Cytoplasmic Androgen Receptor Research Articles

Acute and Chronic Changes in Muscle Androgen Receptor Markers Are Not Associated with Muscle Hypertrophy in Women and Men.

Androgen receptor (AR) expression and signaling have been regarded as a mechanism for regulating muscle hypertrophy. However, little is known about the associations between acute and chronic changes in skeletal muscle total AR, cytoplasmic AR (cAR), nuclear AR (nAR), and AR DNA-binding (AR-DNA) induced by resistance training (RT) and hypertrophy outcomes in women and men. This study aimed to investigate the acute and chronic effects of RT on skeletal muscle total AR, cAR, and nAR contents and AR-DNA in women and men. In addition, we investigated whether these acute and chronic changes in these markers were associated with muscle hypertrophy in both sexes. Nineteen women and 19 men underwent 10 wk of RT. Muscle biopsies were performed at baseline, 24 h after the first RT session, and 96 h after the last session. AR, cAR, and nAR were analyzed using Western blotting, and AR-DNA using an ELISA-oligonucleotide assay. Fiber cross-sectional area (fCSA) was analyzed through immunohistochemistry and muscle cross-sectional area (mCSA) by ultrasound. At baseline, men demonstrated greater nAR than women. Baseline cAR was significantly associated with type II fCSA hypertrophy in men. Acutely, both sexes decreased AR and cAR, whereas men demonstrated greater decreases in nAR. After 10 wk of RT, AR, and nAR remained unchanged, men demonstrated greater cAR compared with women, and both sexes decreased AR-DNA activity. Acute and chronic changes in AR markers did not correlate with muscle hypertrophy (type I/II fCSA and mCSA) in women or men. Baseline cAR content may influence hypertrophy in men, whereas neither RT-induced acute nor chronic changes in AR, cAR, nAR, and AR-DNA are associated with muscle hypertrophy in women or men.

Targeting androgen receptor signaling in glioblastoma: A novel biomarker and therapeutic approach.

e14016 Background: Glioblastoma is a lethal brain cancer. Clinical patterns of disease suggest that androgens influence glioblastoma risk and prognosis, and androgen receptor (AR) transcript levels and protein expression are upregulated in glioblastoma compared to normal brain. Thus, potent brain-penetrant anti-androgen therapies may be an effective treatment strategy in glioblastoma. Methods: Clinical samples and patient-derived glioma xenografts (PDXs) were assessed for AR expression by immunohistochemistry. Glioblastoma cell lines and patient-derived models maintained under stem cell conditions were tested in vitro for their response to anti-androgen therapies (abiraterone, enzalutamide and seviteronel). Change in plasticity marker ZEB1 was measured using immunofluorescence. Stem cell function was assessed using a tumoursphere assay. In an intracranial AR positive glioblastoma PDX model (RN1) in an immunocompromised mouse, the most effective drug in vitro, seviteronel, was tested alone and in combination with temozolomide or radiation. Tumor growth was monitored with weekly bioluminescent imaging, with survival as the primary endpoint. Results: Cytoplasmic AR staining was present in ~55% of glioblastoma samples using immunohistochemistry; this biomarker has shown to be predictive in triple-negative breast cancer. AR positive model RN1 was inhibited by low concentrations of anti-androgen agents, with seviteronel having the lowest half maximal inhibitory concentration (IC50) after 96 h (enzalutamide 52 µM, abiraterone 12 µM, seviteronel 7 µM). AR negative model WK1 was inhibited though with higher IC50 values after 96 h (enzalutamide 63 µM, seviteronel 21 µM). Cell lines U87 and U251 were also inhibited by anti-androgen monotherapy. Immunofluorescence analysis showed downregulation of plasticity marker ZEB1 with anti-androgens (normalized mean fluorescent intensity: abiraterone 0.84, enzalutamide 0.91, seviteronel 0.74). Tumoursphere assays demonstrated that anti-androgens inhibit the tumor-forming ability of cells, with seviteronel and abiraterone showing more inhibition than enzalutamide. In mice intracranially implanted with RN1 cells, seviteronel improved overall survival compared to vehicle alone (36 vs 27.5 days, p=0.03). There was no observed benefit when seviteronel was added to radiotherapy (33 vs 39 days, p=0.19). Adding seviteronel to temozolomide limited tumor growth by IVIS but did not significantly improve overall survival compared to temozolomide alone (63.5 vs 56.5 days, p=0.37). Conclusions: Targeting AR with brain penetrant anti-androgen drugs may be a promising biomarker-directed therapeutic strategy for glioblastoma. To address the limitations of our study, further experiments to optimize translational treatment protocols, address the role of biological sex and examine the role of the immune microenvironment are underway.

