Androgen-dependent LNCaP Cells Research Articles

ABT-737, a small molecule Bcl-2/Bcl-xL antagonist, increases antimitotic-mediated apoptosis in human prostate cancer cells

Castration-resistant prostate cancer (CRPC) expresses high levels of the anti-apoptotic proteins Bcl-2, Bcl-xL and Mcl-1, resulting in resistance to apoptosis and association with poor prognosis. Docetaxel, an antimitotic drug that is the first-line treatment strategy for CRPC, is known to provide a small survival benefit. However, docetaxel chemotherapy alone is not enough to counteract the high levels of Bcl-2/Bcl-xL/Mcl-1 present in CRPC. ABT-737 is a small molecule that binds to Bcl-2/Bcl-xL (but not Mcl-1) with high affinity and disrupts their interaction with pro-apoptotic Bax/Bak, thus enhancing apoptosis. Our results indicate that ABT-737 can sensitize androgen-dependent LNCaP and CRPC PC3 cells to docetaxel- and to the novel antimitotic ENMD-1198-mediated caspase-dependent apoptosis. CRPC DU145 cells, however, are more resistant to ABT-737 because they are Bax null and not because they express the highest levels of anti-apoptotic Mcl-1 (associated with ABT-737 resistance). Knockdown of Bax or Bak in LNCaP indicates that ABT-737-induced antimitotic enhancement of apoptosis is more dependent on the levels of Bax than Bak. Furthermore, we find that the ability of docetaxel to increase cyclin B1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL and decrease Mcl-1 is required for ABT-737 to enhance apoptosis in PC3 cells, as determined by addition of Cdk1 inhibitor purvalanol A and expression of shRNA specific for cyclin B1. Overall, our data suggests that the high levels of anti-apoptotic proteins in Bax-expressing CRPC cells can be overcome by targeting Bcl-2/Bcl-xL with ABT-737 and Mcl-1 with antimitotics.

Brassinin Induces Apoptosis in PC‐3 Human Prostate Cancer Cells through the Suppression of PI3K/Akt/mTOR/S6K1 Signaling Cascades

The oncogenic PI3K/Akt/mammalian target of rapamycin (mTOR) signaling axis and its downstream effector, the ribosomal protein S6 kinase 1 (S6K1) play a key role in mediating cell survival in various tumor cells. Here, we investigated the effects of brassinin (BSN), a phytoalexin first identified as a constituent of cabbage, on the PI3K/Akt/mTOR/S6K1 activation, cellular proliferation, and apoptosis in PC-3 human prostate cancer. BSN exerted a significant dose-dependent cytotoxicity and reduced constitutive phosphorylation of Akt against androgen-independent PC-3 cells as compared to androgen-dependent LNCaP cells. Moreover, knockdown of androgen receptor (AR) by small interfering RNA enhanced the potential effect of BSN on induction of apoptosis in LNCaP cells. BSN clearly suppressed the constitutive activation of PI3K/Akt/mTOR/S6K1 signaling cascade, which correlated with the induction of apoptosis as characterized by accumulation of cells in subG1 phase, positive Annexin V binding, TUNEL staining, loss of mitochondrial membrane potential, down-regulation of antiapoptotic and proliferative proteins, activation of caspase-3, and cleavage of PARP. Additionally, BSN could block broad-spectrum inhibition of PI3K/Akt/mTOR/S6K1 axes, and aberrant Akt activation by pcDNA3-myr-HA-Akt1 plasmid could not prevent the observed suppressive effect of BSN on constitutive mTOR activation. Finally, overexpression of Bcl-2 also attenuated BSN-mediated apoptosis in PC-3 cells. Taken together, our findings suggest that BSN can interfere with multiple signaling cascades involved in tumorigenesis and might be provided as a potential therapeutic candidate for both the prevention and treatment of prostate cancer.

Quercetin synergizes with 2-methoxyestradiol inhibiting cell growth and inducing apoptosis in human prostate cancer cells

Lack of effective treatment options for castration-resistant prostate cancer reinforces the great need to develop novel drug therapies. Quercetin is a plant-derived flavonoid that can induce apoptosis in prostate cancer cells. 2-Methoxyestradiol (2-ME) is an endogenous estrogenic metabolite that also has antineoplastic activity. However, these two agents have limited bioavailability. Herein, we explored the antiproliferative and proapoptotic activities of quercetin combined with 2-ME in both androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cell lines. Compared to quercetin and 2-ME alone, combining quercetin with 2-ME at appropriate concentrations i) showed synergistic antiproliferative and proapoptotic activities; ii) increased G2/M phase population of cells; iii) decreased the ratio of Bcl-2/Bax significantly. The combination of quercetin and 2-ME is a new clinically relevant treatment regimen which has the potential of enhancing the antitumor effect on prostate cancer and lessening the side effect of either quercetin or 2-ME alone.

Abstract 400: Coordinated loss of CD9 and IGSF8 expression on the prostate cancer cell surface contributes to metastasis and hormone responsiveness.

