Rho Guanine Nucleotide Exchange Factor 5 Increases Lung Cancer Cell Tumorigenesis via MMP-2 and Cyclin D1 Upregulation.

Abstract

We sought to elucidate the role of Rho guanine nucleotide exchange factor 5 (ARHGEF5) in tumorigenesis of lung adenocarcinoma cells. ARHGEF5 protein levels were assessed in 91 human lung adenocarcinoma specimens, and A549 and NCI-H1650 cells, by IHC and Western blotting. In addition, ARHGEF5 mRNA expression was evaluated by quantitative reverse transcriptase-PCR. Furthermore, ARHGEF5 long and short isoform coexpression was detected by immunofluorescence. Finally, flow cytometry; CCK8 and wound-healing assays; cell invasion, migration and adhesion; and xenografts were used to evaluate the biologic significance of ARHGEF5. ARHGEF5 was significantly increased in lung adenocarcinoma tissues and cell lines. Interestingly, ARHGEF5 levels were significantly associated with tumor grade and pathologic stage, but not age, gender, T stage, or lymph node metastasis status. ARHGEF5 knockdown by RNAi resulted in dramatically reduced proliferation, adhesion, invasion, and migratory capability of A549 and NCI-H1650 cells. Likewise, protein levels of p-Src, p-Akt, and NF-κB were significantly decreased after ARHGEF5 knockdown. In parallel, increased S-phase population and MMP-2/cyclin D1 expression were observed in the cancer cells, which were not apoptotic. In addition, ARHGEF5 knockdown A549 and NCI-H1650 cells injected s.c. and i.v. into nude mice exhibited decreased xenograft volume and overtly reduced metastasis. Conversely, ARHGEF5 overexpression in A549 and NCI-H1650 cells increased their tumorigenicity in vitro. ARHGEF5 acts as a proto-oncogene in human lung adenocarcinoma cell tumorigenesis.