Progression to metastatic stage in a cellular model of prostate cancer is associated with methylation of the androgen receptor gene and transcriptional suppression of the insulin-like growth factor-I receptor gene

The progression of prostate cancer from an organ-confined, androgen-sensitive disease to a metastatic one is associated with dysregulation of androgen receptor (AR)-regulated target genes and with a decrease in insulin-like growth factor-I receptor (IGF1R) expression. DNA methylation of CpG islands is an epigenetic mechanism associated with gene silencing. Recent studies have demonstrated that methylation occurs early in prostate carcinogenesis and, furthermore, may contribute to androgen independence. The methylation status of the AR and IGF1R genes was evaluated in a series of prostate cancer cell lines corresponding to early (benign) and advanced (metastatic) stages of the disease. Results of 5-Aza-2′-deoxycytidine (5-Aza) experiments, methylation-specific PCR, and sodium bisulfite-direct DNA sequencing revealed that the AR promoter is hypermethylated in metastatic M12, but not in benign P69, cells. On the other hand, no methylation was seen in the IGF1R promoter at any stage of the disease. We show, however, that 5-Aza treatment, which caused demethylation of the AR promoter, led to a significant increase in IGF1R mRNA levels, whereas addition of the AR inhibitor flutamide decreased the IGF1R mRNA levels to basal values measured prior to the 5-Aza treatment. Given that the IGF1R gene has been identified as a downstream target for AR action, our data is consistent with a model in which the AR gene undergoes methylation during progression of the disease, leading to dysregulation of AR targets, including the IGF1R gene, at advanced metastatic stages.     

Interaction of IGF signaling and the androgen receptor in prostate cancer progression

The insulin-like growth factor type I receptor (IGF-IR) has been suggested to play an important role in prostate cancer progression and possibly in the progression to androgen-independent (AI) disease. The term AI may not be entirely correct, in that recent data suggest that expression of androgen receptor (AR) and androgen-regulated genes is the primary association with prostate cancer progression after hormone ablation. Therefore, signaling through other growth factors has been thought to play a role in AR-mediated prostate cancer progression to AI disease in the absence of androgen ligand. However, existing data on how IGF-IR signaling interacts with AR activation in prostate cancer are conflicting. In this Prospect article, we review some of the published data on the mechanisms of IGF-IR/AR interaction and present new evidence that IGF-IR signaling may modulate AR compartmentation and thus alter AR activity in prostate cancer cells. Inhibition of IGF-IR signaling can result in cytoplasmic AR retention and a significant change in androgen-regulated gene expression. Translocation of AR from the cytoplasm to the nucleus may be associated with IGF-induced dephosphorylation. Since fully humanized antibodies targeting the IGF-IR are now in clinical trials, the current review is intended to reveal the mechanisms of potential therapeutic effects of these antibodies on AI prostate cancers.     

Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas

Small cell prostate carcinoma (SCPC) morphology is rare at initial diagnosis but often emerges during prostate cancer progression and portends a dismal prognosis. It does not express androgen receptor (AR) or respond to hormonal therapies. Clinically applicable markers for its early detection and treatment with effective chemotherapy are needed. Our studies in patient tumor–derived xenografts (PDX) revealed that AR–negative SCPC (AR^?SCPC) expresses neural development genes instead of the prostate luminal epithelial genes characteristic of AR–positive castration-resistant adenocarcinomas (AR⁺ADENO). We hypothesized that the differences in cellular lineage programs are reflected in distinct epigenetic profiles. To address this hypothesis, we compared the DNA methylation profiles of AR^? and AR⁺ PDX using methylated CpG island amplification and microarray (MCAM) analysis and identified a set of differentially methylated promoters, validated in PDX and corresponding donor patient samples. We used the Illumina 450K platform to examine additional regions of the genome and the correlation between the DNA methylation profiles of the PDX and their corresponding patient tumors. Struck by the low frequency of AR promoter methylation in the AR^?SCPC, we investigated this region's specific histone modification patterns by chromatin immunoprecipitation. We found that the AR promoter was enriched in silencing histone modifications (H3K27me3 and H3K9me2) and that EZH2 inhibition with 3-deazaneplanocin A (DZNep) resulted in AR expression and growth inhibition in AR^?SCPC cell lines. We conclude that the epigenome of AR^? is distinct from that of AR⁺ castration-resistant prostate carcinomas, and that the AR^? phenotype can be reversed with epigenetic drugs.     

