Androgen-androgen Receptor Signaling Research Articles

Abstract C056: Characterization of prostate cancer secretomes for therapeutic intervention

Abstract Introduction: Prostate cancer (PC) is a common malignancy among men, often driven by dysregulated androgen receptor (AR) signaling. Earlier stages of PC can be favorably treated with androgen deprivation therapies or AR signaling inhibitors. Unfortunately, metastatic PC (mPC) is currently incurable. Neuroendocrine prostate cancer (NEPC) is a subgroup of mPC that may be derived as result of transdifferentiation of adenocarcinomas, after AR-directed treatment. Previous studies using preclinical models have suggested a potentially beneficial relationship between NEPC and AR-active prostate cancer (ARPC) cells. We hypothesize that secreted factors derived from NEPC cells influence local, and distant ARPC cell types to promote treatment resistance. Methods: Five NEPC in vitro cell models: NCI-H660, MSKCC-EF1, LTL331R, LuCaP-49 and LuCaP-173.1 were maintained in serum free media for 24 hours. The conditioned media (CM) was then collected and admixed with growth medium for ARPC cells. The ARPC growth medium was either supplemented with Fetal Bovine Serum (FBS) or Charcoal Stripped fetal bovine Serum (CSS). GFP-labelled ARPC cells: LnCaP and C42B were seeded in the respective media admixed with the CM. The viability of cells was measured using a Cytation5 microplate reader. We implemented a transcriptomic (RNAseq) and mass-spectrometry based proteomic analysis to identify differentially expressing secretory proteins that could have an impact on the treatment resistance of ARPC cell types. Results: All 5 NEPC-CM showed qualitative growth promoting effects towards ARPC C42B cells grown in CSS supplemented media. Only CM from EF1 and H660 promoted the growth of ARPC C42B in FBS supplemented media. NEPC-CM did not affect growth of LnCaP ARPC cells. Mass-spectrometry for secreted proteins was performed on 3 NEPC cell models. Analysis of the proteins against the PANTHER gene ontology database shows: (i) cell communication, and (ii) cellular response to stimulus, as two important biological processes active in NEPC. Genes involved in the two pathways were further compared against a comprehensive transcriptomic dataset spanning distinct mPC phenotypes and genotypes. Neurotrophic growth factors: Brain-derived growth factor (BDNF), Nerve-growth factor (NGF), VGF and neurotrophin-3 (NT-3) emerged as important targets that were differentially expressed in NEPC versus ARPC cells. Conclusions: NEPC cells have the potential to influence ARPC phenotypes that include enhanced proliferation, AR targeting, and potentially other drug resistance programs. As mPC exhibits substantial intra-individual and intra-tumor heterogeneity, understanding a targeting heterotypic cell interactions could lead to new druggable targets and address treatment resistance resulting from complex inter-cellular interactions. (Funding: PC220408) Citation Format: Tarana Arman, Lisa A. Jones, Ilsa M. Coleman, Philip R. Gafken, Peter S. Nelson. Characterization of prostate cancer secretomes for therapeutic intervention [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C056.

Cell-by-cell quantification of the androgen receptor in benign and malignant prostate leads to a better understanding of changes linked to cancer initiation and progression.

The androgen receptor (AR) plays a crucial role in the development and homeostasis of the prostate and is a key therapeutic target in prostate cancer (PCa). The gold standard therapy for advanced PCa is androgen deprivation therapy (ADT), which targets androgen production and AR signaling. However, resistance to ADT develops via AR-dependent and AR-independent mechanisms. As reports on AR expression patterns in PCa have been conflicting, we performed cell-by-cell AR quantification by immunohistochemistry in the benign and malignant prostate to monitor changes with disease development, progression, and hormonal treatment. Prostates from radical prostatectomy (RP) cases, both hormone-naïve and hormone-treated, prostate tissues from patients on palliative ADT, and bone metastases were included. In the normal prostate, AR is expressed in >99% of luminal cells, 51% of basal cells, and 61% of fibroblasts. An increase in the percentage of AR negative (%AR-) cancer cells along with a gradual loss of fibroblastic AR were observed with increasing Gleason grade and hormonal treatment. This was accompanied by a parallel increase in staining intensity of AR positive (AR+) cells under ADT. Staining AR with N- and C-terminal antibodies yielded similar results. The combination of %AR- cancer cells, %AR- fibroblasts, and AR intensity score led to the definition of an AR index, which was predictive of biochemical recurrence in the RP cohort and further stratified patients of intermediate risk. Lastly, androgen receptor variant 7 (ARV7)+ cells and AR- cells expressing neuroendocrine and stem markers were interspersed among a majority of AR+ cells in ADT cases. Altogether, the comprehensive quantification of AR expression in the prostate reveals concomitant changes in tumor cell subtypes and fibroblasts, emphasizing the significance of AR- cells with disease progression and palliative ADT.

