Linking junctional integrity and cell polarity to TGF-beta function in the normal mammary gland

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Transforming growth factor beta (TGF) mediates epithelial homeostasis via a number of context-dependent cellular effects, including cell death/apoptosis, growth arrest, survival, and motility. For instance, TGF mediates mammary gland morphogenesis as a growth inhibitor, and post-lactation involution as an apoptosis inducer, among other effects. TGF signals via canonical (Smad) and non-canonical pathways (e.g. Par6 and PI3K/Akt), both of which facilitate one of the best-documented cellular responses to TGF: the epithelial-mesenchymal transition (EMT). A key feature of EMT is the loss of cell-cell junctions (tight and adherens junctions) and apical-basal polarity. We found that in normal mammary cells undergoing EMT in response to TGF, apoptosis occurs in parallel to the disruption of tight junctions and apical-basal polarity, and an enhanced release of extracellular vesicles. Par6 mediates these processes in association with modulation of PI3K/Akt/FoxO signalling, as well as other signalling pathways. We hypothesize that TGF's function in the normal mammary gland relies on its capacity to modulate junctional integrity, and an interconnected cell polarity-cell survival network. To validate this, we propose 3 objectives: 1) demonstrate the existence of a Par6-PI3K/Akt-FoxO signalling axis and its role in TGF-induced apoptosis, 2) identify additional signalling mediators of the Par6/TJ-cell survival network, 3) address the role of extracellular vesicles in the link between apoptosis and EMT.Research will be conducted on normal mammary epithelial cells, cultured as monolayers or as three-dimensional acini-like structures, which establish proper cell-cell junctions and apical-basal polarity. Variants of these cells, which are susceptible or not to junctional disruption (e.g., cells with overactive or blocked Par6 signalling), will be treated plus or minus TGF, signalling inhibitors and/or silencing RNA. Outcomes will be analyzed by immunoprecipitation, immunoblotting, immunofluorescence imaging, reporter assays and qRT-PCR, to verify protein-protein interactions, activation status of signalling pathways, the effect of treatments on protein levels and sub-cellular localization, and modulation of target genes of interest. Objective 2 will employ a siRNA-based high-throughput screen which, upon dual assessment of TJ loss and apoptosis via immunofluorescence, will identify signalling mediators of survival in association with TJ integrity. Objective 3 will involve isolation, purification, proteomics, and functions characterization of extracellular vesicles released by cells treated under the above-described conditions.This research will provide the first evidence that, by linking normal tissue architecture to cellular survival, cell-cell junctions plays an active role in TGF-dependent mammary gland homeostasis.