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Malignant mesothelioma (MM) frequently exhibits Hippo signaling pathway inactivation (HPI) mainly due to NF2 and/or LATS2 mutations, which leads to the activation of YAP transcriptional co-activator. Here, we show antitumor effects of statin on MM cells with HPI, through the interplay of the mevalonate and Hippo signaling pathways. Statin attenuated proliferation and migration of MM cells harboring NF2 mutation by accelerating YAP phosphorylation/inactivation. CD44 expression was decreased by statin, in parallel with YAP phosphorylation/inactivation. Importantly, we discovered that YAP/TEAD activated CD44 transcription by binding to the CD44 promoter at TEAD-binding sites. On the other hand, CD44 regulated Merlin phosphorylation according to cell density and sequentially promoted YAP transcriptional co-activator, suggesting that CD44 plays two pivotal functional roles as an upstream suppressor of the Hippo pathway and one of downstream targets regulated by YAP/TEAD. Moreover, the YAP/CD44 axis conferred cancer stem cell (CSC)-like properties in MM cells leading to chemoresistance, which was blocked by statin. Together, our findings suggest that YAP mediates CD44 up-regulation at the transcriptional level, conferring CSC-like properties in MM cells, and statin represents a potential therapeutic option against MM by inactivating YAP.

Malignant mesothelioma (MM) is an aggressive neoplasm associated with asbestos exposure. We carried out genome-wide array-based comparative genomic hybridization analysis with 14 MM cell lines. Three cell lines showed overlapping homozygous deletion at chromosome 13q12, which harbored the LATS2 (large tumor suppressor homolog 2) gene. With 6 other MM cell lines and 25 MM tumors, we found 10 inactivating homozygous deletions or mutations of LATS2 among 45 MMs. LATS2 encodes a serine/threonine kinase, a component of the Hippo tumor-suppressive signaling pathway, and we transduced LATS2 in MM cells with its mutation. Transduction of LATS2 inactivated oncoprotein YAP, a transcriptional coactivator, via phosphorylation, and inhibited MM cell growth. We also analyzed LATS2 immunohistochemically and found that 13 of 45 MM tumors had low expression of LATS2. Because NF2 is genetically mutated in 40% to 50% of MM, our data indicate that Hippo pathway dysregulation is frequent in MM cells with inactivation of LATS2 or an upstream regulator of this pathway, Merlin, which is encoded by NF2. Thus, our results suggest that the inactivation of LATS2 is one of the key mechanisms for constitutive activation of YAP, which induces deregulation of MM cell proliferation. Cancer Res; 71(3); 873–83. ©2011 AACR.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with a poor prognosis. Thus, novel therapeutic agents need to be developed for treating it. We recently reported the isolation of the novel anti-MPM compound designated as JBIR-23 from Streptomyces sp. AK-AB27. In this study, JBIR-23 exerted its cytotoxic effect on MPM cells by promotion of tubulin polymerization and G₂/M arrest, which was followed by apoptosis induction via the caspase pathway through phosphorylation of p38 mitogen-activated protein kinase and c-jun N-terminal kinase. Furthermore, in vivo analysis demonstrated that JBIR-23 prevented tumor growth in tumor-bearing nude mice without evident side effects.

Purpose: Malignant pleural mesothelioma (MPM) is a biologically heterogeneous malignant disease with a poor prognosis. We reported previously that the anti–vascular endothelial growth factor (VEGF) antibody, bevacizumab, effectively inhibited the progression of VEGF-high-producing (but not VEGF-low-producing) MPM cells in orthotopic implantation models, indicating the need for novel therapeutic strategies to improve the poor prognosis of this disease. Therefore, we focused on the multi–tyrosine kinase inhibitor E7080 and assessed its therapeutic efficacy against MPM cells with different proangiogenic cytokine production profiles. Experimental Design: The efficacy of E7080 was assayed in orthotopic implantation of severe combined immunodeficient mouse models with three human MPM cell lines (MSTO-211H, NCI-H290, and Y-MESO-14). Results: With regard to proangiogenic cytokine production profiles, MSTO-211H and Y-MESO-14 cells were MPM cells producing high levels of fibroblast growth factor-2 and VEGF, respectively. NCI-H290 cells produced low levels of fibroblast growth factor-2 and VEGF compared with the other two cell lines. E7080 potently suppressed the phosphorylation of VEGF receptor-2 and FGF receptor 1 and, thus, inhibited proliferation of endothelial cells, but not that of the MPM cell lines, in vitro. Orthotopically inoculated MSTO-211H cells produced only thoracic tumors, whereas NCI-H290 and Y-MESO-14 cells also developed pleural effusions. Treatment with E7080 potently inhibited the progression of these three MPM cell lines and markedly prolonged mouse survival, which was associated with decreased numbers of tumor-associated vessels and proliferating MPM cells in the tumor. Conclusions: These results strongly suggest broad-spectrum activity of E7080 against MPM with different proangiogenic cytokine production profiles in humans. (Clin Cancer Res 2009;15(23):7229–37)

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.

Malignant mesothelioma (MM) is one of the most aggressive neoplasms usually associated with asbestos exposure and is highly refractory to current therapeutic modalities. MMs show frequent activation of a transcriptional coactivator Yes-associated protein (YAP), which is attributed to the neurofibromatosis type 2 (NF2)-Hippo pathway dysfunction, leading to deregulated cell proliferation and acquisition of a malignant phenotype. However, the whole mechanism of disordered YAP activation in MMs has not yet been well clarified. In the present study, we investigated various components of the NF2-Hippo pathway, and eventually found that MM cells frequently showed downregulation of LIM-domain protein AJUBA, a binding partner of large tumor suppressor type 2 (LATS2), which is one of the last-step kinases of the NF2-Hippo pathway. Although loss of AJUBA expression was independent of the alteration status of other Hippo pathway components, MM cell lines with AJUBA inactivation showed a more dephosphorylated (activated) level of YAP. Immunohistochemical analysis showed frequent downregulation of AJUBA in primary MMs, which was associated with YAP constitutive activation. We found that AJUBA transduction into MM cells significantly suppressed promoter activities of YAP-target genes, and the suppression of YAP activity by AJUBA was remarkably canceled by knockdown of LATS2. In connection with these results, transduction of AJUBA-expressing lentivirus significantly inhibited the proliferation and anchorage-independent growth of the MM cells that harbored ordinary LATS family expression. Taken together, our findings indicate that AJUBA negatively regulates YAP activity through the LATS family, and inactivation of AJUBA is a novel key mechanism in MM cell proliferation.

Malignant mesothelioma (MM) is an aggressive tumor arising primarily from pleural or peritoneal cavities, which is caused by asbestos exposure after long latency. One of the most frequently mutated genes detected in MM cells is the neurofibromatosis type 2 (NF2) tumor suppressor gene which is located at chromosome 22q12. The NF2 gene encodes Merlin, an ERM (Ezrin/Radixin/Moesin) protein. The underphosphorylated form of Merlin is active and acts as a tumor suppressor by regulating several distinct cellular signaling pathways. One of the downstream pathways regulated by Merlin is the Hippo signaling pathway, which is conserved from Drosophila to mammalian cells and plays important roles in organ size control and cancer development. Recent studies have identified alterations of the components in the Hippo signaling cascade in MM cells, including overexpression of Yes-associated protein (YAP) and inactivation of large tumor suppressor homolog 2 (LATS2). Dysregulation of the Merlin-Hippo signaling cascade is one of the frequent and key events of MM cell development and/or progression. Thus, a strategy to normalize this signaling cascade may be the rationale for developing a new target therapy against MM.