Oncogenic H‐Ras Reprograms Madin‐Darby Canine Kidney (MDCK) Cell‐Derived Midbody Remnant Proteome Following Epithelial‐Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a fundamental, dynamic cellular process involved in embryonic development, metastasis, organ fibrosis, and tissue regeneration. To define the molecular landscape of secreted midbody remnants (MBRs) to the EMT process, a proteome analysis of MBRs released from Madin-Darby canine kidney (MDCK) cells and following oncogenic H-Ras transformation (21D1 cells) was performed. MBRs, a new class of membranous extracellular vesicle (EV) molecularly distinct from exosomes/small EVs, were purified using sequential centrifugation/buoyant density gradient centrifugation. Proteomic profiling revealed MDCK cell-MBRs reflect their epithelial origin (e.g., enriched CDH1, DSP, THBS1, OLCN, EPCAM proteins) and 21D1 cell-MBRs their oncogenic and mesenchymal phenotype (e.g., HRAS, VIM, MMP14, CDH2, WNT5A, and enriched invasive and cell motility protein networks). Validation of proteome cargo revealed key protein networks associated with the EMT process in MBRs, and conserved MBR proteome across different cell types. Prominent findings were the unique expression of the immune checkpoint protein NT5E/CD73 (ecto-5'-nucleotidase) and ser/thr kinases LIMK1/K2 in MBRs from mesenchymal cells following their oncogenic transformation, and enrichment in Wnt signaling network proteins. These data identify the core proteome of MBRs regulated during the dynamic process of EMT and cell transformation over other EV types in context of the EMT process. SUMMARY: Epithelial-to-mesenchymal transition (EMT) is a critical cell biological process that occurs during embryonic development and cancer progression. Our study describes sequential purification of secreted midbody remnants (MBRs) and exosomes/sEVs from the in vitro cell line EMT model Madin-Darby canine kidney (MDCK) cells and MDCK cells transformed with oncogenic H-Ras (21D1 cells): Proteomics identified the repertoire of enriched MDCK-MBR proteins following EMT. MBRs display a proteome profile distinct from sEVs that is enriched with factors of the centralspindlin complex (KIF23.1, KIF4A, INCENP, CEP55, PLK1) and further includes components of the mitochondrial network, cytokinesis, microtubule movement, and intercellular connection. In the context of EMT, our data reveal enriched EMT pathways in MBRs including signaling receptor binding, regulation of cell differentiation, and Wnt, VEGF, and PDGF signaling. We have validated these findings in the context of Wnt signaling in other EV types. We identify several mesenchymal-enriched networks in MBRs associated with focal adhesion, cell matrix, kinase activity, and cell shape/organization, while epithelial-derived MBRs show enriched networks predominantly associated with mitochondrial (processing/transport), midbody, and plasma membrane annotation. Our study sheds light on the proteome architecture of MBRs following oncogenic H-Ras-induced EMT in cell transformation: collectively, our data informs ongoing efforts to delineate oncogenic drivers of cancer initiation, progression, and metastasis.