Overexpressed genes in malignant pleural mesothelioma: possible role in tumorigenesis and evaluation of their use as a prognostic marker

Malignant Pleural Mesothelioma (MPM) is a very aggressive cancer poorly responsive to current therapies. The prognosis is very poor as the development of MPM leads to a median survival of less than one year from the time of diagnosis (Bertino et al. 2009). Moreover, the rapidly growing nature of this cancer, the non-specific onset of symptoms and the lack of an accurate biomarker do not lead to a sufficiently early diagnosis for a radical treatment of the disease. Thus, the identification of novel diagnostic and prognostic biomarkers and therapeutic targets is needed. The knowledge of the genes involved in the triggering/progression of MPM is still limited. In recent years a number of aberrantly expressed genes were suggested, but with a poor consistency among studies. To identify the relevant genes potentially involved in MPM, recently, our group carried out an extensive literature review focused on transcriptome studies whose results were intersected with those from a data mining approach (Melaiu et al. 2012). The results underwent to validation on MPM tissues and cell lines (Melaiu et al. 2015). The study led to the identification of a group of 21 deregulated genes in MPM. Among the 21 genes identified we focused our attention on those up-regulated in MPM tissues and at least in one MPM cell line (i.e. THBS2, CCNO, CFB, TIMP3, SULF1, PDGFRB, ASS1, SOD1, RAN, CDH11, EIF4G1). In addition, we made an extensive literature search to identify the most promising novel diagnostic and therapeutic target for MPM and we undertook the study of IMP3 (Hanley et al.2008; Ikeda et al. 2010; Ikeda et al. 2011; Shi et al.2011; Lee et al. 2013; Okazaki et al. 2013; Minato et a. 2014) , MCT4 (Mogi et al. 2013), MCT1 (Mogi et al. 2013), BSG (Pinheiro et al. 2012) and CAIX (Ramsey et al. 2012; Capkova et al. 2014). In order to investigate the role of these genes in the carcinogenesis of MPM we performed a phenotypic screening of five MPM cell lines (Mero14, Mero25, IstMes2, NCI-H28, REN) and one non-malignant mesothelial cell line (Met5A) following gene-silencing. We analysed the changes in the proliferation rate, in the caspase3-7 activity, in the migration ability, in the colony formation ability, in the level of senescence and in the cell cycle after transient siRNA transfections. The RNAi screening highlighted a role in MPM malignant phenotype for ASS1, CDH11, EIF4G1, IMP3, MCT4, PDGFRB, RAN, SOD1, SULF1, THBS2, TIMP3. Moreover, we evaluated the potential prognostic role of THBS2, CDH11, ITGA4, MCT4, MCT1, BSG and CAIX through a tissue microarray (TMA) immunohistochemistry on a series of 135 MPM samples. None of the target analysed showed a statistical significant association, after multiple correction, between protein expression and the overall survival. The weak staining of THBS2 and the negative staining of MCT4 on six normal pleura samples compared to MPM tissues prompted us to further investigate on protein expression levels: we increased the analysis of protein expression to 15 normal pleura samples. These results highlights the role of a group of genes, overexpressed in MPM, in the carcinogenetic process suggesting potential novel therapeutic approaches for the treatment of MPM.