Androgen Receptor Phosphorylation Research Articles

A plasma membrane-associated form of the androgen receptor enhances nuclear androgen signaling in osteoblasts and prostate cancer cells.

Androgen binding to the androgen receptor (AR) in the cytoplasm induces the AR to translocate to the nucleus, where it regulates the expression of target genes. Here, we found that androgens rapidly activated a plasma membrane-associated signaling node that enhanced nuclear AR functions. In murine primary osteoblasts, dihydrotestosterone (DHT) binding to a membrane-associated form of AR stimulated plasma membrane-associated protein kinase G type 2 (PKG2), leading to the activation of multiple kinases, including ERK. Phosphorylation of AR at Ser515 by ERK increased the nuclear accumulation and binding of AR to the promoter of Ctnnb1, which encodes the transcription factor β-catenin. In male mouse osteoblasts and human prostate cancer cells, DHT induced the expression of Ctnnb1 and CTNN1B, respectively, as well as β-catenin target genes, stimulating the proliferation, survival, and differentiation of osteoblasts and the proliferation of prostate cancer cells in a PKG2-dependent fashion. Because β-catenin is a master regulator of skeletal homeostasis, these results explain the reported male-specific osteoporotic phenotype of mice lacking PKG2 in osteoblasts and imply that PKG2-dependent AR signaling is essential for maintaining bone mass in vivo. Our results suggest that widely used pharmacological PKG activators, such as sildenafil, could be beneficial for male and estrogen-deficient female patients with osteoporosis but detrimental in patients with prostate cancer.

Androgen-repressed lncRNA LINC01126 drives castration-resistant prostate cancer by regulating the switch between O-GlcNAcylation and phosphorylation of androgen receptor.

Prostate cancer (PCa) initially shows satisfactory response to therapies targeting the androgen receptor (AR). However, progression to a castration-resistant stage indicates poor prognosis in PCa patients. AR signalling still plays a central role in most castration-resistant prostate cancers (CRPC). Therefore, unveiling the mechanisms of AR reactivation under androgen-deprived conditions is imperative to discover novel therapeutic targets for CRPC. Using an integrative analysis of the transcriptomics of three independent PCa cohorts and a published landscape of AR-regulated long non-coding RNA (lncRNA), lncRNA LINC01126 was selected as a candidate gene that could drive CRPC progression for further study. Quantitative reverse transcription polymerase chain reaction, in situ hybridisation (ISH) and fluorescent ISH were performed to detect LINC01126 in PCa tissues and cells. The functional role and mechanism of LINC01126 were further investigated using in vitro and in vivo gain and loss of function assays. LINC01126, identified as an AR-repressed lncRNA, was significantly upregulated after AR-targeted therapies. In addition, we found that LINC01126 was upregulated in CRPC and was associated with poor prognosis. We also proved that LINC01126 stabilised AR protein and enhanced AR nuclear translocation and transactivation by promoting the transition from O-GlcNAcylation at threonine 80 to phosphorylation at serine 81 (S81) within the AR protein. Mechanism analysis revealed that LINC01126 facilitates the interaction of CDK9 with AR and impedes the binding of O-linked N-acetylglucosamine (O-GlcNAc) transferase to AR. Consequently, LINC01126 expression was sufficient to activate AR signalling without androgen. LINC01126 overexpression increased, whereas LINC01126 knockdown decreased castration resistance traits in PCa cells in vitro and in vivo. Furthermore, our data showed that LINC01126-targeting antisense oligonucleotides (ASO) substantially inhibited CRPC cells in vitro. Our research expands the functions of AR-regulated lncRNA in sustaining androgen-independent AR activity and promoting CRPC progression and reveals that LINC01126 may be a new therapeutic target for PCa.

Newly developed preclinical models reveal broad-spectrum CDK inhibitors as potent drugs for CRPC exhibiting primary resistance to enzalutamide.

