Mutations in Bcl9 and Pygo genes cause congenital heart defects by tissue-specific perturbation of Wnt/ß-catenin signaling.

Bcl9 and Pygopus (Pygo) are obligate Wnt/ß-catenin cofactors in Drosophila, yet their contribution to Wnt signaling during vertebrate development remains unresolved. Combining zebrafish and mouse genetics, we document a conserved, ß-catenin-associated function for BCL9 and Pygo proteins during vertebrate heart development. Disrupting the ß-catenin-BCL9-Pygo complex results in a broadly maintained canonical Wnt response yet perturbs heart development and proper expression of key cardiac regulators. Our work highlights BCL9 and Pygo as selective ß-catenin cofactors in a subset of canonical Wnt responses during vertebrate development. Moreover, our results implicate alterations in BCL9 and BCL9L in human congenital heart defects.

Pax6-dependent, but ß-catenin-independent, function of Bcl9 proteins in mouse lens development.

Bcl9 and Bcl9l (Bcl9/9l) encode Wnt signaling components that mediate the interaction between ß-catenin and Pygopus (Pygo) via two evolutionarily conserved domains, HD1 and HD2, respectively. We generated mouse strains lacking these domains to probe the ß-catenin-dependent and ß-catenin-independent roles of Bcl9/9l and Pygo during mouse development. While lens development is critically dependent on the presence of the HD1 domain, it is not affected by the lack of the HD2 domain, indicating that Bcl9/9l act in this context in a ß-catenin-independent manner. Furthermore, we uncover a new regulatory circuit in which Pax6, the master regulator of eye development, directly activates Bcl9/9l transcription.

A regulatory receptor network directs the range and output of the Wingless signal.

The potent activity of Wnt/Wingless (Wg) signals necessitates sophisticated mechanisms that spatially and temporally regulate their distribution and range of action. The two main receptor components for Wg - Arrow (Arr) and Frizzled 2 (Fz2) - are transcriptionally downregulated by Wg signaling, thus forming gradients that oppose that of Wg. Here, we analyze the relevance of this transcriptional regulation for the formation of the Wg gradient in the Drosophila wing disc by combining in vivo receptor overexpression with an in silico model of Wg receptor interactions. Our experiments show that ubiquitous upregulation of Arr and Fz2 has no significant effects on Wg output, whereas clonal overexpression of these receptors leads to signaling discontinuities that have detrimental phenotypic consequences. These findings are supported by our in silico model for Wg diffusion and signal transduction, which suggests that abrupt changes in receptor levels causes discontinuities in Wg signaling. Furthermore, we identify a 200 bp regulatory element in the arr locus that can account for the Arr gradient, and we show that this is indirectly negatively controlled by Wg activity. Finally, we analyze the role of Frizzled 3 (Fz3) in this system and find that its expression, which is induced by Wg, contributes to the establishment of the Arr and Fz2 gradients through counteracting canonical signaling. Taken together, our results provide a model in which the regulatory network of Wg and the three receptor components account for the range and shape of this prototypical morphogen system.

MYC promotes immune-suppression in triple-negative breast cancer via inhibition of interferon signaling.

The limited efficacy of immune checkpoint inhibitor treatment in triple-negative breast cancer (TNBC) patients is attributed to sparse or unresponsive tumor-infiltrating lymphocytes, but the mechanisms that lead to a therapy resistant tumor immune microenvironment are incompletely known. Here we show a strong correlation between MYC expression and loss of immune signatures in human TNBC. In mouse models of TNBC proficient or deficient of breast cancer type 1 susceptibility gene (BRCA1), MYC overexpression dramatically decreases lymphocyte infiltration in tumors, along with immune signature remodelling. MYC-mediated suppression of inflammatory signalling induced by BRCA1/2 inactivation is confirmed in human TNBC cell lines. Moreover, MYC overexpression prevents the recruitment and activation of lymphocytes in both human and mouse TNBC co-culture models. Chromatin-immunoprecipitation-sequencing reveals that MYC, together with its co-repressor MIZ1, directly binds promoters of multiple interferon-signalling genes, resulting in their downregulation. MYC overexpression thus counters tumor growth inhibition by a Stimulator of Interferon Genes (STING) agonist via suppressing induction of interferon signalling. Together, our data reveal that MYC suppresses innate immunity and facilitates tumor immune escape, explaining the poor immunogenicity of MYC-overexpressing TNBCs.

TBX3 acts as tissue-specific component of the Wnt/ß-catenin transcriptional complex.

BCL9 and PYGO are ß-catenin cofactors that enhance the transcription of Wnt target genes. They have been proposed as therapeutic targets to diminish Wnt signaling output in intestinal malignancies. Here we find that, in colorectal cancer cells and in developing mouse forelimbs, BCL9 proteins sustain the action of ß-catenin in a largely PYGO-independent manner. Our genetic analyses implied that BCL9 necessitates other interaction partners in mediating its transcriptional output. We identified the transcription factor TBX3 as a candidate tissue-specific member of the ß-catenin transcriptional complex. In developing forelimbs, both TBX3 and BCL9 occupy a large number of Wnt-responsive regulatory elements, genome-wide. Moreover, mutations in Bcl9 affect the expression of TBX3 targets in vivo, and modulation of TBX3 abundance impacts on Wnt target genes transcription in a ß-catenin- and TCF/LEF-dependent manner. Finally, TBX3 overexpression exacerbates the metastatic potential of Wnt-dependent human colorectal cancer cells. Our work implicates TBX3 as context-dependent component of the Wnt/ß-catenin-dependent transcriptional complex.

