FAM64A is an androgen receptor-regulated feedback tumor promoter in prostate cancer

Endocrine therapy for prostate cancer (PCa) mainly inhibits androgen receptor (AR) signaling, due to increased androgen synthesis and AR changes, PCa evolved into castration-resistant prostate cancer (CRPC). The function of Family With Sequence Similarity 64 Member A (FAM64A) and its association with prostate cancer has not been reported. In our research, we first reported that FAM64A is up-regulated and positively associated with poor prognosis of patients with prostate cancer (PCa) by TCGA database and immunohistochemistry staining. Moreover, knockdown of FAM64A significantly suppressed the proliferation, migration, invasion, and cell cycle of PCa cells in vitro. Mechanistically, FAM64A expression was increased by dihydrotestosterone (DHT) through direct binding of AR to FAM64A promoter, and notably promoted the proliferation, migration, invasion, and cell cycle of androgen-dependent cell line of PCa. In addition, abnormal expression of FAM64A affects the immune and interferon signaling pathway of PCa cells. In conclusion, FAM64A was up-regulated by AR through directly binding to its specific promoter region to promote the development of PCa, and was associated with the immune mechanism and interferon signaling pathway, which provided a better understanding and a new potential for treating PCa.Subject terms: Penile cancer, Predictive markers     

Differential targeting of androgen and glucocorticoid receptors induces ER stress and apoptosis in prostate cancer cells: A novel therapeutic modality

Androgen (AR) and glucocorticoid (GR) receptor signaling play opposing roles in prostate tumorigenesis: in prostate, AR acts as an oncogene, and GR is a tumor suppressor. Recently, we found that non-steroidal phyto-chemical compound A (CpdA) is AR/GR modulator acting as anti-inflammatory anti-androgen. CpdA inhibits AR and prevents GR transactivation while enhancing GR transrepression. GR and AR are controlled by proteasomal degradation. We found that prolonged exposure of LNCaP, LNCaP-GR, DU145 and PC3 prostate carcinoma (PCa) cells to proteasome inhibitor Bortezomib (BZ) caused AR degradation and GR accumulation. BZ enhanced CpdA ability to inhibit AR and to augment GR transrepression. We also found that CpdA+BZ differentially regulated GR/AR to cooperatively suppress PCa cell growth and survival and to induce endoplasmic reticulum stress (ERS). Importantly, CpdA+BZ differentially regulated GR-responsive genes. CpdA+BZ blocked activation of glucocorticoid-responsive pro-survival genes, including SGK1, but activated BZ-induced ERS-related genes BIP/HSPA5 and CHOP/GADD153. Using ChIP, we showed that SGK1, BIP/HSPA5 and CHOP regulation was due to effects of CpdA and CpdA+BZ on GR loading on their promoters. We also found that AR and GR are abundant in advanced PCa from patients treated by androgen ablation and/or chemotherapy: 56% of carcinomas from treated patients expressed both receptors, and the other 27% expressed either GR or AR. Overall, our data validate the concept of dual AR/GR targeting in prostate cancer (PC) and suggest that BZ combination with dual-target steroid receptor modulator CpdA has high potential for PC therapy.Key words: prostate cancer, proteasome inhibitor, non-steroidal modulator, apoptosis, ER stress     

mTOR is a fine tuning molecule in CDK inhibitors-induced distinct cell death mechanisms via PI3K/AKT/mTOR signaling axis in prostate cancer cells

Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors that induce cell cycle arrest and apoptosis in various cancer cells. We further hypothesized that co-treatment of CDK inhibitors with rapamycin, an mTOR inhibitor, would be an effective combinatory strategy for the inhibition of prostate cancer regard to androgen receptor (AR) status due to inhibition of proliferative pathway, PI3K/AKT/mTOR, and induction of cell death mechanisms. Androgen responsive (AR+), PTEN^?/? LNCaP and androgen independent (AR?), PTEN^+/? DU145 prostate cancer cells were exposed to purvalanol (20 µM) and roscovitine (30 µM) with or without rapamycin for 24 h. Cell viability assay, immunoblotting, flow cytometry and fluorescence microscopy was used to define the effect of CDK inhibitors with or without rapamycin on proliferative pathway and cell death mechanisms in LNCaP and DU145 prostate cancer cells. Co-treatment of rapamycin modulated CDK inhibitors-induced cytotoxicity and apoptosis that CDK inhibitors were more potent to induce cell death in AR (+) LNCaP cells than AR (?) DU145 cells. CDK inhibitors in the presence or absence of rapamycin induced cell death via modulating upstream PI3K/AKT/mTOR signaling pathway in LNCaP cells, exclusively only treatment of purvalanol have strong potential to inhibit both upstream and downstream targets of mTOR in LNCaP and DU145 cells. However, co-treatment of rapamycin with CDK inhibitors protects DU145 cells from apoptosis via induction of autophagy mechanism. We confirmed that purvalanol and roscovitine were strong apoptotic and autophagy inducers that based on regulation of PI3K/AKT/mTOR signaling pathway. Co-treatment of rapamycin with purvalanol and roscovitine exerted different effects on cell survival and death mechanisms in LNCaP and DU145 cell due to their AR receptor status. Our studies show that co-treatment of rapamycin with CDK inhibitors inhibit prostate cancer cell viability more effectively than either agent alone, in part, by targeting the mTOR signaling cascade in AR (+) LNCaP cells. In this point, mTOR is a fine-tuning player in purvalanol and roscovitine-induced apoptosis and autophagy via regulation of PI3K/AKT and the downstream targets, which related with cell proliferation.     

The role of protein tyrosine phosphatases in prostate cancer biology

Prostate cancer (PCa) is the most frequent malignancy in the male population of Western countries. Although earlier detection and more active surveillance have improved survival, it is still a challenge how to treat advanced cases. Since androgen receptor (AR) and AR-related signaling pathways are fundamental in the growth of normal and neoplastic prostate cells, targeting androgen synthesis or AR activity constitutes the basis of the current hormonal therapies in PCa. However, resistance to these treatments develops, both by AR-dependent and -independent mechanisms. Thus, alternative therapeutic approaches should be developed to target more efficiently advanced disease. Protein tyrosine phosphatases (PTPs) are direct regulators of the protein- and residue-specific phosphotyrosine (pTyr) content of cells, and dysregulation of the cellular Tyr phosphorylation/dephosphorylation balance is a major driving event in cancer, including PCa. Here, we review the current knowledge on the role of classical PTPs in the growth, differentiation, and survival of epithelial prostate cells, and their potential as important players and therapeutic targets for modulation in PCa.     

Identification of Novel AR-Targeted MicroRNAs Mediating Androgen Signalling through Critical Pathways to Regulate Cell Viability in Prostate Cancer

MicroRNAs (miRNAs) have been recognized as significantly involved in prostate cancer (PCa). Since androgen receptor (AR) plays a central role in PCa carcinogenesis and progression, it is imperative to systematically elucidate the causal association between AR and miRNAs, focusing on the molecular mechanisms by which miRNAs mediate AR signalling. In this study, we performed a series of time-course microarrays to observe the dynamic genome-wide expressions of mRNAs and miRNAs in parallel in hormone-sensitive prostate cancer LNCaP cells stimulated by androgen. Accordingly, we introduced Response Score to identify AR target miRNAs, as well as Modulation Score to identify miRNA target mRNAs. Based on theoretical identification and experimental validation, novel mechanisms addressing cell viability in PCa were unravelled for 3 miRNAs newly recognized as AR targets. (1) miR-19a is directly up-regulated by AR, and represses SUZ12, RAB13, SC4MOL, PSAP and ABCA1, respectively. (2) miR-27a is directly up-regulated by AR, and represses ABCA1 and PDS5B. (3) miR-133b is directly up-regulated by AR, and represses CDC2L5, PTPRK, RB1CC1, and CPNE3, respectively. Moreover, we found miR-133b is essential to PCa cell survival. Our study gives certain clues on miRNAs mediated AR signalling to cell viability by influencing critical pathways, especially by breaking through androgen’s growth restriction effect on normal prostate tissue.     

