Chemoresistant Prostate Cancer Research Articles

Abstract 616: Transcription-regulating kinases CDK8 and CDK19 as novel therapeutic targets for advanced prostate cancer

Abstract Most of castration-refractory prostate cancers (CRPC) continue to express androgen receptor (AR) that maintains its activity at low levels or even in the absence of androgen, driving tumor cell proliferation. Androgen-depleting drugs and androgen antagonists have little or no effect against AR+ cancers that are fully androgen-independent (AI). On the other hand, experimental compounds that cause AR degradation prevent AR-mediated repression of tumor-promoting genes, a tumor-suppressive effect of AR. We report a novel druggable target for CRPC, which mediates the induction but not the repression of gene expression by AR and inhibits the growth of AI prostate cancer (PCa) cells. This target is two closely related transcription-regulating kinases CDK8/CDK19 that unlike some other members of the CDK family are not required for cell cycle progression but play critical roles in several transcriptional signaling pathways. CDK8 and CDK19 are highly expressed in AR+ PCa cells and elevated under the conditions of androgen deprivation; CDK19 is overexpressed in AR+ PCa cell lines and in metastatic clinical PCa. A novel selective CDK8/19 kinase inhibitor (Senexin B) blocks ligand-induced AR-mediated transcriptional activation in androgen-dependent (AD) PCa cells but does not affect the AR-mediated gene repression. The CDK8/19 inhibitor also inhibits ligand-independent AR activation and AI cell growth in AI-PCa cells resistant to the anti-androgenic drug enzalutamide, both in vitro and in a mouse xenograft model. We have also discovered that CDK8/19 inhibition decreases NFκB mediated transcriptional activation of a group of tumor-promoting chemokines, such as CXCL1, CXCL2 and IL8, suggesting that CDK8/19 may promote prostate cancer chemoresistance and metastasis through the NFκB pathway. The above results suggest that CDK8/19 is an especially attractive therapeutic target for advanced PCa, which controls both AR-driven cell proliferation and other oncogenic pathways. Citation Format: Mengqian Chen, Vimala Kaza, Jiaxin Liang, Martina McDermott, Igor Roninson. Transcription-regulating kinases CDK8 and CDK19 as novel therapeutic targets for advanced prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 616. doi:10.1158/1538-7445.AM2014-616

Immediate and transient phosphorylation of the heat shock protein 27 initiates chemoresistance in prostate cancer cells.

Drug resistance minimizes the effects of prostate cancer (PC) chemotherapy with docetaxel and is generally considered to be associated with the expression of heat shock protein (HSP) 27 including various cytoprotective pathways. In the present study, we investigated the effects of HSP27 phosphorylation on PC cell growth underlying docetaxel treatment. Cell counting revealed significantly reduced cell growth during docetaxel treatment as a result of both activation of mitogen-activated protein kinase p38 (MAPK p38) and protein kinase D1 (PKD1), and, most importantly, the overexpression of the phosphorylation-mimicking mutant HSP27-3D. Further analysis revealed a docetaxel-dependent induction of HSP27 accompanied by an initial phosphorylation and rapid dephosphorylation of the protein. Based on the data, we can conclude that phosphorylation of HSP27 protein is a crucial mechanism in the initiation of chemoresistance in PC. Moreover, the results indicate a key impact of HSP27 on viability and proliferation of PC cells underlying anticancer therapy. The protective function depends on the initial phosphorylation status of HSP27 and represents a putative co-therapeutic target to prevent chemoresistance during docetaxel therapy.

Cancer Stem Cells in Prostate Cancer Chemoresistance

There is currently no cure for metastatic castration-resistant prostate cancer (CRPC). Chemoresistance and metastatic disease remain the main causes of treatment failure and mortality in CaP patients. Although several advances have been made in the control of CRPC with some newly developed drugs, there is still an urgent need to investigate the mechanisms and pathways of prostate cancer (CaP) metastasis and chemoresistance, identify useful therapeutic targets, develop novel treatment approaches, improve current therapeutic modalities and increase patients' survival. Cancer stem cells (CSCs), a minority population of cancer cells characterised by self-renewal and tumor initiation, have gained intense attention as they not only play a crucial role in cancer recurrence but also contribute substantially to chemoresistance. As such, a number of mechanisms in chemoresistance have been identified to be associated with CSCs. Therefore, a thorough and integral understanding of these mechanisms can identify novel biomarkers and develop innovative therapeutic strategies for CaP treatment. Our recent data have demonstrated CSCs are associated with CaP chemosensitivity. In this review, we discuss the roles of putative CSC markers in CaP chemoresistance and elucidate several CSC-associated signaling pathways such as PI3K/Akt/mTOR, Wnt/β-catenin and Notch pathways in the regulation of CaP chemoresistance. Moreover, we will summarize emerging and innovative approaches for the treatment of CRPC and address the challenging CRPC that is driven by CSCs. Understanding the link between CSCs and metastatic CRPC will facilitate the development of novel therapeutic approaches to overcome chemoresistance and improve the clinical outcomes of CaP patients.