STEM-03. TARGETING ANDROGEN SIGNALING IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma is a lethal brain cancer. Androgen receptor (AR) transcript levels and protein expression are upregulated in glioblastoma compared to normal brain. AR signaling has a role in cancer stem cell function in various cancers. Stem cell activity and cancer cell plasticity represent a key resistance mechanism in glioblastoma. Thus, potent brain-penetrant anti-androgen therapies have the potential to improve treatment outcomes in glioblastoma. METHODS Clinical samples and patient derived xenografts were assessed for AR expression by immunohistochemistry. Cytoplasmic AR+ and AR- glioblastoma patient-derived xenografts maintained under stem cell conditions were tested in vitro for their response to anti-androgen therapies (abiraterone, enzalutamide and seviteronel). Stem cell function was assessed using a tumoursphere assay. Changes in plasticity markers, including ZEB1, were measured with immunofluorescence. RESULTS ~55% of people with glioblastoma have detectable cytoplasmic AR based on immunohistochemistry. AR-positive patient-derived glioma stem cell line RN1 was inhibited by low concentrations of anti-androgen agents, with seviteronel having the lowest half maximal inhibitory concentration (IC50) (at 96 hours: enzalutamide 52µM, abiraterone 12µM, seviteronel 7µM). AR-negative line WK1 was also inhibited though with higher IC50s (at 96 hours: enzalutamide 63µM, seviteronel 21µM). Conventional cell lines U87 and U251 were also inhibited by anti-androgen monotherapy. Tumoursphere assays demonstrated that anti-androgen agents inhibit the tumour-forming ability of cells, with seviteronel and abiraterone showing more inhibition than enzalutamide. Immunofluorescence analysis showed downregulation of plasticity marker ZEB1 with anti-androgen treatment (normalised mean fluorescent intensity: abiraterone 0.84, enzalutamide 0.91, seviteronel 0.74). CONCLUSIONS Glioblastoma is a devastating disease, lacking effective or targeted treatments. Targeting AR with repurposed anti-androgen drugs may be a promising therapeutic strategy, with the potential to abrogate treatment resistance. We are now evaluating these anti-androgen regimens in animal experiments, which will provide a more replicative microenvironment and confirm blood-brain barrier penetrance.

Cytoplasmic Androgen Receptor, CD24 Expression and Smoking Intensity to Urothelial Carcinoma of the Bladder Invasiveness: A Cross-Sectional Study

Cytoplasmic Androgen Receptor, CD24 Expression and Smoking Intensity to Urothelial Carcinoma of the Bladder Invasiveness: A Cross-Sectional Study

The triazole fungicide metconazole inhibits the homodimerization of human androgen receptors to suppress androgen-induced transcriptional activation

We assessed the mechanism of human androgen receptor-mediated endocrine-disrupting effect by a triazole fungicide, metconazole in this study. The internationally validated stably transfected transactivation (STTA) in vitro assay, which was established for determination of a human androgen receptor (AR) agonist/antagonist by using 22Rv1/MMTV_GR-KO cell line, alongside an in vitro reporter-gene assay to confirm AR homodimerization was used. The STTA in vitro assay results showed that metconazole is a true AR antagonist. Furthermore, the results from the in vitro reporter-gene assay and western blotting showed that metconazole blocks the nuclear transfer of cytoplasmic AR proteins by suppressing the homodimerization of AR. These results suggest that metconazole can be considered to have an AR-mediated endocrine-disrupting effect. Additionally, the evidence from this study might help identify the endocrine-disrupting mechanism of triazole fungicides containing a phenyl ring.

Bicalutamide and Trehalose Ameliorate Spinal and Bulbar Muscular Atrophy Pathology in Mice

Spinal and bulbar muscular atrophy (SBMA) is characterized by motor neuron (MN) degeneration that leads to slowly progressive muscle weakness. It is considered a neuromuscular disease since muscle has a primary role in disease onset and progression. SBMA is caused by a CAG triplet repeat expansion in the androgen receptor (AR) gene. The translated poly-glutamine (polyQ) tract confers a toxic gain of function to the mutant AR altering its folding, causing its aggregation into intracellular inclusions, and impairing the autophagic flux. In an in vitro SBMA neuronal model, we previously showed that the antiandrogen bicalutamide and trehalose, a natural disaccharide stimulating autophagy, block ARpolyQ activation, reduce its nuclear translocation and toxicity and facilitate the autophagic degradation of cytoplasmic AR aggregates. Here, in a knock-in SBMA mouse model (KI AR113Q), we show that bicalutamide and trehalose ameliorated SBMA pathology. Bicalutamide reversed the formation of the AR insoluble forms in KI AR113Q muscle, preventing autophagic flux blockage. We demonstrated that apoptosis is activated in KI AR113Q muscle, and that both compounds prevented its activation. We detected a decrease of mtDNA and an increase of OXPHOS enzymes, already at early symptomatic stages; these alterations were reverted by trehalose. Overall, bicalutamide and/or trehalose led to a partial recovery of muscle morphology and function, and improved SBMA mouse motor behavior, inducing an extension of their survival. Thus, bicalutamide and trehalose, by counteracting ARpolyQ toxicity in skeletal muscle, are valuable candidates for future clinical trials in SBMA patients.

