Regulation of the Tumor Suppresser p53 and Survivin by Ras and Ral GTPases:Implications for Malignant Transformation

Abstract Although the critical role of the small GTPases Ras and Ral in oncogenesis has been well documented, much remains to be investigated about the molecular mechanism by which these GTPases regulate malignant transformation. The work under this thesis made two major contributions to this field. The first is the discovery that K-Ras, RalA and/or RalB are required for the maintenance of the high levels of the anti-apoptotic protein survivin in some human cancer cells, and the second is the demonstration that down regulation of K-Ras, RalA and/or RalB, but not Raf-1 or Akt1/2, stabilizes the tumor suppressor p53 and reactivates it to inhibit malignant transformation in human cancer cells with mutant K-Ras and wild type p53. Here we found that depletion of K-Ras leads to decreased survivin levels in human cancer cells that harbor mutant K-Ras but not those with wild type Ras. The mechanism by which this occurs involves ubiquitination and subsequent proteasome-mediated degradation of survivin. The presence of mutant K-Ras alone was not sufficient to predict the effects of RalA/B depletion on survivin levels. Indeed, depletion of RalA and/or RalB reduces survivin levels in human cancer cells with wild type p53 and mutant K-Ras, but not in those with mutant p53 and/or wild type K-Ras. The functional relevance of these findings to malignant transformation was further supported by the demonstration that compromising the expression of survivin by siRNA leads to reduction of mutant K-Ras-driven invasion and anchorage-independent growth. Furthermore, in this thesis, we have discovered that down regulation of K-Ras, RalA and/or RalB using siRNA leads to increased levels of functional p53 that is capable of regulating its target genes. The mechanism by which depleting K-Ras, RalA and RalB increases the levels of p53 involves an increase in the half-life of the p53 protein concurrent with an increase in the phosphorylation of serine-15 of p53, a marker of p53 stability. Finally, we demonstrated that depletion of K-Ras, RalA and/or RalB interferes with cell cycle progression, anchorage-independent growth and invasion in a p53-dependent manner. In summary, the studies suggest that mutant K-Ras contributes to the maintenance of the aberrantly-high survivin levels by regulating its stability, and that the ability of mutant K-Ras to induce malignant transformation is, at least in part, dependent of these high levels of survivin. The work of this thesis also suggests that the expression of K-Ras, RalA and/or RalB proteins is critical to maintain low levels of p53, and that down regulation of these GTPases reactivates p53 by significantly enhancing its stability, and this contributes to suppression of malignant transformation