Establishment and characterization of CRISPR/Cas9-mediated NF2-/- human mesothelial cell line: Molecular insight into fibroblast growth factor receptor 2 in malignant pleural mesothelioma.

Abstract

Malignant pleural mesothelioma (MPM), a highly refractory tumor, is currently incurable due to the lack of an early diagnosis method and medication, both of which are urgently needed to improve the survival and/or quality of life of patients. NF2 is a tumor suppressor gene and is frequently mutated in MPM. Using a CRISPR/Cas9 system, we generated an NF2‐knockout human mesothelial cell line, MeT‐5A (NF2‐KO). In NF2‐KO cell clones, cell growth, clonogenic activity, migration activity, and invasion activity significantly increased compared with those in NF2‐WT cell clones. Complementary DNA microarray analysis clearly revealed the differences in global gene expression profile between NF2‐WT and NF2‐KO cell clones. Quantitative PCR analysis and western blot analysis showed that the upregulation of fibroblast growth factor receptor 2 (FGFR2) was concomitant with the increases in phosphorylation levels of JNK, c‐Jun, and retinoblastoma (Rb) in NF2‐KO cell clones. These increases were all abrogated by the exogenous expression of NF2 in the NF2‐KO clone. In addition, the disruption of FGFR2 in the NF2‐KO cell clone suppressed cell proliferation as well as the phosphorylation levels of JNK, c‐Jun, and Rb. Notably, FGFR2 was found to be highly expressed in NF2‐negative human mesothelioma tissues (11/12 cases, 91.7%) but less expressed in NF2‐positive tissues. Collectively, these findings suggest that NF2 deficiency might play a role in the tumorigenesis of human mesothelium through mediating FGFR2 expression; FGFR2 would be a candidate molecule to develop therapeutic and diagnostic strategies for targeting MPM with NF2 loss.