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Myofibroblasts are a population of highly contractile fibroblasts that express and require the activity of the transcription factor Snail1. Cancer-associated fibroblasts (CAFs) correlate with low survival of cancer patients when present in the stroma of primary tumors. Remarkably, the presence of myofibroblastic CAFs (which express Snail1) creates mechanical properties in the tumor microenvironment that support metastasis. However, therapeutic blockage of fibroblast activity in patients with cancer is a double-edged sword, as normal fibroblast activities often restrict tumor cell invasion. We used fibroblasts depleted of Snail1 or protein arginine methyltransferases 1 and 4 (PRMT1/-4) to identify specific epigenetic modifications induced by TGFβ/Snail1. Furthermore, we analyzed the in vivo efficiency of methyltransferase inhibitors using mouse models of wound healing and metastasis, as well as fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF). Mechanistically, TGFβ-induced Snail1 promotes the epigenetic mark of asymmetrically dimethylated arginine. Critically, we found that inhibitors of methyltransferases prevent myofibroblast activity (but not regular fibroblast activity) in the extracellular matrix, both in cell culture and in vivo. In a mouse breast cancer model, the inhibitor sinefungin reduces both the myofibroblast activity in the tumor stroma and the metastatic burden in the lung. Two distinct inhibitors effectively blocked the exacerbated myofibroblast activity of patient-derived IPF fibroblasts. Our data reveal epigenetic regulation of myofibroblast transdifferentiation in both wound healing and in disease (fibrosis and breast cancer). Thus, methyltransferase inhibitors are good candidates as therapeutic reagents for these diseases.

Snail1 transcriptional factor plays a key role in the control of epithelial to mesenchymal transition and fibroblast activation. As a consequence, Snail1 expression and function is regulated at multiple levels from gene transcription to protein modifications, affecting its interaction with specific cofactors. In this review, we describe the different elements that control Snail1 expression and its activity both as transcriptional repressor or activator. Ministerio de Economía, Industria y Competitividad, Gobierno de España: SAF2016-76461-R.

The transcription factor Snail1 induces epithelial-to-mesenchymal transition (EMT) in tumor epithelial cells, a process associated with the emergence of stemness, invasion and cancer malignancy. Here, we review recent reports indicating that Snail1 also regulates mesenchymal plasticity and paracrine signaling and propose that Snail1 orchestrates the generation of cancer stem cells (CSCs) and cancer-associated fibroblasts (CAFs). Our view supports the current models for tumorigenesis that consider stemness and tumor microenvironment as retroactive actors for metastasis formation, revealing Snail1 as a regulator of these metastatic forces. This view offers new perspectives for understanding and targeting metastasis.

Crosstalk between tumor and stromal cells in the tumor microenvironment alter its properties in ways that facilitate the invasive behavior of tumor cells. Here, we demonstrate that cancer-associated fibroblasts (CAF) increase the stiffness of the extracellular matrix (ECM) and promote anisotropic fiber orientation, two mechanical signals generated through a Snail1/RhoA/αSMA–dependent mechanism that sustains oriented tumor cell migration and invasiveness. Snail1-depleted CAF failed to acquire myofibroblastic traits in response to TGFβ, including RhoA activation, αSMA-positive stress fibers, increased fibronectin fibrillogenesis, and production of a stiff ECM with oriented fibers. Snail1 expression in human tumor–derived CAF was associated with an ability to organize the ECM. In coculture, a relatively smaller number of Snail1-expressing CAF were capable of imposing an anisotropic ECM architecture, compared with nonactivated fibroblasts. Pathologically, human breast cancers with Snail1+ CAF tended to exhibit desmoplastic areas with anisotropic fibers, lymph node involvement, and poorer outcomes. Snail1 involvement in driving an ordered ECM was further confirmed in wound-healing experiments in mice, with Snail1 depletion preventing the anisotropic organization of granulation tissue and delaying wound healing. Overall, our results showed that inhibiting Snail1 function in CAF could prevent tumor-driven ECM reorganization and cancer invasion. Cancer Res; 75(2); 284–95. ©2014 AACR.

Beyond inducing epithelial-to-mesenchymal transcription (EMT), transcriptional factors of the Snail, ZEB and Twist families (EMT-TFs) control global plasticity programmes affecting cell stemness and fate. Literature addressing the reactivation of these factors in adult tumour cells is very extensive, as they enable cancer cell plasticity and fuel both tumour initiation and metastatic spread. Incipient data reveal that EMT-TFs are also expressed in fibroblasts, providing these with additional properties. Here, I will review recent reports on the expression of EMT-TFs in cancer-associated fibroblasts (CAFs). The new model suggests that EMT-TFs can be envisioned as essential metastasis and chemoresistance-promoting molecules, thereby enabling coordinated plasticity programmes in parenchyma and stroma-tumour compartments.