Regulation and Methylation of Tumor Suppressor MiR-124 by Androgen Receptor in Prostate Cancer Cells

Prostate cancer (PCa) is the most frequently diagnosed cancer for men in the developed world. Androgen receptor signaling pathway plays an important role in prostate cancer progression. Recent studies show that microRNA miR-124 exerts a tumor suppressive function in prostate cancer. However, the relationship between AR and miR-124 is unclear. In the present study, we found a negative feedback loop between AR and miR-124 expression. On one hand, miR-124 was a positively regulated target gene of the AR, on the other hand, overexpression of miR-124 inhibited the expression of AR. In addition, we found that miR-124-2 and miR-124-3 promoters were hypermethylated in AR-negative PCa cells. Furthermore, overexpression of miR-124 inhibited proliferation rates and invasiveness capacity of PCa cells in vitro, and suppressed xenograft tumor growth in vivo. Taken together, our results support a negative feedback loop between AR and miR-124 expression. Methylation of miR-124-2 and miR-124-3 may serve as a biomarker for AR-negative PCa cells, and overexpression of miR-124 might be of potential therapeutic value for the treatment of PCa.     

Differential targeting of androgen and glucocorticoid receptors induces ER stress and apoptosis in prostate cancer cells

Androgen (AR) and glucocorticoid (GR) receptor signaling play opposing roles in prostate tumorigenesis: in prostate, AR acts as an oncogene, and GR is a tumor suppressor. Recently, we found that non-steroidal phyto-chemical Compound A (CpdA) is AR/GR modulator acting as anti-inflammatory anti-androgen. CpdA inhibits AR and prevents GR transactivation while enhancing GR transrepression. GR and AR are controlled by proteasomal degradation. We found that prolonged exposure of LNCaP, LNCaP-GR, DU145 and PC3 prostate carcinoma (PCa) cells to proteasome inhibitor Bortezomib (BZ) caused AR degradation and GR accumulation. BZ enhanced CpdA ability to inhibit AR and to augment GR transrepression. We also found that CpdA+BZ differentially regulated GR/AR to cooperatively suppress PCa cell growth and survival and to induce endoplasmic reticulum stress (ERS). Importantly, CpdA+BZ differentially regulated GR-responsive genes. CpdA+BZ blocked activation of glucocorticoid-responsive pro-survival genes, including SGK1, but activated BZ-induced ERS-related genes BIP/HSPA5 and CHOP /GADD153. Using ChIP, we showed that SGK1, BIP/HSPA5 and CHOP regulation was due to effects of CpdA and CpdA+BZ on GR loading on their promoters. We also found that AR and GR are abundant in advanced PCa from patients treated by androgen ablation and/or chemotherapy: 56% of carcinomas from treated patients expressed both receptors, and the other 27% expressed either GR or AR. Overall, our data validate the concept of dual AR/GR targeting in prostate cancer (PC) and suggest that BZ combination with dual-target steroid receptor modulator CpdA has high potential for PC therapy.     

Androgen receptor signaling is required for androgen-sensitive human prostate cancer cell proliferation and survival

Background  

Androgens and androgen receptors (AR) regulate normal prostate development and growth. They also are involved in pathological development of prostatic diseases, including benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Antiandrogen therapy for PCa, in conjunction with chemical or surgical castration, offers initial positive responses and leads to massive prostate cell death. However, cancer cells later appear as androgen-independent PCa. To investigate the role of AR in prostate cell proliferation and survival, we introduced a vector-based small interfering RNA (siRNA). This siRNA targeted 5'-untranslated region of AR mRNA for extended suppression of AR expression in androgen-sensitive human prostate LNCaP cells.     