Abstract C036: Correlation of Kaiso and androgen receptor expression in women of African ancestry with triple-negative breast cancer

Abstract Breast cancer (BCa) is the most frequently diagnosed female cancer and a leading cause of female deaths worldwide, regardless of increased awareness and improved therapies. Triple-negative breast cancer (TNBC) is one of the most challenging BCa subtypes as it is highly heterogeneous, metastatic in nature and has limited targeted therapies. TNBC is most prevalent in young women of African ancestry (WAA), who despite having lower BCa rates, have a disproportionately higher mortality rate compared to women of European ancestry (WEA). However, the cause for this racial disparity is currently unknown, thus highlighting the importance of unraveling the genetic and molecular factors that contribute to TNBC in WAA. Our lab previously showed that the transcription factor Kaiso may be linked to this disparity as it contributes to increased metastasis and mortality in WAA TNBC patients. Interestingly, multiple studies have indicated that WAA TNBC tissues also express less Androgen Receptor (AR) than WEA TNBC tissues, supporting the existence of a novel BCa subtype – quadruple-negative breast cancer (QNBC). Notably, in silico analysis revealed several Kaiso binding sites in the AR promoter region, and that high Kaiso and low AR expression correlated with poorer overall survival in BCa patients. Thus, we hypothesized that high Kaiso and low AR expression could be contributing to the increased mortality in WAA with TNBC. This study seeks to examine the relationship between Kaiso and AR, the clinical significance of this relationship and its role, if any, in TNBC racial disparities. Using tissue microarrays (TMAs) and immunohistochemistry (IHC), we found reduced and cytoplasmic AR expression in WAA compared to WEA. Moreover, preliminary findings from western blot analyses showed an increase in AR expression in response to Kaiso depletion in TNBC cells. These findings suggest that AR could be a bona fide Kaiso target gene and that there may be clinical relevance of high Kaiso, and low AR expression in BCa survival, especially in patients with an African heritage. Ongoing experiments are focused on determining if Kaiso directly associates with the endogenous AR promoter region and discerning the link between high Kaiso and low AR expression in TNBC racial disparities. A significant correlation between Kaiso and AR expression will help to validate the existence of QNBC in WAA and support Kaiso and AR as clinically relevant prognostic markers for TNBC, especially in WAA. Citation Format: Stephanie Ali Fairbairn, Robert Cowan, Juliet M. Daniel, Shawn M. Hercules. Correlation of Kaiso and androgen receptor expression in women of African ancestry with triple-negative breast cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C036.

Downregulation of miR-99b-5p and Upregulation of Nuclear mTOR Cooperatively Promotes the Tumor Aggressiveness and Drug Resistance in African American Prostate Cancer.

Mammalian target of rapamycin (mTOR) regulates various fundamental cellular events including cell proliferation, protein synthesis, metabolism, apoptosis, and autophagy. Tumor suppressive miR-99b-5p has been implicated in regulating PI3K/AKT/mTOR signaling in a variety of types of cancer. Our previous study suggested the reciprocal miR-99b-5p/MTOR (downregulated/upregulated) pairing as a key microRNA-mRNA regulatory component involved in the prostate cancer (PCa) disparities. In this study, we further validated the expression profiles of mTOR and miR-99b-5p in the PCa, colon, breast, and lung cancer specimens and cell lines. The immunohistochemistry (IHC), immunofluorescence, Western blot, and RT-qPCR assays have confirmed that mTOR is upregulated while miR-99b-5p is downregulated in different patient cohorts and a panel of cancer cell lines. Intriguingly, elevated nuclear mTOR expression was observed in African American PCa and other advanced cancers. Transfection of the miR-99b-5p mimic resulted in a significant reduction in nuclear mTOR and androgen receptor (AR), while a slight/moderate to no decrease in cytoplasmic mTOR and AR in PCa and other cancer cells, suggesting that miR-99b-5p inhibits mTOR and AR expression and their nuclear translocation. Moreover, overexpression of miR-99b-5p targets/inhibits AR-mTOR axis, subsequently initiating cell apoptosis and sensitizing docetaxel-induced cytotoxicity in various cancers. In conclusion, our data suggest that reciprocal miR-99b-5p/nuclear mTOR pairing may be a more precise diagnostic/prognostic biomarker for aggressive PCa, than miR-99b-5p/MTOR pairing or mTOR alone. Targeting the AR-mTOR axis using miR-99b-5p has also been suggested as a novel therapeutic strategy to induce apoptosis and overcome chemoresistance in aggressive PCa.

