TAT-RasGAP317-326 Requires p53 and PUMA to Sensitize Tumor Cells to Genotoxins

Abstract

Although chemotherapy has revolutionized cancer treatment, the associated side effects induced by lack of specificity to tumor cells remain a challenging problem. We have previously shown that TAT-RasGAP 317-326 , a cell-permeable peptide derived from RasGAP, specifically sensitizes cancer cells to the action of genotoxins. The underlying mechanisms of this sensitization were not defined however. Here, we report that TAT-RasGAP 317-326 requires p53, but not the Ras effectors Akt and extracellular signal-regulated kinase, to mediate its tumor sensitization abilities. The TAT-RasGAP 317-326 peptide, although not modulating the transcriptional activity of p53 or its phosphorylation and acetylation status, nevertheless requires a functional p53 cellular status to increase the sensitivity of tumor cells to genotoxins. Genes regulated by p53 encode proapoptotic proteins, such as PUMA, and cell cycle control proteins, such as p21. The ability of TAT-RasGAP 317-326 to sensitize cancer cells was found to require PUMA but not p21. TAT-RasGAP 317-326 did not affect PUMA levels, however, but increased genotoxin-induced mitochondrial depolarization and caspase-3 activation. These results indicate that TAT-RasGAP 317-326 sensitizes tumor cells by activating signals that intersect with the p53 pathway downstream of, or at the level of, proapoptotic p53 target gene products to increase the activation of the mitochondrial death pathway. (Mol Cancer Res 2007;5(5):497 -507)

TAT-RasGAP317–326-mediated tumor cell death sensitization can occur independently of Bax and Bak

“…To gain further insight about the manner by which TAT-RasGAP 317-326 exerts its tumor sensitization effect, HeLa, U2OS, and HCT116 cancer cells were treated with additional pro-apoptotic compounds, including TNFα that induces cell death via the extrinsic apoptotic pathway. Figure 1A shows that TATRasGAP 317-326 favored cisplatin-induced apoptosis in the three different cancer cell lines, confirming earlier results 8, 33 . TAT-RasGAP 317-326 sensitized some, but not all, tumor cell lines to UV-or growth factor deprivation-induced apoptosis, despite the fact that these treatments can activate the intrinsic apoptotic pathway 34,35 .…”

“…We showed earlier that TAT-RasGAP 317-326 does not modulate MAPK signaling pathways (p38, ERK and JNK), NF-κB transcriptional activity or Akt protein levels and phosphorylation status 28,29 . Here we show that the activity of Bax can be modulated by TATRasGAP 317-326 in the context of tumor cells sensitization to genotoxin-induced apoptosis.…”

“…Tumor cells sensitized by TAT-RasGAP 317-326 to genotoxin-induced apoptosis show increased caspase-3 activation and more pronounced mitochondrial membrane depolarization 33 . This suggests that the RasGAP-derived peptide acts upstream of cytochrome c release from mitochondria.…”

“…We previously hypothesized that an intact p53/Puma axis was required for efficient genotoxin-induced apoptosis and for TAT-RasGAP 317-326 to favor genotoxin-induced apoptosis in cancer cells 33 . We reasoned that if Puma is necessary and sufficient for the sensitizing activity of the RasGAP-derived peptide, ectopic expression of Puma should render tumor cells sensitive to TAT-RasGAP 317-326 in the absence of cisplatin.…”

“…Taken together these data suggest that TAT-RasGAP 317-326 does not directly amplify the DNAdamage-induced p53/Puma arm. Hence, even though cisplatin requires p53 and Puma to induce apoptosis 33 , this genotoxin may modulate other pro-apoptotic signals that are targeted by TAT-RasGAP 317-326 to induce cell death in cancer cells.…”