The activation of cancer-associated fibroblasts (CAFs) is a key event in tumor progression, and alternative extracellular matrix (ECM) proteins derived from CAFs induce ECM remodeling and cancer cell invasion. Here we found that miR-200 s, which are generally downregulated in activated CAFs in breast cancer tissues and in normal fibroblasts (NFs) activated by breast cancer cells, are direct mediators of NF reprogramming into CAFs and of ECM remodeling. NFs with downregulated miR-200 s displayed the traits of activated CAFs, including accelerated migration and invasion. Ectopic expression of miR-200 s in CAFs at least partially restored the phenotypes of NFs. CAF activation may be governed by the targets of miR-200 s, Fli-1 and TCF12, which are responsible for cell development and differentiation; Fli-1 and TCF12 were obviously elevated in CAFs. Furthermore, miR-200 s and their targets influenced collagen contraction by CAFs. The upregulation of fibronectin and lysyl oxidase directly by miR-200 or indirectly through Fli-1 or TCF12 contributed to ECM remodeling, triggering the invasion and metastasis of breast cancer cells both in vitro and vivo. Thus, these data provide important and novel insights into breast CAF activation and ECM remodeling, which trigger tumor cell invasion.

SummaryCancer stem cells (CSCs) are a subpopulation of neoplastic cells with self‐renewal capacity and limitless proliferative potential as well as high invasion and migration capacity. These cells are commonly associated with epithelial‐mesenchymal transition (EMT), which is also critical for tumor metastasis. Recent studies illustrate a direct link between EMT and stemness of cancer cells. Long non‐coding RNAs (lncRNAs) have emerged as important new players in the regulation of multiple cellular processes in various diseases. To date, the role of lncRNAs in EMT‐associated CSC stemness acquisition and maintenance remains unclear. In this study, we discovered that a set of lncRNAs were dysregulated in Twist‐positive mammosphere cells using lncRNA microarray analysis. Multiple lncRNAs‐associated canonical signaling pathways were identified via bioinformatics analysis. Especially, the Shh‐GLI1 pathway associated lncRNA‐Hh, transcriptionally regulated by Twist, directly targets GAS1 to stimulate the activation of hedgehog signaling (Hh). The activated Hh increases GLI1 expression, and enhances the expression of SOX2 and OCT4 to play a regulatory role in CSC maintenance. Thus, the mammosphere‐formation efficiency (MFE) and the self‐renewal capacity in vitro, and oncogenicity in vivo in Twist‐positive breast cancer cells are elevated. lncRNA‐Hh silence in Twist‐positive breast cells attenuates the activated Shh‐GLI1 signaling and decreases the CSC‐associated SOX and OCT4 levels, thus reduces the MFE and tumorigenesis of transplanted tumor. Our results reveal that lncRNAs function as an important regulator endowing Twist‐induced EMT cells to gain the CSC‐like stemness properties. Stem Cells 2016;34:55–66

Twist, a key regulator of epithelial-mesenchymal transition (EMT), plays an important role in the development of a tumorigenic phenotype. Energy metabolism reprogramming (EMR), a newly discovered hallmark of cancer cells, potentiates cancer cell proliferation, survival, and invasion. Currently little is known about the effects of Twist on tumor EMR. In this study, we found that glucose consumption and lactate production were increased and mitochondrial mass was decreased in Twist-overexpressing MCF10A mammary epithelial cells compared with vector-expressing MCF10A cells. Moreover, these Twist-induced phenotypic changes were augmented by hypoxia. The expression of some glucose metabolism-related genes such as PKM2, LDHA, and G6PD was also found to be upregulated. Mechanistically, activated β1-integrin/FAK/PI3K/AKT/mTOR and suppressed P53 signaling were responsible for the observed EMR. Knockdown of Twist reversed the effects of Twist on EMR in Twist-overexpressing MCF10A cells and Twist-positive breast cancer cells. Furthermore, blockage of the β1-integrin/FAK/PI3K/AKT/mTOR pathway by siRNA or specific chemical inhibitors, or rescue of p53 activation can partially reverse the switch of glucose metabolism and inhibit the migration of Twist-overexpressing MCF10A cells and Twist-positive breast cancer cells. Thus, our data suggest that Twist promotes reprogramming of glucose metabolism in MCF10A-Twist cells and Twist-positive breast cancer cells via activation of the β1-integrin/FAK/PI3K/AKT/mTOR pathway and inhibition of the p53 pathway. Our study provides new insight into EMR.

Cytoplasmic dynein is a protein complex responsible for transporting cellular cargos on microtubules. Dynein's activities are regulated by other proteins including dynactin which mediates dynein-cargo interactions and increases dynein's processivity. The intermediate chain (IC) of dynein binds to dynactin p150Glued subunit, an interaction central to dynein regulation. The N-terminal domain of IC, N-IC is partially disordered, and contains binding site for a coiled-coil domain of p150Glued and a serine-rich region known to undergo phosphorylation in vivo. There are conflicting results to the effect of phosphorylation on dynactin binding. Using different techniques, one group showed phosphorylated S84 and a phosphomimetic mutant S84D abolished p150Glued binding in vitro while another group demonstrated S84D was still able to bind. We use mutagenesis, constructs of varying length, isothermal titration calorimetry (ITC) and NMR spectroscopy to identify the effect of phosphomimetic S84D on structure of IC and its ability to bind p150Glued. Backbone assignments of IC2C1-96 and secondary chemical shifts show residues 4-38 (helix 1) and 52-66 (helix 2) adopt helical structures and the rest of the protein is disordered. NMR and ITC titration studies identify the first 44 residues as sufficient for binding. The phosphomimetic mutant IC2CS84D abolishes p150Glued binding, without causing major changes in structure and dynamics as detected from NMR measurements. Chemical shift comparison between WT and S84D shows long-range effects on residues in the linker between helix 1 and 2, suggesting conformational changes in the linker. Mutations in the linker region restore p150Glued binding. Together, these results demonstrate for the first time phosphorylation in a disordered region affects binding at a distant site in the sequence, and is fine-tuned by changes in a disordered linker separating two structured domains. This regulatory control of dynein-dynactin interactions is key to cargo-and temporal-specific alterations in dynein transport.

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