Androgen-deprivation therapy is a standard treatment for advanced prostate cancer. However, most patients eventually acquire resistance and progress to castration-resistant prostate cancer (CRPC). In this study, we established new CRPC cell lines, AILNCaP14 and AILNCaP15, from LNCaP cells under androgen-deprived conditions. Unlike most pre-existing CRPC cell lines, both cell lines expressed higher levels of androgen receptor (AR) and prostate-specific antigen (PSA) than parental LNCaP cells. Moreover, these cells exhibited primary resistance to enzalutamide. Since AR signaling plays a significant role in the development of CRPC, PSA promoter sequences fused with GFP were introduced into AILNCaP14 cells to conduct GFP fluorescence-based chemical screening. We identified flavopiridol, a broad-spectrum CDK inhibitor, as a candidate drug that could repress AR transactivation of CRPC cells, presumably through the inhibition of phosphorylation of AR on the serine 81 residue (pARSer81 ). Importantly, this broad-spectrum CDK inhibitor inhibited the proliferation of AILNCaP14 cells both in vitro and in vivo. Moreover, a newly developed liver metastatic model using AILNCaP15 cells revealed that the compound attenuated tumor growth of CRPC harboring highly metastatic properties. Finally, we developed a patient-derived xenograft (PDX) model of CRPC and DCaP CR from a patient presenting therapeutic resistance to enzalutamide, abiraterone, and docetaxel. Flavopiridol successfully suppressed the tumor growth of CRPC in this PDX model. Since ARSer81 was found to be phosphorylated in clinical CRPC samples, our data suggested that broad-spectrum CDK inhibitors might be a potent candidate drug for the treatment of CRPC, including those exhibiting primary resistance to enzalutamide.

Abstract A12: Androgen receptor inhibition is involved in Finasteride-reduced AML cell proliferation

Abstract Acute myeloid leukemia (AML) is a top priority type of hematological malignancies to be studied and treated. Androgen receptor (AR) plays important roles in various cancers, such as types of the prostate, breast, liver, and lung. However, the role of AR in leukemia is so far uncertain. Finasteride is a 5 alpha-reductase inhibitor for reduction of cellular 5 alpha-dihydrotestosterone production and commonly used to treat benign prostatic hyperplasia for decades. Here, the data showed that Finasteride treatment significantly decreased the proliferation of AML cell lines, including KG-1 and HL-60. More specific to its impact on androgen’s actions, the authors found that Finasteride inhibited the phosphorylation and activation of AR, while the cell cycle-related proteins such as CDK1, Cyclin A, and p21 were unaffected. Interestingly, the increase of AKT phosphorylation was observed after Finasteride treatment, which is correspondent to the previous findings of mutual correlations between AR inhibition and AKT activation in prostate cancer cells. It implies that AR might play a physiological role in AML cells for proliferation, in which AR closely collaborates with AKT for a balance of regulation. In summary, these results suggest that Finasteride might reduce the proliferation of AML cell lines through AR inhibition. These findings could be helpful to uncover novel AML treatments in the future. Citation Format: Pang-Ting Cheng, Chieh-Lin Jerry Teng, Yu-Chiao Cheng, Mei-Chih Chen, Ho Lin. Androgen receptor inhibition is involved in Finasteride-reduced AML cell proliferation [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A12.

Abstract 3892: EZH2 mediated kinome reprogramming drives AR phosphorylation and activation in receptor tyrosine kinase inhibitors resistant renal carcinoma models

Abstract Systemic treatment of recurrent/metastatic renal cell carcinoma (RCC) largely uses receptor tyrosine kinase inhibitors (RTKI), as single agents or in combination with immune checkpoint inhibitors. Unfortunately, patients commonly develop resistance to RTKI’s, leaving few options for further treatment. Therefore, understanding the mechanisms responsible for drug resistance is critical to improve the clinical outcome in this disease. Our previous studies of acquired resistance to the RTKI sunitinib identified two major regulators, EZH2 and androgen receptor (AR). Inhibition of EZH2 or AR in combination with sunitinib re-sensitizes cells to sunitinib both in vitro and in vivo. To further explore the link between EZH2 and AR in drug resistance, 786-0 RCC cells with acquired resistance to sunitinib (786-0RS) was compared to drug naive 786-0 cells with exogenous AR (786-0AR). Chromatin immunoprecipitation sequencing (ChIP-seq) showed that AR and EZH2 bind the same genomic locations, but only in the resistant strain. Interestingly, treatment of 786-0AR cells with sunitinib caused AR to translocate to the nucleus observed by the presence of AR at EZH2 bound sites. Additionally, mass spec of AR purified from 786-0RS cells and 786-0AR cells found high phosphorylation of S81 and S213, two markers associated with ligand independent activation, in 786-0RS cells, but not in 786-0AR cells. Previously, our lab has reported that 786-0RS cells display a reprogramming of the tyrosine kinome which is dependent on EZH2 likely via a non-canonical mechanism. Consistent with this, EZH2 with a phosphomimetic mutation in Serine 21 (S21D) could promote sunitinib resistance when over-expressed with AR, whereas wild-type and phosphonull (S21A) EZH2 could not. We have also expanded the serine/threonine phosphoproteomic analysis and found extensive rewiring of these signaling networks in 786-0RS cells with an enrichment of EGFR mediated MAPK and AKT signaling when compared to 786-0 cells. Finally, our lab has generated 786-0 strains resistant to other RTKI including pazopanib and lenvatinib, and found that AR is expressed in these resistant models, solidifying AR activation as a conserved feature of RTKI resistance. Overall, our results suggest that AR activation is driven by phosphorylation induced by EZH2 dependent kinome reprogramming. Untangling the cross talk between AR and EZH2 in RTKI resistant RCC will provide the rationale for novel therapeutic strategies to improve the clinical outcome in patients with this disease. Citation Format: Christopher Rupert, Stephanie Metcalf, Saranya Rajendran, Sean Colligan, Peter Hollenhorst, Roberto Pili. EZH2 mediated kinome reprogramming drives AR phosphorylation and activation in receptor tyrosine kinase inhibitors resistant renal carcinoma models. [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 3892.