Adaptation to Hypoxia May Promote Therapeutic Resistance to Androgen Receptor Inhibition in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) surpasses other BC subtypes as the most challenging to treat due to its lack of traditional BC biomarkers. Nearly 30% of TNBC patients express the androgen receptor (AR), and the blockade of androgen production and AR signaling have been the cornerstones of therapies for AR-positive TNBC. However, the majority of women are resistant to AR-targeted therapy, which is a major impediment to improving outcomes for the AR-positive TNBC subpopulation. The hypoxia signaling cascade is frequently activated in the tumor microenvironment in response to low oxygen levels; activation of the hypoxia signaling cascade allows tumors to survive despite hypoxia-mediated interference with cellular metabolism. The activation of hypoxia signaling networks in TNBC promotes resistance to most anticancer drugs including AR inhibitors. The activation of hypoxia network signaling occurs more frequently in TNBC compared to other BC subtypes. Herein, we examine the (1) interplay between hypoxia signaling networks and AR and (2) whether hypoxia and hypoxic stress adaptive pathways promote the emergence of resistance to therapies that target AR. We also pose the well-supported question, “Can the efficacy of androgen-/AR-targeted treatments be enhanced by co-targeting hypoxia?” By critically examining the evidence and the complex entwinement of these two oncogenic pathways, we argue that the simultaneous targeting of androgen biosynthesis/AR signaling and hypoxia may enhance the sensitivity of AR-positive TNBCs to AR-targeted treatments, derail the emergence of therapy resistance, and improve patient outcomes.

Development and Evaluation of a PSMA-Targeted Nanosystem Co-Packaging Docetaxel and Androgen Receptor siRNA for Castration-Resistant Prostate Cancer Treatment.

Primary prostate cancer (PC) progresses to castration-resistant PC (CRPC) during androgen deprivation therapy (ADR) in early stages of prostate cancer. Thus, rather than blocking the androgen-related pathway further, docetaxel (DTX)-based therapy has become the most effective and standard first-line chemotherapy for CRPC. Although the therapy is successful in prolonging the survival of patients with CRPC, chemotherapy resistance develops due to the abnormal activation of the androgen receptor (AR) signaling pathway. Thus, to optimize DTX efficacy, continued maximum suppression of androgen levels and AR signaling is required. Here, we designed a prostate-specific membrane antigen (PSMA)-targeted nanosystem to carry both DTX and AR siRNA (Di-PP/AR-siRNA/DTX) for CRPC treatment. Specifically, DTX was encapsulated into the hydrophobic inner layer, and the AR siRNA was then condensed with the cationic PEI block in the hydrophilic outer layer of the PEI-PLGA polymeric micelles. The micelles were further coated with PSMA-targeted anionic polyethylene glycol-polyaspartic acid (Di-PEG-PLD). In vitro and in vivo results demonstrated that the resulting Di-PP/AR-siRNA/DTX exhibited prolonged blood circulation, selective targeting, and enhanced antitumor effects. Consequently, Di-PP/AR-siRNA/DTX holds great potential for efficient CRPC treatment by combining chemotherapy and siRNA silencing of androgen-related signaling pathways.

Elucidating the Role of Insulin‐induced AKR1C3 and the 11‐oxygenated Androgens in Promoting the Androgen Excess of Polycystic Ovary Syndrome (PCOS)

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age. The androgen excess (AE) of PCOS is believed to promote cardio-metabolic risk, but how this occurs is unknown. As a result, treatment of cardio-metabolic diseases in PCOS women is not entirely effective. AE correlates with insulin resistance (IR) in these women and may be driving lipotoxicity. Aldo-keto reductase family 1 member C3 (AKR1C3) is induced by insulin and may be central in driving the AE of PCOS. Our goal is to understand the role of insulin-induced AKR1C3 in promoting AE and lipotoxicity in PCOS adipocytes. AKR1C3 predominantly catalyzes the conversion of peripheral androgens such Δ4-androstene-3,17-dione (4AD) to testosterone (T) and 5α-androstan-3,17-dione (5AD) to 5α-dihydrotestosterone (DHT). T and DHT are potent agonists for the androgen receptor (AR), which upregulate de novo lipogenesis. Fatty acid synthase (FASN) is known to be induced by androgens and may be responsible for this altered lipid profile in mature adipocytes. AKR1C3 overexpression in adipocytes, in response to insulin, could increase AR signaling and increase de novo lipogenesis. This lipid overflow could exacerbate insulin resistance through a feedforward mechanism and promote the lipotoxic profile seen in PCOS. Additionally, the adrenal 11-oxygenated androgens may be an alternative source of AE and have been reported as the dominant androgens of PCOS. We hypothesize that insulin induction of AKR1C3 and the conversion of 11-oxygenated androgens into potent AR agonists in adipocytes may potentiate AE and lipotoxicity in PCOS. Using differentiated Simpson-Golabi-Behmel Syndrome (SGBS) adipocytes as a model, we have shown that insulin increased AKR1C3 expression by >600 fold at the RNA level. Using pharmacological inhibition, we have preliminary data that shows insulin induction of AKR1C3 to utilize the PI3K/AKT/mTOR pathway as measured by RT-qPCR and immunoblot assays. Using a reverse-phase-HPLC discontinuous assay and post-reaction derivatization with dinitrophenylhydrazine, we determined the kcat and Km for the conversion of 11-ketoandrostene-3,17-dione (11K-4AD) to 11-ketotestosterone (11K-T) and 11-ketoandrostan-3,17-dione (11K-5AD) to 11-ketodihydrotestosterone (11K-DHT) using recombinant AKR1C3. We find that 11K-4AD and 11K-5AD are superior substrates for AKR1C3 when compared to 4AD and 5AD. Furthermore, we have quantified the formation of 11K-T in SGBS cells by stable-isotope-dilution (SID)-LC-MS/MS. This research will elucidate the mechanism of how AKR1C3 potentiates androgen excess and AR signaling to promote lipotoxicity in PCOS. Additionally, this work supports AKR1C3 as a potential therapeutic target in mitigating the androgen excess of PCOS.