Abstract The tetraspanin web is a complex multi-molecular cell structure that tethers cell surface signalling proteins (i.e. integrins) to their intermediate intracellular signalling partners (i.e. small GTPases) and anchors actin filaments. Loss of certain components of the web (CD9, CD82 or CD316) are associated with increased invasive and metastatic behaviors of prostate cancer (PCa) cells and here we demonstrate that two of these tetraspanin web components, CD9 and CD316, also play a role in regulating androgen signaling in PCa cells. Additionally, we utilized human prostate cancer tissue microarrays (TMAs) containing 261 prostate/PCa specimens of differing stages, grades and states of progression (including 60 castration resistant tumors [CRPCs]) to study the relationship between CD9 and CD316 expressions and progression to CRPC. Using androgen-dependent LNCaP cells as a model, we found that androgen deprivation (AD) suppressed expression of CD9 protein and mRNA without affecting CD316 expression. FACS analysis showed that the presence of CD9 on the cell surface was coordinately reduced by AD as was cell surface CD316 which was relocalized to the cytoplasm. This effect was reversed by addition of R1881. Knockdown of CD9 or CD316 in LNCaP cells using siRNA reduced expression of androgen receptor (AR) mRNA but increased expression of PSA, an AR target gene. CD9 or CD316 knockdown also increased LNCaP growth in fetal bovine serum (FBS) containing medium but not in charcoal-stripped FBS (CS-FBS) whereas dual-knockdown further increased growth in FBS as well as in CS-FBS. TMAs immunostained for CD9 or CD316 were assessed for correlation to clinicopathological parameters. For untreated, primary tumors, CD9 and CD316 expression were inversely correlated with Gleason scores (R2=0.9612). CD9 expression was diminished during progression to metastatic (p<0.0038, compared to non-metastatic) and further in CRPC (p<0.040, compared to metastatic non-treated) disease. Conversely, CD316 expression was increased in primary (non-metastatic) as well as in metastatic untreated disease but then reduced (p<0.046) during progression to metastatic CRPC. We propose that androgen deprivation (first line therapy for advanced PCa) may potentially trigger loss of both CD9 and IGSF8 expression on the cell surface that contributes to metastatic behaviors as well as to responsiveness to androgen ablation therapies. This work is supported by the Cancer Research Society (Canada). Citation Format: Na Li, Ladan Fazli, Estelle Li, Ralph Buttyan. Coordinated loss of CD9 and IGSF8 expression on the prostate cancer cell surface contributes to metastasis and hormone responsiveness. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 400. doi:10.1158/1538-7445.AM2013-400

Abstract 418: Polyisoprenylated methylated protein methyl esterase overexpression in prostate cancer: implication in cancer metastasis.

Abstract Objectives: Prostate cancer is the most frequently diagnosed cancer among men in the U.S, with an estimated 241,740 new cases and 28,170 deaths for 2012. The need to identify biomarkers/therapeutic targets for the early/companion diagnosis and development of novel therapies against the recalcitrant form of prostate cancer is imperative. Mutation and overexpression-induced abnormal activities of polyisoprenylated proteins such as of the Ras and Rho families of monomeric G proteins have been implicated in prostate cancer. Also, signaling mediated by polyisoprenylated Rho family of GTPases through the actin cytoskeleton in migratory responses is necessary for cancer cell metastasis. Polyisoprenylated methylated protein methyl esterase (PMPMEase) catalyzes the only reversible and terminal reaction of the polyisoprenylation pathway. We hypothesized that PMPMEase activity may influence the effects of these G proteins on cell viability, metastasis and aggressiveness and thus may be of importance in the detection and treatment of prostate cancer. Method: Prostate cancer tissue microarray was probed for relative PMPMEase expression by immunohistochemistry. Specific activities of PMPMEase and the effect of its inhibition on viability of various prostate cancer cells were also studied. Effect of specific PMPMEase inhibitor L-28 on actin cytoskeleton organization was also determined by phalliodine staining of treated PC-3 cells. Results: PMPMEase specific activities were 4 and 6-fold higher in androgen-dependent 22Rv1 and LNCaP cells and 2- and 17-fold higher in androgen-independent PC-3 and DU 145 prostate cancer cells respectively compared to normal WPE1-NA22 prostate cells. Treatment of the cells for 72 h with L-28, a specific polyisoprenylated PMPMEase inhibitor induced apoptosis with EC50 values ranging from 1.8 to 4.6 μM. The PMPMEase activity of the different cell lines followed a similar profile with IC50 values ranging from 2.3 to 130 μM. L-28 also significantly disrupted the actin filament arrangement. Immunohistochemical analysis of PMPMEase expression revealed intermediate, strong and very strong staining in 94.5% of the 92 adenocarcinoma cases compared to only trace and weak staining in the normal and normal adjacent tissue controls. Conclusion: These results indicate that PMPMEase activity, immunoreactivity and inhibition could serve in the diagnosis and targeted therapeutic management of prostate cancer. Also inhibition of PMPMEase disturbs actin filament organization in cytoskeleton. Citation Format: Rosemary A. Poku, Felix Amissah, Randolph Duverna, Byron J. Aguilar, Nazarius S. Lamango. Polyisoprenylated methylated protein methyl esterase overexpression in prostate cancer: implication in cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 418. doi:10.1158/1538-7445.AM2013-418

Abstract 5387: Integrative analysis of transcriptomic and metabolomic data reveals a critical role for aminosugar metabolism in prostate cancer.

