Systems-level Analysis Reveals Multiple Modulators of Epithelial-mesenchymal Transition and Identifies DNAJB4 and CD81 as Novel Metastasis Inducers in Breast Cancer

Abstract

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.

Proteome Landscape of Epithelial-to-Mesenchymal Transition (EMT) of Retinal Pigment Epithelium Shares Commonalities With Malignancy-Associated EMT

“…Megenta points, which are malignancy associated EMT factors (Fig. 4A) that are altered by SNAI1/TWIST1 overexpression (51)(52)(53)(54), were also identified as being altered following the induction of RPE-EMT.…”

AF10 (MLLT10) prevents somatic cell reprogramming through regulation of DOT1L-mediated H3K79 methylation

“…Reaction was quenched with acidification and the resulting peptides were desalted [ 37 ] and then analyzed with reversed-phase nLC (NanoLC-II, Thermo Scientific) combined with orbitrap mass spectrometer (Q Exactive Orbitrap, Thermo Scientific). The raw files were processed with Proteome Discoverer 1.4 (Thermo Scientific) using human Uniprot database (Release 2015–21,039 entries) as previously described [ 36 , 38 ]. Two technical replicates were performed for each sample.…”

Molecular Chaperones in Cancer Stem Cells: Determinants of Stemness and Potential Targets for Antitumor Therapy

“…The recent proteomic investigations identified another member of the HSP40 subfamily, DnaJB4, as a new driver of EMT in breast cancer [132]. The same researchers showed that the suppression of DnaJB4 in breast cancer cells with a mesenchymal phenotype impaired their migration capacity in vitro and reduced both primary tumor growth and lung metastasis occurrence in vivo.…”