Abstract 1029: The anti-androgen seviteronel sensitizes triple-negative breast cancer to chemotherapy

Abstract Therapeutic strategies that improve survival outcomes for advanced-stage breast cancers have proven a major clinical challenge. Here, we define an androgen receptor (AR) signalling network that governs the maintenance and de novo formation of cancer stem cells in triple-negative breast cancer. In response to chemotherapy, AR activation switches cells into a cancer stem cell state, while AR antagonism suppresses cancer stem cell formation and function. In vivo, we validate that the AR antagonist, seviteronel, significantly improves chemotherapy-mediated inhibition of primary and metastatic tumour growth. Analysing three independent triple-negative breast cancer patient cohorts we identify that cytoplasmic AR expression prognosticates poor survival in treatment-naïve patients and predicts poor response to chemotherapy. Additionally, Phase II clinical trial data demonstrates that seviteronel treatment followed by chemotherapy improved survival outcomes in triple-negative breast cancer patients expressing high cytoplasmic AR levels. Seviteronel combined with chemotherapy represents a promising therapeutic strategy for triple-negative breast cancer patients. Citation Format: Beatriz P. San Juan, Soroor Hediyah-Zadeh, Laura Rangel, Vanina Rodriguez, Heloisa H. Milioli, Felix Kohane, Carley A. Purcell, Therese E. Hickey, Leonard D. Goldstein, John G. Lock, Melissa J. Davis, Christine L. Chaffer. The anti-androgen seviteronel sensitizes triple-negative breast cancer to chemotherapy [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 1029.

Abstract 2572: γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple negative breast cancer (TNBC) MBA-MB-231 and MDA-MB-453 cells is associated with a reduction in proliferation, migration and epithelial-to-mesenchymal transition (EMT)

Abstract Triple negative breast cancer (TNBC) is an aggressive invasive malignancy with the lowest 5-years survival rate. Gene expression profiling indicates that TNBC is a heterogeneous disease and at present, there is no targeted therapy available for treatment. Approximately one third of TNBC expressed androgen receptor (AR) and evaluation of AR positive TNBC primary tumors shows nuclear localization of AR, an indication of transcriptionally active receptors. AR levels are most abundant in the luminal AR (LAR) molecular subtype of TNBC, but other non-LAR molecular TNBC subtypes also display high levels of AR expression and activity. Previous studies have shown that AR inhibition or AR knockdown significantly reduces baseline proliferation, anchorage-independent growth, migration and invasion and increase apoptosis in different TNBC cell lines. γ-Tocotrienol is a natural isoform of vitamin E that displays potent anticancer activity. Studies were conducted to determine if γ-tocotrienol effects on AR levels and activity play a role in mediating γ-tocotrienol induced inhibition of TNBC cell proliferation, migration and epithelial-to-mesenchymal transition (EMT). In vitro studies showed that treatment with 0-12 μM γ-tocotrienol induced a dose-responsive significant decrease in TNBC MDA-MB-231 and MDA-MB-453 cell proliferation and viability. Treatment with 35 nM dihydrotestosterone (DHT) resulted in an increase of AR expression and corresponding increase in the growth of both TNBC cell lines. Treatment of MDA-MB-231 and MDA MB-453 cells with γ-tocotrienol (5 μM and 7 μM, respectively), significantly inhibited DHT-induced proliferation of both TNBC cell lines. Western blot analysis showed that γ-tocotrienol treatment significantly reduced DHT-induced cytoplasmic and nuclear AR expression in MDA-MB-453 cells, but induced only a slight and insignificant reduction in AR expression in MDA-MB-231 cells. Immunocytochemistry studies confirmed that γ-tocotrienol treatment induced a decrease in cytoplasmic and nuclear AR expression in both TNBC cell lines, but this decrease was only found to be significant in MDA-MB-453 cells. Other studies showed that γ-tocotrienol treatment significantly reduced cancer cell migration and this finding was associated with an increased expression in cytokeratin 8 (an epithelial cell biomarker) and decrease in vimentin (a mesenchymal cell biomarker) in both TNBC cell lines, and is an indication of a reversal in EMT. In summary, these results demonstrated that γ-tocotrienol treatment inhibits AR expression as well as DHT-dependent cell proliferation, migration and EMT in TNBC. These findings also suggest that AR may be a potential therapeutic target for the treatment of both LAR and non-LAR TNBC subtypes. Citation Format: Tasmin A. Sultana, Nayef Aldabaan, Paul W. Sylvester. γ-Tocotrienol inhibition of androgen receptor (AR) expression and activation in triple negative breast cancer (TNBC) MBA-MB-231 and MDA-MB-453 cells is associated with a reduction in proliferation, migration and epithelial-to-mesenchymal transition (EMT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2572.

Ligand Binding Prolongs Androgen Receptor Protein Half-Life by Reducing its Degradation.

Androgens are important in female reproduction, but the molecular actions of androgens in female reproductive tissues are not fully understood. We investigated the androgen-responsive transcriptome in human and mouse granulosa cells (GCs) and surprisingly found that the gene-regulation activity of androgen receptor (AR) in these cells is negligible. We then investigated extranuclear actions of AR and found that in human and mouse GCs, as well as in prostate cancer cells, dihydrotestosterone (DHT) dramatically increases the half-life of its own receptor protein. Using the human granulosa-like KGN cells, we show that this effect is not the result of increased AR gene transcription or protein synthesis, nor is it fully abrogated by proteasome inhibition. Knockdown of PTEN, which contributes to degradation of cytoplasmic AR, did not diminish AR accumulation in the presence of DHT. Using immunofluorescence cellular localization studies, we show that nuclear AR is selectively protected from degradation in the presence of DHT. Knockdown of importin 7 expression, a potential regulator of AR nuclear import, does not affect DHT-mediated nuclear accumulation of AR, suggesting importin 7-independent nuclear import of AR in GCs. Further, DNA binding is not required for this protective mechanism. In summary, we show that ligand binding sequesters AR in the nucleus through enhanced nuclear localization independent of DNA binding, thereby protecting it from proteasome degradation in the cytoplasm. This phenomenon distinguishes AR from other sex steroid receptors and may have physiological significance through a positive feedback loop in which androgen induces its own activity in male and female reproductive tissues.