40 p.-6 fig., Snail1 transcriptional factor is essential for triggering epithelial-to-mesenchymal transition (EMT) and inducing tumor cell invasion. We report here an additional, EMTindependent action of Snail1 on tumor invasion: its expression in cancer-associated fibroblasts is necessary for enhancement by these cells on epithelial cells tumor invasion. Snail1 expression in fibroblast requires signals derived from tumor cells such as TGF-; reciprocally, in fibroblasts Snail1 organizes a complex program that favors collective invasion of epithelial cells at least in part by the secretion of diffusible signaling molecules, such as prostaglandin E2. The capability of human or murine tumor-derived cancer associated fibroblasts to promote tumor invasion is associated to Snail1 expression and obliterated by Snail1 depletion. In vivo experiments show that Snail1 depletion in mice prevents the invasion of breast tumors and epithelial tumor cells co-xenografted with Snail1-depleted fibroblasts originate tumors with lower invasion than those transplanted with control fibroblasts. Therefore, these results demonstrate that the role of Snail1 in tumor invasion is not limited to EMT but dependent on its expression in stromal fibroblasts where it orchestrates its activation and the crosstalk with epithelial tumor cells. Moreover, they point to the interference of Snail expression as a promising target for preventing the action of stromal fibroblasts on tumor progression., This work was funded by grants from Fundación Científica de la Asociación Española contra el Cáncer to AGH, JIC and FB, and Ministerio de Economía(SAF2013-48849-C2-1-R) and Fundació La Marató de TV3 (120130) to AGH. We also thank the support from Instituto Carlos III (RD12/0036/005 and RD12/0036/004,1 part of the Plan Nacional I+D+I and cofounded by the ISCIII-Subdirección General de Evaluación and Fondo Europeo de Desarrollo Regional-FEDER) and Generalitat de Catalunya (2014 SGR 32).

Cancer-associated fibroblasts (CAFs) are one of the major players in tumor-stroma crosstalk. Findings in experimental studies suggest important roles for CAFs in regulation of tumor growth, metastasis and drug response (Hanahan and Coussens, 2012). Furthermore, their clinical relevance is supported by new findings from tumor analyses, demonstrating the prognostic and response-predictive significance of CAF-derived markers or gene signatures (Berdiel-hacer et al., 2014; Finak et al., 2008; Navab et al., 2011; Paulsson and Micke, 2014). CAFs are a heterogeneous pool of cell subsets with distinct functions which needs to be better defined by their marker expressions. The development of a methodology for the establishment of fibroblast primary cultures derived from human colon tumors allowed us to characterize their functional and molecular properties (Herrera et al., 2013). In addition, the different molecular mechanisms through which CAFs affect tumor growth and metastasis are still to be clarified. Therefore, functional and molecular characterization of the cancer-associated fibroblasts is essential to fully understand their role in tumor progression., This research is supported by PI12/02037, RD12/0036/0041 and RD12/0036/0021 from the Instituto de Salud Carlos III-FEDER; by the Fundación Científica AECC; by SAF2010-20750 from the Ministerio de Economía y Competitividad of Spain-FEDER; by S2010/BMD-2344 from the Comunidad de Madrid; and by the Fundación Banco Santander. Antonio García de Herreros’ laboratory is supported by RD12/0036/0005 (from the Instituto de Salud Carlos III-FEDER) and SAF2013-48849-C2-1-R (from the Ministerio de Economía y Competitividad of Spain-FEDER). Cristina Peña is a recipient of a Miguel Servet Contract from the Instituto de Salud Carlos III.

Mortality in cancer is strongly associated with the capacity of tumor cells to spread and critically affect other tissues and organs. Genetic mutations accumulated by tumor cells and cross-signaling between tumor and host cells underlie the formation of metastasis. Cancer-activated fibroblasts (CAFs), which are host fibroblasts activated by tumor signaling, can alter tumor cell behavior by both paracrine signaling (secreting diffusible molecules) and mechanical signaling (modifying the composition and organization of the stroma). These fibroblasts resemble myofibroblasts (MFs) of the granulation tissue generated during wound healing, which produce a rigid desmoplastic stroma rich in signaling molecules and cross-linked extracellular fibers. Desmoplasia favors malignant tumor cell properties such as mobility, stemness, and even resistance to pharmacological insults [1].