Abstract Prostate Cancer is the second most common cause of cancer-related death in men in the US. Like other tumors, prostate cancer development and progression is dictated by multiple molecular events that include changes in levels of genes, transcripts, proteins and metabolites. To better understand the biology of prostate cancer development it is essential to integrate these disparate yet related datasets. Our laboratory has identified matched transcriptomic, proteomic and metabolomic changes in localized prostate cancer relative to adjacent benign tissue as well in metastatic disease compared to organ-confined tumor. To study this using a System's Biology approach we have recently embarked on a pilot study aimed at integrating transcriptomic and metabolomic data to obtain biochemical pathways that key to prostate cancer development. Using an in-house network-based enrichment strategy, amino sugar metabolism was found to be significantly enriched in organ-confined prostate cancer but not in metastatic disease. Amino sugar metabolism describes the utilization of glucose-derived carbon and amino acid (mostly glutamine)-derive nitrogen to produce glucosamines. These amino sugars participate in synthesis of immune modulatory compounds as well as in glycosylation cascades. In this study, we describe the molecular analyses of Glucosamine-6 phosphate-N-acetyl Transferase (GNPNAT1), a key enzyme that converts D-glucosamine 6-phosphate to N-acetyl-D-glucosamine 6-phosphate, in prostate cancer. Our results indicate upregulation of GNPNAT1 in organ-confined prostate cancer as compared to benign adjacent tissue as well as metastatic tissue and regulation of the pathway by androgen. Stable knockdown of GNPNAT1 in androgen dependent LNCap cells leads to diminished cell growth and cell cycle arrest. In contrast, growth and cell cycle are not affected by the knockdown of GNPNAT1 in androgen independent C4-2 cells. Knockdown of GNPNAT1 in C4-2 cells enhances invasiveness which is not observed in LNCap knockdown cells. In this work we show that GNPNAT1 is linked to androgen receptor (AR) action and thus is a critical enzyme for the survival of androgen dependent prostate cancer cells. Citation Format: Katrin Panzitt, Ali Shojaie, Nagireddy Putluri, Sumanta Basu, Vasanta Putluri, Susmita Samanta, Michael Ittmann, Ismael Vergara, George Michailidis, Ganesh Palapattu, Arun Sreekumar. Integrative analysis of transcriptomic and metabolomic data reveals a critical role for aminosugar metabolism in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5387. doi:10.1158/1538-7445.AM2013-5387

Abstract 1319: Regulation of AR transcriptional activity and prostate cancer cellular proliferation by DACH1/Eya1/Six1 pathway.

Abstract Prostate cancer progression from androgen responsive to androgen-depletion therapy resistance is a major cause of treatment failures. Novel therapies targeting this patient are a major clinical challenge. We previously reported that the cell fate determination factor DACH1 inhibits androgen signaling though binding the AR and enhancing co-repressor recruitment in the context of local chromatin at AR responsive genes. The repression of AR function of by DACH1 was partly rescued by the co-activators p300 and SRC. The repression was enhanced by ectopic expression of HDACs. We had previously shown that DACH1 inhibited proliferation of androgen-dependent LnCAP cells. To further explore the significance of DACH1 in human prostate cancer, we established stable cell lines expressing DACH1 or its DS domain deleted mutant (ΔDS) in androgen-independent C4-2 cells and AR-negative PC-3 cells by retrovirus infection. Stable expression of DACH1 inhibited cellular proliferation in both cell lines, requiring the DS domain. Ectopic expression of DACH1 inhibited colony formation of the androgen-independent 22RV1 cells. DACH1 inhibited migration, invasion and tumor growth of AR-negative PC-3 cells. Since DACH1, Eya1 and Six1 function together as a network to control cellular differentiation and organ formation during development, we examined whether other components of RDGN pathway, could regulate androgen signaling. In hormone responsive LnCAP cells, ligand stimulation (DHT, 50nM) induced 17 folds activation of MMTV Luc, co-expression of Eya1 reduced AR transactivation, and the repression required the EYA phophatase domain. Expressions of the AR co-repressors, HDAC1, HDAC3 or SirT1 inhibited the ligand-induced AR activity. Eya1 enhanced co-repressor function at the PSA and MMTV promoters. Consistent with our previous publication, DACH1 repressed ligand-induced activity >70%, Eya1 modified DACH1 repression of AR activity. Together, our studies demonstrated that the RDGN regulates both androgen dependent and independent prostate cancer cellular proliferation and tumorigenesis. Citation Format: Jing Wang, Shaoxin Cai, Ke Chen, Sean Li, Richard Pestell, Kongming Wu. Regulation of AR transcriptional activity and prostate cancer cellular proliferation by DACH1/Eya1/Six1 pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1319. doi:10.1158/1538-7445.AM2013-1319