Antagonistic effect of cyclin-dependent kinases and a calcium-dependent phosphatase on polyglutamine-expanded androgen receptor toxic gain of function.

Spinal and bulbar muscular atrophy is caused by polyglutamine (polyQ) expansions in androgen receptor (AR), generating gain-of-function toxicity that may involve phosphorylation. Using cellular and animal models, we investigated what kinases and phosphatases target polyQ-expanded AR, whether polyQ expansions modify AR phosphorylation, and how this contributes to neurodegeneration. Mass spectrometry showed that polyQ expansions preserve native phosphorylation and increase phosphorylation at conserved sites controlling AR stability and transactivation. In small-molecule screening, we identified that CDC25/CDK2 signaling could enhance AR phosphorylation, and the calcium-sensitive phosphatase calcineurin had opposite effects. Pharmacologic and genetic manipulation of these kinases and phosphatases modified polyQ-expanded AR function and toxicity in cells, flies, and mice. Ablation of CDK2 reduced AR phosphorylation in the brainstem and restored expression of Myc and other genes involved in DNA damage, senescence, and apoptosis, indicating that the cell cycle-regulated kinase plays more than a bystander role in SBMA-vulnerable postmitotic cells.

Kindlin-2 promotes Src-mediated tyrosine phosphorylation of androgen receptor and contributes to breast cancer progression

Androgen receptor (AR) signaling plays important roles in breast cancer progression. We show here that Kindlin-2, a focal adhesion protein, is critically involved in the promotion of AR signaling and breast cancer progression. Kindlin-2 physically associates with AR and Src through its two neighboring domains, namely F1 and F0 domains, resulting in formation of a Kindlin-2-AR-Src supramolecular complex and consequently facilitating Src-mediated AR Tyr-534 phosphorylation and signaling. Depletion of Kindlin-2 was sufficient to suppress Src-mediated AR Tyr-534 phosphorylation and signaling, resulting in diminished breast cancer cell proliferation and migration. Re-expression of wild-type Kindlin-2, but not AR-binding-defective or Src-binding-defective mutant forms of Kindlin-2, in Kindlin-2-deficient cells restored AR Tyr-534 phosphorylation, signaling, breast cancer cell proliferation and migration. Furthermore, re-introduction of phosphor-mimic mutant AR-Y534D, but not wild-type AR reversed Kindlin-2 deficiency-induced inhibition of AR signaling and breast cancer progression. Finally, using a genetic knockout strategy, we show that ablation of Kindlin-2 from mammary tumors in mouse significantly reduced AR Tyr-534 phosphorylation, breast tumor progression and metastasis in vivo. Our results suggest a critical role of Kindlin-2 in promoting breast cancer progression and shed light on the molecular mechanism through which it functions in this process.

