Tumor suppressor p16INK4a controls oncogenic K-Ras function in human pancreatic cancer cells.

Pancreatic cancer is characterized by oncogenic activation of K-Ras and inactivation of the cell cycle inhibitor p16(INK4a) . We previously demonstrated that reintroduction of p16(INK4a) reversed anoikis resistance and clonogenicity of human pancreatic cancer cells, properties commonly attributed to the transforming potential of oncogenic K-Ras. Therefore, we aimed to determine the role of Ras after p16(INK4a) re-expression. Here, we show that restitution of p16(INK4a) in pancreatic cancer cell lines elicits a profound suppression of K-Ras activity. A more detailed analysis in p16(INK4a) reconstituted Capan-1 cells indicated selective reduction of both K-Ras activity and protein stability. Re-expression of K-Ras in p16(INK4a) restituted Capan-1 cells reversed the anoikis-sensitive phenotype and increased colony formation, indicating that K-Ras suppression was required for p16(INK4a) -mediated reversion of the transformed phenotype. Inducible expression of p16(INK4a) in DanG cells confirmed inhibition of K-Ras activity as well as an increase in anoikis susceptibility. Thus, our results delineate a novel functional interaction with defined biological consequences for the two most frequent alterations observed in pancreatic cancer.

Frequent RASSF1A promoter hypermethylation and K-ras mutations in pancreatic carcinoma.

Recently, we have characterized the Ras association domain family 1A gene (RASSF1A) at the segment 3p21.3, which is frequently lost in variety of human cancers and epigenetically inactivated in many types of primary tumors, such as lung, breast, kidney, prostate and thyroid carcinomas. Here, we investigated the methylation status of the RASSF1A CpG island promoter in the pathogenesis of pancreatic cancer. RASSF1A hypermethylation was detected in 29 out of 45 (64%) primary adenocarcinomas, in 10 out of 12 (83%) endocrine tumors and in eight out of 18 (44%) pancreatitis samples. In seven out of eight pancreas cancer cell lines, RASSF1A was silenced and was retranscribed after treatment with 5-aza-2'-deoxycytidine. Additionally, we analysed the aberrant methylation frequency of cell cycle inhibitor p16(INK4a) and K-ras gene mutations in the pancreatic samples. p16 inactivation was detected in 43% of adenocarcinomas, in 17% of neuroendocrine tumors, in 18% of pancreatitis and in 63% of pancreas cancer cell lines. K-ras mutations were detected in 16 out of 45 (36%) primary adenocarcinomas. Pancreatic adenocarcinomas with K-ras mutation have significantly less RASSF1A methylation and vice versa (P=0.001, chi(2) test). In conclusion, our data indicate that inactivation of the RASSF1A gene is a frequent event in pancreatic cancer and suggest an inverse correlation between RASSF1A silencing and K-ras activation.