Triptolide inhibits the multidrug resistance in prostate cancer cells via the downregulation of MDR1 expression

Triptolide (TPL) is a diterpenoid triepoxide derived from the Chinese herb Tripterygium wilfordii and possesses anti-tumor activity against a range of cancer cells. However, the effect of TPL on prostate cancer cells and its potential to overcome multidrug resistance (MDR) have not been explored. Therefore, in this study we used prostate cancer cell line DU145 as the experimental model and established DU145/ADM cell line resistant to adriamycin (ADM). Our results showed that TPL inhibited the proliferation and induced the cell cycle arrest and apoptosis of DU145 cells in a dose and time dependent manner. TPL decreased the levels of Cyclin D1 and anti-apoptotic protein Bcl-2, and increased the levels of pro-apoptotic proteins Fas and Bax. Furthermore, we found that TPL restored the sensitivity DU145/ADM cells to ADM in a dose dependent manner, and this was accompanied by the inhibition of MDR1 expression at both mRNA and protein levels. Taken together, these results provide strong evidence that TPL overcomes MDR in prostate cancer cells by downregulating MDR1 expression, and suggest that TPL is a promising agent for prostate cancer therapy, especially for chemoresistant prostate cancer.

Repression of NR4A1 by a chromatin modifier promotes docetaxel resistance in PC-3 human prostate cancer cells

Epigenetic silencing mechanisms play an important role in chemoresistance of human cancer. Here we report the upregulated expression of metastasis-associated protein 1 (MTA1), a component of the nucleosome remodeling deacetylation (NuRD) complex, in chemoresistant prostate cancer (PCa). MTA1 knockdown in PC-3 cells inhibited cell proliferation and enhanced docetaxel (DTX)-induced cell death. Conversely, overexpression of MTA1 promotes DTX chemoresistance in PC-3 cells. MTA1 acted as a potent corepressor of the nuclear receptor NR4A1 transcription by interacting with histone deacetylase 2 (HDAC2). These findings suggest that MTA1 may serve as a novel DTX-resistance promoter in PC-3 cells.

Increased Chemosensitivity via Targeting Testicular Nuclear Receptor 4 (TR4)-Oct4-Interleukin 1 Receptor Antagonist (IL1Ra) Axis in Prostate Cancer CD133+ Stem/Progenitor Cells to Battle Prostate Cancer

Prostate cancer (PCa) stem/progenitor cells are known to have higher chemoresistance than non-stem/progenitor cells, but the underlying molecular mechanism remains unclear. We found the expression of testicular nuclear receptor 4 (TR4) is significantly higher in PCa CD133(+) stem/progenitor cells compared with CD133(-) non-stem/progenitor cells. Knockdown of TR4 levels in the established PCa stem/progenitor cells and the CD133(+) population of the C4-2 PCa cell line with lentiviral TR4 siRNA led to increased drug sensitivity to the two commonly used chemotherapeutic drugs, docetaxel and etoposide, judging from significantly reduced IC50 values and increased apoptosis in the TR4 knockdown cells. Mechanism dissection studies found that suppression of TR4 in these stem/progenitor cells led to down-regulation of Oct4 expression, which, in turn, down-regulated the IL-1 receptor antagonist (IL1Ra) expression. Neutralization experiments via adding these molecules into the TR4 knockdown PCa stem/progenitor cells reversed the chemoresistance, suggesting that the TR4-Oct4-IL1Ra axis may play a critical role in the development of chemoresistance in the PCa stem/progenitor cells. Together, these studies suggest that targeting TR4 may alter chemoresistance of PCa stem/progenitor cells, and this finding provides the possibility of targeting TR4 as a new and better approach to overcome the chemoresistance problem in PCa therapeutics.

Abstract 956: Role of the cell cycle regulator LZTS1 in docetaxel resistance of prostate cancer cells and overcoming the docetaxel resistance by cell cycle pharmacological inhibitors.