Abstract PD1-07: Mutant ESR1 receptors antagonize the tumor suppressor function of androgen receptors

Abstract Background: Acquired ESR1 mutations are a dominant driver of distant metastasis in metastatic breast cancer, inducing a basal-like phenotype that is relatively resistant to ER antagonists with decreased progression free survival. The majority of ER+ patients also express the androgen receptor (AR), and although AR expression is associated with better outcomes, high AR expression has also been associated with resistance to endocrine therapy (ET). Thus, there is currently a conundrum on how best to target AR, and to define when it is participating as a proliferative/metastatic driver, or when it may be a potential tumor suppressor associated with good prognosis. Methods: We generated ESR1 Y537S or D538G homozygous mutations in MCF-7 cells using CRISPR Cas-9 technology. ChIP-Seq, transcriptome analyses, and quantitative analysis of ER ChIP-Seq profiles from patient biopsies were integrated. Nuclear fractionation and immunoblot analyses were performed. Results: Correlation and gene set enrichment analyses demonstrated that the androgen response pathway was significantly reduced in ESR1 mutant, compared to wild-type (WT) ER cells. A dramatic redistribution in AR binding sites to heterochromatin was observed in ESR1 mutant cells. AR and ER co-occupied DNA binding sites were redistributed only to ERE motifs present in a restricted set of gene enhancer regions. Quantitative analysis of ER DNA binding profiles from ER+ patients showed that the highest recruitment of ER to AR/ER binding sites were in patients with metastatic breast cancer. Integration of AR/ER co-bound sites with the differentially-expressed mutant transcriptome identified a gene signature that predicted poor disease-free and overall survival in ER+ primary breast cancer patients from the METABRIC database. Elevated gene expression of these AR/ER co-regulated genes, including NCOA3, BMP7, N4BP3, and FOXA1, were validated in a cohort of metastatic tumors (N=97), compared to primary tumors (N=276). AR protein levels were elevated in ESR1 mutant tumors. Mechanistic studies demonstrated that elevated AR phosphorylation at specific sites (pS515, pS650) and decreased phosphorylation (pS308) occurs in mutant cells. These sites are known to affect AR transcriptional activity, protein stability, and nuclear export. Decreased K48-AR ubiquitination was observed, suggesting that elevated AR protein levels result from altered post-transcriptional modification of AR in mutant cells. Conclusions: We propose a two-prong genomic activation mechanism in ESR1 mutant tumors with AR redistribution to heterochromatin, and genomic co-activation of AR and ER. We hypothesize that the loss of AR tumor suppressor function may help drive metastasis in ESR1 mutant tumors via AR’s collaboration with ESR1 mutant oncogenic activity. Efficacious targeting of AR may require disruption of this driver AR/ER-regulated transcriptional program. Citation Format: Sandra L. Grimm, Guowei Gu, Sarah K. Herzog, Thomas L. Gonzalez, Hangqing Lin, Amanda R. Beyer, Yassine Rechoum, Tasneem Bawa-Khalfe, Ashfia F. Khan, Lili Du, W. Fraser Symmans, Ralf Kittler, Cristian Coarfa, Suzanne A.W. Fuqua. Mutant ESR1 receptors antagonize the tumor suppressor function of androgen receptors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-07.

Phosphorylation of androgen receptor by mTORC1 promotes liver steatosis and tumorigenesis.

Background and AimsAndrogen receptor (AR) has been reported to play an important role in the development and progression of man’s prostate cancer. Hepatocellular carcinoma (HCC) is also male‐dominant, but the role of AR in HCC remains poorly understood. Mechanistic target of rapamycin complex 1 (mTORC1) also has been reported to be highly activated in HCC. In this study, we aimed to explore the role of AR phosphorylation and its relationship with mTORC1 in hepatocarcinogenesis.Approach and ResultsIn vitro experiment, we observed that mTORC1 interacts with hepatic AR and phosphorylates it at S96 in response to nutrient and mitogenic stimuli in HCC cells. S96 phosphorylation promotes the stability, nuclear localization, and transcriptional activity of AR, which enhances de novo lipogenesis and proliferation in hepatocytes and induces liver steatosis and hepatocarcinogenesis in mice independently and cooperatively with androgen. Furthermore, high ARS96 phosphorylation is observed in human liver steatotic and HCC tissues and is associated with overall survival and disease‐free survival, which has been proven as an independent survival predictor for patients with HCC.ConclusionsAR S96 phosphorylation by mTORC1 drives liver steatosis and HCC development and progression independently and cooperatively with androgen, which not only explains why HCC is man‐biased but also provides a target molecule for prevention and treatment of HCC and a potential survival predictor in patients with HCC.

