Nader Al Nakouzi

Stemness markers characterize IGR-CaP1, a new cell line derived from primary epithelial prostate cancer

Deciphering molecular pathways involved in the early steps of prostate oncogenesis requires both in vitro and in vivo models derived from human primary tumors. However the few recognized models of human prostate epithelial cancer originate from metastases. To date, very few models are proposed from primary tumors and immortalizing normal human prostate cells does not recapitulate the natural history of the disease. By culturing human prostate primary tumor cells onto human epithelial extra-cellular matrix, we successfully selected a new prostate cancer cell line, IGR-CaP1, and clonally-derived subclones. IGR-CaP1 cells, that harbor a tetraploid karyotype, high telomerase activity and mutated TP53, rapidly induced subcutaneous xenografts in nude mice. Furthermore, IGR-CaP1 cell lines, all exhibiting negativity for the androgen receptor and PSA, express the specific prostate markers alpha-methylacyl-CoA racemase and a low level of the prostate-specific membrane antigen PSMA, along with the prostate basal epithelial markers CK5 and CK14. More importantly, these clones express high CD44, CD133, and CXCR4 levels associated with high expression of α2β1-integrin and Oct4 which are reported to be prostate cancer stemness markers. RT-PCR data also revealed high activation of the Sonic Hedgehog signalling pathway in these cells. Additionally, the IGR-CaP1 cells possess a 3D sphere-forming ability and a renewal capacity by maintaining their CSC potential after xenografting in mice. As a result, the hormone-independent IGR-CaP1 cellular clones exhibit the original features of both basal prostate tissue and cancer stemness. Tumorigenic IGR-CaP1 clones constitute invaluable human models for studying prostate cancer progression and drug assessment in vitro as well as in animals specifically for developing new therapeutic approaches targeting prostate cancer stem cells.

Abstract 2995: Loss of SHISA3 is an early event of the epithelial-to-mesenchymal transition associated with chemoresistance in prostate cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Despite scientific advances over the past decade, prostate cancer still remains the fifth leading cause of death from cancer in men worldwide. Docetaxel has an established role in the treatment of metastatic castrate-resistant prostate cancer (CRPC). However, malignant cells frequently acquire Docetaxel resistance. Therefore, further research is required to understand the molecular mechanisms underlying Docetaxel-resistance, which could be helpful in formulating alternative and superior therapeutic strategies. Methods: Two Docetaxel-resistant prostate cancer cell lines (IGR-CaP1 and PC3) were obtained by continuous exposure to Docetaxel. By comparing genes and miRs expression profiles established in these two Docetaxel-resistant models, we observed an enrichment of genes involved in the epithelial-to-mesenchymal transition (EMT) in the chemoresistant models and identified new genes potentially implicated in the resistance mechanism. Results: We identified the SHISA3 gene as a highly down-regulated gene in resistant cells. This gene was originally identified as an inhibitor of Wnt and FGF signaling during development in Xenopus. We showed that SHISA3 was lost during the acquisition of resistance to Docetaxel possibly via an EMT mechanism. Knockdown of SHISA3 in the parental IGR-CaP1 and PC3 cells triggered loss of the tight junction protein Occludin, engaged the Cadherin switch and increased the migratory properties of cells in vitro. Loss of SHISA3, along with loss of E-cadherin expression and expression of the mesenchymal marker N-cadherin were also observed in Docetaxel-resistant tumors obtained from IGR-CaP1-R xenografted mice. Phylogenetic comparison of SHISA3 gene sequences suggested that it may correspond to transmembrane adapters capable of regulating the activity of membrane receptors such as growth factor receptors. We are currently studying the binding partners of SHISA3 in our models by a proteomic approach to identify the implicated signaling pathway in prostate cancer. Conclusion: Our results show that loss of SHISA3 is an early event linked to the EMT process associated with chemoresistance and suggest that SHISA3 could be a useful biomarker to identify chemoresistant cells. Moreover, identification of its mechanism of action may lead to the identification of new therapeutic targets to overcome Docetaxel resistance. Citation Format: Nicolas J-p Martin, Sophie Cotteret, Catherine Gaudin, Marine Garrido, Safae Aarab-Terrisse, Nader al Nakouzi, Lucas Gentilini, Daniel Compagno, Vasily Ogryzko, Guillaume Meurice, Karim Fizazi, Anne Chauchereau. Loss of SHISA3 is an early event of the epithelial-to-mesenchymal transition associated with chemoresistance in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2995. doi:10.1158/1538-7445.AM2015-2995

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.

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

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

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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

Abstract 4339: Prostate adenocarcinoma and intensive bone remodeling in a new prostate cancer IGR-CaP1 preclinical model derived from a human primary tumor

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Bone metastasis is a frequent and catastrophic consequence of prostate cancer for which new therapeutics are needed. Although animal models of bone metastatic prostate cancer are necessary for understanding disease mechanism, there is currently no animal model that recapitulates spontaneous clinical bone metastases. We recently obtained the new prostate cell line IGR-CaP1 derived from primary epithelial prostate cancer. In vitro characterization indicates that IGR-CaP1 corresponds to basal epithelial cells expressing cancer stem cell markers and showing TP53 and PTEN mutations. Methods: We realized orthotopic xenografts in male nude mice and used bioluminescence imaging (BLI) that allows the sensitive detection of luciferase-expressing IGR-CaP1 cells to monitor tumor growth. Bone lesions were observed after direct intratibial injections of 5×105 luciferase-expressing IGR-CaP1 cells and were evaluated by both Computerized Tomography (CT) and micro Single-Photon Emission Computed Tomography (SPECT) measuring the bone fixation of 99Tc methylenediphosphonate (m99Tc-MDP). Results: Orthotopic injection of IGR-CaP1 cells yielded an undifferentiated prostate adenocarcinoma capable of metastasizing mainly to the liver and lung. Contrasting with the widely used PC3 preclinical model showing only osteolytic activity after bone injection, high resolution CT scan showed both lytic lesions in the cortical bone and osteoblastic lesions in the trabecular bone in 86% of the IGR-CaP1-injected mice (6/7). The mixed nature of the lesions and the intensive bone remodeling were confirmed by bone scintigraphy with a ∼5 fold increase in m99Tc-MDP fixation in injected tibia versus control tibia (P=0.0013). Conclusion: IGR-CaP1 is a rare model issued from a primary tumor able to reconstitute prostate adenocarcinoma in animals. Characterization of this new model of prostate cancer will provide investigators with an exceptional tool for developing approaches that prevent and treat prostate cancer bone metastases. In this way, micro SPECT/CT imaging provides a useful and quantitative tool for evaluating bone-targeting therapeutic strategies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 4339.