Potential Protective Effects of Testosterone Therapy on Prostate Cancer

: This project, which began with the goal of identifying proteins that bind to the human androgen receptor (hAR) differentially based on the length of the CAG repeat sequence, has been instrumental in revealing new biological findings of the role of the androgen receptor in prostate cancer progression to androgen-independence. Three significant findings were these: 1) Failure to identify any binding proteins for the Gln repeat region of the human androgen receptor. Our conclusion was that the continued search for such binding proteins is not warranted at this time, however further study of ARA24 is warranted. 2) The role of the RB pathway growth arrest in the androgen response in prostate cancer. The occurrence of cell death following the growth arrest, may provide a biology that can be exploited for prostate cancer patient therapy. 3) Bag1L binds to hAR is induced by gain-of-function p53 mutants, and contributes to androgen-independent growth of prostate cancer cells. We believe these findings make important and extremely valuable contributions toward understanding the role of the androgen receptor in hormone-refractory prostate cancer. These multiple findings provide clearer foundations on which to build and propose new projects for study of this important aspect of human prostate cancer.

FOXP1 is a member of the winged helix or forkhead transcription factors. Recent studies have indicated possible roles for FOXP1 as a candidate tumor suppressor gene and a potential estrogen receptor (ER) co-regulator in the development of breast cancer. This study investigated whether FOXP1 has a similar relationship to the androgen receptor (AR) in prostate cancer and how these factors relate to the presence of hypoxia. FOXP1, the AR and various hypoxia-regulated proteins (HIF-1alpha, HIF-2alpha, and VEGF) were measured with immunohistochemistry using a tissue microarray constructed from 167 archival radical prostatectomies. Statistical analyses compared the co-expression of these factors both with each other and conventional parameters including patient age, pre-operative prostate specific antigen (PSA), post-operative Gleason score, capsular invasion, surgical margin status, tumor volume, and PSA recurrence. The influence of hypoxia, dihydrotestosterone, and the AR blocker Casodex was investigated in prostate cell lines VCaP and LNCaP in vitro. Expression of nuclear FOXP1 was significantly positively correlated with AR (P = 0.0001), hypoxia inducible factor 1alpha (HIF-1alpha) (P = 0.01), HIF-2alpha (P = 0.0001), and vascular endothelial growth factor (VEGF) (P = 0.007) expression. A positive significant relationship was also identified with the post-operative Gleason score (P = 0.03) but not with the other variables, including PSA recurrence (P > 0.05). There was no significant change in expression in FOXP1 protein levels under conditions of hypoxia (0.1%), dihydrotestosterone stimulation (10 or 100 nM), or androgen blockade with Casodex (1, 10, or 50 microM). These findings suggest that there may be a hormonal and hypoxia independent regulatory mechanism coordinating the expression of HIFs, the AR, and FOXP1 in prostate tumors.

The PacRim Breast and Prostate Cancer Meeting provided a unique opportunity for an integrated exploration of the translation of biomedical research into novel therapies for two steroid hormone-dependent cancers. The meeting emphasised the central role of the estrogen receptor in breast cancer and of the androgen receptor in prostate cancer. A series of novel approaches, insights and potential therapeutics were discussed although the challenges, particularly in the treatment of hormone-refractory disease, remain substantial. The meeting was held at the Kingfisher Bay Resort on Fraser Island, which lies just off the Queensland Coast. Researchers from Australia, Canada and the US were brought together in an integrated format to identify issues, solutions and collaborations around these two hormone-dependent cancers. If one was to identify a core focus, it would be the role of the estrogen receptor (in the context of breast cancer, we are referring to estrogen receptor-α unless otherwise specified) and of the androgen receptor in breast and prostate cancer, respectively.

Introduction. Through androgen receptors, androgens regulate prostate cellular growth and function, proliferation, differentiation, apoptosis, lipid metabolism and secretory activity, as well as development and progression of prostate cancer. Prostate cancer, and its primary glandular tissue are influenced by hormones, and it is used for therapeutic purposes. Anti-androgen treatment is carried out in patients with metastatic prostatic cancer, in order to block effects of androgens. Immunohistochemical analysis of androgen receptors in the prostate cancer tissue may help us to assume how the tumors will react to the anti-androgen therapy, if they are androgen-positive, -negative, or hormone resistant tumors. Knowledge of the presence of androgen receptors in the tumor tissue may be a prognostic indicator in histopathological analysis. The aim of this study was stereological evaluation of androgen receptor expression in patients with benign prostatic hyperplasia and in patients with prostatic cancer, before therapy. Material and Methods. Immunohistochemical analysis was carried out using anti-human androgen receptor monoclonal antibody 441. The presence and intensity of the androgen receptors were evaluated in 195 patients: 165 with benign prostatic hyperplasia and 30 with prostatic cancer using Weibel?s multi-purpose M 42 stereological test system. Material was obtained by needle biopsy or transurethral resection of the prostate. Results. All secretory cells in patients with benign prostatic hyperplasia were androgen positive, while in patients with prostatic cancer, all tumors were mostly androgen positive, some with foci of negativity. The resulting negative correlation with Gleason score and International Society of Urological Pathology grade was not statistically significant. Conclusion. Study results of stereological analysis of androgen receptors indicate that prior the therapy prostate cancer is androgendependent, with a high level of androgen receptor expression, although slightly lower compared to benign prostatic hyperplasia.

