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Background

Cancer cells are avid extracellular vesicle (EV) producers. EVs transport transforming growth factor-beta (TGF-beta), which is commonly activated under late stages of cancer progression. Nevertheless, whether TGF-beta signaling coordinates EV biogenesis is a relevant topic that remains minimally explored.

Method

We sought after specific TGF-beta pathway mediators that could regulate EV release. To this end, we used a large number of cancer cell models, coupled to EV cell biological assays, unbiased proteomic and transcriptomic screens, followed by signaling and cancer biology analyses, including drug resistance assays.

Results

We report that TGF-beta, by activating its type I receptor and MEK-ERK1/2 signaling, increased the numbers of EVs released by human cancer cells. Upon examining cholesterol as a mediator of EV biogenesis, we delineated a pathway whereby ERK1/2 acted by phosphorylating sterol regulatory element-binding protein-2 that transcriptionally induced 7-dehydrocholesterol reductase expression, thus raising cholesterol abundance at both cellular and EV levels. Notably, inhibition of MEK or cholesterol synthesis, which impaired TGF-beta-induced EV secretion, sensitized cancer cells to chemotherapeutic drugs. Furthermore, proteomic profiling of two distinct EV populations revealed that EVs secreted by TGF-beta-stimulated cells were either depleted or enriched for different sets of cargo proteins. Among these, latent-TGF-beta 1 present in the EVs was not affected by TGF-beta signaling, while TGF-beta pathway-related molecules (e.g., matrix metalloproteinases, including MMP9) were either uniquely enriched on EVs or strongly enhanced after TGF-beta stimulation. EV-associated latent-TGF-beta 1 activated SMAD signaling, even when EV uptake was blocked by heparin, indicating competent signaling capacity from target cell surface receptors. MMP inhibitor or proteinase treatment blocked EV-mediated SMAD signaling, suggesting that EVs require MMP activity to release the active TGF-beta from its latent complex, a function also linked to the EV-mediated transfer of pro-migratory potential and ability of cancer cells to survive in the presence of cytotoxic drugs.

Conclusion

Hence, we delineated a novel signaling cascade that leads to high rates of EV generation by cancer cells in response to TGF-beta, with cholesterol being a key intermediate step in this mechanism.Graphical Abstract center dot TGF-beta increases EV release by activating a MEK-ERK1/2-SREBP2-DHCR7 signaling and transcriptional pathway.center dot TGF-beta-induced DHCR7 expression raises cholesterol abundance that promotes EV release.center dot EVs carry surface latent TGF-beta and MMP9 that can activate TGF-beta receptor signaling on the surface of recipient cells.

Epithelial-to-mesenchymal transition (EMT) is a dynamic process controlling the transition of cells between epithelial and mesenchymal states in various physiological or pathological conditions. In cancer, EMT promotes cell dissemination and metastatic colonization, enriches tumors with stem cell populations and confers resistance to anticancer therapy. Instigators of EMT activate a cohort of transcription factors (EMT-TF) which regulate the expression of each other and confer dynamic chromatin modifications to transcriptionally repress epithelial and induce mesenchymal genes. In this respect, transforming growth factor-β (TGF-β) is a potent inducer of EMT in different types of cancer.

In this study we first identified a link between the EMT-TF SNAI1 that is highly expressed in aggressive triple-negative breast cancers (TNBC), with the establishment of an intermediate epithelial-mesenchymal phenotype and the dual transcriptional induction of FOXA1 and androgen receptor which define mammary epithelial cell differentiation towards the luminal subtype. Studying additional phenotypes of SNAI1 mutant TNBCs, we showed that SNAI1 through TGF-β/SMAD signaling and repression of FOXA1, induces the guanine exchange factor PSD4/EFA6B, driving a vesicular trafficking program that promotes cell-matrix interactions and invasiveness.

Tumor-derived extracellular vesicles (EV) are important mediators of intercellular communication and of microenvironment formation where tumors develop. In this study we showed that MEK/ERK signaling, drives TGF-β promoting EV secretion by regulating cholesterol homeostasis in TNBC cells. Additionally, TGF-β ligands and matrix metalloproteases identified as EV protein contents, conferred pro-invasive attributes and resistance to chemotherapeutic drugs in recipient cells.

Metabolism has a well-documented role in tumor progression and EMT maintenance and here we propose that a hybrid epithelial-mesenchymal state upon knockout of the EMT-TF SNAI2 in TNBC cells, associated with altered expression of genes involved in metabolic pathways. This perturbed cell cycle progression in the mutant cells presumably via the transcription and stem cell factor SOX4.

In conclusion, this study provides insights into the contribution of the SNAIL family EMT-TFs, in the dynamic EMT process and the mechanisms by which this manifests the development of aggressive breast carcinomas. Furthermore, it provides means on the way TGF-β impacts on the biogenesis, secretion, and functional transfer of EV cargo molecules in the context of cancer.

The transcription factor SNAI1 mediates epithelial-mesenchymal transition, fibroblast activation and controls inter-tissue migration. High SNAI1 expression characterizes metastatic triple-negative breast carcinomas, and its knockout by CRISPR/Cas9 uncovered an epithelio-mesenchymal phenotype accompanied by reduced signaling by the cytokine TGFβ. The SNAI1 knockout cells exhibited plasticity in differentiation, drifting towards the luminal phenotype, gained stemness potential and could differentiate into acinar mammospheres in 3D culture. Loss of SNAI1 de-repressed the transcription factor FOXA1, a pioneering factor of mammary luminal progenitors. FOXA1 induced a specific gene program, including the androgen receptor (AR). Inhibiting AR via a specific antagonist regenerated the basal phenotype and blocked acinar differentiation. Thus, loss of SNAI1 in the context of triple-negative breast carcinoma cells promotes an intermediary luminal progenitor phenotype that gains differentiation plasticity based on the dual transcriptional action of FOXA1 and AR. This function of SNAI1 provides means to separate cell invasiveness from progenitor cell de-differentiation as independent cellular programs.