Wild-type N-Ras complements mutant K-Ras in pancreatic cancer cell lines but K-Ras has a specific role in cell cycle independent regulation of G2 cyclins

摘要

Pancreatic Ductal Adenocarcinoma (PDAC) is nearly always associated with mutant K-Ras. Nevertheless, targeting oncogenic K-Ras has so far proved ineffective in treating this form of cancer and pancreatic cancer cell lines can become K-Ras independent. Other forms of Ras are rarely mutated but wild type N-Ras and H-Ras have been shown to be present alongside functional K-Ras mutations and have been demonstrated to increase responsiveness to growth factors. Beyond this little evidence had previously been gathered on the activity or function of N-Ras and H-Ras in PDAC. Therefore, this thesis aimed to determine if other Ras isoforms are active in PDAC cell lines and what effect they may have on controlling cell division, oxidative metabolism, cytokine expression and the phospholipid composition of the membrane. The presence of active N-Ras and K-Ras was identified in three of the four human PDAC cell lines tested. Only active K-Ras was detected in a cell line derived from a mouse model of pancreatic cancer driven by heterologous expression of mutant KRAS. N-Ras was shown to be functioning alongside K-Ras to control the relative level of oxidative metabolism in the Suit-2 and a faster growing variant of the Panc-1 cell lines, but K-Ras acts alone in the slow growing Panc-1 cell line that does not contain N-Ras. N-Ras and K-Ras were shown to have different effects on the levels of cytokines, although K-Ras is largely independent of N-Ras in its regulation of phospholipid composition. A novel N-Ras independent mechanism for K-Ras transcriptional control of cyclin B1 was demonstrated. When K-Ras is depleted cyclin B1 and cyclin A are decreased. Cyclin B1 transcription can be rescued by inhibition of the Proteasome. A model is proposed whereby an unknown protein or proteins activates cyclin B1 transcription in a cell-cycle independent fashion and is protected from proteasomal degradation by K-Ras. These results suggest that mutant K-Ras can act in conjunction with wild-type N-Ras, but also can function independently to regulate G2 cyclins.