Radiosensitivity of human prostate cancer cells can be modulated by inhibition of 12-lipoxygenase

Nearly 30% of prostate cancer (PCa) patients treated with potentially curative doses relapse at the sites of irradiation. How some tumor cells acquire radioresistance is poorly understood. The platelet-type 12-lipoxygenases (12-LOX)-mediated arachidonic acid metabolism is important in PCa progression. Here we show that 12-LOX confers radioresistance upon PCa cells. Treatment with 12-LOX inhibitors baicalein or BMD122 sensitizes PCa cells to radiation, without radiosensitizing normal cells. 12-LOX inhibitors and radiation, when combined, have super additive or synergistic inhibitory effects on the colony formation of both androgen-dependent LNCaP and androgen-independent PC-3 PCa cells. In vivo, the combination therapy significantly reduced tumor growth.

The microRNA -23b/-27b Cluster Suppresses the Metastatic Phenotype of Castration-Resistant Prostate Cancer Cells

MicroRNAs (miRs) are small, endogenous, non-coding RNAs that regulate the stability and/or translation of complementary mRNA targets. MiRs have emerged not only as critical modulators of normal physiologic processes, but their deregulation may significantly impact prostate and other cancers. The expression of miR-23b and miR-27b, which are encoded by the same miR cluster (miR-23b/-27b), are downregulated in metastatic, castration-resistant tumors compared to primary prostate cancer and benign tissue; however, their possible role in prostate cancer progression is unknown. We found that ectopic expression of miR-23b/-27b in two independent castration-resistant prostate cancer cell lines resulted in suppression of invasion and migration, as well as reduced survival in soft agar (a measure of anoikis). However, there was no effect of miR-23b/-27b on cell proliferation suggesting that these miRs function as metastasis (but not growth) suppressors in prostate cancer. Conversely, inhibition of miR-23b/-27b in the less aggressive androgen-dependent LNCaP prostate cancer cell line resulted in enhanced invasion and migration also without affecting proliferation. Mechanistically, we found that introduction of miR-23b/-27b in metastatic, castration-resistant prostate cancer cell lines resulted in a significant attenuation of Rac1 activity without affecting total Rac1 levels and caused increased levels of the tumor suppressor E-cadherin. Inhibition of these miRs had the opposite effect in androgen-dependent LNCaP cells. These results suggest that miR-23b/-27b are metastasis suppressors that might serve as novel biomarkers and therapeutic agents for castration-resistant disease.

Carmustine enhances the anticancer activity of selenite in androgen-independent prostate cancer cells

Apoptosis is one of the major mechanisms targeted in the development of therapies against various cancers, including prostate cancer. Resistance to chemotherapy poses a significant problem for the effective treatment of androgen-independent (hormone-refractory) prostate cancer. Although high concentrations of sodium selenite exert strong anticarcinogenic effects in several cell culture systems and animal models, the therapeutic potential of selenite in patients with advanced or metastatic prostate cancer is extremely limited by the genotoxicity of high-dose selenite. We examined the ability of nontoxic concentrations of selenite to promote apoptosis and inhibit proliferation in carmustine-sensitized androgen-independent human prostate cancer cells. Androgen-dependent LNCaP cells exhibited a significant decrease in cell viability when exposed to nontoxic concentrations of selenite, whereas androgen-independent PC-3 and DU145 cells showed a significant decrease in cell viability only at higher concentrations. Treatment of PC-3 cells with a combination of nontoxic selenite and carmustine resulted in greater increases in cytotoxicity, reactive oxygen species generation, growth inhibition, apoptosis, and DNA double-strand breaks, with concomitant decreases in DNA synthesis, glutathione, glutathione reductase, and antiapoptotic proteins. Combination treatment with carmustine and selenite triggered caspase-dependent apoptosis in PC-3 cells, which was not apparent when these cells were treated with selenite or carmustine alone. Genotoxicity in normal prostate epithelial cells was completely absent in the combination treatment of carmustine and selenite. In addition, carmustine decreased the induction of DNA double strand breaks by high-dose selenite in normal prostate epithelial cells. This is the first study to demonstrate that a nontoxic dose of selenite, in combination with carmustine, significantly induces apoptosis and growth inhibition in androgen-independent prostate cancer cells without causing undesirable genotoxicity in normal prostate epithelial cells, suggesting that this combination therapy may be a promising therapeutic approach in the treatment of patients with metastatic hormone-refractory prostate cancer.

