“Molecular mechanisms induced by p53 reactivating molecules in p53 mutant pancreatic adenocarcinoma cell lines”

TP53 gene mutations compromising p53 transcriptional function occur in more than 50% of human cancers, including pancreatic adenocarcinoma, and render cancer cells more resistant to conventional therapy. In the last few years, many efforts have been addressed to identify p53-reactivating molecules able to restore the wild-type transcriptionally competent conformation of the mutated proteins. In the present thesis, we show that two of these compounds, CP-31398 and RITA, can induce cell growth inhibition, apoptosis, and autophagy by activating p53/DNA binding and p53 phosphorylation (Ser15), without affecting the total amount of p53. These effects occur in both wild-type and mutant p53 (mutp53) pancreatic adenocarcinoma cell lines, whereas they are much less pronounced in normal human primary fibroblasts. Furthermore, CP-31398 and RITA regulate the axis SESN1- 2/AMPK/mTOR by inducing AMPK phosphorylation in Thr172, which has a crucial role in the autophagic response. The protective role of autophagy in cell growth inhibition by CP-31398 and RITA is supported by the finding that the AMPK inhibitor compound C or the autophagy inhibitors chloroquine or 3-methyladenine sensitize pancreatic adenocarcinoma cell lines to the apoptotic response induced by p53-reactivating molecules. Our results demonstrate for the first time a survival role for autophagy induced by p53 reactivating molecules in p53 mutant cancer cells. Mutp53 proteins not only lose their tumor suppressive function but also gain new oncogenic properties known as “gain-of-function” (GOF). Generally, mutp53 proteins are over-expressed in cancer cells and promote GOF activities enhancing the proliferation of cells and their resistance to a variety of chemotherapeutic drugs commonly used in the clinical practice. GOF activities are carried out because mutp53 proteins behave as oncogenic transcription factors by interacting with other transcriptional regulators, such as E2F1, NF-Y and VDR. Moreover, these p53 mutant proteins can also interact with oncosuppressor proteins inhibiting their function. Here, we show that the standard drug gemcitabine (GEM) strongly activates mutp53 by stimulating both its phosphorylation (Ser15) and nuclear translocation. These events result in the stimulation of mutp53 GOF in cancer cells bearing mutant p53, as revealed by the stimulation of cell cycle promoting genes, as Cdk1 and CCNB1, after GEM treatment. Furthermore, we demonstrate that silencing of mutp53 strongly increases sensitivity of cancer cells to GEM and that the addition of CP-31398 or RITA to GEM treatment can synergistically induce apoptotic cell death in both wt and mutant p53 pancreatic adenocarcinoma cell lines, whereas these effects are missing in p53-null cancer cells. This drug combination strongly induces p53 phosphorylation in Ser15 (without affecting the total amount of p53), apoptosis, and autophagosome formation. Furthermore, we demonstrate that autophagy stimulation by GEM/CP-31398 has a protective role for cancer cells. In fact, the addition of the autophagy inhibitors, chloroquine or 3-methyladenine, increases apoptosis induced by GEM/CP-31398 treatment. Our results support the development of an anti-tumoral strategy based on autophagy inhibition associated to the combined treatment of p53-reactivating molecules with standard chemotherapy, for both wild-type and mutant p53 pancreatic adenocarcinoma cell types.