Long-Chain Acyl-CoA Synthetase 4-Mediated Fatty Acid Metabolism Sustains Androgen Receptor Pathway-Independent Prostate Cancer.

Androgen deprivation therapy has led to elevated cases of androgen receptor (AR) pathway-independent prostate cancer with dysregulated fatty acid metabolism. However, it is unclear how prostate cancer cells sustain dysregulated fatty acid metabolism to drive AR-independent prostate cancer. Long-chain acyl-CoA synthetases (ACSL) catalyze the conversion of fatty acids into fatty acyl-CoAs that are required for fatty acid metabolism. In this study, we demonstrate that expression levels of ACSL3 and 4 were oppositely regulated by androgen-AR signaling in prostate cancer cells. AR served as a transcription suppressor to bind at the ACSL4 promoter region and inhibited its transcription. Inhibition of androgen-AR signaling significantly downregulated ACSL3 and PSA, but elevated ACSL4 levels. ACSL4 regulated a broad spectrum of fatty acyl-CoA levels, and its catalytic efficiency in fatty acyl-CoAs biosynthesis was about 1.9- to 4.3-fold higher than ACSL3. In addition, in contrast to ACSL3, ACSL4 significantly regulated global protein myristoylation or myristoylation of Src kinase in prostate cancer cells. Knockdown of ACSL4 inhibited the proliferation, migration, invasion, and xenograft growth of AR-independent prostate cancer cells. Our results suggest that the surge of ACSL4 levels by targeting AR signaling increases fatty acyl-CoAs biosynthesis and protein myristoylation, indicating the opposite, yet complementary or Yin-Yang regulation of ACSL3 and 4 levels in sustaining fatty acid metabolism when targeting androgen-AR signaling. This study reveals a mechanistic understanding of ACSL4 as a potential therapeutic target for treatment of AR-independent prostate cancer. IMPLICATIONS: AR coordinately regulates the expression of ACSL3 and ACSL4, such that AR pathway-independent prostate tumors become dependent on ACSL4-mediated fatty acid metabolism.

295 Poster - Prognostic utility of androgen receptor signaling pathway in invasive breast cancer

Background: Androgen receptor (AR) and AR signaling pathway are thought to play a role in tumour progression and be potentially related to treatment responses and outcomes in invasive breast cancer. In present study, we investigated the clinicopathological significance of AR associated signaling pathway using the large transcriptomic database of early-stage invasive breast cancer.

Abstract B087: Role of GATA-4-Androgen-AR axis in prostate cancer from African American men