Expression of AR-V7 (Androgen Receptor Variant 7) Protein in Granular Cytoplasmic Structures Is an Independent Prognostic Factor in Prostate Cancer Patients.

Simple SummaryThe expression of the androgen receptor (AR) and its splice variant AR-V7 is crucial for prostate cancer (PCa) biology. An immunohistochemical staining was performed on a tissue microarray with specimens from 410 PCa patients. AR staining, neither in the nucleus nor in the cytoplasm was associated with prognosis. AR-V7 staining of the general cytoplasm was associated with a shorter relapse free survival (RFS), whereas AR-V7 staining of cytoplasmic granules was associated with a longer RFS. Further subgroup stratification for AR-V7 granular staining revealed it as an independent prognostic factor in younger patients (age ≤ 65), patients with negative CK20 staining and patients with perineural invasion. Altogether, AR-V7 protein detected in granular cytoplasmic structures is an independent prognostic factor for RFS in PCa patients.Prostate cancer (PCa) is the second most common cancer, causing morbidity and mortality among men world-wide. The expression of the androgen receptor (AR) and its splice variants is a crucial factor of prostate cancer biology that has not been comprehensively studied in PCa tumors. The aim of this study was to characterize the protein expression of the AR and its splice variant, AR-V7, and their subcellular distributions in PCa by immunohistochemistry and to correlate the results to the clinicopathological data and prognosis. Immunohistochemical staining for AR and AR-V7 was performed on a tissue microarray (TMA) with specimens from 410 PCa patients using an immunoreactive score (IRS) or only the percentage of AR-V7 staining in cytoplasmic granules. Nuclear or cytoplasmic AR staining was not associated with prognosis. AR-V7 staining was only occasionally observed in the nucleus. However, AR-V7 staining in the cytoplasm or in cytoplasmic granules was associated with relapse-free survival (RFS). AR-V7 staining of the cytoplasm was associated with a shorter RFS, whereas AR-V7 staining of cytoplasmic granules was associated with a longer RFS. In a multivariate Cox’s regression analysis, only negative (<5%) AR-V7 staining of cytoplasmic granules remained an independent prognostic factor for RFS (HR = 5.3; p = 0.006). In a further subgroup analysis by multivariate Cox’s regression analysis, AR-V7 was an independent prognostic factor in the following groups: age ≤ 65 (HR = 9.7; p = 0.029), negative CK20 staining (HR = 7.0; p = 0.008), and positive perineural invasion (HR = 3.7; p = 0.034). Altogether, AR-V7 protein in granular cytoplasmic structures is an independent prognostic factor for RFS in PCa patients.

Inflammatory infiltration is associated with AR expression and poor prognosis in hormone naïve prostate cancer.

Tumor microenvironment inflammatory infiltration is proposed as a protumorigenic mechanism for prostate cancer with proinflammatory cytokines stimulating androgen receptor (AR) activity. However, association with patient prognosis remains unclear. This study derives an inflammatory gene signature associated with AR expression and investigates CD3+ and CD8+ T-lymphocyte infiltration association with AR and prognosis. Gene profiling of inflammatory related genes was performed on 71 prostate biopsies. Immunohistochemistry on 243 hormone-naïve prostate cancers was performed for CD3, CD8, AR, and phosphorylated AR tumor expression. Multiple proinflammatory genes were differentially expressed in association with high AR expression compared with low AR expression including PI3KCA and MAKP8 (adjusted P < .05). High CD3+ and high CD8+ infiltration associated with reduced cancer-specific survival (P = .018 and P = .020, respectively). High CD3+ infiltration correlated with high tumor cytoplasmic AR expression and if assessed together, they associated with reduced cancer-specific and 5-year survival from 90% to 56% (P = .000179). High CD8+ cytotoxic infiltration associated with high androgen-independent tumor nuclear AR serine 213 phosphorylation (correlation coefficient = 0.227; P = .003) and when assessed together associated with poor clinico-pathological features including perineural invasion (P = .001). Multiple genes involved in proinflammatory signaling pathways are upregulated in high AR expressing prostate samples. T-lymphocyte infiltration in hormone-naïve disease associates with androgen-independent driven disease and provides possible therapeutic targets to reduce transformation from hormone-naïve to castrate-resistant disease.

