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 0 -deoxycytidine. Additionally, we analysed the aberrant methylation frequency of cell cycle inhibitor p16INK4a 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 ¼ 0.001, v 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.

Pancreatic cancer is characterized by oncogenic activation of K-Ras and inactivation of the cell cycle inhibitor p16INK4a . We previously demonstrated that reintroduction of p16INK4a 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 p16INK4a re-expression. Here, we show that restitution of p16INK4a in pancreatic cancer cell lines elicits a profound suppression of K-Ras activity. A more detailed analysis in p16INK4a reconstituted Capan-1 cells indicated selective reduction of both K-Ras activity and protein stability. Re-expression of K-Ras in p16 INK4arestituted Capan-1 cells reversed the anoikis-sensitive phenotype and increased colony formation, indicating that K-Ras suppression was required for p16INK4a -mediated reversion of the transformed phenotype. Inducible expression of p16INK4a 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. (Cancer Sci 2012; 103: 169-175)

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Background:Pancreatic cancer is a deadly disease characterised by high incidence of TP53 mutations. Restoration of TP53 function is perceived as a highly attractive therapeutic strategy, whose effects are not well characterised.Methods:The current work adapted an inducible strategy of stage-specific reexpression of wild-type (wt) TP53 in an in vivo orthotopic mouse model of pancreatic cancer.Results:The reconstitution of wt TP53 function in TP53-mutant DanG and MiaPaCa-2 cells caused G1 cell cycle arrest but no evidence of apoptosis induction. Consistent with subcutaneous xenograft models, we found that wt TP53 reduced primary tumour growth. Wt TP53 reexpression during early tumour growth led to significant increase in vascularisation. This correlated with an unexpectedly high rate of micro-metastases in lymph nodes of animals with wt TP53 induction, despite the 90% decrease in median primary tumour weight. Reexpression of wt TP53 later in tumour development did not significantly affect the number of CD31-reactive vessels, but increased lymphatic vessel density.Conclusion:The increased number of lymphatic vessels and micro-metastases suggests that wt TP53 induction complexly affected the biology of different tumour constituents of pancreatic cancer. Our observation suggests that combination of the inducible system with an orthotopic model can yield important insights into in vivo pancreatic cancer biology.