Jae Yeon Chun

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.

Interleukin-6 increases prostate cancer cells resistance to bicalutamide via TIF2

The standard treatment for advanced, androgen-responsive prostate cancer is androgen deprivation therapy with or without a nonsteroidal antiandrogen, such as bicalutamide. Although maximal androgen blockade exhibits favorable responses in the majority of patients, prostate cancer eventually progresses to an androgen-refractory stage. The mechanism underlying bicalutamide resistance in the course of prostate cancer progression is incompletely understood. However, interleukin-6 (IL-6) plays a critical role in the development and progression of CRPC. Herein, we explored an association between IL-6 and bicalutamide resistance. To study this, series of lower and higher passages of LNCaP cell sublines generated by long-term exposure to IL-6 were used. The cells from higher passages of LNCaP treated with IL-6 developed resistance to bicalutamide treatment compared with parental LNCaP cells. The levels of transcriptional intermediary factor 2 (TIF2) in IL-6-treated LNCaP cells were found to be significantly higher than parental LNCaP cells. Down-regulation of TIF2 expression via short hairpin RNA in IL-6-treated LNCaP cells sensitized these cells to bicalutamide treatment, whereas overexpression of TIF2 in the parental LNCaP cells increased resistance to bicalutamide. Furthermore, overexpression of IL-6 attenuated bicalutamide-mediated blockage of androgen-induced androgen receptor nuclear translocation and recruitment. These results show that overexpression of IL-6 increases the resistance of prostate cancer cells to bicalutamide via TIF2. Overexpression of IL-6 not only plays an important role in prostate cancer progression but also contributes to bicalutamide resistance. Our studies suggest that bicalutamide-IL-6-targeted adjunctive therapy may lead to a more effective intervention than bicalutamide alone. [Mol Cancer Ther 2009;8(3):665–71]

Andrographolide, an Herbal Medicine, Inhibits Interleukin-6 Expression and Suppresses Prostate Cancer Cell Growth

Elevated interleukin-6 (IL-6), a major mediator of the inflammatory response, has been implicated in androgen receptor (AR) activation, cellular growth and differentiation, plays important roles in the development and progression of prostate cancer, and is a potential target in cancer therapy. Through drug screening using human prostate cancer cells expressing IL-6 autocrine loop, we found that andrographolide, a diterpenoid lactone isolated from a traditional Chinese and Indian medicinal plant Andrographis paniculata, could inhibit IL-6 expression and suppress IL-6-mediated signals. Andrographolide inhibits IL-6 expression at both mRNA and protein levels in a dose-dependent manner. Andrographolide suppresses both IL-6 autocrine loop- and paracrine loop-induced cell signaling including Stat3 and Erk phosphorylation. Furthermore, andrographolide inhibits cell viability and induces apoptotic cell death in both androgen-stimulated and castration-resistant human prostate cancer cells without causing significant toxicity to normal immortalized prostate epithelial cells. Moreover, treatment of andrographolide to mice bearing castration-resistant DU145 human prostate tumors that express constitutive IL-6 autocrine loop significantly suppresses tumor growth. Taken together, these results demonstrate that andrographolide could be developed as a therapeutic agent to treat both androgen-stimulated and castration-resistant prostate cancer possibly by suppressing IL-6 expression and IL-6-induced signaling.

Mechanisms of selenium down-regulation of androgen receptor signaling in prostate cancer

Prevention trials showed that selenium reduced prostate cancer incidence by 50%, establishing selenium as a promising chemopreventive agent for prostate cancer. Selenium inhibited human prostate cancer cell growth, blocked cell cycle progression at multiple transition points, and induced apoptotic cell death. Previous studies showed a novel mechanism of selenium anticancer action in which selenium markedly reduces androgen signaling and androgen receptor (AR)–mediated gene expression, including prostate-specific antigen (PSA), in human prostate cancer cells. The molecular mechanisms of selenium-mediated down-regulation of AR signaling are not clear. In this study, a systemic approach was taken to examine the modification of androgen signaling by selenium in human prostate cancer cells. In addition to reduced AR mRNA expression, selenium was found to initially increase the stability of AR mRNA within 6 hours while decreasing the stability of AR mRNA after 8 hours. Selenium increased AR protein degradation and reduced AR nuclear localization. Scatchard analysis indicated that selenium did not affect ligand binding to AR in LNCaP cells. Chromatin immunoprecipitation analyses showed that DHT increased the recruitment of AR and coactivators, such as SRC-1 and TIF-2, to the promoter of the PSA gene, and that recruitment was greatly diminished in the presence of 5 μmol/L selenium. On the other hand, selenium enhanced the recruitment of corepressors, such as SMRT, to the promoter of the PSA gene. Taken together, these results suggest that selenium disrupts AR signaling at multiple stages, including AR mRNA expression, mRNA stability, protein degradation, nuclear translocation, and recruitment of coregulators. [Mol Cancer Ther 2006;5(4):913–8]