Abstract Background: Docetaxel is the standard treatment for metastatic castration-resistant prostate cancer (CRPC) since 2004 and overall survival benefit in CRPC has been demonstrated in docetaxel-treated patients. In spite of this benefit, a drug resistance is eventually observed in all patients, leaving few therapeutic options. Therefore, it is crucial to identify predictive markers that enable selection of patients who will respond to treatment. Methods: Three docetaxel-resistant prostate cancer cell lines (LNCaP, PC3 and IGR-CaP1) were obtained by continuous exposure to Docetaxel. A high-density genomic profiling by cDNA microarrays (Agilent technologies) was performed to compare sensitive and chemoresistant cell lines and a signature of 99 highly differentially expressed genes (with Fold Change >5) potentially implicated in chemoresistance was generated. Results: We focused on the role of the cell cycle regulator LZTS1, which was strongly under-expressed in all the docetaxel-resistant cell lines. This underexpression was due to a stretch of 20 highly methylated CpGs in the region encompassing the exon 1 of the LZTS1 promoter in resistant cells. Knockdown of LZTS1 in IGR-CaP1 parental cells with siRNA showed that LZTS1 plays an important role in the acquisition of the resistant phenotype. Importantly, immunohistochemical staining showed a loss of LZTS1 expression in 33% of diagnostic biopsies obtained from patients with metastatic CRPC. Furthermore, we observed that targeting Cdc25C, a partner of LZTS1, with the Cdc25 pharmacological inhibitor NSC 663284 killed specifically the docetaxel-resistant cells. There are currently no CDC25C inhibitors tested in clinical trials, therefore we are currently investigating the role of other kinases that are involved in the G2/M checkpoint and in the regulation of CDC25C. Importantly, inhibitors of these kinases are currently being assessed in clinical trials. We wish to determine if targeting CDC25C and/or other kinases could kill docetaxel-resistant cells and if the use of such inhibitors could be a promising strategy to overcome docetaxel resistance in prostate cancer. Conclusion: High-density microarray genomic analyses comparing chemo-resistant versus sensitive prostate cancer cell lines were used to identify signatures of genes and microRNAs, and signaling pathways potentially implicated in Docetaxel resistance. Our findings identify an important role of LZTS1 in docetaxel resistance in prostate cancer through its regulation of CDC25C. It could also provide the framework for formulation of novel combined therapies that may improve taxane therapy efficacy or prevent chemoresistance in men with prostate cancer. Citation Format: Sophie Cotteret, Nader Al Nakouzi, Catherine Gaudin, Frederic Commo, Shanna Rajpar, Sandra Lejuste, Nicolas Martin, Karim Fizazi, Anne Chauchereau. Role of the cell cycle regulator LZTS1 in docetaxel resistance of prostate cancer cells and overcoming the docetaxel resistance by cell cycle pharmacological inhibitors. [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 956. doi:10.1158/1538-7445.AM2013-956

A novel semisynthetic inhibitor of the FRB domain of mammalian target of rapamycin blocks proliferation and triggers apoptosis in chemoresistant prostate cancer cells.

The mammalian target of rapamycin (mTOR) is a key regulator of cell growth and its uncontrolled activation is a hallmark of cancer. Moreover, mTOR activation has been implicated in the resistance of cancer cells to many anticancer drugs, rendering this pathway a promising pharmacotherapeutic target. Here we explored the capability of a semisynthetic compound to intercept mTOR signaling. We synthesized and chemically characterized a novel, semisynthetic triterpenoid derivative, 3-cinnamoyl-11-keto-β-boswellic acid (C-KβBA). Its pharmacodynamic effects on mTOR and several other signaling pathways were assessed in a number of prostate and breast cancer cell lines as well as in normal prostate epithelial cells. C-KβBA exhibits specific antiproliferative and proapoptotic effects in cancer cell lines in vitro as well as in PC-3 prostate cancer xenografts in vivo. Mechanistically, the compound significantly inhibits the cap-dependent transition machinery, decreases expression of eukaryotic translation initiation factor 4E and cyclin D1, and induces G(1) cell-cycle arrest. In contrast to conventional mTOR inhibitors, C-KβBA downregulates the phosphorylation of p70 ribosomal S6 kinase, the major downstream target of mTOR complex 1, without concomitant activation of mTOR complex 2/Akt and extracellular signal-regulated kinase pathways, and independently of protein phosphatase 2A, liver kinase B1/AMP-activated protein kinase/tuberous sclerosis complex, and F12-protein binding. At the molecular level, the compound binds to the FKBP12-rapamycin-binding domain of mTOR with high affinity, thereby competing with the endogenous mTOR activator phosphatidic acid. C-KβBA represents a new type of proapoptotic mTOR inhibitor that, due to its special mechanistic profile, might overcome the therapeutic drawbacks of conventional mTOR inhibitors.

SiRNA-mediated Silencing of Notch-1 Enhances Docetaxel Induced Mitotic Arrest and Apoptosis in PCa Cells

Notch is an important signaling pathway that regulates cell fate, stem cell maintenance and the initiation of differentiation in many tissues. It has been reported that activation of Notch-1 contributes to tumorigenesis. However, whether Notch signaling might have a role in chemoresistance of prostate cancer is unclear. This study aimed to investigate the effects of Notch-1 silencing on the sensitivity of prostate cancer cells to docetaxel treatment. siRNA against Notch-1 was transfected into PC-3 prostate cancer cells. Proliferation, apoptosis and cell cycle distribution were examined in the presence or absence of docetaxel by MTT and flow cytometry. Expression of p21(waf1/cip1) and Akt as well as activation of Akt in PC-3 cells were detected by Western blot and Real-time PCR. Silencing of Notch-1 promoted docetaxel induced cell growth inhibition, apoptosis and cell cycle arrest in PC-3 cells. In addition, these effects were associated with increased p21(waf1/cip1) expression and decreased Akt expression and activation in PC-3 cells. Notch-1 promotes chemoresistance of prostate cancer and could be a potential therapeutic target.

