Downregulation of PAR-4, a pro-apoptotic gene, in pancreatic tumors harboring K-ras mutation

Oncogenic ras is known to inhibit cell death and growth inhibitory genes and activate prosurvival genes. Proapoptotic gene PAR-4, has been found to be downregulated by oncogenic ras. Since pancreatic tumors harbor a high incidence of K-ras point mutations, we hypothesized that oncogenic K-ras might influence the function and expression of PAR-4. PAR-4 expression levels were analyzed in 4 established pancreatic tumor cell lines, 10 normal pancreatic tissues, 44 frozen tumor tissues and 25 paraffin-embedded pancreatic adenocarcinoma samples by Real Time RT-PCR, Western blot analysis and immunohistochemistry. K-ras mutational status was analyzed by allele-specific oligonucleotide-hybridization. Expression levels of PAR-4 were correlated with the K-ras mutational status and clinical characteristics. Further, modulation of endogenous PAR-4 was tested by transiently expressing oncogenic ras in a wild-type K-ras pancreatic cancer cell line, BxPC-3. Three cell lines with K-ras mutations showed low levels of PAR-4 when compared to a normal pancreatic tissue. Of 44 frozen tumors, 16 showed appreciable upregulation of Par mRNA and 27 showed significant downregulation of PAR-4 mRNA when compared to normal pancreatic tissue and 1 had levels equivalent to normal pancreatic tissue. Of 25 paraffin-embedded tumors, 9 showed downregulation of PAR-4 protein and this downregulation of PAR-4 correlated significantly with K-ras mutational status (p < 0.00002). In addition, the presence of PAR-4 mRNA or protein expression in pancreatic tumors correlated with prolonged survival. Transient overexpression of oncogenic ras in wild-type K-ras BxPC-3 cells significantly downregulated the endogenous PAR-4 protein levels and conferred accelerated growth. Thus, downregulation or loss of PAR-4 expression by oncogenic ras may provide a selective survival advantage for pancreatic tumors, through inhibition of proapoptotic pathway mediated bv PAR-4.