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.     

The heat shock protein 70 inhibitor VER155008 suppresses the expression of HSP27, HOP and HSP90β and the androgen receptor,induces apoptosis,and attenuates prostate cancer cell growth

Heat shock proteins (HSPs) are molecular chaperones that play a pivotal role in correct folding, stabilization and intracellular transport of many client proteins including those involved in oncogenesis. HSP70, which is frequently overexpressed in prostate cancer (PCa), has been shown to critically contribute to tumor cell survival, and might therefore represent a potential therapeutic target. We treated both the androgen receptor (AR)-positive LNCaP and the AR-negative PC-3 cell lines with the pharmacologic HSP70 inhibitor VER155008. Although we observed antiproliferative effects and induction of apoptosis upon HSP70 inhibition, the apoptotic effect was more pronounced in AR-positive LNCaP cells. In addition, VER155008 treatment induced G1 cell cycle arrest in LNCaP cells and decreased AR expression. Further analysis of the HSP system by Western blot analysis revealed that expression of HSP27, HOP and HSP90β was significantly inhibited by VER155008 treatment, whereas the HSP40, HSP60, and HSP90α expression remained unchanged. Taken together, VER155008 might serve as a novel therapeutic option in PCa patients independent of the AR expression status.     

Androgen receptor expression reduces stemness characteristics of prostate cancer cells (PC3) by repression of CD44 and SOX2

Studies have shown that a subgroup of tumor cells possess stemness characteristics having self-renewal capacity and the ability to form new tumors. We sought to identify the plausible stemness factor that determines the “molecular signature” of prostate cancer (PCa) cells derived from different metastases (PC3, PCa2b, LNCaP, and DU145) and whether androgen receptor (AR) influences the maintenance of stemness features. Here we show sex-determining region Y (SRY)-box 2 (SOX2) as a putative stem cell marker in PC3 PCa cells and not in DU145, PCa2b, or LNCaP cells. PCa2b and PC3 cells were derived from bone metastases. PCa2b cells which are positive for the AR failed to demonstrate the expression of either cluster of differentiation 44 (CD44) or SOX2. Knockdown (KD) of AR in these cells did not affect the expression of either CD44 or SOX2. Conversely, PC3 cells, which are negative for AR, expressed both CD44 and SOX2. However, the expression of AR downregulated the expression of both CD44 and SOX2 in PC3 cells. CD44 regulates SOX2 expression as KD of CD44 and reduces SOX2 levels considerably. SOX2 KD attenuated not only the expression of SNAIL and SLUG but also the migration and tumorsphere formation in PC3 cells. Collectively, our findings underscore a novel role of CD44 signaling in the maintenance of stemness and progression of cancer through SOX2 in AR-independent PC3 cells. SOX2 has a role in the regulation of expression of SNAIL and SLUG. SOX2 could be a potential therapeutic target to thwart the progression of SOX2-positive cancer cells or recurrence of androgen-independent PCa.     

A competitive inhibitor that reduces recruitment of androgen receptor to androgen-responsive genes

The androgen receptor (AR) has a critical role in the growth and progression of androgen-dependent and castration-resistant prostate cancers. To identify novel inhibitors of AR transactivation that block growth of prostate cancer cells, a luciferase-based high-throughput screen of ~160,000 small molecules was performed in cells stably expressing AR and a prostate-specific antigen (PSA)-luciferase reporter. CPIC (1-(3-(2-chlorophenoxy) propyl)-1H-indole-3-carbonitrile) was identified as a small molecule that blocks AR transactivation to a greater extent than other steroid receptors. CPIC inhibited AR-mediated proliferation of androgen-sensitive prostate cancer cell lines, with minimal toxicity in AR-negative cell lines. CPIC treatment also reduced the anchorage-independent growth of LAPC-4 prostate cancer cells. CPIC functioned as a pure antagonist by inhibiting the expression of AR-regulated genes in LAPC-4 cells that express wild-type AR and exhibited weak agonist activity in LNCaP cells that express the mutant AR-T877A. CPIC treatment did not reduce AR levels or alter its nuclear localization. We used chromatin immunoprecipitation to identify the site of action of CPIC. CPIC inhibited recruitment of androgen-bound AR to the PSA promoter and enhancer sites to a greater extent than bicalutamide. CPIC is a new therapeutic inhibitor that targets AR-mediated gene activation with potential to arrest the growth of prostate cancer.     

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.     

Celastrol Induces Autophagy by Targeting AR/miR-101 in Prostate Cancer Cells

Autophagy is an evolutionarily conserved process responsible for the degradation and recycling of cytoplasmic components through autolysosomes. Targeting AR axis is a standard strategy for prostate cancer treatment; however, the role of AR in autophagic processes is still not fully understood. In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy. Knockdown of AR in AR-positive prostate cancer cells resulted in enhanced autophagy. Ectopic expression of AR in AR-negative prostate cancer cells, or gain of function of the AR signaling in AR-positive cells, led to suppression of autophagy. Since miR-101 is an inhibitor of autophagy and its expression was decreased along with AR in the process of celastrol-induced autophagy, we hypothesize that AR inhibits autophagy through transactivation of miR-101. AR binding site was defined in the upstream of miR-101 gene by luciferase reporter and ChIP assays. MiR-101 expression correlated with AR status in prostate cancer cell lines. The inhibition of celastrol-induced autophagy by AR was compromised by blocking miR-101; while transfection of miR-101 led to inhibition of celastrol-induced autophagy in spite of AR depletion. Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR. Finally, autophagy inhibition by miR-101 mimic was found to enhance the cytotoxic effect of celastrol in prostate cancer cells. Our results demonstrate that AR inhibits autophagy via transactivation of miR-101, thus combination of miR-101 mimics with celastrol may represent a promising therapeutic approach for treating prostate cancer.     

