iProXProteomicsLibing SongHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0004710000songlb@sysucc.org.cnSun Yat-sen University Cancer CenteriProXHuman epidermal growth factor receptor 2-targeted (HER2-targeted) therapy is the mainstay of treatment for HER2+ breast cancer. However, the proteolytic cleavage of HER2, or HER2 shedding, induces the release of the target epitope at the ectodomain (ECD) and the generation of a constitutively active intracellular fragment (p95HER2), impeding the effectiveness of anti-HER2 therapy. Therefore, identifying key regulators in HER2 shedding might provide promising targetable vulnerabilities against resistance. In the current study, we found that upregulation of dolichyl-phosphate N-acetylglucosaminyltransferase (DPAGT1) sustained high-level HER2 shedding to confer trastuzumab resistance, which was associated with poor clinical outcomes. Upon trastuzumab treatment, the membrane-bound DPAGT1 protein was endocytosed via the caveolae pathway and retrogradely transported to the ER, where DPAGT1 induced N-glycosylation of the sheddase - ADAM metallopeptidase domain 10 (ADAM10) - to ensure its expression, maturation, and activation. N-glycosylation of ADAM10 at N267 protected itself from ER-associated protein degradation and was essential for DPAGT1-mediated HER2 shedding and trastuzumab resistance. Importantly, inhibition of DPAGT1 with tunicamycin acted synergistically with trastuzumab treatment to block HER2 signaling and reverse resistance. These findings reveal a prominent mechanism for HER2 shedding and suggest that targeting DPAGT1 might be a promising strategy against trastuzumab-resistant breast cancer.Inhibition of DPAGT1 suppresses HER2 shedding and trastuzumab resistance in human breast cancer.Yang Muwen M, Li Yue Y, Kong Lingzhi L, Huang Shumei S, He Lixin L, Liu Pian P, Mo Shuang S, Lu Xiuqing X, Lin Xi X, Xiao Yunyun Y, Shi Dongni D, Huang Xinjian X, Chen Boyu B, Chen Xiangfu X, Ouyang Ying Y, Li Jun J, Lin Chuyong C, Song Libing LfalseIdentification of ADAM10 interactors and N-glycosylation sites in SK-BR-3 cellsImmunoprecipitation of ADAM10-WT and ADAM10-4NQ interacting proteins were identified by mass spectrometry(MS));purified ADAM10 protein was subjected to Coomassie Blue Staining and LC-MS/MS in order to identify the N-glycosylation sitesSat Jul 16 00:00:00 BST 2022PXD035378960637463446