NF‐κB2/p52 enhances androgen‐independent growth of human LNCaP cells via protection from apoptotic cell death and cell cycle arrest induced by androgen‐deprivation

Androgen‐deprivation therapy only causes a temporary regression of prostate cancer, as all tumors will eventually progress to refractory to hormonal therapy after 1–3 years of treatment. The underlying mechanisms of prostate cancer androgen refractory progression are incompletely understood. In this study, we employed in vitro as well as in vivo models to examine the role of NF‐κB2/p52 in prostate cancer growth and androgen independent progression.

Andrographolide targets androgen receptor pathway in castration-resistant prostate cancer

Androgen receptor (AR) signaling not only plays a pivotal role in the development of androgen-dependent prostate cancer but is also important in the growth and survival of castration-resistant prostate cancer (CRPC). The first line of treatment of androgen-dependent prostate cancer is the use of androgen deprivation therapy. However, most patients will eventually relapse due to development of CRPC. Thus, development of a strategy to target AR for treatment of CRPC is urgently needed. The authors have previously identified andrographolide as an inhibitor of interleukin-6, which can suppress tumor growth of prostate cancer cells by screening compounds from the Prestwick Natural compound library. In this study, they identified that andrographolide can inhibit AR expression and prostate cancer cell growth and induce apoptosis. Andrographolide is able to down-regulate AR expression at both mRNA and protein levels, prevents its nuclear translocation, and inhibits transactivation of its target genes. Andrographolide prevents the binding of Hsp90 to AR, resulting in proteasome-mediated AR degradation. Furthermore, andrographolide inhibits castration-resistant C4-2 cell growth by reducing AR expression and activity. Thus, andrographolide can be developed as a potential therapeutic agent for prostate cancer by inhibition of androgen receptor signaling.

Interleukin‐4 activates androgen receptor through CBP/p300

Aberrant activation of androgen receptor (AR) plays an important role in the progression of castration resistant prostate cancer. Interleukin‐4 (IL‐4) enhances AR activation in the absence of androgen and stimulates castration resistant growth of androgen‐sensitive prostate cancer cells. However, the mechanism of IL‐4 mediated AR activation has not yet been revealed.

Abstract 23: Sanguinarine suppresses survivin expression and inhibits the growth of prostate cancer cells

Background: Prostate cancer is a frequently occurring disease and is the second leading cause of cancer related deaths of men in the United States. Current treatments have proved inadequate in curing or controlling prostate cancer and a search for agents for the management of this disease is urgently needed. Survivin plays an important role in both progression of castration-resistant prostate cancer and resistance to chemotherapy. Altered expression of survivin in prostate cancer cells is associated with cancer progression, drug/radiation resistance, poor prognosis and short patient survival. Methods: We performed a cell based rapid screen of the Prestwick Chemical Library consisting of 1120 FDA-approved compounds with known safety and bioavailability in humans to identify potential inhibitors of survivin and anti-cancer agents for prostate cancer. Results: Sanguinarine, a benzophenanthridine alkaloid derived primarily from the bloodroot plant, was identified as a novel inhibitor of survivin that selectively kills prostate cancer cells over “normal” prostate epithelial cells. We found that sanguinarine inhibits survivin protein expression through protein degradation via the ubiquitin-proteasome system. Sanguinarine induces apoptosis and inhibits tumor formation and growth of human prostate cancer cells. Administration of sanguinarine, beginning 3 days post ectopic implantation of DU145 human prostate cancer cells, reduces both tumor weight and volume by more than 70% compared to the control treated mice with no apparent toxicity. In addition, sanguinarine sensitized prostate cancer cells to paclitaxel-mediated growth inhibition and apoptosis, offering a potential therapeutic strategy for overcoming taxol resistance. Conclusions: These results suggest that sanguinarine may be developed as an agent either alone or in combination with taxol for treatment of castration-resistant prostate cancer overexpressing survivin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 23.