A transcriptional target of androgen receptor,miR-421 regulates proliferation and metabolism of prostate cancer cells

Prostate cancer is one of the most common malignancies, and microRNAs have been recognized to be involved in tumorigenesis of various kinds of cancer including prostate cancer (PCa). Androgen receptor (AR) plays a core role in prostate cancer progression and is responsible for regulation of numerous downstream targets including microRNAs. This study identified an AR-repressed microRNA, miR-421, in prostate cancer. Expression of miR-421 was significantly suppressed by androgen treatment, and correlated to AR expression in different prostate cancer cell lines. Furthermore, androgen-activated AR could directly bind to androgen responsive element (ARE) of miR-421, as predicted by bioinformatics resources and demonstrated by ChIP and luciferase reporter assays. In addition, over-expression of miR-421 markedly supressed cell viability, delayed cell cycle, reduced glycolysis and inhibited migration in prostate cancer cells. According to the result of miR-421 target genes searching, we focused on 4 genes NRAS, PRAME, CUL4B and PFKFB2 based on their involvement in cell proliferation, cell cycle progression and metabolism. The expression of these 4 downstream targets were significantly repressed by miR-421, and the binding sites were verified by luciferase assay. Additionally, we explored the expression of miR-421 and its target genes in human prostate cancer tissues, both in shared microarray data and in our own cohort. Significant differential expression and inverse correlation were found in PCa patients.     

Prostate cancer: a model of integration of genomic and non-genomic effects of the androgen receptor in cell lines model

Androgens and the androgen receptor (AR) are involved both in early tumorigenesis of prostate cancer (PCa) and in androgen-refractory disease. The role of AR signalling has also been highlighted by the fusion gene TMPRSS2:ERG recently identified in the majority of PCa. Several data indicate that re-expression of AR in PCa cell lines confers a less aggressive phenotype. We observed that re-expression of AR in the AR-negative cells PC3 decreases anchorage-independent growth and Matrigel invasiveness of PC3-AR cells where plasma membrane interaction between AR and EGFR led to an interference with downstream signalling and internalization of activated EGFR. Our data evidenced a shift of EGFR internalization pathway from the clathrin-coated pit one mediating signalling and recycling of EGFR to the lipid raft-mediated one mainly involved in lysosomal degradation of EGFR. These effects involved an altered recruitment to EGFR of the adaptor proteins Grb2 and c-Cbl followed by a reduced ubiquitination of EGFR. Our preliminary results suggest that in PC3-AR cells a pool of classical AR is located within cholesterol-rich membrane microdomains (namely as lipid rafts) and a population of EGFR is within cholesterol-rich membrane microdomains too. However, AR and EGFR membrane interaction that is increased by rapid androgen signalling is not within cholesterol-rich membrane microdomains. Our data enlighten that the crosstalk between genotropic and non-genotropic AR signalling interferes with signalling of EGFR in response to ligand leading to a lower invasive phenotype of AR-positive PCa cells.     

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.     

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.     

ARTIK-52 induces replication-dependent DNA damage and p53 activation exclusively in cells of prostate and breast cancer origin

The realization, that the androgen receptor (AR) is essential for prostate cancer (PC) even after relapse following androgen deprivation therapy motivated the search for novel types of AR inhibitors. We proposed that targeting AR expression versus its function would work in cells having either wild type or mutant AR as well as be independent of androgen synthesis pathways. Previously, using a phenotypic screen in androgen-independent PC cells we identified a small molecule inhibitor of AR, ARTIK-52. Treatment with ARTIK-52 caused the loss of AR protein and death of AR-positive, but not AR-negative, PC cells. Here we present data that ARTIK-52 induces degradation of AR mRNA through a mechanism that we were unable to establish. However, we found that ARTIK-52 is toxic to breast cancer (BC) cells expressing AR, although they were not sensitive to AR knockdown, suggesting an AR-independent mechanism of toxicity. Using different approaches we detected that ARTIK-52 induces replication-dependent double strand DNA breaks exclusively in cancer cells of prostate and breast origin, while not causing DNA damage, or any toxicity, in normal cells, as well as in non-PC and non-BC tumor cells, independent of their proliferation status. This amazing specificity, combined with such a basic mechanism of toxicity, makes ARTIK-52 a potentially useful tool to discover novel attractive targets for the treatment of BC and PC. Thus, phenotypic screening allowed us to identify a compound, whose properties cannot be predicted based on existing knowledge and moreover, uncover a barely known link between AR and DNA damage response in PC and BC epithelial cells.