Anti-metastatic effect of GV1001 on prostate cancer cells; roles of GnRHR-mediated G\u03b1s-cAMP pathway and AR-YAP1 axis

BackgroundGonadotropin-releasing hormone receptor (GnRHR) transmits its signal via two major Gα-proteins, primarily Gαq and Gαi. However, the precise mechanism underlying the functions of Gαs signal in prostate cancer cells is still unclear. We have previously identified that GV1001, a fragment of the human telomerase reverse transcriptase, functions as a biased GnRHR ligand to selectively stimulate the Gαs/cAMP pathway. Here, we tried to reveal the potential mechanisms of which GV1001-stimulated Gαs-cAMP signaling pathway reduces the migration and metastasis of prostate cancer (PCa) cells.MethodsThe expression of epithelial-mesenchymal transition (EMT)-related genes was measured by western-blotting and spheroid formation on ultra-low attachment plate was detected after GV1001 treatment. In vivo Spleen-liver metastasis mouse model was used to explore the inhibitory effect of GV1001 on metastatic ability of PCa and the transwell migration assay was performed to identify whether GV1001 had a suppressive effect on cell migration in vitro. In order to demonstrate the interaction between androgen receptor (AR) and YAP1, co-immunoprecipitation (co-IP), immunofluorescence (IF) staining, chromatin immunoprecipitation (ChIP) were performed in LNCaP cells with and without GV1001 treatment.ResultsGV1001 inhibited expression of EMT-related genes and spheroid formation. GV1001 also suppressed in vivo spleen-liver metastasis of LNCaP cells as well as cell migration in vitro. GV1001 enhanced the phosphorylation of AR and transcription activity of androgen response element reporter gene through cAMP/protein kinase A pathway. Moreover, GV1001 increased Ser-127 phosphorylation of YAP1 and its ubiquitination, and subsequently decreased the levels of AR-YAP1 binding in the promoter region of the CTGF gene. In contrast, both protein and mRNA levels of NKX3.1 known for tumor suppressor gene and AR-coregulator were upregulated by GV1001 in LNCaP cells. YAP1 knockout using CRISPR/Cas9 significantly suppressed the migration ability of LNCaP cells, and GV1001 did not affect the cell migration of YAP1-deficient LNCaP cells. On the contrary, cell migration was more potentiated in LNCaP cells overexpressing YAP5SA, a constitutively active form of YAP1, which was not changed by GV1001 treatment.ConclusionsOverall, this study reveals an essential role of AR-YAP1 in the regulation of PCa cell migration, and provides evidence that GV1001 could be a novel GnRHR ligand to inhibit metastasis of PCa via the Gαs/cAMP pathway.

Androgen receptor phosphorylated at Ser815: The expression and function in the prostate and tumor-derived cells

Androgen is beneficial for the prostate with normal functions but creates a risk for prostate cancer progression. How androgen receptor (AR) mediates these various androgen actions remains elusive. AR conserves a phosphorylation motif within its ligand-binding domain throughout species. Here, we have found AR phosphorylated at Ser815 (P-AR) is expressed in normal tissues of both human and mouse prostates. P-AR begins expression in association with prostatic development and castration decreases its expression levels in the mouse prostate. Functional analysis of AR in prostate cancer PC-3 cells showed ligand-induced AR nuclear translocation and transactivation were disturbed by its phosphorylation at Ser815. Moreover, P-AR suppressed oncogenic AKT signaling suggesting a suppressive function for prostate cancer development. In fact, AR phosphorylation levels progressively decrease in human prostates as cancer worsens. These findings showed androgen might utilize P-AR to self-antagonize oncogenic signals and cancer progression believed to be regulated by non-phosphorylated AR (NonP-AR). By differing its target genes and signal regulations from those of NonP-AR, P-AR co-expression with NonP-AR may be the molecular basis for androgen to balance its actions and to control disease developments.

PIM1 phosphorylation of the androgen receptor and 14-3-3 \u03b6 regulates gene transcription in prostate cancer

PIM1 is a serine/threonine kinase over-expressed in prostate cancer. We have previously shown that PIM1 phosphorylates the androgen receptor (AR), the primary therapeutic target in prostate cancer, at serine 213 (pS213), which alters expression of select AR target genes. Therefore, we sought to investigate the mechanism whereby PIM1 phosphorylation of AR alters its transcriptional activity. We previously identified the AR co-activator, 14-3-3 ζ, as an endogenous PIM1 substrate in LNCaP cells. Here, we show that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, and that they extensively occupy the same sites on chromatin in an AR-dependent manner. Their occupancy at a number of genes involved in cell migration and invasion results in a PIM1-dependent increase in the expression of these genes. We also use rapid immunoprecipitation and mass spectrometry of endogenous proteins on chromatin (RIME), to find that select AR co-regulators, such as hnRNPK and TRIM28, interact with both AR and 14-3-3 ζ in PIM1 over-expressing cells. We conclude that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, which in turn recruits additional co-regulatory proteins to alter AR transcriptional activity.