Krüppel-like factor 8 is a novel androgen receptor co-activator in human prostate cancer

Krüppel-like factor 8 (KLF8) plays important roles in cell cycle and oncogenic transformation. On other hand, androgen receptor (AR) is crucial in development of both androgen-dependent and independent prostatic malignancies. The aim of this study is to investigate the role of KLF8 in prostate cancer (PCa) and the relationship between KLF8 and AR. Eight human PCa cell lines, including androgen-dependent LNCap cells and androgen-independent 22Rv1 cells, as well as human PCa samples were studied. LNCap cells and 22Rv1 cells were transfected with plasmids encoding full-length wild-type KLF8 or KLF8 shRNA. The expression of KLF8 protein was detected using Western blotting or immunohistochemical staining. Cell proliferation in vitro was measured with MTT assay, and in vivo in a xenograft nude mouse model. Yeast two-hybrid screening, co-immunoprecipitation and pull down assays were used to examine the binding of KLF8 to AR. Luciferase reporter gene assay was used to measure the transcriptional activity of the genes targeted by AR. In 133 human PCa samples, KLF8 protein staining was observed in 92.65% (63/68) of high-grade PCa, 66.15% (43/65) of low-grade PCa, and 6.82% (3/44) of adjacent normal tissues. The expression of KLF8 was significantly associated with poorer overall survival. Overexpression of KLF8 enhanced the proliferation of both LNCap and 22Rv1 cells, while knockdown of endogenous KLF8 suppressed the proliferation. These manipulations exerted similar effects on the tumor volumes in the xenograft nude mouse model. Yeast two-hybrid screening revealed that KLF8 was a novel AR-interacting protein. With pull down assay and co-immunoprecipitation assay, we demonstrated that KLF8 bound directly to AR, and KLF8 enhanced AR target gene transcription. The results demonstrate that KLF8 is a novel AR transcriptional co-activator that is overexpressed in PCa and may play a role in progression of hormone-refractory PCa.

7-epi-nemorosone from Clusia rosea induces apoptosis, androgen receptor down-regulation and dysregulation of PSA levels in LNCaP prostate carcinoma cells

The aim of this work was to characterize the antitumoral activity of the plant compound 7-epi-nemorosone in prostate carcinoma cell lines. Prostate cancer is the most frequently diagnosed malignancy and the second-leading cause of cancer death in men. In spite of the current therapeutic options for this cancer entity, many patients die due to metastases in distant organs and acquired chemotherapy resistance. Thus, approaches to provide improvements in outcome and quality of life for such patients are urgently needed. Recently, the polyisoprenylated benzophenone 7-epi-nemorosone, originally collected by honeybees from Clusia rosea and Clusia grandiflora (Clusiaceae), has been described to be a potent antitumoral agent. Here, its activity in prostate carcinoma is reported. 7-epi-nemorosone was isolated from Caribbean propolis employing RP-HPLC techniques. Its cytotoxicity was assessed using the MTT proliferation assay in human androgen-dependent prostate carcinoma LNCaP cells including an MDR1+ sub-line. No cross-resistance was detected. FACS-based cell cycle analysis revealed a significant increase in the sub-G0/G1, G1, and depletion in the S phase populations. A concomitant down-regulation of cyclins D1/D3 and CDK 4/6 in LNCaP cells was detected by Western blot. Annexin-V-FITC labeling and caspase-3 cleavage assays showed that 7-epi-nemorosone induced apoptotic events. Major signal transduction elements such as p38 MAPK and Akt/PKB as well as androgen receptor AR and PSA production were found to be down-regulated after exposure to the drug. ERK1/2 protein levels and phosphorylation status were down-regulated accompanied by up-regulation but inhibition of the activity of their immediate upstream kinases MEK1/2. Additionally, Akt/PKB enzymatic activity was effectively inhibited at a similar concentration as for MEK1/2. Here, we demonstrate for the first time that 7-epi-nemorosone exerts cytotoxicity in an androgen-dependent prostate carcinoma entity by targeting the MEK1/2 signal transducer.

Embelin inhibits growth and induces apoptosis through the suppression of Akt/mTOR/S6K1 signaling cascades

Akt/mTOR/S6K1 signaling cascades play an important role both in the survival and proliferation of tumor cells. In the present study, we investigated the effects of embelin (EB), identified primarily from the Embelia ribes plant, on the Akt/mTOR/S6K1 activation, associated gene products, cellular proliferation, and apoptosis in human prostate cancer cells. EB exerted significant cytotoxic and suppressive effects on Akt and mTOR activation against androgen-independent PC-3 cells as compared to androgen-dependent LNCaP cells. Moreover, EB suppressed the constitutive activation of Akt/mTOR/S6K1 signaling cascade, which correlated with the induction of apoptosis as characterized by accumulation of cells in subG1 phase, positive Annexin V binding, down-regulation of anti-apoptotic (Bcl-2, Bcl-xL, survivin, IAP-1, and IAP-2) and proliferative (cyclin D1) proteins, activation of caspase-3, and cleavage of PARP. We also observed that EB can significantly enhance the apoptotic effects of a specific pharmacological Akt inhibitor when used in combination and also caused broad inhibition of all the three kinases in Akt/mTOR/S6K1 signaling axis in PC-3 cells. EB inhibits multiple signaling cascades involved in tumorigenesis and can be used as a potential therapeutic candidate for both the prevention and treatment of prostate cancer.