Chemoresistance in Prostate Cancer Cells Is Regulated by miRNAs and Hedgehog Pathway

Many prostate cancers relapse due to the generation of chemoresistance rendering first-line treatment drugs like paclitaxel (PTX) ineffective. The present study aims to determine the role of miRNAs and Hedgehog (Hh) pathway in chemoresistant prostate cancer and to evaluate the combination therapy using Hh inhibitor cyclopamine (CYA). Studies were conducted on PTX resistant DU145-TXR and PC3-TXR cell lines and clinical prostate tissues. Drug sensitivity and apoptosis assays showed significantly improved cytotoxicity with combination of PTX and CYA. To distinguish the presence of cancer stem cell like side populations (SP), Hoechst 33342 flow cytometry method was used. PTX resistant DU145 and PC3 cells, as well as human prostate cancer tissue possess a distinct SP fraction. Nearly 75% of the SP cells are in the G0/G1 phase compared to 62% for non-SP cells and have higher expression of stem cell markers as well. SP cell fraction was increased following PTX monotherapy and treatment with CYA or CYA plus PTX effectively reduced their numbers suggesting the effectiveness of combination therapy. SP fraction cells were allowed to differentiate and reanalyzed by Hoechst staining and gene expression analysis. Post differentiation, SP cells constitute 15.8% of total viable cells which decreases to 0.6% on treatment with CYA. The expression levels of P-gp efflux protein were also significantly decreased on treatment with PTX and CYA combination. MicroRNA profiling of DU145-TXR and PC3-TXR cells and prostate cancer tissue from the patients showed decreased expression of tumor suppressor miRNAs such as miR34a and miR200c. Treatment with PTX and CYA combination restored the expression of miR200c and 34a, confirming their role in modulating chemoresistance. We have shown that supplementing mitotic stabilizer drugs such as PTX with Hh-inhibitor CYA can reverse PTX chemoresistance and eliminate SP fraction in androgen independent, metastatic prostate cancer cell lines.

P21-Activated kinase 4 promotes prostate cancer progression through CREB

Prostate cancer is initially androgen-dependent but, over time, usually develops hormone- and chemo-resistance. The present study investigated a role for p21-activated kinase 4 (PAK4) in prostate cancer progression. PAK4 activation was markedly inhibited by H89, a specific protein kinase A (PKA) inhibitor, and PAK4 was activated by the elevation of cAMP. The catalytic subunit of PKA interacted with the regulatory domain of PAK4, and directly phosphorylated PAK4 at serine 474 (S474). Catalytically active PAK4 enhanced the transcriptional activity of CREB independent of S133 phosphorylation. Stable knockdown of PAK4 in PC-3 and DU145 prostate cancer cells inhibited tumor formation in nude mice. Decreased tumorigenicity correlated with decreased expression of CREB and its targets, including Bcl-2 and cyclin A1. Additionally, in androgen-dependent LNCap-FGC cells, PAK4 regulated cAMP-induced neuroendocrine differentiation, which is known to promote tumor progression. Finally, PAK4 enhanced survival and decreased apoptosis following chemotherapy. These results suggested that PAK4 regulates progression toward hormone- and chemo-resistance in prostate cancer, and this study identified both a novel activation mechanism and potential downstream effector pathways. Therefore, PAK4 may be a promising therapeutic target in prostate cancer.

Abstract 841: CCL2 and interleukin-6 regulate EMT-mediated chemo-resistance in prostate cancer

