Abnormal alternative splicing occurs in cancer, resulting in the production of novel transcript variants. Understanding the diverse mechanisms by which splicing dysregulation contributes to human disease will generate new perspectives for drug development and biomarkers identification. The overall aim of this work is the generation of libraries of alternative splicing events that are deregulated in cancer and during anti-cancer treatments. The goal of these libraries is to be interrogated for the identification of novel biomarkers, allowing to monitor disease status, progression/relapse, and specificity/selectivity of drug response. The platform used is Exonhit's Genome Wide SpliceArray, a new generation of microarray that extends transcriptomic profiling to the monitoring of alternative splicing, thereby increasing the discriminatory power of the analyses. Here, we profiled different cancers to identify novel targets and markers that are either commonly regulated across multiple cancers or specific of a given cancer type. Alternatively spliced transcripts were isolated from breast, colon, and lung tumors and their corresponding adjacent normal tissues (20 each). Different splicing patterns were evidenced in tumoral versus normal tissues and from specificity analysis performed across a pool of 20 normal organs. Events of interest, focused on splicing events that generate potential novel amino acid sequences, were selected based on combination of statistical analysis of probe sets deregulations, protein knowledge and pathway analyses. The events were subsequently validated by QPCR analysis in these 3 cancers. These validated events will be used to identify novel cell surface epitopes for antibody development with therapeutic or diagnostic usefulness. Following this same approach, we also performed a comparative analysis of the transcriptomic response of endothelial cells to tubulin polymerization inhibitors, in order to test the feasibility of identifying differential drug response markers. To do so, we profiled various tubulin inhibitors in an in vitro angiogenesis inhibition assay. HUVEC cells were induced to form neovessels in vitro in the presence or absence of the tested tubulin inhibitors. Then, microarray profiling was performed, followed by a detailed analysis with Ingenuity, focusing on up-regulated genes with biomarker potential. Our results demonstrate that alternative RNA splicing offers a currently underexploited source of biological information for cancer research. Platforms dedicated to alternative splicing such as the SpliceArray can be integrated into discovery processes to allow identification of novel targets for drug discovery and biomarkers identification. Further, such platforms may also provide guidance in the selection and follow-up of patients in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B29.
Alternative RNA splicing is a key molecular mechanism for the generation of functional protein diversity. Abnormal alternative splicing can occur in cancer, resulting in the production of novel transcript variants. Therefore, development of platform technologies aimed at studying alternative splicing will help to understand the diverse mechanisms by which splicing dysregulation contribute to human disease. The impact of alternative splicing in disease and its regulation by drug actions have opened up new perspectives for target discovery, drug development and drug response monitoring. ExonHit has generated this type of discovery platform, the Genome Wide SpliceArrayTM, and is currently building libraries of alternative splicing events that are deregulated in cancer and in cases of therapy resistance. These libraries can be interrogated to answer questions such as drug response monitoring, specificity and selectivity of drug response, diagnostics for monitoring disease status, progression or relapse. Using ExonHit's SpliceArrayTM, we previously reported a significant contribution of splicing to cancer development and drug response (1). Here, we demonstrated the capability of ExonHit's platforms 1/ to identify novel cell surface epitopes for antibody development and 2/ to study drug resistance. Based on SpliceArray™ microarray analysis, transcripts alternatively spliced were isolated from breast tumor tissues for the epitope study. Different splicing patterns were evidenced in tumoral versus normal tissues and specificity analysis was performed too. Based on combination of statistical analysis and protein knowledge, most relevant events were selected as alternatively spliced transcripts encoding novel amino acid sequence that can be used to target monoclonal antibodies. Finally, QPCR expression analysis validate the specificity of the selected events identified by the probe sets that emerged from the genome-wide splicing analysis. For the drug resistance study, two types of Imatinib-resistant leukemia cell lines were established. Distinct splicing patterns were evidenced using principal component analysis, through statistical analysis of differential splicing and pathway analyses. We also implemented selectivity filters allowing to identify a drug resistance signature and a set of pathways/genes involved in drug resistance. These signatures will need to be confirmed in subsequent studies. Our results demonstrate that alternative RNA splicing offers a currently underexploited source of novel disease targets. Platforms dedicated to studying alternative splicing can be integrated into discovery processes to allow identification of novel targets for drug discovery and we have began to identify genomic expression profiles related to drug treatment that will ultimately help monitoring drug response and clinical trial design. 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 4860. doi:10.1158/1538-7445.AM2011-4860
Alternative splicing plays a major role in gene regulation, both in normal tissues as well as in disease. In cancer, abnormal alternative splicing, resulting in the production of novel transcript variants, has an impact on cellular processes related to tumor progression, including inhibition of apoptosis, tumor invasion, metastasis and angiogenesis. The identification of consistent molecular changes underlying the initiation and the progression of cancers is critical to better understand the mechanisms underlying tumorigenesis. Such molecular changes represent promising candidates for diagnostic and therapeutic applications. Exonhit has developed the Genome Wide SpliceArray™, a new generation of microarray that extends transcriptomic profiling to the monitoring of alternative splicing, thereby increasing the discriminatory power of the analyses. Through this novel discovery platform, libraries of alternative splicing events that are deregulated in cancer have been generated in order to be interrogated for the identification of novel biomarkers, allowing to monitor disease status, progression/relapse, and specificity/selectivity of drug response. Here we describe the identification of novel markers and transmembrane targets, arising in different cancers due to alternative splicing. Alternatively spliced transcripts were isolated from breast, colon, and lung tumors and their corresponding adjacent normal tissues (20 each). Different splicing patterns were evidenced in tumoral versus normal tissues and from specificity analysis performed across a pool of 20 normal organs. Events of interest, focused on splicing events that generate potential novel amino acid sequences, were selected based on combination of statistical analysis of probe sets deregulations, protein knowledge and pathway analyses. The events were used to identify novel cell surface epitopes for antibody development with therapeutic or diagnostic usefulness and were subsequently validated by QPCR analysis. With our microarray profiling performed here, focusing on up-regulated genes with biomarker potential, we identified and successfully validated genes that exhibit differential splicing in different cancers compared to normal adjacent tissue. We concentrated our analyses on genes involved in key processes of cancer progression such as epithelial-to-mesenchymal transition (EMT) and extracellular matrix remodeling. Alternative RNA splicing offers a currently underexploited source of biological information for cancer research. Platforms dedicated to alternative splicing such as the SpliceArray™ can be integrated into discovery processes to allow identification of biomarkers and novel targets. Further, such platforms may also provide guidance in the selection and follow-up of patients in clinical trials. 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 1260. doi:1538-7445.AM2012-1260
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