KLF6 Loss of Function in Human Prostate Cancer Progression Is Implicated in Resistance to Androgen Deprivation

Inactivation of the transcription factor/tumor suppressor Krüppel-like factor 6 (KLF6) has been described in prostate cancer (PC). This study investigated the prevalence and significance of KLF6 exon 2 mutations and splice variants (SVs) in different stages of human PC progression. By using laser-capture microdissection and recombinant clone isolation of DNA sequences to enhance sensitivity, base changes were found in 20 (24.7%) of 81 PC tissues versus 1 (4%) of 25 normal prostate tissues (P = 0.02). Of 26 base changes, 54% produced nonsynonymous mutations. Only three mutations had driver characteristics (PCs, 4%; NPs, 0%). By using microdissection of fresh-frozen tissues and recombinant isolation of RNA sequences, SVs were found in 39 (75%) of 52 PCs and in 10 (45%) of 22 NPs (P = 0.01). Sixteen different SVs, including 13 unique SVs, were identified that used cryptic splicing sites and encoded nonfunctional KLF6 proteins. PCs that had survived hormone (androgen)-deprivation therapy (n = 21) had a significantly higher (P < 0.05) incidence, number, and expression level of nonfunctional SVs than either NPs (n = 22) or hormone-naïve PCs (n = 25). Forced expression of nonfunctional SVs conferred a survival advantage of androgen-dependent LNCaP cells under castration-simulated culture conditions. Together, these data suggest that decreased availability of functional KLF6 contributes to clinical PC progression. This decrease arises infrequently by somatic mutation and more commonly by the acquisition of SVs that provide a survival advantage under castrate conditions, enabling resistance to hormone therapy.

Monascuspiloin Induces Apoptosis and Autophagic Cell Death in Human Prostate Cancer Cells via the Akt and AMPK Signaling Pathways

Monascus pigments have been reported to possess anticancer effects in various cancer cells; however, the molecular mechanisms of their anticancer properties remain largely unknown. Monascuspiloin is an analogue of the Monascus pigment monascin, and its anticancer growth activity against human prostate cancer cells was evaluated using in vitro and in vivo models. Monascuspiloin effectively inhibits the growth of both androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cells. Monascuspiloin preferentially induces apoptosis in LNCaP cells by attenuating the PI3K/Akt/mTOR pathway. In androgen-independent PC-3 cells, monascuspiloin induces G2/M arrest and autophagic cell death by an AMPK-dependent pathway. Induction of autophagy in PC-3 cells further sensitizes cells to apoptosis induced by monascuspiloin. Monascuspiloin inhibits tumor growth in nude mice bearing PC-3 xenografts through induction of apoptosis and autophagy. This study is the first to demonstrate that monascuspiloin has therapeutic potential for the treatment of both androgen-dependent and -independent human prostate cancers.

Abstract A12: Identification of Nrdp1 as a novel androgen receptor transcription target differentially regulated in androgen-dependent and independent prostate cancer

Abstract Background: The ErbB receptor tyrosine kinase family regulates proliferation and survival in prostate cancer (PCa). We recently showed that ErbB3 plays a significant role in increasing androgen receptor (AR) transcriptional activity and in causing castration-resistant PCa (CRPC). Reciprocally, AR maintained castration sensitivity by suppressing ErbB3 levels through transcriptional regulation of E3 ubiquitin ligase Nrdp1, while loss of AR regulation of Nrdp1 resulted in an unrestricted surge in ErbB3 levels, and cell growth. Here, we investigate whether Nrdp1 is a transcriptional target of the AR in PCa cells. Results: The promoter region of Nrdp1 contains three androgen response elements (AREs) - one located 215 aa upstream of the transcriptional start site (ARE.03), and two within an internal promoter (ARE.01 and ARE.02). Chromatin immunoprecipitation (ChIP) studies revealed AR binding in PCa cells to ARE.03. AR binding to ARE.03 was found to be androgen regulated in androgen-dependent LNCaP cells, whereas no AR binding to ARE.01 or ARE.02 has been detected in this cell line. Luciferase assay to determine AR transcriptional activity on ARE.03 showed significant response to androgens, whereas full mutation of ARE.03 abolished AR transcriptional activity. However, the AR failed to bind to ARE.03 in C4-2 and LNCaP-AI cell lines, CRPC sublines of LNCaP cells, although no mutations in these regions were identified, while AR continued to bind the AR to PSA ARE in these cells, indicating that the AR was still active. Thus the AR binds to PSA ARE in both androgen-dependent and –independent cells, but to ARE.03 only in androgen-dependent but not –independent cells. Luciferase assay in LNCaP-AI cells showed decreased AR transcriptional activity on ARE.03 when compared to LNCaP, supporting decreased AR binding to this region. Hence we investigated the cause for the differential binding of the AR in these two areas. The structural protein Filamin A (FlnA) was earlier shown to cleave to a 90 kDa fragment (FlnA16-24) whose nuclear localization maintained androgen dependence. We investigated whether this protein played a role in regulation of AR binding to ARE.03 in PCa cells. Transfection of FlnA16-24 in C4-2 cells resulted in localization of FlnA 16-24 to the nucleus, and restored AR binding to ARE.03. FlnA 16-24 significantly increased luciferase activity on ARE.03, while suppressing AR activity on PSA indicating that this AR binding protein is required for AR-mediated transcription of Nrdp1. Conclusions: Our results indicated that Nrdp1 is regulated in androgen sensitive cells, but not in CRPC cells, by AR binding to ARE.03, whereas no such difference in AR binding to PSA ARE exists. We show that AR binding to ARE.03 is abolished in CRPC cells because of a decreased expression of nuclear FlnA, and that restoration of nuclear FlnA will restore androgen-sensitive cell growth. Our data indicate that the AR-coregulator FlnA is required for AR binding to the Nrdp1 promoter and that loss of this protein results in the failure of AR to regulate Nrdp1 transcription, resulting in unrestricted increase in ErbB3. These data identified Nrdp1 as a novel target of AR transcriptional activity in androgen-dependent but not in CRPC cells.