Abstract Prostate cancer (PCa) is one of the most common cancers and the second leading cause of cancer-related death in western countries. In China, the incidence has increased, unexpectedly and sharply, for the past 20 years. Chemo-resistance, in various types of tumor including PCa, is one of the critical problems in cancer therapy. In PCa, for example, the resistance to taxanes (DTX) accounts for tumor relapse and progression resulting in skeletal metastasis and high mortality. Epithelial-mesenchymal transition (EMT) has been suggested as a key process that dominates chemo-resistance through unknown mechanisms. In the current study, we reported that CCL2 and interleukin-6 (IL-6) serve as key molecules in EMT-mediated chemo-resistance in PCa cells using previously established paclitaxel-resistant cells, DU145-TxR and PC-3-TxR. Parental DU145 and PC-3 cells were used as controls. We observed that DU145-TxR and PC-3-TxR cells produced significantly higher amount of CCL2 and IL-6 compared to their parental cells, determined by cytokine array. In addition, DU145-TxR and PC-3-TxR cells demonstrated EMT phenotypes including hall maker changes in E-cadherin and Vimentin expression and transcriptional factor (Snail, ZEB1, and Slug) alterations. These changes were reversed partially by adding neutralizing antibodies against either CCL2 or interleukin-6. Interestingly, synergistic effects were observed by adding both reagents. Finally DU145-TxR, PC-3-TxR, or their parental cells were implanted into SCID mice and the mice were given DTX (10mg/kg, biweekly) and tumor growth was monitored for 4 weeks. Both DU145-TxR and PC-3-TxR tumors grew faster than the parental tumors. Tumor tissues were collected and total RNAs were isolated. Levels of CCL2 expression in these tumors were significantly higher in DU145-TxR and PC-3-TxR tumors. IHC analysis confirmed corresponding staining of EMT markers such as E-cadherin and Vimentin expression. Together we concluded that CCL2 and IL-6 regulates EMT-mediated chemo-resistance in prostate cancer. These results are of high impact of human prostate cancer chemotherapy. Supported by U.S. Department of Defense PC061231 (J. Zhang), National Natural Sciences Foundation Key Project (81130046, J. Zhang) and National Natural Science Foundation Project (81171993, Y. Lu). 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 841. doi:1538-7445.AM2012-841

Abstract 775: Targeting the transcriptional coactivator LEDGF/p75 to overcome chemoresistance in prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer death among men in the US, with African American men presenting a disproportionately high incidence and mortality. Reducing this mortality disparity requires developing novel strategies for overcoming prostate tumor chemoresistance. Currently, chemotherapy with Docetaxel (DTX), the standard of care for advanced PCa, is limited because most patients treated with the drug ultimately develop chemoresistance and succumb to the disease. Circumventing this resistance is therefore essential for increasing the effectiveness of DTX and other anti-PCa drugs. Our group has established previously that lens ephitelium-derived growth factor p75 (LEDGF/p75), a stress survival oncoprotein, is overexpressed in human prostate tumors, and that its overexpression in PCa cell models promotes resistance to DTX via inhibition of lysosomal cell death. The present study was designed to evaluate if overexpression of LEDGF/p75 in PCa cells also promotes resistance to other chemotheurapeutic drugs, and if its depletion sensitizes chemoresistant PCa cells to drug-induced cell death. First, we treated PC-3 cells (with and without stable LEDGF/p75 overexpression) with increasing concentrations of DTX, paclitaxel, doxorubicin, and TRAIL for up to 48 hrs. We then assessed cell viability by MTT assays and morphological examination. LEDGF/p75 overexpressing PC-3 cells showed higher viability than parental PC-3 cells in response to DTX, paclitaxel, and doxorubicin. However, LEDGF/p75 did not protect against TRAIL-induced cell death, indicating selectivity in the ability of this stress oncoprotein to induce chemoresistance. We also observed by immunoblotting and quantitative PCR that metastatic PCa cell lines selected for DTX resistance, designated PC3-DR and DU-145DR, naturally express high levels of endogenous LEDGF/p75 relative to their sensitive parental cell lines. To assess the contribution of elevated LEDGF/p75 expression to DTX resistance in these two cell lines, we knocked down this protein with a specific small inhibitory RNA (si-p75). Immunoblots showed that si-p75 depleted cells of LEDGF/p75. Cell viability significantly decreased in both PC3-DR and DU-145DR cell lines in response to combined treatment with DTX and si-p75, relative to scrambled siRNA control. Our preliminary results suggest that LEDGF/p75 overexpression promotes chemoresistance in cellular models of advanced PCa, and that its inhibition increases sensitization to DTX. These promising results have implications for the development of novel theraupetic strategies for chemoresistant PCa. Further studies are in progress to determine the spectrum of anti-PCa drugs antagonized by LEDGF/p75, and if depletion of this protein sensitizes PCa cells to a wide variety of antitumor drugs. 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 775. doi:1538-7445.AM2012-775

Abstract 823: Integration of microRNA and gene expression signatures from several models of Docetaxel-resistant prostate cancer cell lines

