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

Abstract

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 [H-3]-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.