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

RENNA F; VACCARO MI

Miami

Congreso; American Pancreatic Association Meeting 2021; 2021

American Pancreatic Association

Autophagy is a tightlyregulated catabolic process involved in the degradation and recycling ofproteins and organelles. This process has been linked to the resistance ofpancreatic cancer stem cells to anticancer drugs. Ubiquitination plays animportant role in the regulation of autophagy. VMP1 is an essential autophagy proteinwhose expression in pancreatic cancer stem cells, carrying activated KRAS,enables therapeutic resistance. Using biochemical and cellular approaches weinvestigated the role of VMP1 ubiquitination in the regulation of autophagy inpancreatic cancer stem cells PANC1 and MIA PaCa-2. In silico analysis of VMP1structure revealed two high-confidence ubiquitination sites.Co-immunoprecipitation of VMP1-Ub from cells concomitantly transfected withVMP1-EGFP and Ub-Flag expression plasmids, as well as co-distribution of VMP1and ubiquitin in LC3-labeled autophagosomes, confirmed VMP1 ubiquitinationduring autophagy. In order to understand whether ubiquitination marks VMP1 fordegradation, we inhibited the proteasome using MG132 and the lysosome withChloroquine. We found that VMP1 levels were not affected after proteasomeinhibition nor after lysosome inactivation, suggesting that VMP1 is neitherdegraded by the ubiquitin-proteasome system nor is it a cargo of autophagy. Toinvestigate whether VMP1 ubiquitination regulates its role in autophagy, weperformed distribution analyses for VMP1 and markers of autophagic structures.We found significant colocalization between VMP1 and ubiquitin in ATG13- andLC3-labeled omegasomes and autophagosomes, as well as in LAMP1-labeledautolysosomes. Finally, we developed VMP1-K404R and VMP1-K406R mutants lackingubiquitination sites and found that these mutants are unable to inducerecruitment of ATG13, LC3, or LAMP1 and complete the autophagic flux. Ourresults indicate that VMP1 ubiquitination is required for the regulation of theautophagic pathway in pancreatic cancer cells. We conclude that ubiquitinationis a regulatory mechanism in VMP1-induced autophagy and suggest a possibleclinical relevance of modulating VMP1 ubiquitination among therapeuticstrategies in pancreatic cancer. @font-face{font-family:"Cambria Math";panose-1:2 4 5 3 5 4 6 3 2 4;mso-font-charset:0;mso-generic-font-family:roman;mso-font-pitch:variable;mso-font-signature:3 0 0 0 1 0;}@font-face{font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;mso-font-charset:0;mso-generic-font-family:swiss;mso-font-pitch:variable;mso-font-signature:-536859905 -1073732485 9 0 511 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal{mso-style-unhide:no;mso-style-qformat:yes;mso-style-parent:"";margin:0cm;line-height:115%;mso-pagination:widow-orphan;font-size:11.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:Arial;mso-ansi-language:#000A;mso-fareast-language:EN-US;}p.MsoTitle, li.MsoTitle, div.MsoTitle{mso-style-priority:10;mso-style-unhide:no;mso-style-qformat:yes;mso-style-link:"Título Car";mso-style-next:Normal;margin-top:0cm;margin-right:0cm;margin-bottom:3.0pt;margin-left:0cm;line-height:115%;mso-pagination:widow-orphan lines-together;page-break-after:avoid;font-size:26.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:Arial;mso-ansi-language:#000A;mso-fareast-language:EN-US;}span.TtuloCar{mso-style-name:"Título Car";mso-style-priority:10;mso-style-unhide:no;mso-style-locked:yes;mso-style-link:Título;mso-ansi-font-size:26.0pt;mso-bidi-font-size:26.0pt;}.MsoChpDefault{mso-style-type:export-only;mso-default-props:yes;font-size:11.0pt;mso-ansi-font-size:11.0pt;mso-bidi-font-size:11.0pt;font-family:"Arial",sans-serif;mso-ascii-font-family:Arial;mso-fareast-font-family:Arial;mso-hansi-font-family:Arial;mso-bidi-font-family:Arial;mso-ansi-language:#000A;mso-fareast-language:EN-US;}.MsoPapDefault{mso-style-type:export-only;line-height:115%;}div.WordSection1{page:WordSection1;}