The Ubiquitin System is a critical regulator of VMP1-dependent autophagy in human pancreatic cancer cells

TAMARA ORQUERA; MARIANA TADIC; FELIPE JAVIER RENNA; CAROLINA VECINO

Berna

Congreso; Swiss Apoptosis & Autophagy Meeting (SA2M) 2021; 2021

Life Sciences Switzerland Autophagy

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, Maria Manifava, Mario Rossi, Alejandro Ropolo, Nicholas T. Ktistakis, Maria I. VaccaroUniversidad de Buenos Aires, CONICET, Instituto de Bioquímica y Medicina Molecular (IBIMOL). Universidad Austral, CONICET, Instituto de Investigaciones en Medicina Traslacional (IIMT). Signalling Programme, The Babraham Institute.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 the cell lines HEK 293T, HeLa and PANC1. 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 know 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 highly significant colocalization between VMP1 and ubiquitin in ATG13-labeled omegasomes and in LC3-labeled autophagosomes, as well as in LAMP1-marked autolysosomes. In Atg5 -/- MEF cells we found VMP1 distributed in dots which colocalizes with ubiquitin but not with LC3, suggesting that VMP1 ubiquitination occurs upstream to LC3 conjugation. Moreover, in some Atg5 wt cells we found big membrane vesicles with VMP1 signal that do not colocalize with ubiquitin nor LC3 indicating VMP1 is ubiquitinated when it is part of the autophagy structures. Finally, by proteomic analysis and co-immunoprecipitation assay we found the subunit Cdt2 of the E3 ligase CRL4/Cdt2 as an interactor of VMP1, suggesting VMP1 ubiquitination reaction is catalyzed by this enzymatic complex. Our results suggest 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.