Abstract Background: Docetaxel is the standard treatment for metastatic castration-resistant prostate cancer (CRPC) since 2004 and overall survival benefit in CRPC has been demonstrated in docetaxel-treated patients. In spite of this benefit, a drug resistance is eventually observed in all patients, leaving few therapeutic options. Therefore, it is crucial to identify predictive markers that enable selection of patients who will respond to treatment. Methods: Three docetaxel-resistant prostate cancer cell lines (LNCaP, PC3 and IGR-CaP1) were obtained by continuous exposure to Docetaxel. A high-density genomic profiling by cDNA microarrays (Agilent technologies) was performed to compare sensitive and chemoresistant cell lines. The differential expression levels of 377 microRNAs between resistant and sensitive parental cell lines were measured by Taqman Low Density Array (TLDA, Applied Biosystems technology). Each determination was generated from biological replicates in the absence of drug. Results: A gene expression signature of 583 genes was generated by the bootstrap method (Fold change>2 for each doses of Docetaxel, P value <10-5) in IGR-CaP1 cells. Based on the hypothesis of a biological effect due to increasing drug, a second microarray expression profile was identified using a 5-parameters logistic regression model. This analysis led to the identification of 486 genes associated with resistance to increasing doses of docetaxel (with a P value ≤ 10-5 and a fold change between the first and the last dose of drug ≤ 2). 45 genes were common in the two analyses. A 65 miRs expression profile of docetaxel resistance was determined in all three cell lines using two reference miRs. Interestingly, we identified the under-expression of three clusters of miRs in the resistant cells. In particular, the under-expression of the miR 141-200c cluster was inversely correlated to the over-expression of its target genes Jagged1 (JAG1) and dll1(DLL1), which were identified in the gene expression signature. JAG1 and DLL1 are ligands for Notch receptors, thus we are currently exploring the role of the miR-200c/ZEB1/JAG1 axis and the Notch signalling pathway in mechanisms of Docetaxel resistance. Conclusion: High-density microarray genomic and microRNA profiling analyses comparing chemo-resistant versus sensitive prostate cancer cell lines were used to identify signatures of genes and microRNAs, and signaling pathways potentially implicated in Docetaxel resistance. Ultimately, integration of data from gene and microRNA expression will allow the identification of biomarkers to select patients that could benefit from Docetaxel chemotherapy. It could also provide the framework for formulation of novel therapies that may improve taxane therapy efficacy in men with prostate cancer. 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 823. doi:1538-7445.AM2012-823

Experience in the use of sunitinib given as a single agent in metastatic chemoresistant and castration-resistant prostate cancer patients

Objective: Vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) correlate with poor prognosis in castration-resistant prostate cancer (CRPC). Sunitinib has shown activity in CRPC and at the time of this analysis there was no standard therapy for docetaxel-refractory CRPC.Methods: We present a case series data collection of 19 patients with a median age of 73 years, CRPC and rising prostate-specific antigen (PSA). Patients received sunitinib 37.5 mg continuous daily dose. One cycle comprised a 4-week period. Patients were evaluated by CT scan every 8 weeks and PSA was monitored every 4 weeks.Results: Median Eastern Cooperative Oncology Group (ECOG) performance status score was 2. Patients received a median of two previous treatment lines for the hormone-refractory setting. Baseline median PSA was 280 ng/ml. Patients received a median of 16 weeks of therapy (4 – 48+). One patient achieved a partial response (5%) and 12 (66.7%) achieved stable disease for at least 3 months according to RECIST criteria. Median progression-free survival was 4 months. PSA declined > 50% in 5/19 (26.3%) and stabilized in 7/19 (37%) patients. Frequent adverse events were grade 3 asthenia (21%), grade 3 diarrhea (10%) and grade 3 hand-foot syndrome (15.7%).Conclusions: Activity with sunitinib was observed in highly pretreated docetaxel-refractory CRPC with acceptable tolerability. Additional studies should confirm the role of antiangiogenic agents in this setting.

Abstract 5052: N-cadherin monoclonal antibody as a therapeutic agent against chemoresistance in prostate cancer

