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Autophagy induced by p53-reactivating molecules protects pancreatic cancer cells from apoptosis

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Abstract

TP53 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. Here, we show that two of these compounds, CP-31398 and RITA, induce cell growth inhibition, apoptosis, and autophagy by activating p53/DNA binding and p53 phosphorylation (Ser15), without affecting the total p53 amount. These effects occur in both wild-type and mutant p53 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 on 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 both 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, supporting the development of an anti-cancer therapy based on autophagy inhibition associated to p53 activation.

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Acknowledgments

We thank Dr. Silvia di Agostino (Regina Elena Cancer Institute, Rome, Italy) who kindly provided us with the p53-null non-small cell lung cancer cell line. This work was supported by Associazione Italiana Ricerca Cancro (AIRC), Milan, Italy; Fondazione CariPaRo, Padova, Italy; Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR), Rome, Italy; Progetti di Ricerca di Interesse Nazionale (PRIN, MIUR), Rome, Italy.

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Life and Reproduction Sciences, Section of Biochemistry, University of Verona, Strada Le Grazie 8, 37134, Verona, Italy

    Claudia Fiorini, Marta Menegazzi, Chiara Padroni, Ilaria Dando, Elisa Dalla Pozza, Alex Gregorelli, Chiara Costanzo, Marta Palmieri & Massimo Donadelli

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Correspondence to Massimo Donadelli.

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Supplementary Fig. 1

Effect of CP-31398 or RITA on p53-null cancer cells (H1299). a H1299 cells were seeded in 100-mm diameter culture dishes, incubated overnight, and treated with 40 μM CP-31398 or RITA for 16 h. Nuclear extracts were used to perform electrophoretic mobility shift assay (EMSA), as described in “Materials and methods” section. Nuclear extracts of Panc1 cells treated with 40 μM CP-31398 for 16 h were used as positive control. b H1299 cells were seeded in 96-well plates, incubated overnight, and treated with increasing concentrations of CP-31398 or RITA for 24 h. Cell proliferation was determined using the Crystal Violet colorimetric assay. Values are the means (±SD) of three independent experiments each performed in triplicate. c H1299 cells were seeded in 96-well plates, incubated overnight, and treated with increasing concentrations of CP-31398 or RITA for 24 h. Apoptosis was analyzed using the annexinV binding assay. Values are the means (±SD) of three independent experiments each performed in triplicate. d H1299 cells were seeded in 96-well plates, incubated overnight, and treated with increasing concentrations of CP-31398 or RITA for 24 h. Autophagosomes formation assay was analyzed using the incorporation of monodansylcadaverine (MDC) probe. Values are the means (±SD) of three independent experiments each performed in triplicate. Supplementary material 1 (TIFF 127 kb)

Supplementary Fig. 2

Effect of CP-31398 or RITA on p53 phosphorylation (Ser15) and p53 expression. Normal fibroblasts were seeded in 100-mm diameter culture dishes, incubated overnight, and treated with 40 μM CP-31398 or RITA for 24 h. Whole-cell extracts were used for Western blot analysis Supplementary material 2 (TIFF 83 kb)

Supplementary Fig. 3

Effect of Compound C on AMPK/mTOR axis. a Cells were seeded in 100-mm diameter culture dishes, incubated overnight, and treated with 20 μM Compound C for 24 h. Whole-cell extracts were used for western blot analyses. b and c Quantitative analyses of P-AMPK/P-p70S6K and AMPK/p70S6K ratios. The bands were scanned as digital peaks and the areas of the peaks were calculated in arbitrary units, as described in “Materials and methods” section. The value of Ponceau S dye was used as a normalizing factor. Values are the means of three independent experiments (±SD). Statistical analysis: (*) p < 0.05 Compound C versus CTRL Supplementary material 3 (TIFF 137 kb)

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Fiorini, C., Menegazzi, M., Padroni, C. et al. Autophagy induced by p53-reactivating molecules protects pancreatic cancer cells from apoptosis. Apoptosis 18, 337–346 (2013). https://doi.org/10.1007/s10495-012-0790-6

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