Genes involved in the carcinogenetic mechanisms underlying malignant pleural mesothelioma (MPM)

Genes involved in the carcinogenetic mechanisms underlying malignant pleural mesothelioma (MPM) are still poorly characterized. the pleural cavity triggered by asbestos exposure. Patients with MPM have a poor prognosis with overall survival typically ranging between 6 and 13 months. The carcinogenetic mechanisms underlying MPM and the genes involved are still poorly characterized although so far has aroused the most interest. The human gene encodes a ~71 kDa precursor protein of 622 amino acids. The precursor is processed by a removal of 33 N-terminal residues. Moreover the C-terminal residues departing from Ser598 are replaced with glycosyl-phosphatidyl-inositol (GPI) facilitating the anchoring of the peptide to the cell membrane. Then the precursor is cleaved at Arg295 by the endoprotease furin into a ~31 kDa soluble peptide called “megakaryocyte potentiating factor” (from aminoacid Ser34 to Arg286) [1] and a ~40 kDa GPI-anchored membrane-bound glycoprotein (mature mesothelin MSLN starting from Glu296) [2] [3] [4]. It was found that MSLN is present at low levels in a restricted set of normal adult tissues including the mesothelium but it Tonabersat is overexpressed aberrantly by several cancers such as MPM and pancreatic (PC) and ovarian carcinomas (OC) [5] [6]. Moreover a soluble form of MSLN (soluble mesothelin related Rabbit Polyclonal to Cytochrome P450 26A1. peptide Tonabersat SMRP) is known lacking the C-terminal GPI-membrane anchor binding segment [7]. Interestingly the levels of SMRP are elevated in the sera of MPM PC or OC patients but not in patients with other types of cancer or inflammatory diseases or in healthy controls [8] [9] [10]. Unfortunately since knock-out mice did not exhibit any adverse pathology the exact function of MSLN remains unclear [11]. Recent studies highlighted the possible mechanisms by which MSLN could play an active role in cancer progression; it was shown to interact with MUC16 [12] and to activate the p38 pathway leading to the selective induction of matrix metalloproteinase (MMP)-7 [13]. MSLN could also increase cancer cell survival and proliferation via the activation of the NF-κB signaling pathway [14]. Finally it was suggested that MSLN could exert its role in the malignant transformation of human cells through the β-catenin pathway an important molecule for the epithelial-mesenchymal transition [15]. For all these reasons MSLN was considered a good target for immunotherapeutic strategies. In fact it was used to deliver immunotoxins to specific cancer cells [16] [17] [18] [19] [20] [21] or such as for the case of the monoclonal antibody MORAb-009 to arrest cancer progression by direct inhibition (e.g. disrupting the interaction with MUC16) [22]. Although the use of monoclonal antibodies could provide several advantages (indeed MORAb-009 is currently under clinical trial) target-specific drugs or novel inhibitors (such as antisense oligonucleotides) acting at gene-level could be an alternative for complete inhibition. To date direct inhibition of mesothelin with non-immune strategies has been attempted in a very limited number of studies using silencing RNA (siRNA) approaches. One study on the Eker Tonabersat (Tsc2 mutant) rat model of hereditary renal cancer showed tumor growth inhibition following the use of siRNA microspheres designed against Erc which is considered the rat homologue of using 1μg of total RNA in a final volume of 20μl (Bio-Rad Laboratories Hercules CA). Quantitative Real-Time PCR (RT-qPCR) Pre-designed TaqMan probes (Life Technologies Monza Italy) were employed. For the TaqMan assay the reaction mixture consisted of 2 μl of cDNA template 7 μl of deionized H2O 1 μl of specific TaqMan Assay probe and primer mixture and 10 μl of Tonabersat TaqMan? Gene Expression Master Mix (Life Technologies Monza Italy). The thermal cycling conditions were: 15 min at 95°C followed by 15 s at 95°C and 60 s at 60°C (40 cycles). TaqMan ID assays are reported in Table S1. Tonabersat Seven housekeeping genes and (aa 360-2230) was kindly donated by Dr. Uehara (Kansai Medical University Japan); the empty vector pcDNA3.1 employed as control was donated by Dr. Giamas (Imperial College London). siRNA and plasmid transfections siMSLN-1 and-2 were purchased from Qiagen (Qiagen S.p.A Milano Italy). The “AllStars Negative Control siRNA” (SI03650318) was used as non-targeting control (siRNA-Ctrl). siRNA oligonucleotides were re-suspended in the provided buffer at a final stock concentration of 20 μM. siRNA transfection was performed with the HiPerfect transfection reagent (Qiagen S.p.A Milano Italy) according to the manufacturer’s instructions..