Androgen Receptors in Prostate Cancer

Background: Prostate cancer (PCa) is a slowly progressing disease that affects nearly 50% of males over the age of 60. Current therapies for patients diagnosed with advanced PCa include Androgen Deprivation Therapy (ADT) and anti-androgen receptor (Anti-AR) treatment. PCa treatment primarily targets the androgen receptor (AR), a ligand-activated transcription factor, which is a key driver of PCa tumor growth. Our previous research has demonstrated that ABI1, acts as a tumor suppressor in PCa. ABI1 is a scaffold protein and an integral member of the WAVE Regulatory Complex (WRC), a nucleation promoting factor. UCSC database indicates there is an AR binding site within the ABI1 gene, suggesting AR plays a role in the transcriptional regulation of ABI1. In this study, we aim to characterize the ABI1-dependent AR regulation in PCa. Method: We generated an ABI1 KO cell line model in LNCaP cells using CRISPR-Cas9. The efficiency of CRISPR-Cas9 was evaluated by Western Blotting and genomic alterations confirmed by DNA sequencing. In addition, we have generated ABI1 Isoform-specific rescue cell lines in our LNCaP ABI1 KO cell line with a binding mutation in the SH3 domain (ABI1-W485N) and an SH3 domain deleted (Abi1-ΔSH3) ABI1 protein. To understand AR and ABI1 dependent pathways we performed qPCR, co-immunoprecipitation, Western Blotting, ChIP, in vitro fluorescence spectra assay, immunofluorescence, proximity ligation assays. Results: In vitro binding assays indicate the interaction of AR, NTD poly-proline region, with the ABI1 SH3 domain. Expression of ABI1- W485N in our ABI1 CRISPR KO cell line showed decrease binding, while ABI1-ΔSH3 showed no binding to AR in co-IP assays compared to our control. Consequently, we saw decreased AR nuclear localization compared to our ABI1-WT control. Furthermore, decreased nuclear localization in our ABI1-W485N mutant was associated with decreased mRNA expression of hallmark AR target genes, Prostate-Specific-Antigen (KLK3), and TMPRSS2. ABI1 is an AR responsive gene and was confirmed with ChIP assays in an AR overexpression cell line. Further, the stimulation of AR transcriptional activity increased cell-cell adhesion in an ABI1 dependent system. Conclusions: Our preliminary studies demonstrate that AR and ABI1 have a negative feedback pathway. The loss of ABI1 resulted in a decrease of nuclear AR and a subsequent decrease in mRNA expression of AR target genes. AR can also modulate ABI1 expression on a transcriptional level as needed for this pathway to function. Future studies will investigate if anti-AR treatments could lead to the dysregulation of ABI1 and promote EMT through STAT3 activation until AR transcriptional regulation on ABI1 is restored. These findings will allow for novel insights into the mechanisms underlying AR and ABI1 relationship in neoplastic progression. Citation Format: Baylee A. Porter, Alaji Bah, Alfonso Urbanucci, Fan Zhang, Sonia Kung, Ladan Fazli, Martin Gleave, Gennady Bratslavsky, Leszek Kotula. Defining the reciprocal regulation of Abi1 and the androgen receptor in prostate cancer [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 2470.

Reciprocal regulation between DNMT3A/3B and microRNAs miRs-299-3p/-30e is a causal factor for the downregulation of microRNAs targeting androgen receptor in prostate cancer.

Disclosure: K.M. Hasan: None. H. Khambati: None. R.R. Huerta: None. J. Vadgama: None. A.P. Sinha-Hikim: None. T.C. Friedman: None. Prostate cancer (PCa) is the second most prevalent cancer among men worldwide. Since PCa onset is androgen dependent, androgen deprivation therapy (ADT) is the first treatment choice for a primary tumor or metastatic PCa. However, ADT eventually leads to the development of castration resistant PCa (CRPC), which is the leading cause of higher mortality in PCa. Thus, there is a need to identify the underlying mechanisms of CRPC to manage this aggressive disease. Increased androgen receptor (AR) expression is one of the most important factors contributing to the development of CRPC. It has been shown that tumor suppressor p53 negatively regulates the expression of AR. So, loss of p53 functions has been associated with increased expression of AR in advanced PCa. However, the mechanism of p53-mediated inhibition of AR is not well understood. In this study, we demonstrated that CARF (CDKN2AIP), a p53 pathway regulatory protein, is highly expressed in PCa, but its significance in PCa development is unknown. Since, like AR, CARF is negatively regulated by p53, we hypothesize that a reciprocal relationship between CARF and p53 could regulate AR expression in PCa cells. Indeed, we found that activation of p53 by DNA damage stress inhibited both CARF and AR in LnCap and 22RV1, AR-positive, and wtp53 cell lines. Our result indicated that CARF and AR were decreased dose-dependent manner, and presumably, that could trigger cell death. Consistent with this speculation, we found that silencing CARF by ShRNA in LnCap triggered apoptosis and inhibition of cell proliferation. With great interest, we further demonstrated that silencing of CARF downregulated the AR level in LnCap and 22RV1 cells suggesting that CARF regulates the expression of AR in PCa. RT-PCR results showed that AR transcripts remained unchanged in CARF-depleted LnCap cells. This result indicated that CARF could regulate the AR stability but not the transcription. Intriguingly, we found that the expression of NEDD4, one of the AR-targeting E3 ligases, was increased in CARF-depleted cells. Therefore, by enhancing the stability, CARF could positively regulate the expression of AR in PCa cells. Further studies are in progress to know more details about the CARF-mediated AR regulation and its impact on androgen-independent PCa development and progression. Presentation: Thursday, June 15, 2023