Insulin-like growth factor 1 stimulation of androgen receptor activity requires β(1A) integrins

Despite the findings that β1 integrins play a vital role in the regulation of cell proliferation and survival, the mechanisms through which they operate and lead to cancer progression remain elusive. Previously, our laboratory has shown that β(1A) integrins support insulin-like growth factor 1 (IGFI)-mediated mitogenic and transforming activities. Here, we report that β(1A) integrins regulate basal levels of IGF-IR, although they are not critical for maintaining cancer cell morphology. Upon transfection of β(1A) siRNA and consequent downregulation of IGF-IR, we show inhibition of anchorage-independent growth of prostate cancer cells, a function which is dependent on IGF-IR expression. In addition, we demonstrate that IGFI-mediated activation of androgen receptor (AR), known to occur in prostate cancer cells, requires expression of β(1A) integrins as evaluated by luciferase reporter assays and immunoblotting analysis. Since β(1A) integrin levels are increased by R1881 or dihydrotestosterone (DHT), our results imply that β(1A) integrins support an androgen-enhanced feedback loop that regulates the expression of IGF-IR. β(1A) integrins also regulate inducible levels of IGF-IR in cells stimulated by androgen or by a combination of androgen and IGFI, as evaluated by flow cytometric analysis and immunoblotting. Furthermore, upon transfection of β(1A) siRNA and consequent downregulation of IGF-IR, neither activation of AKT, an effector of IGF-IR, nor AR levels are affected. We conclude that β(1A) integrin expression is critical for maintaining the regulatory crosstalk between IGF-IR and AR.     

Peroxiredoxin 2 in the nucleus and cytoplasm distinctly regulates androgen receptor activity in prostate cancer cells

Currently, few therapies are effective against castration-resistant prostate cancer. Increased activation of the androgen/androgen receptor (AR) signaling pathway is thought to promote castration-resistant prostate cancer. Herein, we report that peroxiredoxin (Prx) gene expression in castration-resistant prostate cancer and hydrogen peroxide-resistant cells was upregulated. Prx2 was overexpressed in castration-resistant prostate cancer at the mRNA and protein levels and was localized to the nucleus and cytoplasm. Overexpression of Prx2 increased AR transactivation, whereas Prx2 overexpression in the nucleus suppressed AR transactivation. These effects of Prx2 on AR activity were abolished by the introduction of function-disrupting mutations into Cys⁵¹ and Cys¹⁷². Silencing Prx2 reduced the expression of androgen-regulated genes and suppressed the growth of AR-expressing prostate cancer cells by inducing cell-cycle arrest at the G1 phase. Furthermore, Prx2 knockdown also suppressed cell growth in castration-resistant prostate cancer cells. These findings indicate that Prx2 is involved in the proliferation of AR-expressing prostate cancer cells by modulating AR activity. Designing therapeutics targeting Prx2 may offer a novel strategy for developing treatments for prostate cancer, including castration-resistant prostate cancer, which is dependent on AR signaling.     

Androgen receptor is up-regulated by a BLT2-linked pathway to contribute to prostate cancer progression

The androgen receptor (AR) plays a central role in the development and progression of prostate cancer. AR expression is maintained throughout the progression of prostate cancer and is also associated with an aggressive, castration-resistant (CR) phenotype. Despite the critical roles of AR expression in prostate cancer progression, the exact signaling mechanism regulating AR expression remains unclear. In this study, we demonstrated that AR expression was increased by a low-affinity leukotriene B(4) receptor (BLT2)-linked pathway. We found that BLT2 was overexpressed in AR-positive prostate cancer cells, such as LNCaP cells, and BLT2 inhibition, using an inhibitor or siRNA knockdown, clearly attenuated AR expression and triggered apoptosis in these cells. These results suggest a role for BLT2 in AR expression and the survival of AR-positive prostate cancer cells. Moreover, we found that the NADPH oxidase family protein, Nox4, lay downstream of BLT2 and mediated the production of reactive oxygen species (ROS) and subsequent NF-κB stimulation, thereby inducing AR expression. Taken together, our results demonstrate that BLT2 plays a critical role in AR expression via a Nox4-ROS-NF-κB-linked pathway, thereby mediating the survival of AR-positive prostate cancer cells. Our findings point to BLT2 as a key regulator of AR expression and will contribute to the development of novel therapies for AR-positive prostate cancers, including androgen-responsive and CR prostate cancers.     