Abstract 5451: Longitudinal proteomic assessment of patient with metastatic apocrine adenocarcinoma reveals evolutionary selection for androgen-receptor-dependence and therapeutic response

Abstract Combined use of new molecular diagnostic and imaging methods could improve care of individual patients with rare cancers. A 57-year-old developed apocrine adenocarcinoma of the left axilla with regional lymph node metastasis detected at initial surgery. After adjuvant regional radiotherapy, metastases developed and over the subsequent 7 years he received serial systemic therapy with 11 different regimens. Expression of 128 proteins/phosphoproteins was evaluated in 8 tissue samples from diagnosis, progressive adenopathy at 45 months, brain metastases at 60 months, and abdominal metastases at 63 months by CLIA-standardized reverse phase phosphoprotein arrays (RPPA). Tumor burden was estimated by quantifying the volume of individual lesions with semi-automated segmentation algorithms on serially collected CT images. Next-generation sequencing (NGS) revealed a PIK3CA K111T mutation, prompting treatment with sirolimus 28 months after diagnosis. Seventeen months later, new neoplastic adenopathy emerged. NGS revealed a new PIK3CA M610V mutation. Semiquantitative scoring of RPPA demonstrated relative increase in AKT (from 0.72 to 0.9) and mTOR (from 0.55 to 0.8) pathway activation. The relative estimated tumor burden (RETB) was 21,145 mm3. Over 6 months of docetaxel therapy the RETB decreased to 12,077 mm3. The patient subsequently received pembrolizumab with increased RETB (71,308 mm3). Gemcitabine appeared to stabilize the RETB (73,168 mm3), but brain metastases emerged. The patient underwent craniotomy, began paclitaxel therapy, and then RETB declined to 35,623 mm3. But new symptomatic abdominal metastases prompted surgical resection. The patient then received the PI3K inhibitor taselisib; however, after an initial decrease, RETB rose to 31,208 mm3. At this point, the patient began bicalutamide therapy. NGS of the primary mass and the initial metastatic adenopathy revealed no amplification of the androgen receptor (AR). But RPPA demonstrated inversion of the ratio of measured phosphorylated AR S81 to AR S650, suggesting primarily cytoplasmic AR in tumor through 45 months but nuclear AR in samples resected at 60 and 63 months. Immunohistochemistry revealed 3+ nuclear AR expression in the latter metastatic tissue. After 9 months of bicalutamide, enzalutamide and leuprolide were administered for 7 months (final RETB = 1,555 mm3). Treatment continued beyond the study period. Tumor burden assessment by new CT imaging measurement methods, combined with RPPA could improve adaptive, responsive, therapy for patients with rare tumors. We identified the phenotypic evolution of androgen-driven growth of apocrine adenocarcinoma after cytotoxic and PI3K-mTOR inhibitor therapy. Protein-based diagnostics revealed an effective treatment strategy in late metastatic disease that was not indicated by NGS testing. Citation Format: Erik Dvergsten, Anthony Serritella, Danielle Kimble, Malcom McIver, Sanja Karovic, Vasiliki Thomeas-McEwing, Hao Yang, Manish R. Sharma, Thomas P. Conrads, Mariaelena Pierobon, Peter Pytel, Binsheng Zhao, Lawrence H. Schwartz, Emanuel F. Petricoin, Russell Szmulewitz, Michael L. Maitland. Longitudinal proteomic assessment of patient with metastatic apocrine adenocarcinoma reveals evolutionary selection for androgen-receptor-dependence and therapeutic response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5451.

Increased nuclear factor I/B expression in prostate cancer correlates with AR expression.

Most prostate cancers express androgen receptor (AR), and our previous studies have focused on identifying transcription factors that modify AR function. We have shown that nuclear factor I/B (NFIB) regulates AR activity in androgen-dependent prostate cancer cells in vitro. However, the status of NFIB in prostate cancer was unknown. We immunostained a tissue microarray including normal, hyperplastic, prostatic intraepithelial neoplasia, primary prostatic adenocarcinoma, and castration-resistant prostate cancer tissue samples for NFIB, AR, and synaptophysin, a marker of neuroendocrine differentiation. We interrogated publically available data sets in cBioPortal to correlate NFIB expression and AR and neuroendocrine prostate cancer (NEPCa) activity scores. We analyzed prostate cancer cell lines for NFIB expression via Western blot analysis and used nuclear and cytoplasmic fractionation to assess where NFIB is localized. We performed co-immunoprecipitation studies to determine if NFIB and AR interact. NFIB increased in the nucleus and cytoplasm of prostate cancer samples versus matched normal controls, independent of Gleason score. Similarly, cytoplasmic AR and synaptophysin increased in primary prostate cancer. We observed strong NFIB staining in primary small cell prostate cancer. The ratio of cytoplasmic-to-nuclear NFIB staining was predictive of earlier biochemical recurrence in prostate cancer, once adjusted for tumor margin status. Cytoplasmic AR was an independent predictor of biochemical recurrence. There was no statistically significant difference between NFIB and synaptophysin expression in primary and castration-resistant prostate cancer, but cytoplasmic AR expression was increased in castration-resistant samples. In primary prostate cancer, nuclear NFIB expression correlated with cytoplasmic NFIB and nuclear AR, while cytoplasmic NFIB correlated with synaptophysin, and nuclear and cytoplasmic AR. In castration-resistant prostate cancer samples, NFIB expression correlated positively with an AR activity score, and negatively with the NEPCa score. In prostate cancer cell lines, NFIB exists in several isoforms. We observed NFIB predominantly in the nuclear fraction of prostate cancer cells with increased cytoplasmic expression seen in castration-resistant cell lines. We observed an interaction between AR and NFIB through co-immunoprecipitation experiments. We have described the expression pattern of NFIB in primary and castration-resistant prostate cancer and its positive correlation with AR. We have also demonstrated AR interacts with NFIB.

