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Malignant pleural mesothelioma (MPM) is an aggressive neoplasm associated with asbestos exposure. Although expression and activation of receptor tyrosine kinases (RTKs), including MET, have been reported in most MPM, specific RTK inhibitors showed less than the expected response in MPM cells. To determine whether the lack of response of MET inhibitors was due to cooperation with other RTKs, we determined activation status of MET and other RTKs, including epidermal growth factor receptor (EGFR) family of 20 MPM cell lines, and tested whether dual RTK inhibition is an effective therapeutic strategy. We detected MET upregulation and phosphorylation (thus indicating activation) in 14 (70%) and 13 (65%) cell lines, but treatment with MET-specific inhibitors showed weak or modest effect of suppression in most of the cell lines. Phospho-RTK array analysis revealed that MET was simultaneously activated with other RTKs, including EGFR, ErbB2, ErbB3 and platelet-derived growth factor receptor-beta. Combination of MET and EGFR inhibitors triggered stronger inhibition on cell proliferation and invasion of MPM cells than that of each in vitro. These results indicated that coactivation of RTKs was essential in mesothelioma cell proliferation and/or survival, thus suggesting that simultaneous inhibition of RTKs may be a more effective strategy for the development of molecular target therapy for MPM.

We previously reported the results of bacterial artificial chromosome array comprehensive genomic hybridization of malignant pleural mesotheliomas (MPMs), including two cases with high-level amplification in the 11q22 locus. In this study, we found that the YAP1 gene encoding a transcriptional coactivator was localized in this amplified region and overexpressed in both cases, suggesting it as a candidate oncogene in this region. We analyzed the involvement of YAP1 in MPM proliferation, as well as its functional and physical interaction with Merlin encoded by the neurofibromatosis type 2 (NF2) tumor suppressor gene, which is frequently mutated in MPMs. YAP1-RNA interference suppressed growth of a mesothelioma cell line NCI-H290 with NF2 homozygous deletion, probably through cell-cycle arrest and apoptosis induction, whereas YAP1 transfection promoted the growth of MeT-5A, an immortalized mesothelial cell line. We also found that the introduction of NF2 into NCI-H290 induced phosphorylation at serine 127 of YAP1, which was accompanied by reduction of nuclear localization of YAP1, whereas nuclear localization of a YAP1 S 127A mutant was not affected. Furthermore, results of immunoprecipitation and in vitro pull-down assays indicated a physical interaction between Merlin and YAP1. These results suggest that YAP1 is involved in mesothelial cell growth and that the transcriptional coactivator activity of YAP1 is functionally inhibited by Merlin through the induction of phosphorylation and cytoplasmic retention of YAP1. This is the first report of negative regulatory signaling from Merlin to YAP1 in mammalian cells. Future studies of transcriptional targets of YAP1 in MPMs may shed light on the molecular mechanisms of MPM development and lead to new therapeutic strategies.