Epithelial-mesenchymal transition (EMT) is driven by complex signaling events that induce dramatic biochemical and morphological changes whereby epithelial cells are converted into cancer cells. However, the underlying molecular mechanisms remain elusive. Here, we used mass spectrometry based quantitative proteomics approach to systematically analyze the post-translational biochemical changes that drive differentiation of human mammary epithelial (HMLE) cells into mesenchymal. We identified 314 proteins out of more than 6,000 unique proteins and 871 phosphopeptides out of more than 7,000 unique phosphopeptides as differentially regulated. We found that phosphoproteome is more unstable and prone to changes during EMT compared to the proteome and multiple alterations at proteome level are not thoroughly represented by transcriptional data highlighting the necessity of proteome level analysis. We discovered cell state specific signaling pathways, such as Hippo, sphingolipid signaling, and unfolded protein response (UPR) by modeling the networks of regulated proteins and potential kinase-substrate groups. We identified two novel factors for EMT whose expression increased upon EMT induction: DnaJ heat shock protein family (Hsp40) member B4 (DNAJB4) and cluster of differentiation 81 (CD81). Suppression of DNAJB4 or CD81 in mesenchymal breast cancer cells resulted in decreased cell migration in vitro and led to reduced primary tumor growth, extravasation, and lung metastasis in vivo. Overall, we performed the global proteomic and phosphoproteomic analyses of EMT, identified and validated new mRNA and/or protein level modulators of EMT. This work also provides a unique platform and resource for future studies focusing on metastasis and drug resistance.

CLIC members are required for the progression of cytokinesis by coupling the plasma membrane and cortical actin network at the cleavage furrow and polar cortex.

Cell migration requires spatiotemporally coordinated activities of multicomponent structures including the actomyosin cortex, plasma membrane, adhesion complexes and the polarity proteins. How they function together to drive this complex dynamic process remains an outstanding question. Here, we show that a member of the protocadherin family, PCDH7 displays a polarized localization in migratory cells with a dynamic enrichment at the leading and rear edges. Perturbation of PCDH7 interferes with the migration of nontransformed retinal pigment epithelial cells and invasion of cancer cells. The overexpression of PCDH7 enhances the migration capability of cortical neurons in vivo. PCDH7 interacts with the myosin phosphatase subunits MYPT1 and PP1cβ and it enhances the phosphorylation of regulatory light chain and ERM at the leading and rear edges of migratory cells. The chemical inhibition of phosphatase activity recovers migration phenotypes of PCDH7 knockout cells. We propose that PCDH7 regulate phosphorylation thus activity of myosin and ERM at the polarized cortex by quenching myosin phosphatase that results in a higher persistence of migrating cells. Collectively, our study suggests a new mechanism for the spatial coordination of plasma membrane and the cortex during cell migration.