ATRA inhibits the proliferation of DU145 prostate cancer cells through reducing the methylation level of HOXB13 gene

All-trans retinoic acid (ATRA) has been widely investigated for treatments of many cancers including prostate cancer. HOXB13, silenced in androgen receptor-negative (AR(-)) prostate cancer cells, plays a role in AR(-) prostate cancer cell growth arrest. In this study we intended to elucidate the mechanisms that are involved in the proliferation inhibition of AR(-) prostate cancer cells triggered by ATRA. We discovered that ATRA was able to induce the growth arrest and to increase HOXB13 expression in AR(-) prostate cancer cells. Both EZH2 and DNMT3b participated in the repression of HOXB13 expression through an epigenetic mechanism involving DNA and histone methylation modifications. Specifically, EZH2 recruited DNMT3b to HOXB13 promoter to form a repression complex. Moreover, ATRA could upregulate HOXB13 through decreasing EZH2 and DNMT3b expressions and reducing their interactions with the HOXB13 promoter. Concurrently, the methylation level of the HOXB13 promoter was reduced upon the treatment of ATRA. Results from this study implicated a novel effect of ATRA in inhibition of the growth of AR(-) resistant human prostate cancer cells through alteration of HOXB13 expression as a result of epigenetic modifications.     

Androgens as therapy for androgen receptor-positive castration-resistant prostate cancer

Prostate cancer is the most frequently diagnosed non-cutaneous tumor of men in Western countries. While surgery is often successful for organ-confined prostate cancer, androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. Shortening the period of androgen ablation therapy may benefit prostate cancer patients. Intermittent Androgen Deprivation therapy improves quality of life, reduces toxicity and medical costs, and delays disease progression in some patients. Cell culture and xenograft studies using androgen receptor (AR)-positive castration-resistant human prostate cancers cells (LNCaP, ARCaP, and PC-3 cells over-expressing AR) suggest that androgens may suppress the growth of AR-rich prostate cancer cells. Androgens cause growth inhibition and G1 cell cycle arrest in these cells by regulating c-Myc, Skp2, and p27^Kip via AR. Higher dosages of testosterone cause greater growth inhibition of relapsed tumors. Manipulating androgen/AR signaling may therefore be a potential therapy for AR-positive advanced prostate cancer.     

Interleukin-6 is a potent inducer of S100P, which is up-regulated in androgen-refractory and metastatic prostate cancer

Elevated circulating interleukin-6 (IL6) and up-regulated S100P in prostate cancer (PCa) specimens correlate independently with progression to androgen-independent and metastatic PCa. The cause of up-regulated S100P levels in advanced PCa remains to be determined. We investigated the possibility that IL6 is an inducer of S100P. Determination of mRNA and protein levels by real-time PCR and Western blotting revealed that IL6 is a more potent inducer of S100P than the synthetic androgen, R1881, in the LNCaP/C4-2B model of PCa progression. IL6 did not require androgen to induce S100P in these cells, which express a functional androgen receptor (AR). Like R1881, IL6 was unable to induce S100P in PC3 cells that lack a functional AR. IL6 did not strongly induce the AR-dependent genes PSA and KLK2 and, contrary to R1881, down-regulated Cyr61/CCN1, a potential marker that is down-regulated in PCa. Epidermal growth factor (EGF), which like IL6 is a non-androgen activator of the AR, did not induce S100P. The data identifies a unique gene-induction profile for IL6 and suggests that IL6 may require a functional AR for S100P induction. A link between elevated IL6 and up-regulated S100P in androgen-refractory and metastatic PCa is postulated.     

Pomegranate polyphenols down-regulate expression of androgen-synthesizing genes in human prostate cancer cells overexpressing the androgen receptor

Prostate cancer is dependent on circulating testosterone in its early stages and is treatable with radiation and surgery. However, recurrent prostate tumors advance to an androgen-independent state in which they progress in the absence of circulating testosterone, leading to metastasis and death. During the development of androgen independence, prostate cancer cells are known to increase intracellular testosterone synthesis, which maintains cancer cell growth in the absence of significant amounts of circulating testosterone. Overexpression of the androgen receptor (AR) occurs in androgen-independent prostate cancer and has been proposed as another mechanism promoting the development of androgen independence. The LNCaP-AR cell line is engineered to overexpress AR but is otherwise similar to the widely studied LNCaP cell line. We have previously shown that pomegranate extracts inhibit both androgen-dependent and androgen-independent prostate cancer cell growth. In this study, we examined the effects of pomegranate polyphenols, ellagitannin-rich extract and whole juice extract on the expression of genes for key androgen-synthesizing enzymes and the AR. We measured expression of the HSD3B2 (3beta-hydroxysteroid dehydrogenase type 2), AKR1C3 (aldo-keto reductase family 1 member C3) and SRD5A1 (steroid 5alpha reductase type 1) genes for the respective androgen-synthesizing enzymes in LNCaP, LNCaP-AR and DU-145 human prostate cancer cells. A twofold suppression of gene expression was considered statistically significant. Pomegranate polyphenols inhibited gene expression and AR most consistently in the LNCaP-AR cell line (P=.05). Therefore, inhibition by pomegranate polyphenols of gene expression involved in androgen-synthesizing enzymes and the AR may be of particular importance in androgen-independent prostate cancer cells and the subset of human prostate cancers where AR is up-regulated.