Abstract 4001: Lin28/miR-let-7c axis controls the growth and tumorigenicity of prostate cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: MicroRNAs (miRNA) are small ∼22 nt non-coding RNAs that regulate gene expression by binding to and inducing degradation of target mRNAs. Deregulated expression of several miRNAs has been found in prostate cancer cells. Let-7 is an evolutionarily conserved family of miRNAs that have been shown to regulate expression of several oncogenes like HMGA2, RAS and Myc. Expression of members of the let-7 family has been shown to be downregulated in many human cancers including lung, breast, ovarian and prostate. In this study, we examined the relative expression levels of let-7c and its master regulator, Lin28 in prostate cancer cells and clinical specimens. We also examined the role of the let-7/Lin28 axis in controlling proliferation in prostate cancer cells. Methods: Expression levels of Let-7c and Lin28 in prostate cancer cells and clinical specimens were measured by real-time qRT-PCR, Northern and Western blotting. Prostate cancer cell lines with stable expression of let-7c or Lin28 were generated and their growth characteristics, clonogenic ability and colony forming abilities in soft agar were examined. Expression of let-7c was downregulated in LNCaP cells using antisense oligonucleotides and effects on growth were examined. Lentiviruses containing let-7c were injected into tumors generated by implantation of prostate cancer cells in nude mice and tumor volumes were measured. Results: Expression levels of let-7c were found to be downregulated in castration-resistant prostate cancer cells and clinical specimens. These levels were found to be inversely correlated to expression levels of Lin28. Overexpression of let-7c reduced prostate cancer cell growth, clonogenic ability and ability to form colonies in soft agar, while downregulation of let-7c resulted in increased cell growth. Conversely, overexpression of Lin28 enhanced prostate cancer cell growth and clonogenic ability. In addition, intratumoral injection of lentiviruses containing let-7c reduced tumor volumes significantly in vivo in nude mice compared to control viruses. Conclusions: Levels of miR-let-7c are reduced in CRPC compared to androgen-dependent prostate cancer. Levels of Lin28, which regulates processing of let-7c into its mature form, are elevated in CRPC. Overexpression of let-7c reduces prostate cancer cell growth and tumorigenic ability. These results suggest that miR-let-7c is a tumor suppressor and may be developed as a therapeutic agent against CRPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4001. doi:10.1158/1538-7445.AM2011-4001

Abstract 4554: Regulation of intracrine androgen synthesis by NF-kappaB2/p52

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Benign prostatic hyperplasia and the initial stages of prostate cancer (CaP) exhibit androgen dependence, but androgen ablation results only in temporary regression of CaP, with progression to castration-resistant prostate cancer (CRPC). Androgen receptor (AR) signaling remains active in CRPC due to aberrant activation of the AR. Synthesis of intracrine androgens has emerged as one of the mechanisms by which AR is activated in CRPC after androgen ablation. Even though levels of androgen biosynthetic enzymes have been shown to be elevated in CRPC, their regulation is not completely understood. In this study, we examined the role of NF-kappaB2/p52 in intracrine androgen synthesis and castration-resistant progression of CaP. Methods: Expression levels of androgen biosynthetic enzymes were measured in CaP cells with or without expression of NF-kappaB2/p52 using real-time RT-PCR and western blotting. Regulation by NF-kappaB2/p52 was examined using luciferase reporter assays and plasmids containing regulatory elements of androgen biosynthetic enzymes. Intracrine levels of androgens were measured using EIA in tumors obtained from castrated mice. Results: Expression levels of androgen biosynthetic enzymes including AKR1C3, CYP17A1, HSD3B2, and SRD5A1 were found to be elevated in CaP cells expressing NF-kappaB2/p52. Luciferase assays showed that NF-kappaB2/p52 regulates their expression directly by binding to their promoters and inducing transcription. The levels of total testosterone in CaP cells expressing NF-kappaB2/p52 were approximately 3-fold higher than control cells as measured using EIA. Intraprostatic androgen levels were found to be at 1002 ± 232 pg/g tissue, compared to 377.8 ± 105 pg/g tissue in control tumors obtained by orthotopic implantation of CaP cells expressing NF-kappaB2/p52. These data suggest that CaP cells synthesize detectable levels of testosterone in the absence of exogenous steroid precursors and overexpression of NF-kappaB2/p52 can increase this process, possibly by enhancing the expression of genes encoding steroidogenic enzymes. Conclusions: Intraprostatic androgen synthesis in recurrent prostate tumors contributes significantly to resistance to androgen ablation and development of CRPC. NF-kappaB2/p52 regulates the expression levels of steroidogenic enzymes and thereby enhances synthesis of intracrine androgens and aberrant activation of the AR. Coupled with our previous studies, these data suggest that antagonizing NF-kappaB2/p52 signaling may prove beneficial in CRPC therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4554. doi:10.1158/1538-7445.AM2011-4554