Androgen Receptor Activation in Glioblastoma Can Be Achieved by Ligand-Independent Signaling through EGFR-A Potential Therapeutic Target.

Androgen receptor (AR) is a ligand-mediated transcription factor that belongs to the superfamily of steroid receptors. AR is overexpressed in most glioblastomas and is a potential therapeutic target. In prostate and breast cancers, AR activation can be achieved also by a ligand-independent signaling through receptor tyrosine kinases such as epidermal growth factor receptor (EGFR). Considering its major role in glioblastoma, we explored whether EGFR is involved in AR signaling in this tumor. Analysis of mRNA expression in 28 glioblastoma samples with quantitative real-time reverse-transcription polymerase chain reaction revealed a positive and significant correlation between AR and EGFR mRNA expression levels (R = 0.47, p = 0.0092), which was validated by The Cancer Genome Atlas dataset (n = 671) analysis (R = 0.3, p = 0.00006). Using Western blotting and immunofluorescence staining, we showed that the transduced overexpression of EGFR or its variant EGFRvIII in the U87MG cells induced AR protein overexpression and nuclear translocation and Protein kinase B (AKT) S473 and AR S210/213 phosphorylation. The EGFR kinase inhibitor afatinib and the AKT inhibitor MK2206 reduced AR nuclear translocation. Afatinib diminished AKT phosphorylation at 30 min and 6 h in the EGFR- and EGFRvIII-overexpressing cells, respectively, and decreased AR phosphorylation in EGFR-overexpressing cells at 4 h. Afatinib or MK2206 combination therapy with the AR antagonist enzalutamide in the EGFR and EGFRvIII-overexpressing cells had synergistic efficacy. Our findings suggest that EGFR signaling is involved in AR activation in glioblastoma and buttresses the concept of combining an EGFR signaling inhibitor with AR antagonists as a potential glioblastoma treatment.

3,5,6-trichloro-2-pyridinol intensifies the effect of chlorpyrifos on the paracrine function of Sertoli cells by preventing binding of testosterone and the androgen receptor

3,5,6-Trichloro-2-pyridinol (TCP) is an important biomarker and one of the final metabolites of chlorpyrifos (CPF). TCP inhibits secretion of sex hormones. Similar to CPF, TCP can bind to sex steroid hormone receptors and decrease the secretion of sex hormones. However, little attention has been paid to the ability of TCP and CPF to interfere with androgen receptor (AR) in Sertoli cells. This study aimed to explain how TCP promotes the inhibitory effect of CPF on the paracrine function of Sertoli cells. Western blotting indicated that after 20 weeks of exposure, expression of AR in testes was significantly reduced by CPF. An in vitro assay measured the cytotoxicity of CPF, TCP and diethylphosphate (DEP) on viability of Sertoli cells by Cell Counting Kit-8. CPF cytotoxicity was greater than that of TCP, and TCP cytotoxicity was greater than that of DEP at concentrations of 1000 μmol/L. Western blotting indicated that TCP and CPF both decreased expression of AR and cAMP-response element binding protein phosphorylation, while DEP had no effect in Sertoli cells, which are important in regulating paracrine function of Sertoli cells. The fluorescence measurements and docking studies revealed that testosterone, CPF and TCP showed four types of intermolecular interactions with AR, highlighting alkyl bonds with some of the same amino acids. Compared with testosterone, CPF and TCP also showed significant synergistic interaction with AR. CPF interacted with more amino acids and interaction energy than TCP did. This research elucidates TCP in the antiandrogenic effect of CPF on the paracrine function and suggests that TCP or chemicals with a trichloropyridine structure must be considered during reproductive toxicity assessment of potential environmental pollutants.

FKBP51 and FKBP52 regulate androgen receptor dimerization and proliferation in prostate cancer cells.

