CONICET | Buscador de Institutos y Recursos Humanos

The Ubiquitin System is a critical regulator of VMP1-dependent autophagy in human pancreatic cancer cells.Felipe J. Renna, Mariana Tadic, Tamara Orquera, Carolina Vecino, Juliana Enriqué Steinberg, Mario Rossi, Alejandro Ropolo, Maria I. VaccaroUniversidad de Buenos Aires, CONICET, Instituto de Bioquímica y Medicina Molecular (IBIMOL). Universidad Austral, CONICET, Instituto de Investigaciones en Medicina Traslacional (IIMT).Autophagy is a tightly regulated catabolic process involved in the degradation and recycling of proteins and organelles. This process has been linked to the resistance of pancreatic cancer stem cells to anticancer drugs. Ubiquitination plays an important role in the regulation of autophagy. VMP1 is an essential autophagy protein whose expression in pancreatic cancer stem cells, carrying activated KRAS, enables therapeutic resistance. Using biochemical and cellular approaches we investigated the role of VMP1 ubiquitination in the regulation of autophagy in pancreatic cancer stem cells PANC1 and MIA PaCa-2. In silico analysis of VMP1 structure revealed two high-confidence ubiquitination sites. Co-immunoprecipitation of VMP1-Ub from cells concomitantly transfected with VMP1-EGFP and Ub-Flag expression plasmids, as well as co-distribution of VMP1 and ubiquitin in LC3-labeled autophagosomes, confirmed VMP1 ubiquitination during autophagy. In order to understand whether ubiquitination marks VMP1 for degradation, we inhibited the proteasome using MG132 and the lysosome with Chloroquine. We found that VMP1 levels were not affected after proteasome inhibition nor after lysosome inactivation, suggesting that VMP1 is neither degraded by the ubiquitin-proteasome system nor is it a cargo of autophagy. To investigate whether VMP1 ubiquitination regulates its role in autophagy, we performed distribution analyses for VMP1 and markers of autophagic structures. We found significant colocalization between VMP1 and ubiquitin in ATG13- and LC3-labeled omegasomes and autophagosomes, as well as in LAMP1-labeled autolysosomes. Finally, we developed VMP1-K404R and VMP1-K406R mutants lacking ubiquitination sites and found that these mutants are unable to induce recruitment of ATG13, LC3, or LAMP1 and complete the autophagic flux. Our results indicate that VMP1 ubiquitination is required for the regulation of the autophagic pathway in pancreatic cancer cells. We conclude that ubiquitination is a regulatory mechanism in VMP1-induced autophagy and suggest a possible clinical relevance of modulating VMP1 ubiquitination among therapeutic strategies in pancreatic cancer.