Abstract 1529: RASAL2 promotes tumor progression and metastasis in colorectal cancer

Abstract

Abstract Background: Metastatic colorectal cancer (mCRC) is a heterogeneous disease with a poor prognosis. By using high resolution array comparative genomic hybridization (aCGH) and mRNA gene expression microarray of mCRC samples, we found up-regulation of RASAL2, a RAS GAP gene that is located on chromosome 1q, in colorectal tumors. Oncogenic RAS represents one of the most common molecular changes in human colorectal adenocarcinoma. However, the role of RASAL2 in colorectal adenocarcinoma metastasis and the related mechanisms are still unclear. We analyzed its aberrant expression, clinical significance and biological effects in colorectal cancer. Methods: Expression of RASAL2 was examined by QRT-PCR, western blot and immunohistochemistry in CRC cell lines, primary and metastatic CRC and the corresponding normal colonic mucosa. The biological effects of RASAL2 on proliferation, apoptosis, cell cycle, and cell motility and invasiveness were evaluated by siRNA knock down and RASAL2 reconstitution in CRC cell lines. Results: Up-regulation of RASAL2 mRNA was observed in CRC cell lines (n = 9) than normal colon mucosal tissues. Interestingly, significantly higher RASAL2 mRNA was found in cell lines derived from advanced stage tumors (n = 4, Dukes’ C and D) than those from early stage tumors (n = 5, Dukes’ A and B) (P = 0.0317). Up-regulation of RASAL2 mRNA was also found in primary CRCs (n = 77) compared with normal colon mucosa (n = 18, P<0.0001). In 15 cases that paired primary and metastatic tumors were available, 12 (80%) demonstrated higher RASAL2 in metastatic tumors than their primary counterparts. RASAL2 protein was detected in 37% (81 of 219) of CRC by immunohistochemistry. Whereas in the paired normal colonic mucosa, the positive rate is 14% (20 of 142, P<0.0001). We knocked down RASAL2 by siRNA in 2 CRC cell lines (DLD1 and HCT116) with high endogenous RASALs level. Successful knockdown was demonstrated by western blot analysis. SiRASAL2 significantly inhibited cell proliferation (P<0.05), reduced colony formation (P<0.05) and repressed cell invasion and migration ability (P<0.05) in both cell lines. Flow cytometry analysis revealed G1 arrest in cells treated with siRASAL2. Ectopic expression of RASAL2 was performed in 2 CRC cell lines with low endogenous RASAL2 (SW480 and LoVo). Over expression of RASAL2 did not change the cell proliferation and anchorage-dependent growth of these cell lines but effectively enhanced cell invasiveness by transwell invasion assay. Conclusions: Up-regulation of RASAL2 was frequently found in CRC cell lines and primary and metastatic tumors. Our experimental findings suggested that RASAL2 might play an oncogenetic role in CRC and promotes tumor invasion and metastasis. A better understanding of the molecular mechanism of RASAL2 in promoting CRC cell metastasis may lead to a more effective management of mCRC patients. Citation Format: Yi PAN, Joanna Hung Man TONG, Raymond Wai Ming LUNG, Samantha Wei Man LUN, Kwok Wai LO, Anthony Wing Hung CHAN, Ka Fai TO. RASAL2 promotes tumor progression and metastasis in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1529.