Abstract Background: African American (AA) men are 1.7 times more likely to develop and 2.4 times more likely to die of prostate cancer (PrCa) than their European American (EA) counterparts, however the underlying biology causing this disparity is unknown. Here, we show how epigenetic dysregulation of calcium signaling genes affect GATA-4, androgen receptor (AR), androgen metabolism and associate with clinical outcomes in AA men with PrCa. Methods: Using Illumina arrays we identified DNA methylation differences between 32 EA and AA PrCa patients. We used RNA-sequencing to discover transcriptomic changes potentially mediated by DNA methylation. We measured GATA-4 and AR protein expression in a tissue microarray (TMA) of 95 EA and 92 AA PrCa patients. We measured testosterone (T) and dihydrotestosterone (DHT) in serum of the same AA and EA PrCa patients. In vitro, we treated MDA PCa 2a (2a) and LaPC4 cells derived from AA and EA prostate tumors, respectively with a calcium chelator (BAPTA-AM) and T. We measured GATA-4 and AR protein expression in control and treated cells. Results: Our DNA methylation analysis showed that AA but not EA prostate tumors enriched for hypermethylated sites in calcium sensing genes was associated with worse disease-free time (21.6 vs 46.7 months, p<0.05). DNA hypermethylation of calcium signaling genes, which correlated with decreased transcript levels, can potentially raise intracellular calcium. Calcium levels regulate stability of proteins including AR and GATA-4. Therefore, we tested potential calcium mediated changes in GATA-4 and AR expression in vitro and in patient samples. In vitro, depleting calcium in the presence of T increased GATA-4 protein expression by 1.5 times and AR expression only in AA prostate tumor derived 2a cells. DNA hypermethylation of calcium sensing genes was associated with lower GATA-4 and AR expression in PrCa and adjacent non-tumor tissues of AA men. The role of GATA-4 in PrCa is unknown, but it is a known transcriptional regulator of androgen metabolizing enzymes (AME). AMEs are important for androgen metabolism that result in T and DHT production through the primary and alternative pathways. AME transcripts in the primary and alternative pathways were one of the top 5 transcriptionally dysregulated pathways in AA compared to EA prostate tumors. We found that T but not DHT is significantly lower (p<0.05) in serum samples from AA (3.64ng/ml) compared to EA (3.99ng/ml) PrCa patients. T and DHT bioavailability in prostate tumors affect AR expression and function. In our studies, AA adjacent non-tumor tissues had higher percent AR positive nuclei (p<0.05) compared to EA adjacent non-tumor tissues. Conclusion: Epigenetic alteration of calcium signaling can affect GATA-4 mediated changes in androgen metabolism and AR signaling that are well-established therapeutic targets in PrCa. This molecular alteration, unique to AA prostate tumors, can be used to rationalize specific androgen deprivation and AR-targeted therapies in these patients. Citation Format: Swathi Ramakrishnan, Xuan Peng, Eduardo Cortes Gomez, Kristopher Attwood, Li Yan, John Wilton, Gissou Azabdaftari, James Mohler, Jianmin Wang, Anna Woloszynska. Role of GATA-4-Androgen-AR axis in prostate cancer from African American men [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B087.

KLF5 Is Crucial for Androgen-AR Signaling to Transactivate Genes and Promote Cell Proliferation in Prostate Cancer Cells.

Androgen/androgen receptor (AR) signaling drives both the normal prostate development and prostatic carcinogenesis, and patients with advanced prostate cancer often develop resistance to androgen deprivation therapy. The transcription factor Krüppel-like factor 5 (KLF5) also regulates both normal and cancerous development of the prostate. In this study, we tested whether and how KLF5 plays a role in the function of AR signaling in prostate cancer cells. We found that KLF5 is upregulated by androgen depending on AR in LNCaP and C4-2B cells. Silencing KLF5, in turn, reduced AR transcriptional activity and inhibited androgen-induced cell proliferation and tumor growth in vitro and in vivo. Mechanistically, KLF5 occupied the promoter of AR, and silencing KLF5 repressed AR transcription. In addition, KLF5 and AR physically interacted with each other to regulate the expression of multiple genes (e.g., MYC, CCND1 and PSA) to promote cell proliferation. These findings indicate that, while transcriptionally upregulated by AR signaling, KLF5 also regulates the expression and transcriptional activity of AR in androgen-sensitive prostate cancer cells. The KLF5-AR interaction could provide a therapeutic opportunity for the treatment of prostate cancer.

Estrogen Receptors in Epithelial-Mesenchymal Transition of Prostate Cancer.

Prostate cancer (PC) remains a widespread malignancy in men. Since the androgen/androgen receptor (AR) axis is associated with the pathogenesis of prostate cancer, suppression of AR-dependent signaling by androgen deprivation therapy (ADT) still represents the primary intervention for this disease. Despite the initial response, prostate cancer frequently develops resistance to ADT and progresses. As such, the disease becomes metastatic and few therapeutic options are available at this stage. Although the majority of studies are focused on the role of AR signaling, compelling evidence has shown that estrogens and their receptors control prostate cancer initiation and progression through a still debated mechanism. Epithelial versus mesenchymal transition (EMT) is involved in metastatic spread as well as drug-resistance of human cancers, and many studies on the role of this process in prostate cancer progression have been reported. We discuss here the findings on the role of estrogen/estrogen receptor (ER) axis in epithelial versus mesenchymal transition of prostate cancer cells. The pending questions concerning this issue are presented, together with the impact of the available data in clinical management of prostate cancer patients.

Abstract 2026: Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant prostate cancer

