Human ARHGEF5 ELISA Matched Antibody Pair

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-kappa B 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. (C) 2015 AACR.

Epithelial tumor cells often acquire malignant properties, such as invasion/metastasis and uncontrolled cell growth, by undergoing epithelial-mesenchymal transition (EMT). However, the mechanisms by which EMT contributes to malignant progression remain elusive. Here we show that the Rho guanine nucleotide exchange factor (GEF) ARHGEF5 promotes tumor malignancy in a manner dependent on EMT status. We previously identified ARHGEF5, a member of the Dbl family of GEFs, as a multifunctional mediator of Src-induced cell invasion and tumor growth. In the present study, ARHGEF5 was upregulated during tumor growth factor-beta-induced EMT in human epithelial MCF10A cells, and promoted cell migration by activating the Rho-ROCK pathway. ARHGEF5 was necessary for the invasive and in vivo metastatic activity of human colorectal cancer HCT116 cells. These findings underscore the crucial role of ARHGEF5 in cell migration and invasion/metastasis. An in vivo tumorigenesis assay revealed that ARHGEF5 had the potential to promote tumor growth via the phosphatidylinositol 3-kinase (PI3K) pathway. However, ARHGEF5 was not required for tumor growth in epithelial-like human colorectal cancer HCT116 and HT29 cells, whereas the growth of mesenchymal-like SW480 and SW620 cells depended on ARHGEF5. Induction of EMT by tumor necrosis factor-a or Slug in HCT116 cells resulted in the dependence of tumor growth on ARHGEF5. In these mesenchymal-like cells, Akt was activated via ARHGEF5 and its activity was required for tumor growth. Analysis of a transcriptome data set revealed that the combination of ARHGEF5 upregulation and E-cadherin downregulation or Snail upregulation was significantly correlated with poor prognosis in patients with colorectal cancers. Taken together, our findings suggest that EMT-induced ARHGEF5 activation contributes to the progression of tumor malignancy. ARHGEF5 may serve as a potential therapeutic target in a subset of malignant tumors that have undergone EMT.