Nagalakshmi Nadiminty

Niclosamide Inhibits Androgen Receptor Variants Expression and Overcomes Enzalutamide Resistance in Castration-Resistant Prostate Cancer

Purpose: Enzalutamide, a second-generation antiandrogen, was recently approved for the treatment of castration-resistant prostate cancer (CRPC) in patients who no longer respond to docetaxel. Despite these advances that provide temporary respite, resistance to enzalutamide occurs frequently. Androgen receptor (AR) splice variants such as AR-V7 have recently been shown to drive castration-resistant growth and resistance to enzalutamide. This study was designed to identify inhibitors of AR variants and test its ability to overcome resistance to enzalutamide. Experimental Design: The drug screening was conducted using luciferase activity assay to determine the activity of AR-V7 after treatment with the compounds in the Prestwick Chemical Library, which contains about 1,120 FDA-approved drugs. The effects of the identified inhibitors on AR-V7 activity and enzalutamide sensitivity were characterized in CRPC and enzalutamide-resistant prostate cancer cells in vitro and in vivo. Results: Niclosamide, an FDA-approved antihelminthic drug, was identified as a potent AR-V7 inhibitor in prostate cancer cells. Niclosamide significantly downregulated AR-V7 protein expression by protein degradation through a proteasome-dependent pathway. Niclosamide also inhibited AR-V7 transcription activity and reduced the recruitment of AR-V7 to the PSA promoter. Niclosamide inhibited prostate cancer cell growth in vitro and tumor growth in vivo. Furthermore, the combination of niclosamide and enzalutamide resulted in significant inhibition of enzalutamide-resistant tumor growth, suggesting that niclosamide enhances enzalutamide therapy and overcomes enzalutamide resistance in CRPC cells. Conclusions: Niclosamide was identified as a novel inhibitor of AR variants. Our findings offer preclinical validation of niclosamide as a promising inhibitor of AR variants to treat, either alone or in combination with current antiandrogen therapies, patients with advanced prostate cancer, especially those resistant to enzalutamide. Clin Cancer Res; 20(12); 3198–210. ©2014 AACR.

MicroRNA let-7c Suppresses Androgen Receptor Expression and Activity via Regulation of Myc Expression in Prostate Cancer Cells

Background: Let-7c is a microRNA down-regulated in prostate cancer. Results: Let-7c suppresses androgen receptor expression by targeting its transcription via c-Myc. Suppression of AR by let-7c leads to decreased cell proliferation and tumor growth. Conclusion: Let-7c suppresses androgen receptor expression. Significance: Our study demonstrates that let-7c plays an important role in regulation of androgen signaling and prostate cancer proliferation. Castration-resistant prostate cancer continues to rely on androgen receptor (AR) expression. AR plays a central role in the development of prostate cancer and progression to castration resistance during and after androgen deprivation therapy. Here, we identified miR-let-7c as a key regulator of expression of AR. miR-let-7c suppresses AR expression and activity in human prostate cancer cells by targeting its transcription via c-Myc. Suppression of AR by let-7c leads to decreased cell proliferation of human prostate cancer cells. Down-regulation of Let-7c in prostate cancer specimens is inversely correlated with AR expression, whereas the expression of Lin28 (a repressor of let-7) is correlated positively with AR expression. Our study demonstrates that the miRNA let-7c plays an important role in the regulation of androgen signaling in prostate cancer by down-regulating AR expression. These results suggest that reconstitution of miR-let-7c may aid in targeting enhanced and hypersensitive AR in advanced prostate cancer.

NF-κB2/p52 Induces Resistance to Enzalutamide in Prostate Cancer: Role of Androgen Receptor and Its Variants

Resistance of prostate cancer cells to the next-generation antiandrogen enzalutamide may be mediated by a multitude of survival signaling pathways. In this study, we tested whether increased expression of NF-κB2/p52 induces prostate cancer cell resistance to enzalutamide and whether this response is mediated by aberrant androgen receptor (AR) activation and AR splice variant production. LNCaP cells stably expressing NF-κB2/p52 exhibited higher survival rates than controls when treated with enzalutamide. C4-2B and CWR22Rv1 cells chronically treated with enzalutamide were found to express higher levels of NF-κB2/p52. Downregulation of NF-κB2/p52 in CWR22Rv1 cells chronically treated with enzalutamide rendered them more sensitive to cell growth inhibition by enzalutamide. Analysis of the expression levels of AR splice variants by quantitative reverse transcription PCR and Western blotting revealed that LNCaP cells expressing p52 exhibit higher expression of AR splice variants. Downregulation of expression of NF-κB2/p52 in VCaP and CWR22Rv1 cells by short hairpin RNA abolished expression of splice variants. Downregulation of expression of either full-length AR or the splice variant AR-V7 led to an increase in sensitivity of prostate cancer cells to enzalutamide. These results collectively demonstrate that resistance to enzalutamide may be mediated by NF-κB2/p52 via activation of AR and its splice variants. Mol Cancer Ther; 12(8); 1629–37. ©2013 AACR.

