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Mesotheliomas are frequently characterized by disruption of Hippo pathway due to deletion and/or mutation in genes, such as neurofibromin 2 (NF2). Hippo disruption attenuates yes-associated protein (YAP) phosphorylation allowing YAP to translocate to the nucleus and regulate gene expression. The role of disrupted Hippo pathway in maintenance of established mesotheliomas has been extensively investigated using cell lines; however, its involvement in development of human mesothelioma has not been explored much. Here, we employed immortalized human mesothelial cells to disrupt Hippo pathway. YAP phosphorylation was reduced on NF2 knockdown and the cells exhibited altered growth in vitro, developing tumors when transplanted into nude mice. Similar results were obtained from enforced expression of wildtype or constitutively active (S127A) YAP, indicating the crucial role of activated YAP in the transformation of mesothelial cells. Gene expression analysis comparing control- and YAP-transduced immortalized human mesothelial cells revealed phospholipase-C beta 4 (PLCB4) to be among the genes highly upregulated by YAP. PLCB4 was upregulated by YAP in immortalized human mesothelial cells and downregulated on YAP knockdown in Hippo-disrupted mesothelioma cell lines. PLCB4 knockdown attenuated the growth of YAP-transduced immortalized mesothelial cells and YAP-active, but not YAPnonactive, mesothelioma cell lines. Our model system thus provides a versatile tool to investigate the mechanisms underlying mesothelioma development. We suggest that PLCB4 may be an attractive drug target for the treatment of mesothelioma. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]