Abstract B54: Identification of the PI3K‐mTOR pathway as regulator of mutant p53 turnover in human cancer cells

The tumor suppressor p53 plays a crucial role in our cells to induce cell cycle arrest or apoptosis in response to different kinds of genotoxic stresses. In 50% of all solid tumors, p53 is mutated by a single point mutation within the DNA‐binding domain and thereby loses its activity as a tumor suppressor. However, at the same time, mutant p53 actively contributes to chemoresistance and tumor progression through a gain of function (Dittmer et al., 1993). It was previously shown that mutant p53 highly accumulates in tumors of mice with targeted germ line mutations of the p53 gene (knock‐in), whereas normal cells of these mice express it at low levels (Lang et al., 2004; Terzian et al., 2008). This argues that the deregulation of tumor‐specific signaling cascades leads to an accumulation of mutant p53 and subsequently to tumor progression. Our study aims at identifying these signaling pathways that contribute to the regulation of mutant p53 expression, and to describe their mechanisms. We performed quantitative analysis of mutant p53 protein levels in a cancer cell line after the individual knock‐down of all known human kinases using automated microscopy (high content cell screening). The bladder carcinoma derived cell line 5637 (carrying the p53 R280T mutation) was transfected with three different synthetic siRNAs targeting each known kinase or kinase regulator in a 96‐well format, and p53 was detected by automated immunofluorescence microscopy. In each of the resulting 2400 cell populations the p53 expression levels were determined for at least 500 individual cells. Alterations in the distribution of p53 staining intensities were assessed for each kinase relative to non‐targeting control siRNAs. The results of the kinases, that lead to the strongest up‐ or down‐regulations of mutant p53 in the screen, were confirmed in different cell lines (U251 and U373, both derived from glioma, with the p53 hot spot mutation R273H), by immunofluorescence and immunoblot analysis. We identified a group of kinases currently known to act in the PI3‐kinase → mTOR pathway as potential regulators of mutant p53. The knock‐down of these kinases resulted in a decrease of mutant p53 expression. Studying the mechanism of mutant p53 regulation, we performed quantitative real‐time PCR after siRNA transfection and found no significant impairment of the p53 transcription. On the contrary, posttranslational modifications (phosphorylation and acetylation) of mutant p53 were decreased after knock‐down of the identified kinases, even more strongly than the overall levels of the protein. These observations support the idea that the degradation of mutant p53 is decreased in cancer cells through alterations of PI3K‐mTOR pathway associated kinases, resulting in p53 accumulation and oncogenic gain of function. Citation Information: Cancer Res 2009;69(23 Suppl):B54.