MicroRNA let-7c is downregulated in prostate cancer and suppresses prostate cancer growth.

Purpose Prostate cancer (PCa) is characterized by deregulated expression of several tumor suppressor or oncogenic miRNAs. The objective of this study was the identification and characterization of miR-let-7c as a potential tumor suppressor in PCa. Experimental Design Levels of expression of miR-let-7c were examined in human PCa cell lines and tissues using qRT-PCR and in situ hybridization. Let-7c was overexpressed or suppressed to assess the effects on the growth of human PCa cell lines. Lentiviral-mediated re-expression of let-7c was utilized to assess the effects on human PCa xenografts. Results We identified miR-let-7c as a potential tumor suppressor in PCa. Expression of let-7c is downregulated in castration-resistant prostate cancer (CRPC) cells. Overexpression of let-7c decreased while downregulation of let-7c increased cell proliferation, clonogenicity and anchorage-independent growth of PCa cells in vitro. Suppression of let-7c expression enhanced the ability of androgen-sensitive PCa cells to grow in androgen-deprived conditions in vitro. Reconstitution of Let-7c by lentiviral-mediated intratumoral delivery significantly reduced tumor burden in xenografts of human PCa cells. Furthermore, let-7c expression is downregulated in clinical PCa specimens compared to their matched benign tissues, while the expression of Lin28, a master regulator of let-7 miRNA processing, is upregulated in clinical PCa specimens. Conclusions These results demonstrate that microRNA let-7c is downregulated in PCa and functions as a tumor suppressor, and is a potential therapeutic target for PCa.

Interleukin-6 Regulates Androgen Synthesis in Prostate Cancer Cells

Purpose: The standard systemic treatment for prostate cancer patients is androgen deprivation therapy. Although serum testosterone concentrations were significantly reduced after androgen deprivation therapy, levels of intraprostatic androgens are reproducibly measured at concentrations sufficient to activate androgen receptor and stimulate tumor growth, suggesting that prostate cancer cells may survive androgen deprivation therapies by increasing intracrine androgen synthesis within the prostate. However, factors that regulate de novo intracrine androgen synthesis have not been identified. Interleukin-6 (IL-6) has been implicated in the modulation of androgen receptor activation and growth and differentiation in prostate cancer. In this study, we investigate whether IL-6 regulates intraprostatic androgen synthesis in prostate cancer cells. Experimental Design: Quantitative reverse transcription-PCR and Western blotting were done to detect expression levels of steroidogenic enzymes. AKR1C3 promoter reporter was constructed and analyzed for IL-6–mediated AKR1C3 transcriptional activity. IL-6–mediated signaling was knocked down using small interfering RNA specific to IL-6 receptor and gp130, and the effect on AKR1C3 expression was examined. Intraprostatic androgen levels in prostate cancer cells in culture and in tumors were measured by an enzyme immunoassay (Testosterone EIA kit). Results: We found that IL-6 increases the expression of genes encoding many steroidogenic enzymes, including HSD3B2 and AKR1C3, involved in androgen biosynthesis. Down-regulation of IL-6 receptor and gp130 expression using specific small interfering RNA abolished IL-6–mediated AKR1C3 expression, suggesting that IL-6 signaling is responsible for AKR1C3 expression. IL-6 increases AKR1C3 promoter activity, indicating that the increase in IL-6–mediated AKR1C3 expression is in part at the transcriptional level. Treatment of IL-6 increased testosterone level in LNCaP cells. The tumor testosterone levels were detected at 378 pg/g in tumors generated from IL-6–overexpressing LNCaP-IL6+ cells inoculated orthotopically into the prostates of castrated male nude mice. Conclusions: These results suggest that IL-6 increases levels of intracrine androgens through enhanced expression of genes mediating androgen metabolism in prostate cancer cells.

Intracrine Androgens and AKR1C3 Activation Confer Resistance to Enzalutamide in Prostate Cancer