Expression of Snail transcriptional factor is a determinant in the acquisition of a mesenchymal phenotype by epithelial tumor cells. However, the regulation of the transcription of this gene is still unknown. We describe here the characterization of a human SNAIL promoter that contains the initiation of transcription and regulates the expression of this gene in tumor cells. This promoter was activated in cell lines in response to agents that induce Snail transcription and the mesenchymal phenotype, as addition of the phorbol ester PMA or overexpression of integrin-linked kinase (ILK) or oncogenes such as Ha-ras or v-Akt. Although other regions of the promoter were required for a complete stimulation by Akt or ILK, a minimal fragment (-78/+59) was sufficient to maintain the mesenchymal specificity. Activity of this minimal promoter and SNAIL RNA levels were dependent on ERK signaling pathway. NFkappaB/p65 also stimulated SNAIL transcription through a region located immediately upstream the minimal promoter, between -194 and -78. These results indicate that Snail transcription is driven by signaling pathways known to induce epithelial to mesenchymal transition, reinforcing the role of Snail in this process.

Alterations in the transcriptional activity of the beta-catenin-Tcf complex have been associated with the earlier stages of colonic transformation. We show here that the activation of protein kinase C by the phorbol ester PMA in several intestinal cell lines increases the levels of beta-catenin detected in the nucleus and augments the transcriptional activity mediated by beta-catenin. The response to PMA was not related to modifications in the cytosolic levels of beta-catenin and was observed not only in cells with wild-type adenomatous polyposis coli protein (APC) but also in APC-deficient cells. Binding assays in vitro revealed that PMA facilitates the interaction of the beta-catenin with the nuclear structure. Our results therefore show that beta-catenin-mediated transcription can be regulated independently of the presence of APC.

Snail1 is a transcriptional repressor of E-cadherin that triggers epithelial–mesenchymal transition (EMT). Here, we report assisted Snail1 interaction with the promoter of a typical mesenchymal gene, fibronectin (FN1), both in epithelial cells undergoing EMT and in fibroblasts. Together with Snail1, the p65 subunit of NF-κB and PARP1 bound to the FN1 promoter. We detected nuclear interaction of these proteins and demonstrated the requirement of all three for FN1 transcription. Moreover, other genes involved in cell movement mimic FN1 expression induced by Snail1 or TGF-β1 treatment and recruit p65NF-κB and Snail1 to their promoters. The molecular cooperation between Snail1 and NF-κB in transcription activation provides a new insight into how Snail1 can modulate a variety of cell programs.

The epithelial to mesenchymal transition (EMT) consists of a rapid change of cell phenotype, characterized by the loss of epithelial traits and the acquisition of a more motile phenotype reminiscent of a fibroblast. The study of this process has received considerable attention because of its potential role in the acquisition of several cancer traits, particularly in cell invasion. In this article we describe the current knowledge of the molecular mechanisms governing this transition. In particular we discuss how initiation of EMT is dependent on the mutually exclusive levels of the transmembrane cell to cell adhesion molecule E-cadherin and its transcriptional repressor Snail1 and how Snail1 and other E-cadherin transcriptional repressors drive the EMT process. We focus on several new aspects of Snail1 regulation and propose a model for understanding the initiation and progression of this transition, based on the existence of feed-back mechanisms that limit or amplify the response to extracellular cues.

Fibronectins are cell-secreted glycoproteins that modulate cell attachment, spreading, migration, morphology, differentiation and oncogenic transformation. Fibronectin expression is activated during EMT (epithelial–mesenchymal transition) and is a hallmark of mesenchymal cells. It is shown in the present study that a transcription factor previously unrelated with EMT, TFCP2c/LSF/LBP-1c, was translocated to the nucleus and bound to the fibronectin promoter upon EMT induction by Snail1. Consequently, the interference of TFCP2c/LSF/LBP-1c's activity prevented fibronectin expression. Moreover, TFCP2c/LSF/LBP-1c was detected in nuclei of embryonic dermal mesenchymal cells adjacent to the hair bud, a cell population that expresses endogenous nuclear Snail1 and fibronectin. Therefore we indicate a new molecular role for TFCP2c/LSF/LBP-1c in fibronectin expression.

A431 cells were treated with epidermal growth factor (EGF) to study the mechanism by which this factor accelerates the glycolytic flux. After EGF treatment, fructose-2,6-bisphosphate (Fru-2,6-P2) levels rose up to 2-fold. This change correlated with an increase in phosphofructokinase-2 activity, which was not due to a change in the transcription or translation of the enzyme, neither in the amount of enzyme. PK-C does not appear to be involved in the signalling mechanism since EGF was equally potent in PK-C depleted cells than in control cells. The increase in Fru-2,6-P2 levels was lower and more transient in cells treated with EGF in a calcium-free medium than in the presence of the cation, and it was restored by the addition of calcium to the medium. These results suggest a possible role for calcium-mediated pathways in the control of Fru-2,6-P2 levels in A431 cells.