 23 patients with OSCC and five OSCC cell lines were used for analyzing AR expression. The effects of AR agonist and antagonist were used to examine the role of AR in regulating the migration of OSCC cells.Results: Ten of 23 tumors from patients with OSCC were AR positive. There was no significant difference in total EGFR (tEGFR) expression between AR-positive tumors and AR-negative tumors. However, the expression of phosphorylated EGFR (pEGFR) in AR-positive tumors was significantly higher than that in AR-negative tumors (p<0.01). Stimulation of AR by dihydrotestosterone in SCC9 (AR-positive OSCC cell) caused an increase in pEGFR and pAKT expression and promoted cell migration without changed tEGFR expression, whereas treatment with bicalutamide led to a decrement in pEGFR expression and pAKT and inhibited cell migration. No effects were found in SCC25 cell line (AR-negative) either treated by dihydrotestosterone or bicalutamide. Furthermore, SCC9 cell line treated by EGF or cetuximab (EGFR inhibitor) significantly promoted or inhibited cell migration.Conclusion: Our data indicate that OSSC tumors and OSCC cell lines express AR which is critical for promoting cell migration by increasing EGFR phosphorylation.

Phosphorylation of HSP90 by protein kinase A is essential for the nuclear translocation of androgen receptor

The androgen receptor (AR) is often activated in prostate cancer patients undergoing androgen-ablative therapy because of the activation of cellular pathways that stimulate the AR despite low androgen levels. In many of these tumors, the cAMP-dependent protein kinase A (PKA) pathway is activated. Previous studies have shown that PKA can synergize with low levels of androgen to enhance androgen signaling and consequent cell proliferation, leading to castration-resistant prostate cancer. However, the mechanism by which PKA causes AR stimulation in the presence of low/no androgen is not established yet. Here, using immunofluorescence immunoblotting assays, co-immunoprecipitation, siRNA-mediated gene silencing, and reporter gene assays, we demonstrate that PKA activation is necessary for the phosphorylation of heat shock protein (HSP90) that binds to unliganded AR in the cytoplasm, restricting its entry into the nucleus. We also found that PKA-mediated phosphorylation of the Thr89 residue in HSP90 releases AR from HSP90, enabling AR binding to HSP27 and its migration into the nucleus. Substitution of the Thr89 in HSP90 prevented its phosphorylation by PKA and significantly reduced AR transactivation and cellular proliferation. We further observed that the transcription of AR target genes, such as prostate-specific antigen (PSA), is also lowered in the HSP90 Thr89 variant. These results suggest that using a small-molecule inhibitor against the HSP90 Thr89 residue in conjunction with existing androgen-ablative therapy may be more effective than androgen-ablative therapy alone in the treatment of prostate cancer patients.

Abstract 1088: Defining bone metastatic potential of Abi1 CRISPR prostate cancer cell lines

Abstract Prostate cancer is the second most common cancer in American men, and it is the third leading cause of cancer-related deaths in American men. Distant stage and metastatic disease is often treated with androgen-deprivation therapy (ADT); however, this treatment ultimately fails, leading to castration-resistant prostate cancer (CRPC). This further emphasizes the need for better therapeutic targets. Our work focuses on understanding the role of the Abi1 gene in prostate cancer tumor progression. Our current in vitro data demonstrate an invasive phenotype with our CRISPR-mediated Abi1 knockout (KO) cell lines. In total, using CRISPR-mediated technology we have generated four Abi1 gene KO cell lines, PC-3, DU145, LnCaP, and RWPE-1. Importantly, the androgen-dependent cell line, LNCaP, demonstrated upregulation of Abi1 to the androgen receptor stimulation and the opposite response to the receptor inhibition upon treatment with enzalutamide. These data indicated that low Abi1 level could be induced by ADT therapies and are consistent with our previous observation of low Abi1/WAVE complex in prostate tumor samples. To learn more about this mechanism, we set out to investigate the role of Abi1 in castrated mice. The overall goal of the experiment is to compare the ability of cells lacking the Abi1 gene to engraft in bone under androgen deprivation condition and compare it to the control cells and examine any change in the engraftment phenotype. It is hoped that this research will lead to better understanding of the Abi1/WAVE complex role in CRPC. Supported by NCI R01 CA16101. Citation Format: Alexander Nappi, Dawn Post, Disharee Das, Ishita Joshi, Megan Oest, Gennady Bratslavsky, Leszek Kotula. Defining bone metastatic potential of Abi1 CRISPR prostate cancer cell lines [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 1088.

Molecular Mechanism of Androgen Receptor Stimulation of Insulin Secretion in Male ß Cells

Androgen deprivation therapy increases the risk of type 2 diabetes mellitus (T2DM) in men. We previously showed that male pancreatic β-cell specific androgen receptor knockout (βARKOMIP) mice develop glucose intolerance because AR potentiates glucose-stimulated insulin secretion (GSIS) through increasing cyclic AMP (cAMP) production and amplifies the insulinotropic effect of glucagon-like peptide-1 (GLP-1). We show that male βARKOMIP mice exhibit impaired intraperitoneal (IP) glucose tolerance- because of impaired IP-GSIS- without alteration in oral glucose tolerance, suggesting that AR amplifies the islet-derived, but not the gut-derived GLP-1 to potentiate GSIS. Using male insulin-secreting β-cell line 832/3 transduced with exchange factor directly activated by a cAMP (EPAC)-based fluorescence resonance energy transfer (FRET) sensor, we observe that AR agonist dihydrotestosterone (DHT) allows GLP-1, glucagon and gastric inhibitory polypeptide (GIP) to increase cAMP production above level of the individual hormones and to a similar level to forskolin control. Accordingly, DHT increases the insulinotropic effect of forskolin, GLP-1, glucagon and GIP in mouse islets. The insulinotropic effect of DHT is abolished using EPAC and PKA inhibitors as well as rapamycin indicating that DHT amplifies the effect of GPCRs coupled to adenylate cyclase and stimulates GSIS via a cAMP/PKA/EPAC pathway and activation of mTOR. Disclosure W. Xu: None. F.B. Ashford: None. D. Hodson: None. F. Mauvais-Jarvis: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc..

Delineating the regulation of estrogen and androgen receptor expression by sex steroids during rat spermatogenesis

Estrogen receptors (ERα and β) and androgen receptor (AR) regulate various critical processes during spermatogenesis. Since spermatogenesis is very sensitive to hormonal stimuli and perturbations, it is important to understand the regulation of expression of these receptors by sex steroid hormones. Although many studies have reported deregulation of steroid receptors on endocrine disruption, there is no consensus on the regulation of their expression by steroid hormones during spermatogenesis, and a lack of clear understanding of the mechanism of regulation. Here, we evaluated the receptor expressions in a well-established exogenous estradiol administration model. We then investigated the mechanisms by which the individual receptors regulate their expression by binding to the respective hormone response elements upstream of these receptor genes. By further employing in vitro and in vivo models of ER and AR stimulation or antagonism, we delineated their regulation in a receptor subtype-specific manner. Our results indicate that ERα positively regulates expression of both the ERs; whereas, ERβ and AR negatively regulate expression of both ERβ and AR by direct binding to upstream regulatory regions. The perturbations in the levels of steroid receptors could be an important factor contributing to spermatogenic defects and male sub-fertility after estradiol and ER agonist treatment. Our study delineates the direct contribution of the individual steroid receptors in the regulation of their own expression.