Tumor suppressor REIC/Dkk-3 and its interacting protein SGTA inhibit glucocorticoid receptor to nuclear transport.

REIC/Dkk-3 is a tumor suppressor, and its expression is significantly downregulated in a variety of human cancer types. A previous study performed yeast two-hybrid screening and identified the small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), known as a negative modulator of cytoplasmic androgen receptor (AR) signaling, which is a novel interacting partner of REIC/Dkk-3. The previous study also indicated that the REIC/Dkk-3 protein interferes with the dimerization of SGTA and then upregulates the AR transport and signaling in human prostate cancer PC3 cells. Since the transport of some steroid receptors to nucleus is conducted similarly by dynein motor-dependent way, the current study aimed to investigate the role of SGTA and REIC/Dkk-3 in the transport of other glucocorticoid receptors (GR). In vitro reporter assays for the cytoplasmic GR transport were performed in human prostate cancer PC3 cells and 293T cells. As for the SGTA protein, a suppressive effect on the GR transport to the nucleus was observed in the cells. As for the REIC/Dkk-3 protein, an inhibitory effect was observed for the GR transport in PC3 cells. Under the depleted condition of SGTA by short-hairpin (sh)RNA, the downregulation of GR transport by REIC/Dkk-3 was significantly enhanced compared with the non-depleted condition in PC3 cells, suggesting a compensatory role of REIC/Dkk-3 in the SGTA mediated inhibition of GR transport. The current study therefore demonstrated that SGTA inhibited the cytoplasmic transport of GR in 293T and PC3 cells, and REIC/Dkk-3 also inhibited the cytoplasmic transport of GR in PC3 cells. These results may be used to gain novel insight into the GR transport and signaling in normal and cancer cells.

Targeting prostate cancer cells with enzalutamide-HDAC inhibitor hybrid drug 2-75.

The progression of castration-resistant prostate cancer (CRPC) still relies on the function of androgen receptor (AR), achieved by evolving mechanisms to reactivate AR signaling under hormonal therapy. Histone deacetylase inhibitors (HDACis) disrupt cytoplasmic AR chaperone heat shock protein 90 (Hsp90) via HDAC6 inhibition, leading to AR degradation and growth suppression of prostate cancer (PCa) cells. However, current HDACis are not effective in clinical trials treating CRPC. We designed hybrid molecules containing partial chemical scaffolds of AR antagonist enzalutamide (Enz) and HDACi suberoylanilide hydroxamic acid (SAHA) as new anti-PCa agents. We previously demonstrated that Enz-HDACi hybrid drug 2-75 targets both AR and Hsp90, which inhibits the growth of Enz-resistant C4-2 cells. In the current study, we further investigate the molecular and cellular actions of 2-75 and test its anti-PCa effects in vivo. Compared with Enz, 2-75 had greater AR antagonistic effects by decreasing the stability, transcriptional activity, and nuclear translocation of intracellular AR. In addition to inhibition of full-length AR (FL AR), 2-75 downregulated the AR-V7 variant in multiple PCa cell lines. Mechanistic studies indicated that the AR affinity of 2-75 retains the drug in the cytoplasm of AR + PCa cells and further directs 2-75 to the AR-associated protein complex, which permits localized effects on AR-associated Hsp90. Further, unlike pan-HDACi SAHA, the cytoplasm-retaining property allows 2-75 to significantly inhibit cytoplasmic HDAC6 with limited impact on nuclear HDACs. These selective cytoplasmic actions of 2-75 overcome the unfavorable resistance and toxicity properties associated with classical AR antagonists, HDACis, and Hsp90 inhibitors. Finally, 2-75 showed greater antitumor activities than Enz in vivo on SQ xenografts derived from LNCaP cells. Novel therapeutic strategy using newly designed 2-75 and related AR antagonist-HDACi hybrid drugs has great potential for effective treatment of CRPC.