Abstract A52: Comparison of the efficacy of different mTOR inhibitors for the treatment of prostate cancer

Abstract Background: We previously showed that the mTOR pathway is activated in castration resistant prostate cancer (CRPC) cell lines while its inhibition resulted in upregulation of androgen receptor (AR) signaling, resulting in resistance to mTOR-based therapy (Wang et al., 2008, Oncogene, 27(56):7106-17). mTOR forms two complexes: activation of mTORC1 (mTOR, raptor) increases mRNA translation by phosphorylation of the downstream molecules p70-S6K (S6K) and eIF4E binding protein 1 (4E-BP1), while activation of mTORC2 (mTOR, rictor) leads to activation of Akt, which is also activated by phosphatidylinositol 3-kinase (PI3K). A number of drugs targeting these complexes are currently available, among them, RAD001, which inhibits mTORC1 alone, BEZ235, a dual mTORC1/PI3K inhibitor, and Torin1, an ATP-competitive inhibitor which prevents activation of both mTORC1 and mTORC2. The goal of the present study was to identify the drug that is most effective against PCa either alone or in combination with the AR inhibitor bicalutamide. Results: The drugs were used in a number of cell lines including C4-2 and LNCaP-AI, two CRPC sublines of androgen-dependent LNCaP cells, as well as CWR-R1 and CWR22Rv1, two lines derived from relapsed CWR22 tumors in castrated mice. Another androgen-sensitive cell line, PC-346C, was also used. RAD001 dose responsibly inhibited growth in C4-2 cells, but the other cell lines were resistant to this therapy, either alone or in combination with bicalutamide. In contrast, LNCaP-AI and CWR-R1 cells were also sensitive to BEZ235 and Torin1, but PC-346C cells were resistant to all three. Hence we investigated the molecular mechanism underlying the resistance of these cells to mTOR inhibitors. Clonogenic assays revealed that 20 nM BEZ235 and 25 nM Torin1 are optimal doses that prevent growth of PCa cells, effect comparable of that of 10 nM of RAD001 (physiological concentration in blood of patients under treatment with this drug). Comparison of the downstream targets of the various drugs at these doses revealed that 4E-BP1 and Akt are highly phosphorylated in both C4-2 and PC-346C cells despite RAD001 treatment, whereas BEZ235 and Torin1 inhibited 4E-BP1 phosphorylation. This is despite the fact that AR levels, but not AR transcriptional activity, increased in BEZ235 treated cells, but not in those treated with RAD001 and Torin1. However, phosphorylation of its binding partner eIF4E increased in PC-346C cells but not in C4-2 cells. Conclusion: Phosphorylation of 4E-BP1 and eIF4E, as well as Akt, needs to be downregulated in order for the drug to be effective. The effects of BEZ235 are likely complicated by the increase in AR levels despite the use of bicalutamide. Therefore, a stronger AR inhibitor, such as MDV3100, in combination with this drug, may be required. Acknowledgments: We would like to thank Dr. David Sabatini for the drug Torin1, Novartis Pharmaceuticals for providing BEZ235 and RAD001, and Astra Zeneca, Cheshire, UK, for the drug bicalutamide. CWR-R1 cells were generously provided by Dr Elizabeth Wilson (University of North Carolina), while PC-346C cells were from Dr. W.M. van Weerden, Josephine Nefkens Institute, Erasmus MC, Rotterdam, The Netherlands.