The introduction of enzalutamide and abiraterone has led to improvement in the treatment of metastatic castration-resistant prostate cancer. However, acquired resistance to enzalutamide and abiraterone therapies frequently develops within a short period in many patients. In the present study, we developed enzalutamide-resistant prostate cancer cells in an effort to understand the mechanisms of resistance. Global gene-expression analysis showed that the steroid biosynthesis pathway is activated in enzalutamide-resistant prostate cancer cells. One of the crucial steroidogenic enzymes, AKR1C3, was significantly elevated in enzalutamide-resistant cells. In addition, AKR1C3 is highly expressed in metastatic and recurrent prostate cancer and in enzalutamide-resistant prostate xenograft tumors. LC/MS analysis of the steroid metabolites revealed that androgen precursors such as cholesterol, DHEA and progesterone, as well as androgens are highly upregulated in enzalutamide-resistant prostate cancer cells compared to the parental cells. Knockdown of AKR1C3 expression by shRNA or inhibition of AKR1C3 enzymatic activity by indomethacin resensitized enzalutamide-resistant prostate cancer cells to enzalutamide treatment both in vitro and in vivo. In contrast, overexpression of AKR1C3 confers resistance to enzalutamide. Furthermore, the combination of indomethacin and enzalutamide resulted in significant inhibition of enzalutamide-resistant tumor growth. These results suggest that AKR1C3 activation is a critical resistance mechanism associated with enzalutamide resistance; targeting intracrine androgens and AKR1C3 will overcome enzalutamide resistance and improve survival of advanced prostate cancer patients.

Functional p53 determines docetaxel sensitivity in prostate cancer cells.

Docetaxel is the first line treatment for castration resistant prostate cancer (CRPC). However, docetaxel resistance rapidly develops. Identifying the critical mechanisms giving rise to docetaxel resistance is the major challenge in advanced prostate cancer.

Inhibition of Stat3 activation by sanguinarine suppresses prostate cancer cell growth and invasion

Signal transducer and activator of transcription 3 (Stat3) is an oncogenic transcriptional factor that plays a critical role in carcinogenesis and cancer progression and is a potential therapeutic target. Sanguinarine, a benzophenanthridine alkaloid derived primarily from the bloodroot plant, was identified previously as a novel inhibitor of survivin that selectively kills prostate cancer cells over “normal” prostate epithelial cells.

Selenium Disrupts Estrogen Signaling by Altering Estrogen Receptor Expression and Ligand Binding in Human Breast Cancer Cells

Cancer prevention studies suggest that selenium is effective in reducing the incidence of cancers including prostate, colon, and lung cancers. Previous reports showed that selenium inhibits premalignant human breast MCF-10AT1 and MCF10AT3B cell growth in vitro and reduces mammary tumor incidence after exposure to carcinogens in tumor models. Because estrogen is critical to the development and differentiation of estrogen target tissues, including the breast, the present study was designed to examine the effect of selenium on estrogen receptor (ER) expression and activation using methylseleninic acid (MSA), an active form of selenium in vitro. Selenium decreased the levels of expression of ERalpha mRNA and protein and reduced the binding of labeled estradiol to estrogen receptor in MCF-7 cells. Selenium inhibited the trans-activating activity of estrogen receptor in MCF-7 cells expressing functional estrogen receptor using a luciferase reporter construct linked to estrogen responsive element. Selenium decreased the binding of estrogen receptor to the estrogen responsive element site using an electrophoretic mobility gel shift assay. Selenium suppressed estrogen induction of the endogenous target gene c-myc. In contrast to the effect on ERalpha in MCF-7 cells, selenium increased ERbeta mRNA expression in MDA-MB231 human breast cancer cells. Thus, differential regulation of ERalpha and ERbeta in breast cancer cells may represent a novel mechanism of selenium action and provide a rationale for selenium breast cancer prevention trial.

Prostate-Specific Antigen Modulates Genes Involved in Bone Remodeling and Induces Osteoblast Differentiation of Human Osteosarcoma Cell Line SaOS-2

Purpose: The high prevalence of osteoblastic bone metastases in prostate cancer involves the production of osteoblast-stimulating factors by prostate cancer cells. Prostate-specific antigen (PSA) is a serine protease uniquely produced by prostate cancer cells and is an important serologic marker for prostate cancer. In this study, we examined the role of PSA in the induction of osteoblast differentiation. Experimental Design: Human cDNA containing a coding region for PSA was transfected into human osteosarcoma SaOS-2 cells. SaOS-2 cells were also treated with exogenously added PSA. We evaluated changes in global gene expression using cDNA arrays and Northern blot analysis resulting from expression of PSA in human osteosarcoma SaOS-2 cells. Results: SaOS-2 cells expressing PSA had markedly up-regulated expression of genes associated with osteoblast differentiation including runx-2 and osteocalcin compared with the controls. Consistent with these results, the stable clones expressing PSA showed increased mineralization and increased activity of alkaline phosphatase in vitro compared with controls, suggesting that these cells undergo osteoblast differentiation. We also found that osteoprotegerin expression was down-regulated and that the receptor activator of NF-κB ligand expression was up-regulated in cells expressing PSA compared with controls. Conclusions: Modulation of the expression of osteogenic genes and alteration of the balance between osteoprotegerin–receptor activator of NF-κB ligand by PSA suggests that PSA produced by metastatic prostate cancer cells may participate in bone remodeling in favor of the development of osteoblastic metastases in the heterogeneous mixture of osteolytic and osteoblastic lesions. These findings provide a molecular basis for understanding the high prevalence of osteoblastic bone metastases in prostate cancer.