Abstract Introduction: The development of chemoresistance is a formidable obstacle in realizing effective therapy against cancer. Docetaxel is a common therapeutic agent given to men with castration-resistant prostate cancer (CRPC). However, a number of patients show poor response or eventually develop resistance to the treatment. N-cadherin, a calcium-dependent cell-cell adhesion surface protein associated with Epithelial to mesenchymal transition (EMT), is implicated in castration resistance and metastasis in prostate cancer. We have shown in previous studies that monoclonal antibodies raised against the extracellular domain of N-cadherin have potential therapeutic effects in castration-resistant tumor growth and metastasis. In this study, our objective is to determine the role of N-cadherin expression in chemoresistance and whether treatment with monoclonal antibodies restores chemosensitivity. Methods: N-cadherin was ectopically expressed in N-cadherin negative prostate cancer cells (LNCaP, MDA PCa 2b). For in vitro experiments, these models were used to evaluate cytotoxicity and apoptosis in the presence of docetaxel. Endogenously expressing N-cadherin were either treated with monoclonal antibodies raised against the extracellular domain of N-cadherin or N-cadherin expression was reduced by siRNA knockdown. For in vivo studies, monoclonal antibodies raised against N-cadherin were used in combination with docetaxel to treat N-cadherin positive xenografts, and tumor growth monitored over time. Results: Ectopic expression of N-cadherin in N-cadherin negative cells resulted in >25-fold increase in resistance to cytotoxicity by docetaxel treatment when compared with control cells. N-cadherin knockdown by siRNA of cells endogenously expressing N-cadherin resulted in an increase of cytotoxicity in response to docetaxel when compared with cells transfected with scramble siRNA. Treatment with monoclonal antibody also resulted in increase of cytotoxicity in response to docetaxel. In vitro studies also show that increase in N-cadherin leads to upregulation in NFkB (>2.5-fold ± 0.17 increase in activity) and Toll-like receptor signaling, which are implicated in chemoresistance. Preliminary in vivo studies show some additive effect monoclonal antibodies against N-cadherin in combination with docetaxel on growth of xenograft tumors expressing N-cadherin. Conclusions: These studies suggest that N-cadherin is involved in the development of chemoresistance in prostate cancers. Therapeutic targeting of N-cadherin with monoclonal antibodies, in combination with therapeutic agents such as docetaxel may have significant clinical benefit. Future experiments involve further elucidating the pathways involved and downstream targets, such as those involved in NFkB and Toll-like receptor signaling. 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 5052. doi:10.1158/1538-7445.AM2011-5052

Abstract 661: Efficiency of the multivalent pseudopeptide nucant 6L in castration- and chemo-resistant prostate cancers

Abstract Introduction: Targeted therapies on advanced prostate cancer, resistant to castration and chemotherapy, offer significant hope for future treatments. NucAnt 6L (N6L) is a ligand of surface nucleolin. This protein is overexpressed at the cell surface of tumoral and endothelial cells, and is involved in tumor growth and associated angiogenesis. It has been demonstrated that N6L displays, in vitro and in vivo, a double anti-tumoral activity. It inhibits growth of various human tumor cell lines derived from mammary and colorectal carcinoma, melanoma, glioblastoma and lymphoma. In addition, it can compromise associated angiogenesis. In this context, N6L might be of value for therapeutic intervention. Previous studies have demonstrated drug's efficacy of this agent in prostate cancer cell lines. However, its effect on therapeutic resistant lines has yet to be documented. Using in vivo and in vitro models, the aim of this work is to evaluate N6L activity on castration and chemo-resistant prostate cancers. Methods: When maintained in medium deprived of androgens, androgen-responsive LNCaP cells undergo a “transdifferentiation” process giving rise to neuroendocrine (NE) like LNCaP cells which are resistant to androgen deprivation and chemotherapeutic agents such as docetaxel. Here, the anti-tumoral activity of N6L has been evaluated, in vitro, on human LNCaP-NE cells. Cell viability, as assessed by MTT test, and soft agar assays were performed at 72 hours after treatment with N6L (range of 0.1 to 100 µM). In vivo, anti-tumoral activity was then confirmed using VCaP ectopic xenografts into athymic nude mice, a condition that mimics clinical evolution of advanced prostate cancers in human. Mice bearing tumors of about 600 mm3 were castrated. Mice were treated with either vehicle or N6L at 10 mg/kg by the intraperitoneal route at different times of tumor growth. Tumors volumes were monitored at different time points using the caliper method. Results: N6L induces death of LNCaP-NE cells with a IC50 of 16.2 µM. These findings were supported by soft agar assays in which LNCaP-NE cells were found to be sensitive to N6L treatment whereas they were resistant to docetaxel. In vivo, N6L inhibits VCaP tumor growth at different stages of tumor progression, the castration-sensitive state and also the castration resistant state. Conclusions: N6L, which is currently in I/IIb phase of clinical trials, might represent a new targeted approach to treat advanced prostate cancers resistant to castration and chemotherapy. 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 661. doi:10.1158/1538-7445.AM2011-661

Abstract 4379: Role of Nanog in resistance of prostate cancer cells toward chemotherapy