Chemopreventive Effects ofRubus coreanusMiquel on Prostate Cancer

The growing incidence of prostate cancer and the traditional use of Rubus coreanus Miquel (RCM) for prostate health led us to compare RCM extracts and to test their efficacy in inhibiting the growth of prostate cancer cells differing in androgen dependency. Ethanol extracts of unripe RCM (EUR) were more effective in reducing cell viability than water extracts or ripe RCM. EUR-induced growth inhibition, as indicated by significant reductions in numbers of proliferating cells and decreases in the protein levels of proliferating cell nuclear antigen (PCNA), cyclin D1 and CDK4, was greater in the androgen-dependent LNCaP cells than in the androgen-independent DU145 cells. EUR also induced mitochondrial-mediated apoptosis in prostate cancer cells by reducing Bcl-2 and Bcl-(X)L levels, but increased Bax levels. Nevertheless, the LNCaP cells were more sensitive to EUR-induced apoptosis and displayed sub-G1 and late apoptotic cell populations, whereas the DU145 cells did not. Our findings suggest that EUR suppresses the growth of prostate cancer cells by anti-proliferative and/or pro-apoptotic effects, and that these effects are stronger in androgen-dependent cells.

Abstract LB-300: The role of WNT7B in prostate cancer bone metastasis

Abstract Prostate cancer is the most common cancer for men in the US. It is well known that the androgen receptor (AR), a master transcription factor in prostate, plays a critical role in all stages of prostate cancer. There has been a dedicated interest in identification of downstream AR target genes responsible for prostate cancer growth and progression. Using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq), we identified WNT7B as a direct AR target gene in both androgen-dependent LNCaP and androgen-independent C4-2B cells. Knockdown of WNT7B by RNA interference inhibits the growth of LNCaP and C42B cells, accompanied by a corresponding increase in apoptosis as determined by Caspase-3 and -7 activities. Overexpression of WNT7B promotes the growth of C4-2B cells in the absence of androgen. One of the major challenges in prostate cancer research is the propensity for bone metastasis, which displays characteristic osteoblastic lesions. Since WNT7B plays an important role in bone formation, we asked whether prostate cancer-produced WNT7B induces osteoblast differentiation using an in vitro model of the murine osteoblast precursor ST2 cell line. We found that in a co-culture system, overexpression of WNT7B in LNCaP and C42B cells induces osteoblastic differentiation of ST2 cells, as indicated by increased alkaline phosphatase production, bone sialoprotein (an early marker for osteoblast differentiation) expression, and mineralization. On the other hand, knockdown of WNT7B in prostate cancer cells inhibits the differentiation of ST2 cells. These results indicate that WNT7B can stimulate the growth of prostate cancer cells as an autocrine agent and induce osteoblastic differentiation in bone metastases as a paracrine agent. WNT7B may be a potential therapeutic target for prostate cancer bone metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-300. doi:1538-7445.AM2012-LB-300

Abstract 2407: Snail 1 regulates invasion through uPA-uPAR and MMP-9 signaling in prostate cancer cells

Abstract Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of N-cadherin, while epithelial-associated genes like E-cadherin are lost. This enables the cells to be more invasive, migratory and metastatic. Factors that can induce EMT include growth factors like transforming growth factor -α (TGF-β) and epidermal growth factor (EGF), and transcription factors like Snail1. Snail1-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by urokinase (uPA) and its receptor (uPAR) activities. uPA, a serine protease, may be activated by integrin engagement to bind to its receptor, uPAR, and initiate a signaling cascade that regulates a variety of biological pathways including invasion. Since Snail1 can induce matrix metalloproteinases (MMP 2 and 9) which are also involved in cell invasion, we also examined MMP activity. Androgen-dependent LNCaP, and 22Rv1 and androgen-independent ARCaP human prostate cancer (PC) cells were stably transfected with empty vector control (Neo) or constitutively active Snail1 which led to increased cell migration and invasion. The levels of Snail1, uPA, and uPAR were measured by Western Blot analysis. uPA activity in conditioned media was measured using the Elisa uPA activity assay kit. Additionally, cells were treated with MAPK inhibitor, UO126, at set time points. Gelatin zymography analysis was utilized to detect MMP-9 activity in the conditioned media. Currently, ARCaP-Snail cells are being transiently transfected with uPAR siRNA to assay for the effects of uPAR knockdown on uPA and MMP activity. The data suggests that overexpression of Snail1 increased uPA and uPAR protein levels, while uPA activity was elevated in conditioned media from LNCaP, 22Rv1 and ARCaP cells. Also, MAPK inhibited MAPK expression in the cell and decreased uPA activity. In addition gelatin zymography showed higher MMP 9 activity in ARCaP-Snail overexpressing cells that was inhibited by MAPK inhibitor. Therefore, Snail-mediated cell migration and invasion in human prostate cancer cells may occur via the regulation of uPA and uPAR activities in addition to MMPs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2407. doi:1538-7445.AM2012-2407