Project description:To investigate the transcriptional remodelling during EMT, we transfected normal murine mammary gland epithelial cells during a 4d TGFbeta treatment individually with siRNA against 46 transcription (co)factors or with 13 mature miRNA, all factors that blocked EMT in a phenotypic microscopy-based EMT screen upon RNAi . As a control, cells were transfected with siRNA/miRNA control followed by 4d TGFbeta treatment (mesenchymal control) or were left untreated (epithelial control). miRNA-sequencing together with mRNA-sequencing of these EMT perturbations in combination with transcription factor binding and miRNA target prediction enabled us to build an interaction map between these EMT factors.
Project description:To investigate the transcriptional remodelling during EMT, we transfected normal murine mammary gland epithelial cells during a 4d TGFbeta treatment individually with siRNA against 46 transcription (co)factors or with 13 mature miRNA, all factors that blocked EMT in a phenotypic microscopy-based EMT screen upon RNAi . As a control, cells were transfected with siRNA/miRNA control followed by 4d TGFbeta treatment (mesenchymal control) or were left untreated (epithelial control). miRNA-sequencing together with mRNA-sequencing of these EMT perturbations in combination with transcription factor binding and miRNA target prediction enabled us to build an interaction map between these EMT factors.
Project description:To investigate the transcriptional remodelling during EMT, we transfected normal murine mammary gland epithelial cells during a 4d TGFbeta treatment individually with siRNA against 46 transcription (co)factors or with 13 mature miRNA, all factors that blocked EMT in a phenotypic microscopy-based EMT screen upon RNAi . As a control, cells were transfected with siRNA/miRNA control followed by 4d TGFbeta treatment (mesenchymal control) or were left untreated (epithelial control). miRNA-sequencing together with mRNA-sequencing of these EMT perturbations in combination with transcription factor binding and miRNA target prediction enabled us to build an interaction map between these EMT factors.
Project description:Painting a holistic picture of disease etiology will require longitudinal systems-scale reconstruction of the multitiered architecture of eukaryotic signaling. As opposed to ‘one omic at a time’, which provides an incomplete view on disease mechanisms, here we developed an experimental and analytics framework, PAMAF, to simultaneously acquire and analyze twelve omic modalities from the same set of samples, i.e., protein abundance from whole-cells, nucleus, exosomes, secretome and membrane; peptidome; N-glycosylation, phosphorylation; metabolites; mRNA, miRNA; and, in parallel, single-cell transcriptomes. We applied PAMAF in a well-studied in vitro model of TGFβ-induced EMT to generate the EMT-ExMap dataset, cataloguing >61,000 expression profiles (>10,000 differential) over 12 days. PAMAF revealed that EMT is more complex than currently understood and identified numerous stage-specific mechanisms and vulnerabilities not captured in literature. Broad application of PAMAF will provide unprecedented insights into multifaceted biological processes relevant to human health and disease.
Project description:<p>The involvement of membrane-bound solute carriers (SLCs) in neoplastic transdifferentiation processes is poorly defined. Here, we examined changes in the SLC landscape during epithelial-mesenchymal transition (EMT) of pancreatic cancer cells. We show that two SLCs from the organic anion/cation transporter family, SLC22A10 and SLC22A15, favor EMT via interferon (IFN) α and γ signaling activation of receptor tyrosine kinase-like orphan receptor 1 (ROR1) expression. In addition, SLC22A10 and SLC22A15 allow tumor cell accumulation of glutathione to support EMT via the IFNα/γ-ROR1 axis. Moreover, a pan-SLC22A inhibitor lesinurad reduces EMT-induced metastasis and gemcitabine chemoresistance to prolong survival in mouse models of pancreatic cancer, thus identifying new vulnerabilities for human PDAC.</p>
Project description:We combine state-of-the-art data acquisition platforms and bioinformatics tools to devise PAMAF, a workflow that simultaneously examines twelve omics modalities, i.e., protein abundance from whole-cells, nucleus, exosomes, secretome and membrane; N-glycosylation, phosphorylation; metabolites; mRNA, miRNA; and, in parallel, single-cell transcriptomes. Here we apply PAMAF in an established in vitro model of TGFβ-induced epithelial to mesenchymal transition (EMT) to quantify >61,000 molecules from 12 omics and 10 timepoints over 12 days. Bioinformatics analysis of this EMT-ExMap resource allowed us to identify; –unexpected topological coupling between omics, –four distinct cell states during EMT (E, E/M-1, E/M-2, M), –omics-specific kinetic paths, –stage-specific multi-omics characteristics, –distinct regulatory classes of genes, –ligand–receptor mediated intercellular crosstalk using an innovative pipeline integrating scRNAseq and subcellular proteomics, and –novel combinatorial drug targets (e.g., Hedgehog signaling and CAMK-II) to inhibit EMT, which we validate using a 3D mammary duct-on-a-chip platform. Overall, while this study provides an unprecedented resource on TGFβ signaling and EMT, PAMAF-like workflows can be applied to generate comprehensive molecular landscapes of other multifaceted biological processes.