Impaired Nuclear Export of Polyglutamine-Expanded Androgen Receptor in Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by polyglutamine (polyQ) expansion in the androgen receptor (AR). Prior studies have highlighted the importance of AR nuclear localization in SBMA pathogenesis; therefore, in this study, we sought to determine the role of AR nuclear export in the pathological manifestations of SBMA. We demonstrate here that the nuclear export of polyQ-expanded AR is impaired, even prior to the formation of intranuclear inclusions of aggregated AR. Additionally, we find that promoting AR export with an exogenous nuclear export signal substantially reduces its aggregation and blocks hormone-induced toxicity. Moreover, we show that these protective effects are conferred by destabilization of the mutant protein due to an increase in proteasomal degradation of the cytoplasmic AR. Despite a growing body of evidence that global disruption of nucleo/cytoplasmic transport occurs in ALS and HD, our data suggest that no such global disruption occurs in models of SBMA; rather, AR-specific mechanisms, including reduced phosphorylation at Serine 650, are likely responsible for the impaired nuclear export of polyQ-expanded AR.

The Activity of Urolithin A and M4 Valerolactone, Colonic Microbiota Metabolites of Polyphenols, in a Prostate Cancer In Vitro Model.

The gut microbiota-derived metabolites of ellagitannins and green tea catechins, urolithin A (uroA) and 5-(3',4',5'-trihydroxyphenyl)-γ-valerolactone (M4), respectively, are among the main compounds absorbed into human system after ingestion of these polyphenols. The aim of this study was to establish the effects of M4, uroA, and their combinations on LNCaP cells, an androgen dependent prostate cancer in vitro model.. The LNCaP cells were incubated with increasing concentrations of tested metabolites. The cell proliferation was determined by measurement of DNA-bisbenzimide H 33 258 complexes fluorescence. The isobolographic analysis was used to establish the type of interaction between metabolites. The apoptosis, androgen receptor (AR) localization, and phosphorylation of Akt kinase were measured by flow cytometry. Prostate-specific antigen (PSA) secretion was determined by ELISA. M4 showed modest antiproliferative activity in LNCaP cells (IC50 = 117 µM; CI: 81 - 154). UroA decreased proliferation (IC50 = 32.7 µM; CI: 24.3 - 41.1) and induced apoptosis of LNCaP cells. The mixture of M4 with uroA had synergistic antiproliferative effect. Moreover, M4 potentiated inhibition of PSA secretion and enhanced retention of AR in cytoplasm caused by uroA. Interestingly, uroA increased levels of pSer473 Akt in LNCaP cells. These results show that colonic metabolites may contribute to chemoprevention of prostate cancer by varied polyphenol-rich diet or composite polyphenol preparations.

Abstract A024: Prostate cancer neoadjuvant intensive androgen deprivation therapy selects for tumor foci with diverse oncogenic alterations

Abstract Based on the hypothesis that early use of androgen deprivation therapy (ADT) may improve outcomes, we conducted a phase 2 trial of neoadjuvant leuprolide for 24 weeks in combination with abiraterone acetate plus prednisone (referred to subsequently as leuprolide plus AAP) for 12 or 24 weeks prior to radical prostatectomy (RP). As reported recently, we confirmed that the addition of AAP further markedly reduced intraprostatic androgen levels and appeared to improve responses relative to historical controls using single-agent GnRH agonists. Nonetheless, residual prostate cancer (PCa) was found in the majority of patients, with only a small number of patients demonstrating complete pathologic responses. Moreover, substantial nuclear and cytoplasmic AR expression was detected by immunohistochemistry in most cases, suggesting that AR activity still may persist and contribute to residual disease. Residual PCa foci in RPs from 18 men treated with neoadjuvant intensive androgen deprivation therapy (leuprolide plus AAP) were microdissected and analyzed for resistance mechanisms. Transcriptome profiling showed reduced but persistent androgen receptor (AR) activity in residual tumors, with no increase in neuroendocrine differentiation. Unexpectedly, proliferation was negatively correlated with AR activity, but positively correlated with decreased RB1 expression, and whole-exome sequencing (WES) further showed enrichment for RB1 genomic loss. In 14 cases where two tumor foci were microdissected, WES confirmed a common origin, but identified multiple oncogenic alterations unique to one focus. Primary PCa can have extensive microheterogeneity, but its contribution to the later emergence of metastatic castration-resistant PCa (mCRPC) has not been clear. These findings indicate that neoadjuvant intense androgen deprivation therapy selects for subclonal genomic alterations, including RB1 loss, which may be the origin for metastatic castration-resistant PCa. This study indicates that subclonal RB1 loss may be more common than previously appreciated in intermediate- to high-risk primary PCa, and may be an early event, independent of neuroendocrine differentiation, in the development of mCRPC. Comprehensive molecular analyses of primary PCa may detect aggressive subclones, and possibly inform on adjuvant strategies to prevent the emergence of mCRPC. Citation Format: Adam G. Sowalsky, Huihui Ye, Manoj Bhasin, Eliezer M. Van Allen, Massimo Loda, Rosina T. Lis, Laleh Montaser, Carla Calagua, Fen Ma, Joshua W. Russo, Rachel J. Schaefer, Olga S. Voznesensky, Zhenwei Zhang, Glenn J. Bubley, Robert B. Montgomery, Elahe A. Mostaghel, Peter S. Nelson, Mary-Ellen Taplin, Steven P. Balk. Prostate cancer neoadjuvant intensive androgen deprivation therapy selects for tumor foci with diverse oncogenic alterations [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A024.