Abstract Well-differentiated prostate cancer (PCa) cells are generally androgen/androgen receptor (AR)-dependent, i.e. AR signaling regulates cell cycle and differentiation. Loss of AR signaling after androgen deprivation therapy (ADT) triggers AR-independent outgrowth, generating poorly differentiated uncontrollable PCa cells. To prevent development and progression of castration-resistant prostate cancer (CRPC), we hypothesize that preservation of AR signaling after ADT is an important target. Tumor stroma surrounding PCa cells is enriched in fibroblasts secreting AR-activating factors, e.g. EGF, IGF1, and IL-6. Thus, we investigated the role of fibroblasts in AR activation of human prostate cancer LNCaP sublines differing in androgen sensitivity under androgen deprivation. Androgen-low-sensitive E9 and F10 cells were obtained from the parental androgen-sensitive LNCaP cell population through use of a limiting dilution method in regular culture condition. In contrast, androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging under hormone-depleted condition. Original fibroblasts pcPrFs (-M5, -M6, -M7) were isolated from needle biopsy samples of patients with PCa. The PCa cells alone or PCa cells plus pcPrF-M5 were grafted beneath the renal capsule of male athymic nude mice, and then mice were castrated on 14 days post transplantation. Tumor volume of E9 cells + pcPrF-M5 became diminished post castration but that of F10 and AIDL cells + pcPrF-M5 was gradually increased even post castration. In E9 cells + pcPrF-M5 tumors, serum PSA became detected on day 21 post castration. Interestingly, serum PSA was gradually increased even in F10 cells inoculated alone. In AIDL tumors with or without pcPrF-M5, serum PSA was not detected because of mutated AR in AIDL cells. Cell growth of E9 and AIDL cells was directly increased by treating with EGF, whereas that in F10 cells was not affected. Phosphorylation of AKT and ERK1/2 in E9 and AIDL cells was also significantly increased by treating with EGF, whereas that in F10 cells was not affected. In the condition of co-cultures with fibroblasts, PSA production was directly increased in E9 cells but not in F10 and AIDL cells. Protein expression of AR splice variant 7 (AR-V7) was only detected in F10 cells. The reduced AR-dependency of PCa cells is an important clinical development because of its association with the cell’ progression to CRPC. Here our results showed that fibroblasts secreting AR-activating factors preserved AR signaling in E9 cells after ADT, indicating that this type of PCa cells can be controlled by ADT. In contrast, we suggest that loss of fibroblasts-dependent AR activation in F10 and AIDL cells may be responsible for development and progression of CRPC. Citation Format: Kenichiro Ishii, Izumi Matsuoka, Takeshi Sasaki, Manabu Kato, Kohei Nishikawa, Hideki Kanda, Yoshifumi Hirokawa, Kazuhiro Iguchi, Kiminobu Arima, Masatoshi Watanabe, Yoshiki Sugimura. Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant prostate cancer [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 2026.

Abstract CT097: Phase I trial of apalutamide plus abiraterone acetate, docetaxel, and prednisone in patients with metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background: Prostate cancer is driven by the androgen receptor (AR) pathway, which may be targeted by inhibition of androgen synthesis (e.g. abiraterone = Abi), AR signaling (e.g. apalutmide = Apa), and/or translocation of AR to the nucleus (e.g. docetaxel = Doce). The combination of AR inhibition plus taxane chemotherapy has already demonstrated clinical benefit (CHAARTED, STAMPEDE), lending weight to the theory that combining drugs targeting different points in the AR signaling pathway is effective. We hypothesize that the combination of Apa, Abi, and Doce will be safe and efficacious. Methods: Men with progressive mCRPC, intact organ function, and no prior exposure to Apa (ever) or Doce within 3 years were enrolled. Standard Abi 1000 mg daily, Doce 75 mg/m2 q3 weeks, and prednisone 5 mg BID was administered with different doses of Apa. Cohort 1 = Apa 120 mg daily, Cohort 2 = Apa 240 mg daily, Cohort 3 (if necessary) = Apa 180 mg daily in modified 3+3 design. The primary endpoint of the Phase I dose-escalation portion of the study was determination of dose-limiting toxicity (over 6 weeks) and recommended Phase II dose (RP2D). Results: Nine men (3 Apa 120 mg, 6 Apa 240 mg) with mCRPC and median age 69, median PSA 8.18 (range 0.07 - 278.5) were treated. 67% with bone, 78% LN, 11% lung, 11% other metastases. No DLT occurred with Apa 120 mg and 1 of 6 with Apa 240 mg had possibly related grade 3 hypertension. All had >95% PSA decline (78% with > 99% decline). Of 5 with measureable disease, 100% had RECIST response. 6 of 7 with post-treatment CTC counts were undetectable. Conclusions: The combination of apalutamide plus abiraterone, docetaxel, and prednisone at full doses is tolerable, with the RP2D of the regimen Apa 240 mg daily, Abi 1000 mg daily, docetaxel 75 mg/m2 q3 wks, and prednisone 5 mg BID. The combination appears to be associated with a high rate of PSA decline, measurable disease response, and CTC count control. Tumor tissue, plasma ctDNA, and CTCs are being collected for analysis. Enrollment to the expansion phase of study to further refine efficacy and toxicity at PCCTC sites is ongoing including assessment of response and resistance biomarkers [NCT02913196] Citation Format: Ana Molina, Paul Christos, Amy Hackett, Luke Nordquist, Edward Gelmann, Mark Stein, Cora Sternberg, Himisha Beltran, Lauren Gracey, Giuseppe Galletti, Paraskevi Giannakakou, David M. Nanus, Scott T. Tagawa. Phase I trial of apalutamide plus abiraterone acetate, docetaxel, and prednisone in patients with metastatic castration-resistant prostate cancer (mCRPC) [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 CT097.

