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Scale bars=1000?m. serum withdrawal induces mesenchymal breast cancer Syringin cells to undergo VM and that knockdown of the epithelial-to-mesenchymal transition (EMT) regulator, Zinc finger E-box binding homeobox 1 (ZEB1), or overexpression of the ZEB1-repressed microRNAs (miRNAs), miR-200c, miR-183, miR-96 and miR-182 inhibits this process. We find that secreted proteins Fibronectin 1 (FN1) and serine protease inhibitor (serpin) family E member 2 (SERPINE2) are essential for VM in this system. These secreted factors are upregulated in mesenchymal cells in response to serum withdrawal, and overexpression of VM-inhibiting miRNAs abrogates this upregulation. Intriguingly, the receptors for these secreted proteins, low-density lipoprotein receptor-related protein 1 (LRP1) and Syringin Integrin beta 1 (ITGB1), will also be focuses on of the VM-inhibiting miRNAs, suggesting that autocrine signaling stimulating VM is definitely controlled by ZEB1-repressed miRNA clusters. Collectively, these data provide mechanistic insight into the rules of VM and suggest that miRNAs repressed during EMT, in addition to suppressing migratory and stem-like properties of tumor cells, also inhibit endothelial phenotypes of breast cancer cells used in response to a nutrient-deficient microenvironment. Intro As solid tumors increase in size, they deplete the local microenvironment of nutrients and oxygen. Tumor cells canonically respond to this stress by eliciting the recruitment of vasculature via sprouting angiogenesis.1 While the nascent tumor vessels are often disorganized and dysfunctional, they nevertheless resupply the oxygen and nutrients necessary to facilitate tumor growth, as well as provide a route by which tumor cells can metastasize.2 Syringin Inhibitors of angiogenesis have been developed for the treatment of tumors, but these have shown only modest effects on survival in many tumor types, including breast cancer.3 This has been linked, at least in part, to effects of the nutrient- and oxygen-deficient microenvironment resulting from these treatments on tumor cell phenotypes. For example, improved hypoxia following anti-angiogenic therapy can travel an epithelial-to-mesenchymal transition, which induces migratory and stem-like phenotypes in malignancy cells.4, 5, 6 More recently, treatment with anti-angiogenic therapies has also been shown to increase alternate modes of vascularization, including tumor cell adoption of endothelial phenotypes through vascular mimicry (VM) and/or endothelial transdifferentiation.7, 8 While such alternate methods of tumor vascularization have been shown to effect tumor growth and metastasis,7, 9, 10 the extrinsic signals that drive and the intrinsic pathways that regulate these processes are poorly understood. Here we utilize a manipulable, model of VM to show that mesenchymal breast cancer cells form endothelial-like networks when plated on Matrigel (BD Biosciences, San Jose, CA, USA) in the absence of serum. This VM is definitely accompanied by enrichment, under network-forming conditions, of gene signatures indicated by endothelial cells in response to vascular endothelial growth element or hypoxia. We find that knockdown of ZEB1 or re-expression of ZEB1-repressed microRNA (miRNA) clusters, miR-200c.141 or miR-183.96.182, is sufficient to inhibit VM and further, that transient manifestation of miR-200c, -183, -96 and -182, but not miR-141, blocks VM. We determine FN1 and the COPII secretory pathway machinery protein SEC23A as focuses on of miR-200c but not miR-141 in this system, suggesting a role for the malignancy cell secretome in VM. We display that obstructing COPII-mediated secretion through knockdown of SEC13 Syringin is sufficient to inhibit VM and, finally, we uncover a pathway whereby mesenchymal breast tumor cells, in response to serum withdrawal, upregulate MGC126218 secreted proteins FN1 and SERPINE2 (also called protease nexin 1) that are critical for VM. Specific miRNAs repressed during epithelial-to-mesenchymal transition can inhibit these secreted proteins as well as their cellular receptors ITGB1 and LRP1. Large expression of the autocrine signaling factors involved in VM: FN1, ITGB1, SERPINE2, and LRP1, is found in claudin-low malignancy cell lines and is significantly correlated with decreased survival in breast tumor individuals. Collectively our data show that in addition to regulating migratory and stem-like properties of tumor cells, ZEB1-repressed miRNAs can also inhibit autocrine signaling that contributes to the ability of malignancy cells to undergo VM in response to a nutrient-deficient microenvironment. Results Serum deprivation induces VM in mesenchymal breast tumor cell lines VM has been associated with restorative resistance and metastasis in breast cancer.10, 11 To examine the intrinsic and extrinsic requirements for breast cancer cells to undergo VM, we utilized an VM assay that assesses cell network formation on Matrigel.12 As triple-negative (ER?PR?HER2?) breast cancer.