Anti-androgen receptor ASC-J9 versus anti-androgens MDV3100 (Enzalutamide) or Casodex (Bicalutamide) leads to opposite effects on prostate cancer metastasis via differential modulation of macrophage infiltration and STAT3-CCL2 signaling

Despite androgen deprivation therapy (ADT) suppression of prostate cancer (PCa) growth, its overall effects on PCa metastasis remain unclear. Using human (C4-2B/THP1) and mouse (TRAMP-C1/RAW264.7) PCa cells–macrophages co-culture systems, we found currently used anti-androgens, MDV3100 (enzalutamide) or Casodex (bicalutamide), promoted macrophage migration to PCa cells that consequently led to enhanced PCa cell invasion. In contrast, the AR degradation enhancer, ASC-J9, suppressed both macrophage migration and subsequent PCa cell invasion. Mechanism dissection showed that Casodex/MDV3100 reduced the AR-mediated PIAS3 expression and enhanced the pSTAT3-CCL2 pathway. Addition of CCR2 antagonist reversed the Casodex/MDV3100-induced macrophage migration and PCa cell invasion. In contrast, ASC-J9 could regulate pSTAT3-CCL2 signaling using two pathways: an AR-dependent pathway via inhibiting PIAS3 expression and an AR-independent pathway via direct inhibition of the STAT3 phosphorylation/activation. These findings were confirmed in the in vivo mouse model with orthotopically injected TRAMP-C1 cells. Together, these results may raise the potential concern about the currently used ADT with anti-androgens that promotes PCa metastasis and may provide some new and better therapeutic strategies using ASC-J9 alone or a combinational therapy that simultaneously targets androgens/AR signaling and PIAS3-pSTAT3-CCL2 signaling to better battle PCa growth and metastasis at castration-resistant stage.     

Ligand-dependent corepressor acts as a novel androgen receptor corepressor, inhibits prostate cancer growth, and is functionally inactivated by the Src protein kinase

The activated androgen receptor (AR) promotes prostate cancer (PCa) growth. AR antagonists repress the AR by recruitment of corepressors. Not much is known about the inactivation of AR by corepressors in the presence of agonists (androgens). Here we show that the corepressor LCoR acts as an androgen-dependent corepressor that represses human PCa growth in vivo. In line with this, progressive decrease of ligand-dependent corepressor expression was observed in the PCa TRAMP mouse model with increasing age. LCoR interacts with AR and is recruited to chromatin in an androgen-induced manner. Unexpectedly, the LXXLL motif of LCoR is dispensable for interaction with the AR. Rather, the data indicate that LCoR interacts with the AR DNA binding domain on DNA. Interestingly, the interaction of LCoR with AR is inhibited by signaling pathways that are associated with androgen-independent PCa. Here we also show that the Src kinase inactivates the corepressive function of LCoR. Interfering with endogenous Src function by a dominant negative Src mutant, the growth inhibitory activity of LCoR is enhanced in vivo in a xenograft mouse model system. Thus, our studies indicate a role of LCoR as an AR corepressor and a tumor suppressor. Further, the decreased expression or inactivation of LCoR is as an important step toward PCa carcinogenesis in vivo.     

Targeting the Unique Methylation Pattern of Androgen Receptor (AR) Promoter in Prostate Stem/Progenitor Cells with 5-Aza-2′-deoxycytidine (5-AZA) Leads to Suppressed Prostate Tumorigenesis