Utility of ankyrin 3 as a prognostic marker in androgen-receptor-positive breast cancer.

Androgen receptor (AR) and AR signaling pathways are thought to play a role in breast cancer (BC) and are potentially related to treatment responses and outcomes. Ankyrin 3 (ANK3) is associated with AR stability in cancer cells. In the present study, we investigated the clinicopathological utility of ANK3 expression with emphasis on AR and its associated signalling pathway at transcriptomic and proteomic phases. The Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort (n = 1980) and The Cancer Genome Atlas (TCGA) dataset (n = 1039) were used to assess the expression and significance of ANK3 mRNA and other AR signalling pathway-associated gene signature. Using immunohistochemistry, ANK3 protein expression was evaluated in large (n = 982) cohort of early-stage BC with long-term follow-up and compared with clinicopathological characteristics and its prognostic value in the whole cohort and the subgroups stratified by AR protein expression. An AR-related gene signature was developed, comprising 20 genes, which included ANK3. This AR-related gene signature was significantly associated with AR mRNA expression, oestrogen receptor, human epidermal growth factor receptor 2 (HER2) status and the patients' outcomes. In tumours with high AR protein expression (n = 614), high ANK3 protein expression was significantly associated with progesterone receptor positivity and it was independently associated with the good outcomes (p = 0.025). This study indicates that ANK3 is related to AR signalling pathway and is associated with BC prognosis.

Suppressive Role of Androgen/Androgen Receptor Signaling via Chemokines on Prostate Cancer Cells.

Androgen/androgen receptor (AR) signaling is a significant driver of prostate cancer progression, therefore androgen-deprivation therapy (ADT) is often used as a standard form of treatment for advanced and metastatic prostate cancer patients. However, after several years of ADT, prostate cancer progresses to castration-resistant prostate cancer (CRPC). Androgen/AR signaling is still considered an important factor for prostate cancer cell survival following CRPC progression, while recent studies have reported dichotomic roles for androgen/AR signaling. Androgen/AR signaling increases prostate cancer cell proliferation, while simultaneously inhibiting migration. As a result, ADT can induce prostate cancer metastasis. Several C-C motif ligand (CCL)-receptor (CCR) axes are involved in cancer cell migration related to blockade of androgen/AR signaling. The CCL2-CCR2 axis is negatively regulated by androgen/AR signaling, with the CCL22-CCR4 axis acting as a further downstream mediator, both of which promote prostate cancer cell migration. Furthermore, the CCL5-CCR5 axis inhibits androgen/AR signaling as an upstream mediator. CCL4 is involved in prostate carcinogenesis through macrophage AR signaling, while the CCL21-CCR7 axis in prostate cancer cells is activated by tumor necrotic factor, which is secreted when androgen/AR signaling is inhibited. Finally, the CCL2-CCR2 axis has recently been demonstrated to be a key contributor to cabazitaxel resistance in CRPC.

Melatonin up-regulates the expression of the GATA-4 transcription factor and increases testosterone secretion from Leydig cells through RORα signaling in an in vitro goat spermatogonial stem cell differentiation culture system.

Because androgen function is regulated by its receptors, androgen-androgen receptor signaling is crucial for regulating spermatogenesis. Androgen is mainly testosterone secreted by testis. Based on the results of early studies in goats, the administration of melatonin over an extended period of time increases steroid production, but the underlying mechanism remains unclear. Here, we report the expression of the melatonin membrane receptors MT1 and MT2 and the retinoic acid receptor-related orphan receptor-alpha (RORα) in the goat testis. An in vitro differentiation system using spermatogonial stem cells (SSCs) cultured in the presence of testicular somatic cells was able to support the formation of sperm-like cells with a single flagellum. The addition of 10-7 M melatonin to the in vitro culture system increased RORα expression and considerably improved the efficiency of haploid cell differentiation, and the addition of the RORα agonist CGP52608 significantly increased the testosterone concentration and expression of GATA binding factor 4 (GATA-4). Furthermore, inhibitors of melatonin membrane receptors and a RORα antagonist (T0901317) also led to a considerable reduction in the efficiency of haploid spermatid formation, which was coupled with the suppression of GATA-4 expression. Based on these results, RORα may play a crucial role in enhancing melatonin-regulated GATA-4 transcription and steroid hormone synthesis in the goat spermatogonial stem cell differentiation culture system.