e22141 Background: Prostate cancer (PRCA) is driven by the Androgen Receptor (AR), a cytoplasmic protein that translocates to the nucleus to activate a transcriptional program critical to PRCA tumorigenesis. The AR is the major therapeutic target in PRCA, with multiple drugs that improve survival and quality of life for men with advanced PRCA. However, few biomarkers can assay for AR activity to identify those most likely to benefit from AR-targeting therapies. Methods: Utilizing a novel microfluidic technology known as the VERSA (Vertical Exclusion-based Rare Sample Analysis) platform, CTCs were captured and purified from men with advanced PRCA who had failed androgen deprivation therapy. CTCs were then stained and imaged via epiflourescent microscopy for EpCAM, AR, Hoechst and CD45. The enhanced optical imaging of the VERSA platform enabled subcellular localization of the AR and calculation of the relative amount of AR localized to the nucleus as a biomarker of resistance to AR-targeting therapies. Results: CTCs were prospectively collected and analyzed for AR nuclear localization. Significant heterogeneity in AR localization was found between patients who had failed androgen deprivation therapy with the mean nuclear AR ranging from 20-57% of total AR. Significant intra-patient heterogeneity was also identified with the percentage of AR localized to the nucleus ranging from 11-77% of total AR. DNA and mRNA have been isolated from these cells to analyze for underlying mechanisms of nuclear localization. Conclusions: The VERSA platform is able to analyze subcellular localization of proteins in CTCs followed by total nucleic acid extraction. We show that among men whose disease has progressed through androgen deprivation, a significant portion of the AR is localized to the nucleus as a possible biomarker of resistance to AR targeting therapies. We also identify significant heterogeneity in AR localization among CTCs, suggesting resistant PRCA clones that have greater proliferative potential.

Pre-chemotherapy abiraterone acetate. A proposal of a treatment algorithm in castration resistant prostate cancer

TUMOR NECROSIS FACTOR-α REPRESSES ANDROGEN SENSITIVITY IN THE LNCaP PROSTATE CANCER CELL LINE

Mithramycin suppresses DNA damage repair via targeting androgen receptor in prostate cancer.

Prostate Cancer in Men 70 Years Old or Older, Indolent or Aggressive: Clinicopathological Analysis and Outcomes

Androgen receptor plays a pivotal role in prostate cancer progression, and androgen deprivation therapy to intercept androgen receptor signal pathway is an indispensable treatment for most advanced prostate cancer patients to delay cancer progression. However, the emerging of castration-resistant prostate cancer reminds us the alteration of androgen receptor, which includes androgen receptor mutation, the formation of androgen receptor variants, and androgen receptor distribution in cancer cells. In this review, we introduce the process of androgen receptor and also its variants' formation, translocation, and function alteration by protein modification or interaction with other pathways. We dissect the roles of androgen receptor in prostate cancer from molecular perspective to provide clues for battling prostate cancer, especially castration-resistant prostate cancer.

BackgroundThis study aimed to preliminarily assess the relationship between erythropoietin‐producing hepatocellular carcinoma receptor A3 (EphA3) and androgen receptor (AR) protein expression levels and prognosis in prostate cancer (PCa) to better understand the role of EphA3 in the prognosis and progression of PCa.MaterialsWe investigated the expression of EphA3 and AR in human PCa by immunohistochemistry.ResultsEphA3 and AR were both significantly upregulated in PCa, with expression mainly localized to the nucleus. A high level of AR expression was found in 48.4% of 64 tumor samples, which was significantly more than in the adjacent tissue samples (15.6%) (P < 0.01). The percentage of samples expressing a high level of EphA3 was significantly greater in the PCa samples (54.7%) than in the adjacent tissue samples (20.3%) for the 64 tumors (P < 0.01). The high levels of EphA3 and AR expression in the PCa tissue samples were both correlated with the pathological stage, bladder and rectal invasion, distant metastasis, and preoperative PSA level (both P < 0.05). The survival time was significantly shorter in high levels of AR expression of patients. (P < 0.01). A high level of EphA3 in PCa patients suggests a poor prognosis (P < 0.05). Biochemical recurrence, distant metastasis, and the final scores of EphA3 and AR expression were significantly correlated with the prognosis of PCa (P < 0.05).ConclusionsIncreased EphA3 expression is an independent prognostic factor for a poor outcome and decreased survival in PCa.