Androgen receptor (AR) expression surveys found that normal prostate/prostate cancer (PCa) stem/progenitor cells, but not embryonic or mesenchymal stem cells, expressed little AR with high methylation in the AR promoter. Mechanism dissection revealed that the differential methylation pattern in the AR promoter could be due to differential expression of methyltransferases and binding of methylation binding protein to the AR promoter region. The low expression of AR in normal prostate/PCa stem/progenitor cells was reversed after adding 5-aza-2′-deoxycytidine, a demethylating agent, which could then lead to decreased stemness and drive cells into a more differentiated status, suggesting that the methylation in the AR promoter of prostate stem/progenitor cells is critical not only in maintaining the stemness but also critical in protection of cells from differentiation. Furthermore, induced AR expression, via alteration of its methylation pattern, led to suppression of the self-renewal/proliferation of prostate stem/progenitor cells and PCa tumorigenesis in both in vitro assays and in vivo orthotopic xenografted mouse studies. Taken together, these data prove the unique methylation pattern of AR promoter in normal prostate/PCa stem/progenitor cells and the influence of AR on their renewal/proliferation and differentiation. Targeting PCa stem/progenitor cells with alteration of methylated AR promoter status might provide a new potential therapeutic approach to battle PCa because the PCa stem/progenitor cells have high tumorigenicity.     

Oxidative stress and androgen receptor signaling in the development and progression of castration-resistant prostate cancer

Aberrant androgen receptor (AR) signaling plays a critical role in androgen-dependent prostate cancer (PCa), as well as in castration-resistant PCa (CRPC). Oxidative stress seems to contribute to the tumorigenesis and progression of PCa, as well as the development of CRPC, via activation of AR signaling. This notion is supported by the fact that there is an aberrant or improper regulation of the redox status in these disorders. Additionally, androgen-deprivation-induced oxidative stress seems to be involved in the pathogenesis of several disorders caused by androgen-deprivation therapy (ADT), including osteoporosis, neurodegenerative disease, and cardiovascular disease. Oxidative stress can be suppressed with antioxidants or via a reduction in reactive oxygen species production. Thus, developing new therapeutic agents that reduce oxidative stress might be useful in preventing the conversion of androgen-dependent PCa into CRPC, as well as reducing the adverse effects associated with ADT. The objective of this review is to provide an overview regarding the relationship between oxidative stress and AR signaling in the context of PCa and especially CRPC. Additionally, we discuss the potential use of antioxidant therapies in the treatment of PCa.     

White button mushroom (Agaricus bisporus) disrupts androgen receptor signaling in human prostate cancer cells and patient-derived xenograft

White button mushroom (WBM) (Agaricus bisporus) is a potential prostate cancer (PCa) chemo-preventative and therapeutic agent. Our clinical phase І trial of WBM powder in patients with biochemically recurrent PCa indicated that WBM intake reduced the circulating levels of prostate-specific antigen (PSA). We hypothesized that WBM exerts its effects on PCa through the androgen receptor (AR) signaling axis. Therefore, we conducted a reverse translational study with androgen-dependent PCa cell lines (LNCaP and VCaP) and patient-derived-xenografts (PDX) from a prostate tumor (TM00298). In both LNCaP and VCaP cells, western blots and qRT-PCR assays indicated that WBM extract (6–30 mg/mL) suppressed DHT-induced PSA expression and cell proliferation in a dose-dependent manner. Immunofluorescence analysis of AR revealed that WBM extract interrupted the AR nuclear-cytoplasmic distribution. PSA promotor-luciferase assay suggested that WBM extract inhibited DHT-induced luciferase activity. RNA-Seq on WBM-treated LNCaP cells confirmed that WBM treatment suppressed the androgen response pathways and cell-cycle control pathways. Our PDX showed that oral intake of WBM extract (200 mg/kg/d) suppressed tumor growth and decreased PSA levels in both tumors and serum. In the present study, we also identified a conjugated linoleic acid isomer (CLA-9Z11E) as a strong AR antagonist by performing LanthaScreen TR-FRET AR Coactivator Interaction Assays. The inhibitory effect of CLA-9Z11E (IC50: 350 nM) was nearly two times stronger than the known AR antagonist, cyproterone acetate (IC50: 672 nM). The information gained from this study improves the overall understanding of how WBM may contribute to the prevention and treatment of PCa.