Abstract 4492: Androgen receptor affects GLI activity by promoting transcriptionally active states of GLI upon androgen stimulation

Abstract Introduction: While “castration” therapies benefit metastatic prostate cancer (PCa) patients, their effects are transient and progression to castration resistant disease (CRPC) remains a vexing clinical problem. Despite low levels of circulating androgens in treated patients, CRPC typically remains dependent on androgen/androgen receptor (AR) signaling for further growth. Previously we showed that Gli protein binding to AR co-activates AR transcriptional activity and supports androgen-independent growth. Here we show evidence that the interaction of AR with Gli proteins, particularly Gli3, allows for a “non-canonical” activation of Hedgehog signaling that contributes to PCa cell growth and progression to CRPC. Methods: Androgen growth-dependent (LNCaP) or -independent (LNCaP-AI, LN95) PCa cells were transfected with a Gli-luciferase (Gli-luc) reporter and were treated with R1881, enzalutamide (Enz), or a combination. Luciferase activity was measured. R1-D567 (harboring a genomic deletion of AR) served as a negative control. Knockdown of AR-FL with siRNA or overexpression of a small decoy peptide from the Gli2 AR-binding domain (Gli-DP) was also performed in above cells. Western blot and real-time PCR confirmed both AR and Gli target gene/protein expressions in LNCaP and LNCaP-AI cells (-/+ R1881). Sonic hedgehog (Shh) ligand-responsive 3T3-E1 cells were transiently expressed with AR-FL to confirm the involvement of canonical Shh-Gli activation pathway in AR co-activation of Gli transcription. Co-immunoprecipitation (co-IP) was performed to verify the blocking efficiency of Gli-DP in AR-Gli3 interaction in LN95 cells and AR-overexpressing 293FT. Results: Androgen (R1881) treatment strongly induced Gli transcription activity in AR+ PCa cells while Enz reversed this effect; AR-FL-specific knockdown or exogenous Gli-DP expression suppressed androgen-induced Gli reporter expression as well as expression of endogenous Gli-responsive genes. R1881 treatment promoted Gli2/3 protein processing to transcriptionally active states (Gli3-FullLength and Gli2-Active) and enhanced the expression of Gli-target genes, Gli1/Ptch1, at protein and/or mRNA levels. Co-IP revealed that exogenous Gli-DP blocked the interaction between AR and transcriptionally active Gli3-FL in PCa cells. Overexpression of AR in 3T3-E1 cells confirmed that AR interaction with Gli stabilizes the active Gli3-FL form by suppressing degradation of Gli3 in a manner independent of the canonical Shh-Gli activation pathway. Conclusions: Our findings further identify the importance of AR-Gli interaction in progression of PCa to CRPC and shows that cooperative binding of AR to Gli proteins enhances both AR and Hedgehog signaling in PCa cells. Interference with the AR-Gli interaction using a decoy peptide provides a means to suppress AR activation of Hedgehog in PCa cells. Citation Format: Sarah Truong, Na Li, Mannan Nouri, Ralph Buttyan. Androgen receptor affects GLI activity by promoting transcriptionally active states of GLI upon androgen stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4492. doi:10.1158/1538-7445.AM2017-4492

6BIO Enhances Oligonucleotide Activity in Cells: A Potential Combinatorial Anti-androgen Receptor Therapy in Prostate Cancer Cells

Approximately 15%-25% of men diagnosed with prostate cancer do not survive their disease. The American Cancer Society estimated that for the year 2016 the number of prostate cancer deaths will be 26,120. Thus, there is a critical need for novel approaches to treat this deadly disease. Using high-throughput small-molecule screening, we found that the small molecule 6-bromo-indirubin-3'-oxime (6BIO) significantly improves the targeting of antisense oligonucleotides (ASOs) delivered by gymnosis (i.e., in the absence of any transfection reagents) in both the cell cytoplasm and the nucleus. Furthermore, as a single agent, 6BIO had the unexpected ability to simultaneously downregulate androgen receptor (AR) expression and AR signaling in prostate cancer cells. This includes downregulating levels of the AR-V7, a drug-resistance-related AR splice variant that is important in the progression of prostate cancer. Combining 6BIO and an anti-AR oligonucleotide (AR-ASO) can augment the downregulation of AR expression. We also demonstrated that 6BIO enhances ASO function and represses AR expression through the inhibition of the two main glycogen synthase kinase 3 (GSK-3) isoforms: GSK-3α and GSK-3β activity. Our findings provide a rationale for the use of 6BIO as a single agent or as part of a combinatorial ASO-based therapy in the treatment of human prostate cancer.

Abstract 4387: Alterations of TP53 mediate resistance to abiraterone in castration-resistant prostate cancer

