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
Bone metastases have a devastating impact on quality of life and bone pain in patients with prostate cancer and decrease survival. Animal models are important tools in investigating the pathogenesis of the disease and in developing treatment strategies for bone metastases, but few animal models recapitulate spontaneous clinical bone metastatic spread. In the present study, IGR-CaP1, a new cell line derived from primary prostate cancer, was stably transduced with a luciferase-expressing viral vector to monitor tumor growth in mice using bioluminescence imaging. The IGR-CaP1 tumors grew when subcutaneously injected or when orthotopically implanted, reconstituted the prostate adenocarcinoma with glandular acini-like structures, and could disseminate to the liver and lung. Bone lesions were detected using bioluminescence imaging after direct intratibial or intracardiac injections. Anatomic bone structure assessed using high-resolution computed tomographic scans showed both lytic and osteoblastic lesions. Technetium Tc 99m methylene diphosphonate micro single-photon emission computed tomography confirmed the mixed nature of the lesions and the intensive bone remodeling. We also identified an expression signature for responsiveness of IGR-CaP1 cells to the bone microenvironment, namely expression of CXCR4, MMP-9, Runx2, osteopontin, osteoprotegerin, ADAMTS14, FGFBP2, and HBB. The IGR-CaP1 cell line is a unique model derived from a primary tumor, which can reconstitute human prostate adenocarcinoma in animals and generate experimental bone metastases, providing a novel means for understanding the mechanisms of bone metastasis progression and allowing preclinical testing of new therapies.
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
3880 Introduction: Around 10 human prostate cancer cell lines are available to-date and thereof only five are currently used. Most of these models have been obtained from prostate tumor metastases or have been artificially established in vitro. Therefore it is still a challenge to obtain new cancer models that may better reflect the mechanisms of local and distant tumor progression. Methods: Taking advantage of the natural extra cellular matrix we recently developed, one new cell line (IGR-CaP1) was obtained from 3 processed samples derived from patients with clinically localized prostate cancer. Extensive characterization of this cell line was performed using immunoblotting and immunofluorescence analysis, quantitative real-time PCR, cytogenetic analysis and tumorigenicity in mice. Results: IGR-CaP1 cells were cultured for at least 35 passages. They showed epithelial-like morphology although CK8, CK19, EpCAM were expressed at low levels. Immunoblotting studies showed that IGR-Cap1 cells have lost expression of epithelial cell adhesion molecule E-Cadherin and possess high levels of the mesenchymal protein vimentin suggesting that the cells are already engaged in the tumor invasion process. Further, these cells showed a tetraploid karyotype, a high expression of p53 protein expression, and a high telomerase activity. They express neither PSA nor androgen receptor. However, the tight-junction protein ZO-1 was mainly localized at the membrane and the cells do not exhibit mesenchymal markers such as STRO-1, CD105 and CD73. In vivo study reveals that subcutaneous injection of IGR-CaP1 cells in immune-deprived nude mice induce tumor formation after 3 weeks. The effect of orthotopic injections of IGR-CaP1 cells on metastases is currently studied. Conclusion: The IGR-CaP1 cell line is one of the only in vitro and in vivo models obtained from localized prostate cancer. In contrast to previously established models from metastases tissue, IGR-CaP1 may be a suitable model to study molecular pathways implicated in the early steps of the oncogenic development of prostate cancer. Furthermore, its high tumorigenic properties make a potential model for both tumor progression and drug assessment in animals.
