Ras association domain family 1 isoform A suppresses colonic tumor cell growth through p21 ^WAF1 activation in a p53-dependent manner

Background and Aim: Despite the frequent loss of Ras association domain family 1 isoform A (RASSF1A) expression in various cancers, the precise mechanism underlying its tumor-suppressive effect is not fully understood. To elucidate the growth-inhibitory role for RASSF1A in colorectal tumorigenesis, this study investigated the RASSF1A regulation of the p53-p21 ^WAF1 pathway. Methods: Ras association domain family 1 isoform A effect on cellular growth was tested in three human colon cancer cell lines by flow cytometry, cell counting, and [ ³ H]-thymidine incorporation assay. HCT116 p53 ^+/+ and p53 ^−/− isogenic sublines were utilized to determine the p53 dependence of RASSF1A effect on p21 ^WAF1 . Cycloheximide chase experiment and immunoprecipitation assay were carried out to define RASSF1A effect on p53 stability and mouse double minute 2 (MDM2) homolog ubiquitination. Results: Ras association domain family 1 isoform A expression inhibits colonic cell proliferation by preventing the G1 to S phase transition of the cell cycle. The RASSF1A-induced G1 cell cycle arrest is accompanied by the increase in the level of p21 ^WAF1 mRNA expression. The p21 ^WAF -inducing activity of RASSF1A was substantially higher in HCT116 p53 ^+/+ cell compared with isogenic p53 ^−/− cells. The cycloheximide chase assay revealed that RASSF1A expression leads to p53 stabilization and MDM2 homolog degradation. Using p53 ^−/− and p21 ^WAF1−/− subline cells, this study finally validated a crucial role of the p53-p21 ^WAF1 axis in RASSF1A-mediated growth inhibition. Conclusions: RASSF1A suppresses colonic tumor growth through the activation of the p53-p21 ^WAF1 pathway. This finding supports that RASSF1A could be a valuable marker for the assessment of colorectal cancer development and progression.