Abstract Drug resistance is a major barrier to efficacious chemotherapy. Cancer stem cells (CSCs) may pose resistance to chemo- or radio-therapy, but the mechanism remains unknown. Nanog is a transcription factor essential for self-renewal of embryonic stem cells. The expression of Nanog has been reported in some cancer cells but its role in resistance to chemotherapy has not been convincingly demonstrated. Herein, we report that Nanog is expressed in tumorigenic prostate cancer cells and further it contributes to chemoresistance. Nanog expression was detected in prostate cancer cells by Western blot and RT-PCR and confirmed by sequencing. In the surviving fractions of prostate cancer cells after chemo-treatments, we found increased levels of Nanog expression and activities when compared to vehicle control-treated group. To determine the role of Nanog in resistance of prostate cancer cells, we marked and enriched Nanog-expressing prostate cancer DU145 using a reporter gene under control of 2.5 kb hNanog1 promoter. When compared to the control, the prospectively enriched Nanog-expressing cells had enhanced endogenous Nanog expression, increased positivity for stem/progenitor markers (CD44 and CD133), and presented increased resistance to Taxol, vinblastine, and doxorubicin, but not camptothecin (CPT). In addition, Nanog-enriched DU145 cells showed reduced apoptosis in the response to Taxol treatment, whereas control cells had increased activation of effector caspases after treatment with Taxol. Colony formation assay revealed that prostate tumor cells with enriched Nanog expression displayed increased recurrent ability after chemotherapy. Knockdown of Nanog by tet-inducible shRNA sensitized prostate tumor cells to multiple drug treatments, suggesting its essential role in prostate cancer chemo-resistance. Profiling of genes in drug resistance and metabolism revealed a marked increase in the mRNA levels of ATP-binding cassette (ABC) efflux transporters B1 and G2 in tumor cells enriched with endogenous Nanog expression. The increased expression of ABCB1 and ABCG2 in Nanog expressing cells was confirmed by Western blot and immunocytochemistry. Inhibition of ABCB1 activities or knockdown of ABCB1 expression sensitized Nanog expressing cells toward Taxol and vinblastine, but not doxorubicin. Knockdown of ABCG2 sensitized Nanog expressing cells toward doxorubicin, and to lesser extent, vinblastine, but not Taxol. Knockdown of Nanog reduced the expression of ABCB1 and ABCG2 and reduced the resistance of tumor cells toward chemotherapy. In summary, Nanog expression is associated with tumor cells positive for stem/progenitor markers and resistant to chemotherapy. The gain-of-function and loss-of-function studies suggest an essential role of Nanog for prostate cancer cells to resist chemotherapy via regulating the expression of ABC transporters. 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 4379. doi:10.1158/1538-7445.AM2011-4379

Calpain activation through galectin-3 inhibition sensitizes prostate cancer cells to cisplatin treatment

Prostate cancer will develop chemoresistance following a period of chemotherapy. This is due, in part, to the acquisition of antiapoptotic properties by the cancer cells and, therefore, development of novel strategies for treatment is of critical need. Here, we attempt to clarify the role of the antiapoptotic molecule galectin-3 in prostate cancer cells using siRNA and antagonist approaches. The data showed that Gal-3 inhibition by siRNA or its antagonist GCS-100/modified citrus pectin (MCP) increased cisplatin-induced apoptosis of PC3 cells. Recent studies have indicated that cisplatin-induced apoptosis may be mediated by calpain, a calcium-dependent protease, as its activation leads to cleavage of androgen receptor into an androgen-independent isoform in prostate cancer cells. Thus, we examined whether calpain activation is associated with the Gal-3 function of regulating apoptosis. Here, we report that Gal-3 inhibition by siRNA or GCS-100/MCP enhances calpain activation, whereas Gal-3 overexpression inhibits it. Inhibition of calpain using its inhibitor and/or siRNA attenuated the proapoptotic effect of Gal-3 inhibition, suggesting that calpain activation may be a novel mechanism for the proapoptotic effect of Gal-3 inhibition. Thus, a paradigm shift for treating prostate cancer is suggested whereby a combination of a non-toxic anti-Gal-3 drug together with a toxic chemotherapeutic agent could serve as a novel therapeutic modality for chemoresistant prostate cancers.

Cathepsin E enhances anticancer activity of doxorubicin on human prostate cancer cells showing resistance to TRAIL-mediated apoptosis

We previously described that cathepsin E specifically induces growth arrest and apoptosis in several human prostate cancer cell lines in vitro by catalyzing the proteolytic release of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from the tumor cell surface. It also prevents tumor growth and metastasis in vivo through multiple mechanisms, including induction of apoptosis, angiogenesis inhibition and enhanced immune responses. Using the prostate cancer cell line PPC-1, which is relatively resistant to cell death by doxorubicin (40-50% cytotoxicity), we first report that a combination treatment with cathepsin E can overcome resistance of the cells to this agent. In vitro studies showed that combined treatment of PPC-1 cells with the two agents synergistically induces viability loss, mainly owing to down-regulation of a short form of the FLICE inhibitory protein FLIP. The enhanced antitumor activity was corroborated by in vivo studies with athymic mice bearing PPC-1 xenografts. Intratumoral application of cathepsin E in doxorubicin-treated mice results in tumor cell apoptosis and tumor regression in xenografts by enhanced TRAIL-induced apoptosis through doxorubicin-induced c-FLIP down-regulation and by a decrease in tumor cell proliferation. These results indicate that combination of cathepsin E and doxorubicin is sufficient to overcome resistance to TRAIL-mediated apoptosis in chemoresistant prostate cancer PPC-1 cells, thus indicating therapeutic potential for clinical use.