Abstract 569: Inverse relationship between p53 and phospho-Chk1 (Ser317) protein expression in UVB-induced skin tumors in SKH-1 mice.

Abstract

Previous data demonstrated that the topical application of caffeine inhibited UVB-induced skin tumor formation. Studies showed that caffeine significantly diminished phospho (p)-Chk1 (Ser317) staining and increased the number of mitotic cells that expressed both cyclin B1 and caspase 3 in tumors, suggesting that caffeine induces apoptosis selectively in tumors by inhibiting the ATR/Chk1 pathway and promoting lethal mitosis. We also found that caffeine attenuated the UVB-induced decrease in mitotic cells with cyclin B1 to a greater extent in p53 knockout (KO) mice compared with p53 wild-type (WT) littermates. Therefore, we sought to investigate the mechanism by which caffeine could selectively sensitize p53-deficient cells to apoptosis following UVB exposure by inhibiting ATR/Chk1 pathway. Performing immunohistochemistry on stored tumor paraffin samples, we found an almost exclusive inverse relationship (>95%) between p53 expression and p-Chk1 (Ser317), but not p-Chk1 (Ser345), in all 42 keratoacanthomas and 11 squamous cell carcinomas examined. Tumors that expressed high levels and large areas of p53 protein had no detectable p-Chk1 (Ser317) expression, whereas tumors that expressed high levels and large areas of p-Chk1 (Ser317) protein had no detectable p53 expression. Squamous cell carcinomas that demonstrated heterogeneous p53 and p-Chk1 (Ser317) expression within the same tumor showed that the areas that expressed p53 were negative for p-Chk1 (Ser317) and the areas that expressed p-Chk1 (Ser317) were negative for p53. Similar patterns were observed for keratoacanthomas. Not only was the inverse relationship demonstrated in heterogeneous tumors that showed clusters of positive and negative cells, it was also demonstrated in heterogeneous tumors that showed mixtures of single cells either positive or negative for p53. These findings were consistent for the epidermis away from tumors that demonstrated no p-Chk1 (Ser317), but appreciable p53 protein in the basal layer. As expected, the inverse relationship could not be observed by western blotting tumors that expressed both proteins. However, tumors from p53 KO mice had elevated levels of p-Chk1 (Ser317) compared with tumors from p53 wild-type SKH-1 controls. A similar pattern was observed in normal epidermis from p53 KO mice after a single exposure to UVB, whereas p53 WT littermates induced p-Chk1 (Ser317), but to a much lesser extent. Moreover, we demonstrated that UVB-induced p-Chk1 (Ser317) was inhibited by caffeine in p53 KO epidermal cells in vitro. To our knowledge, this striking phenotypic negative association between two proteins has never been observed in skin tumors. These data illustrate the dynamic regulation of checkpoint function, suggesting that phosphorylation of Chk1 on Serine 317 is regulated by p53 status and that p53 may act as a molecular on and off switch for the phosphorylation of Chk1 on Serine 317. Citation Format: Jamie J. Bernard, You-Rong Lou, Sarah Peng, Tao Li, Paul Nghiem, Allan H. Conney, Yao-Ping Lu. Inverse relationship between p53 and phospho-Chk1 (Ser317) protein expression in UVB-induced skin tumors in SKH-1 mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 569. doi:10.1158/1538-7445.AM2013-569