The Wnt canonical ligands elicit the activation of β-catenin transcriptional activity, a response dependent on, but not limited to, β-catenin stabilization through the inhibition of GSK3 activity. Two mechanisms have been proposed for this inhibition, one dependent on the binding and subsequent block of GSK3 to LRP5/6 Wnt coreceptor and another one on its sequestration into multivesicular bodies (MVBs). Here we report that internalization of the GSK3-containing Wnt-signalosome complex into MVBs is dependent on the dissociation of p120-catenin/cadherin from this complex. Disruption of cadherin-LRP5/6 interaction is controlled by cadherin phosphorylation and requires the previous separation of p120-catenin; thus, p120-catenin and cadherin mutants unable to dissociate from the complex block GSK3 sequestration into MVBs. These mutants substantially inhibit, but do not completely prevent, the β-catenin upregulation caused by Wnt3a. These results, besides elucidating how GSK3 is sequestered into MVBs, support this mechanism as cause of β-catenin stabilization by Wnt.

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Snail1 transcriptional repressor is a major inducer of epithelial-to mesenchymal transition but is very limitedly expressed in adult animals. We have previously demonstrated that Snail1 is required for the maintenance of mesenchymal stem cells (MSCs), preventing their premature differentiation. Now, we show that Snail1 controls the tumorigenic properties of mesenchymal cells. Increased Snail1 expression provides tumorigenic capabilities to fibroblastic cells; on the contrary, Snail1 depletion decreases tumor growth. Genetic depletion of Snail1 in MSCs that are deficient in p53 tumor suppressor downregulates MSC markers and prevents the capability of these cells to originate sarcomas in immunodeficient SCID mice. Notably, an analysis of human sarcomas shows that, contrarily to epithelial tumors, these neoplasms display high Snail1 expression. This is particularly clear for undifferentiated tumors, which are associated with poor outcome. Together, our results indicate a role for Snail1 in the generation of sarcomas.

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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.

Canonical Wnt signaling induces the stabilization of β-catenin, its translocation to the nucleus and the activation of target promoters. This pathway is initiated by the binding of Wnt ligands to the Frizzled receptor, the association of the LRP5/6 coreceptor and the formation of a complex comprising Dvl-2, Axin and protein kinases CK1α, ε, γ and GSK3. Among these, activation of CK1ε, constitutively bound to LRP5/6 through p120-catenin, is required for the association of the rest of the components. We describe here that CK1ε is activated by the PP2A/PR61ε phosphatase. Binding of Wnt ligands promotes the interaction of LRP5/6-associated CK1ε with Frizzled-bound PR61ε regulatory subunit, facilitating the access of PP2A catalytic subunit to CK1ε and its activation, what enables the recruitment of Dvl-2 to the receptor complex and the initiation of the Wnt pathway. Our results uncover the mechanism of activation of the canonical Wnt pathway by its ligands.

Canonical and noncanonical Wnt pathways share some common elements but differ in the responses they evoke. Similar to Wnt ligands acting through the canonical pathway, Wnts that activate the noncanonical signaling, such as Wnt5a, promote Disheveled (Dvl) phosphorylation and its binding to the Frizzled (Fz) Wnt receptor complex. The protein kinase CK1ε is required for Dvl/Fz association in both canonical and noncanonical signaling. Here we show that differently to its binding to canonical Wnt receptor complex, CK1ε does not require p120‐catenin for the association with the Wnt5a co‐receptor Ror2. Wnt5a promotes the formation of the Ror2–Fz complex and enables the activation of Ror2‐bound CK1ε by Fz‐associated protein phosphatase 2A. Moreover, CK1ε also regulates Ror2 protein levels; CK1ε association stabilizes Ror2, which undergoes lysosomal‐dependent degradation in the absence of this kinase. Although p120‐catenin is not required for CK1ε association with Ror2, it also participates in this signaling pathway as p120‐catenin binds and maintains Ror2 at the plasma membrane; in p120‐depleted cells, Ror2 is rapidly internalized through a clathrin‐dependent mechanism. Accordingly, downregulation of p120‐catenin or CK1ε affects late responses to Wnt5a that are also sensitive to Ror2, such as SIAH2 transcription, cell invasion, or cortical actin polarization. Our results explain how CK1ε is activated by noncanonical Wnt and identify p120‐catenin and CK1ε as two critical factors controlling Ror2 function.

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, TGFβinduced 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.