The growth of prostate cancer is dependent on the androgen receptor (AR), which serves as a ligand‐specific transcription factor. Although two immunophilins, FKBP51 and FKBP52, are known to regulate AR activity, the precise mechanism remains unclear. We found that depletion of either FKBP51 or FKBP52 reduced AR dimer formation, chromatin binding, and phosphorylation, suggesting defective AR signaling. Furthermore, the peptidyl‐prolyl cis/trans isomerase activity of FKBP51 was found to be required for AR dimer formation and cancer cell growth. Treatment of prostate cancer cells with FK506, which binds to the FK1 domain of FKBPs, or with MJC13, an inhibitor of FKBP52–AR signaling, also inhibited AR dimer formation. Finally, elevated expression of FKBP52 was associated with a higher rate of prostate‐specific antigen recurrence in patients with prostate cancer. Collectively, these results suggest that FKBP51 and FKBP52 might be promising targets for prostate cancer treatment through the inhibition of AR dimer formation.

Androgen and glucocorticoid receptor phosphorylation following resistance exercise and pre-workout supplementation

PurposeConsumption of caffeine or caffeine containing pre-workout supplements (SUPP) augments steroid hormone responses to resistance exercise (RE). However, the activation of glucocorticoid (GR) and androgen receptors (AR) following RE SUPP has not been investigated. The purpose of this study was to determine the influence of a pre-workout supplement on AR and GR phosphorylation following RE. Methods: In a randomized, counter-balanced, double-blind, placebo-controlled, within-subject crossover study, ten resistance-trained males ((X¯±SD, age = 22 ± 2.4 yrs, hgt = 175 ± 7 cm, body mass = 84.1 ± 11.8 kg) performed four sets of 8 repetitions of barbell back squats at 75% of their 1-repetition maximum (1-RM) with two minutes of rest between sets and a fifth set of barbell back squats at 60% of 1-RM until concentric failure. A SUPP or flavor and color matched placebo (PL) was consumed 60-minutes prior to RE. Vastus lateralis muscle biopsies were obtained prior to supplementation at rest (BL), and ten minutes post-exercise (POST). Biopsies were analyzed for phosphorylated GR (ser134, ser211, and ser226) and phosphorylated AR (ser81, ser213, ser515, ser650) via western blotting. Results: pGRser134 decreased, and pGRser226 increased following RE (p < 0.05) with no difference between conditions (p > 0.05). pGRser211 was unchanged after RE (p > 0.05). pARser515 increased, and total AR expression decreased after RE (p < 0.05) in SUPP only. Testosterone and cortisol were not different between SUPP and PL at POST (p > 0.05). Conclusion: RE influences AR and GR phosphorylation, and SUPP minimally influences this response in the early recovery period.

Lycium barbarum Polysaccharide Ameliorates Heat-Stress-Induced Impairment of Primary Sertoli Cells and the Blood-Testis Barrier in Rat via Androgen Receptor and Akt Phosphorylation.

Male infertility induced by heat stress has been attracting more and more attention. Heat stress not only causes apoptosis of spermatocytes but also has adverse effects on Sertoli cells, further damaging spermatogenesis. Lycium barbarum polysaccharide (LBP) is the main bioactive component of Lycium barbarum, which has a protective effect on male reproduction, but its mechanism is still unclear. In this study, our results proved that LBP blocked the inhibitory effect on the proliferation activity of Sertoli cells after heat stress, reversed the dedifferentiation of Sertoli cells induced by heat stress, and ameliorated the structural integrity of the blood-testis barrier. In addition, it increased the expression of the androgen receptor and activated Akt signaling pathway to resist heat-stress-induced injury of Sertoli cells.

