Patients with long-standing ulcerative colitis (UC) are at high risk of developing colitis-associated cancer (CAC). Although chronic persistent inflammation with sustained activation of NF-κB system in cells by stimulation of inflammatory cytokines such as TNFα and IL-6 was involved in CAC, the exact mechanism of CAC development is not completely elucidated. Small GTPases are molecular switches of cellular signaling. They are activated by GDP/GTP exchange mediated by the guanine nucleotide exchange factors (GEFs) and inactivated by GTP hydrolysis mediated by the GTPase activating proteins (GAPs). It is widely known that a small GTPase Ras is continuously activated by the point mutation in many human cancer cells. Small GTPase Ral, which is regulated by Ral-specific GTPase-activating protein (RalGAP), is a member of Ras family proteins and plays an important role in NF-κB induced inflammation. Recently, it is reported that Ral regulates tumorigenesis and invasion/metastasis of bladder cancers and hepatocellular carcinoma. However, there has been no report on the association between Ral and the development of CAC. The aim of this study is to elucidate the role of Ral in CAC with RalGAPα2 knockout (KO) mice that could enhance Ral-GTP expression.Methods:
(1) CAC model was prepared as following: Wild type (WT) C57BL/6J mice or RalGAPα2 KO mice received a single intraperitoneal injection of the carcinogen azoxymethane (AOM) (12.5 μg/kg), followed by 3 rounds of 2.0% dextran sulfate sodium (DSS)-induced colitis. We compared the histology between both mice with AOM/DSS-induced colorectal cancer. (2) We transfected RalGAPα2 siRNA into Colon26 cells to examine the involvement of Ral in migratory capacity and invasion capacity of colonic cancer cells by using wound healing assay and cell invasion assay. (3) Furthermore, we performed microarray analysis with epithelial cells isolated from colonic tissues of both mice to identify genes associated with cancer cell invasion, and performed RT-PCR to compare the expression of the identified genes between tumors of WT mice and of RalGAPα2 KO mice.Results:
(1) Despite no significant differences in the number and size of tumors between WT mice and RalGAPα2 KO mice, RalGAPα2 KO mice showed a tendency to higher number and larger size of tumors. (2) Significant differences in the percentage of SM invasive tumors were observed between RalGAPα2 KO mice and WT mice (45.5% versus 8.3%, P = 0.04). (3) The migratory capacity and invasion capacity of Colon26 cells transfected with RalGAPα2 siRNA was significantly increased compared to those with control siRNA. (4) Microarray analysis demonstrated that at least 2-fold and significant up-regulation genes in RalGAPα2 KO mice versus WT mice were MMP-9 and MMP-13. (5) Quantitative analysis of gene expression showed that significant up-regulation of MMP-9 and MMP-13 gene in RalGAPα2 KO mice was observed in compared with WT mice.Conclusions:
This study firstly demonstrated that sustained Ral activation was strongly involved in the invasive capacity of CAC with up-regulation of MMP-9 and MMP-13.