Abstract Prostate cancer remains the second-leading cause of cancer death in men. The first line treatment for advanced prostate cancer is hormone deprivation therapy (ADT), which although it is initially effective, usually leads to progression to more aggressive and often lethal castration resistant prostate cancer (CRPC). Nonetheless, CRPC remains dependent on androgen receptor (AR) signaling through various mechanisms. Therefore, drugs that target androgen synthesis or AR signaling, such as abiraterone acetate and enzalutamide, have been employed as second line therapy. Despite the fact that both drugs improve the overall survival for CRPC patients, neither of them is curative, suggesting intrinsic or acquired resistance. Here we have focused on elucidating resistance mechanisms for abiraterone acetate, an inhibitor of androgen biosynthesis, using preclinical analyses of relevant genetically engineered mouse models (GEMMs). Given the high frequency of co-alteration in PTEN and p53 in mCRPC, we generated conditional GEMMs based on deletion of either PTEN alone or PTEN plus p53 to obtain the NP and NPp53 GEMMs, respectively. Both NP and NPp53 mice develop CRPC, and cross-species gene set enrichment analysis (GSEA) suggests that they have molecular programs that are well-conserved with human CRPC. In preclinical studies, we find that the NP mice are highly responsive to tumor inhibition by abiraterone, whereas the NPp53 mice do not respond. In fact, the NPp53 mice display a marked acceleration of tumor progression following abiraterone treatment. The disparate phenotypic responses of the NP and NPp53 mice to abiraterone are evident by analyses of their tumor histology, immunohistochemical staining, as well as the change in tumor volume by MRI imaging. We further validated the results in a genetically modified human prostate cancer cell line. In particular, a xenograft model based on 22RV1 cells that are depleted for both PTEN and p53 also displayed an acceleration of the tumor phenotype following abiraterone treatment. Notably, expression profiling of the abiraterone- or vehicle-treated “responder” NP tumors versus “non-responder” NPp53 tumors identified a unique set of cancer driver genes that are enriched for genes found in human neuroendocrine prostate cancer (NEPC). These findings were confirmed by immunohistochemistry, which demonstrated a significant increase in synaptophysin-expressing cells in CRPC in the NPp53 mice but not the NP mice. Our findings suggest that loss of p53 in the context of PTEN deletion promotes resistance to abiraterone while CRPC in the NPp53 mice partially resemble human NEPC. Given the high prevalence of co-alteration of PTEN and p53 in advanced human mCRPC, and observed occurrence of treatment induced NEPC-like phenotypes observed following treatment with AR targeting agents in patients, our finding suggest that abiraterone may not be suitable for patients with alterations in both PTEN and p53. Citation Format: Min Zou, Roxanne Toivanen, Antonina Mitrofanova, Michael M. Shen, Cory Abate-Shen. Alterations of TP53 mediate resistance to abiraterone in castration-resistant prostate cancer. [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 4387.

Oncolytic adenovirus-mediated therapy for prostate cancer.

Prostate cancer is a leading cause of cancer-related death and morbidity in men in the Western world. Tumor progression is dependent on functioning androgen receptor signaling, and initial administration of antiandrogens and hormone therapy (androgen-deprivation therapy) prevent growth and spread. Tumors frequently develop escape mechanisms to androgen-deprivation therapy and progress to castration-resistant late-stage metastatic disease that, in turn, inevitably leads to resistance to all current therapeutics, including chemotherapy. In spite of the recent development of more effective inhibitors of androgen–androgen receptor signaling such as enzalutamide and abiraterone, patient survival benefits are still limited. Oncolytic adenoviruses have proven efficacy in prostate cancer cells and cause regression of tumors in preclinical models of numerous drug-resistant cancers. Data from clinical trials demonstrate that adenoviral mutants have limited toxicity to normal tissues and are safe when administered to patients with various solid cancers, including prostate cancer. While efficacy in response to adenovirus administration alone is marginal, findings from early-phase trials targeting local-ized and metastatic prostate cancer suggest improved efficacy in combination with cytotoxic drugs and radiation therapy. Here, we review recent progress in the development of multimodal oncolytic adenoviruses as biological therapeutics to improve on tumor elimination in prostate cancer patients. These optimized mutants target cancer cells by several mechanisms including viral lysis and by expression of cytotoxic transgenes and immune-stimulatory factors that activate the host immune system to destroy both infected and noninfected prostate cancer cells. Additional modifications of the viral capsid proteins may support future systemic delivery of oncolytic adenoviruses.

The Epidemiological, Mechanistic and Potential Clinical Role of Androgen Receptor (AR) in Urothelial Carcinoma.

The androgen receptor (AR) is a ligand-inducible transcription factor that regulates target gene expression. Androgen signaling has been considered a putative explanation for gender differences in urothelial carcinoma (UC) incidence. In the absence of established risk factors, men still experience a threefold risk of UC as compared to women. Multiple investigations to modulate the AR have been performed with in vitro and in vivo models of UC. Down-regulation of the AR has been shown to inhibit UC growth through increased apoptosis, decreased cell proliferation, and decreased cell migration. AR activation up-regulates EGFR and HER2/neu expression contributing to UC progression. UC is more easily induced in male than female models and the incidence of chemically-induced UC is decreased by castration and the addition of estrogens; it is increased by testosterone. Epithelial to mesenchymal transition (EMT) has been postulated to be androgen-driven in UC and affects chemotherapy sensitivity. UC has not achieved the same therapeutic advances that have been seen in other tumor types in recent years. Androgen-driven events may account for some of the treatment resistance seen in this tumor type. Novel agents which disrupt androgen synthesis and/or AR signaling are in development and some (abiraterone, enzalutamide) are approved for advanced prostate cancer. Biomarker AR-driven clinical trials of highly effective anti-androgen therapy (HEAT) agents in UC present a promising picture.