Abstract 1262: Targeting transcriptional addiction in anti-androgen refractory castration-resistant prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related mortality in men of the western world. Androgen receptor (AR) is the primary driver alteration in metastatic castration-resistant prostate cancer (mCRPC). Advanced PCa treated with first line androgen-deprivation therapy (ADT) eventually relapse in a hormone refractory or castration-resistant (CR) form. Relapsed disease is highly aggressive and poses an increased risk of morbidity and death. This refractory state of CRPC is characterized by a strong addiction to AR-mediated transcription driven by the synergy between a variety of genomic alterations and epigenetic remodeling. Despite the success of recently approved therapies targeting AR signaling, such as abiraterone and second-generation anti-androgens including enzalutamide, durable responses are limited, owing to acquired resistance. Epigenetic therapies targeting coactivators within the AR transcription machinery have shown promise as effective options for the treatment of CRPC in both its naive and Enzalutamide Resistant state (Enza-Res). Using basic proteomic and next generation genomic approaches, we have identified CDK7, as an indispensable component of the AR-transcription machinery and a novel target in CRPC. The phosphorylation of MED1 (T1457) by CDK7 leads to the formation of the chromatin bound AR-MED1 complex and is essential for its stability and onset of AR-mediated transcription. Loss of CDK7 activity either through genetic silencing or through treatment with the CDK7 inhibitor THZ1 led to de-recruitment of chromatin bound MED1, loss of AR-target gene expressions, increased apoptosis, and a drastic reduction in cell proliferation. Here, we present evidence for the efficacy of targeting CDK7 in curtailing AR signaling in the Enza-Res state. Further, we observed hyper-phosphorylation of MED1 and AR, and a concomitant decrease in the catalytic-subunit (alpha-isoform) of the protein phosphatase 2A (PP2ACα) in enzalutamide resistant cell lines and metastatic biopsies. This hyper-phosphorylated state was strongly dependent on the catalytic activity of CDK7, wherein treatment of Enza-Res cells with THZ1 led to complete loss of phosphorylation of MED1 and AR, and death in Enza-res cells. These findings point to the indispensable role of CDK7 in driving the AR oncogenic transcription program in anti-androgen refractory CRPC and its viability as a potential therapeutic target. Citation Format: Reyaz ur Rasool, Ramakrishnan Natesan, Qu Deng, Shweta Aras, Samuel Sander Effron, Samuel Sander Effron, Erick Mitchell Velasquez, Jessica M. Posimo, Priti Lal, Donita C. Brady, Irfan A. Asangani. Targeting transcriptional addiction in anti-androgen refractory castration-resistant prostate cancer [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 1262.

AR phosphorylation and CHK2 kinase activity regulates IR-stabilized AR-CHK2 interaction and prostate cancer survival.

We have previously demonstrated that checkpoint kinase 2 (CHK2) is a critical negative regulator of androgen receptor (AR) transcriptional activity, prostate cancer (PCa) cell growth, and androgen sensitivity. We have now uncovered that the AR directly interacts with CHK2 and ionizing radiation (IR) increases this interaction. This IR-induced increase in AR-CHK2 interactions requires AR phosphorylation and CHK2 kinase activity. PCa associated CHK2 mutants with impaired kinase activity reduced IR-induced AR-CHK2 interactions. The destabilization of AR - CHK2 interactions induced by CHK2 variants impairs CHK2 negative regulation of cell growth. CHK2 depletion increases transcription of DNAPK and RAD54, increases clonogenic survival, and increases resolution of DNA double strand breaks. The data support a model where CHK2 sequesters the AR through direct binding decreasing AR transcription and suppressing PCa cell growth. CHK2 mutation or loss of expression thereby leads to increased AR transcriptional activity and survival in response to DNA damage.

Androgen receptor phosphorylation at serine 81 and serine 213 in castrate-resistant prostate cancer.

Despite increases in diagnostics and effective treatments, over 300,000 men die from prostate cancer highlighting the need for specific and differentiating biomarkers. AR phosphorylation associates with castrate-resistance, with pARser213 promoting transcriptional activity. We hypothesise that combined pARser81 and pARser213 reduces survival and would benefit from dual-targeting androgen-dependent and Akt-driven disease. Immunohistochemistry and immunofluorescence were performed on matched hormone-naive and castrate-resistant prostate cancer samples. TempO-Seq gene profiling was analysed using DESeq2 package. LNCaP-AI cells were stimulated with DHT or EGF. WST-1 assays were performed to determine effects of Enzalutamide and BKM120 on cell viability. Following the development of castrate-resistance, pARser81 expression reduced and pARser213 expression increased. Castrate-resistance pARser81 expression was not associated with survival but high pARser213 expression was associated with reduced survival from relapse. Combined high pARser81 and pARser213 was associated with reduced survival from relapse. pARser81 expression was induced by 10 nM DHT or 10 nM EGF and pARser213 expression was induced by treatment with 10 nM EGF in LNCaP-AI cells. Cell viability was reduced following treatment with 10 nM Enzalutamide and 10 nM BKM120. Eight genes were differentially expressed between hormone-naive and castrate-resistant tumours and twenty-five genes were differentially expressed between castrate-resistant tumours with high and low pARser213 expression. Combined pARser81 and pARser213 provides a novel prognostic biomarker for castrate-resistant disease and a potential predictive and therapeutic target for prostate cancer. Further studies will be required to investigate the combined effects of targeting AR and PI3K/AKT signalling.