Concurrent depletion of Vps37 proteins evokes inflammatory response and cell growth inhibition via ESCRT-I destabilization.
Ample evidence points to the contribution of endocytosis to oncogenesis at the cellular level but molecular details still remain obscure. Our analysis of The Cancer Genome Atlas data involving cohorts of colorectal cancer (CRC) patients revealed stage-dependent alterations in the expression of 113 genes whose function is related to endocytic transport. Among differentially expressed genes we observed decreased transcription of VPS37B and to a lesser extent VPS37A in the advanced stages of CRC. One member of the Vps37 protein family (Vps37A, Vps37B, Vps37C, Vps37D) together with Tsg101 and Vps28 forms the core of the Endosomal Sorting Complex Required for Transport (ESCRT)-I mediating membrane-remodeling processes. Our analysis of an independent cohort of CRC patients corroborated that both VPS37B and VPS37A genes are downregulated at mRNA and protein levels. Depletion of both Vps37A and Vps37B proteins elicits multifaceted transcriptional alterations, including cell growth arrest and induction of sterile inflammatory response, which are further potentiated by co-silencing of VPS37C. Among analyzed silencing condition, concurrent knockdown of VPS37A, VPS37B and VPS37C evokes the greatest magnitude of p21-mediated inhibition of cell proliferation and activation of Nuclear Factor-?B and mitogen-activated protein kinases signaling. These processes are independently induced after co-silencing of VPS37 paralogs. We further show that the type and magnitude of transcriptional responses correlate with the ESCRT-I stability, which is differentially affected upon individual and concurrent Vps37 depletion. Our study provides novel insights into cancer cell biology by describing a cellular stress response that is associated with ESCRT-I destabilization, which might occur in CRC patients Overall design: The aim of the study was to analyze transcriptional changes upon single, double and triple depletion of VPS37 paralogs (VPS37A, VPS37B, VPS37C) in human colorectal DLD1 cell line. For RNA-seq analysis 3 biological replicates were prepared, each encompassing following samples: 1) non-transfected DLD1 (NT), 2) DLD1 transfected with a combination of non -targeting siRNA (siCTRL#1), 3) DLD1 transfected with siRNA targeting VPS37A, sequence #1 (siVPS37A#1), 4) DLD1 transfected with siRNA targeting VPS37B, sequence #1 (siVPS37B#1), 5) DLD1 transfected with siRNA targeting VPS37C, sequence #1 (siVPS37C#1), 6) DLD1 transfected with a combination of siRNAs targeting VPS37A, sequence #1 and VPS37B, sequence #1 (referred thereafter as siVPS37AB#1), 7) DLD1 transfected with a combination of siRNAs targeting VPS37A, sequence #1 and VPS37C, sequence #1 (referred thereafter as siVPS37AC#1), 8) DLD1 transfected with a combination of siRNAs targeting VPS37B, sequence #1 and VPS37C, sequence #1 (referred thereafter as siVPS37BC#1), 9) DLD1 transfected with a combination of siRNAs targeting VPS37A, sequence #1, VPS37B, sequence #1 and VPS37C, sequence #1 (referred thereafter as siVPS37ABC#1). Non-transfected and siCTRL#1-transfected cells served as reference samples.