Reactivation of a developmental Bmp2 signaling center is required for therapeutic control of the murine periosteal niche.

Two decades after signals controlling bone length were discovered, the endogenous ligands determining bone width remain unknown. We show that postnatal establishment of normal bone width in mice, as mediated by bone-forming activity of the periosteum, requires BMP signaling at the innermost layer of the periosteal niche. This developmental signaling center becomes quiescent during adult life. Its reactivation however, is necessary for periosteal growth, enhanced bone strength, and accelerated fracture repair in response to bone-anabolic therapies used in clinical orthopedic settings. Although many BMPs are expressed in bone, periosteal BMP signaling and bone formation require only Bmp2 in the Prx1-Cre lineage. Mechanistically, BMP2 functions downstream of Lrp5/6 pathway to activate a conserved regulatory element upstream of Sp7 via recruitment of Smad1 and Grhl3. Consistent with our findings, human variants of BMP2 and GRHL3 are associated with increased risk of fractures.

WNT ligands control initiation and progression of human papillomavirus-driven squamous cell carcinoma.

Human papillomavirus (HPV)-driven cutaneous squamous cell carcinoma (cSCC) is the most common cancer in immunosuppressed patients. Despite indications suggesting that HPV promotes genomic instability during cSCC development, the molecular pathways underpinning HPV-driven cSCC development remain unknown. We compared the transcriptome of HPV-driven mouse cSCC with normal skin and observed higher amounts of transcripts for Porcupine and WNT ligands in cSCC, suggesting a role for WNT signaling in cSCC progression. We confirmed increased Porcupine expression in human cSCC samples. Blocking the secretion of WNT ligands by the Porcupine inhibitor LGK974 significantly diminished initiation and progression of HPV-driven cSCC. Administration of LGK974 to mice with established cSCC resulted in differentiation of cancer cells and significant reduction of the cancer stem cell compartment. Thus, WNT/ß-catenin signaling is essential for HPV-driven cSCC initiation and progression as well as for maintaining the cancer stem cell niche. Interference with WNT secretion may thus represent a promising approach for therapeutic intervention.

EZH2-Mediated Primary Cilium Deconstruction Drives Metastatic Melanoma Formation.

Human melanomas frequently harbor amplifications of EZH2. However, the contribution of EZH2 to melanoma formation has remained elusive. Taking advantage of murine melanoma models, we show that EZH2 drives tumorigenesis from benign BrafV600E- or NrasQ61K-expressing melanocytes by silencing of genes relevant for the integrity of the primary cilium, a signaling organelle projecting from the surface of vertebrate cells. Consequently, gain of EZH2 promotes loss of primary cilia in benign melanocytic lesions. In contrast, blockade of EZH2 activity evokes ciliogenesis and cilia-dependent growth inhibition in malignant melanoma. Finally, we demonstrate that loss of cilia enhances pro-tumorigenic WNT/ß-catenin signaling, and is itself sufficient to drive metastatic melanoma in benign cells. Thus, primary cilia deconstruction is a key process in EZH2-driven melanomagenesis.

Pharmacological interventions in the Wnt pathway: inhibition of Wnt secretion versus disrupting the protein-protein interfaces of nuclear factors.

Mutations in components of the Wnt pathways are a frequent cause of many human diseases, particularly cancer. Despite the fact that a causative link between aberrant Wnt signalling and many types of human cancers was established more than a decade ago, no Wnt signalling inhibitors have made it into the clinic so far. One reason for this is that no pathway-specific kinase is known. Additionally, targeting the protein-protein interactions needed to transduce the signal has not met with success so far. Complicating the search for and use of inhibitors is the complexity of the cascades triggered by the Wnts and their paramount biological importance. Wnt/ß-catenin signalling is involved in virtually all aspects of embryonic development and in the control of the homeostasis of adult tissues. Encouragingly, however, in recent years, first successes with Wnt-pathway inhibitors have been reported in mouse models of disease. In this review, we summarize possible roads to follow during the quest to pharmacologically modulate the Wnt signalling pathway in cancer. LINKED ARTICLES: This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc.

A cytoplasmic role of Wnt/ß-catenin transcriptional cofactors Bcl9, Bcl9l, and Pygopus in tooth enamel formation.

Wnt-stimulated ß-catenin transcriptional regulation is necessary for the development of most organs, including teeth. Bcl9 and Bcl9l are tissue-specific transcriptional cofactors that cooperate with ß-catenin. In the nucleus, Bcl9 and Bcl9l simultaneously bind ß-catenin and the transcriptional activator Pygo2 to promote the transcription of a subset of Wnt target genes. We showed that Bcl9 and Bcl9l function in the cytoplasm during tooth enamel formation in a manner that is independent of Wnt-stimulated ß-catenin-dependent transcription. Bcl9, Bcl9l, and Pygo2 localized mainly to the cytoplasm of the epithelial-derived ameloblasts, the cells responsible for enamel production. In ameloblasts, Bcl9 interacted with proteins involved in enamel formation and proteins involved in exocytosis and vesicular trafficking. Conditional deletion of both Bcl9 and Bcl9l or both Pygo1 and Pygo2 in mice produced teeth with defective enamel that was bright white and deficient in iron, which is reminiscent of human tooth enamel pathologies. Overall, our data revealed that these proteins, originally defined through their function as ß-catenin transcriptional cofactors, function in odontogenesis through a previously uncharacterized cytoplasmic mechanism, revealing that they have roles beyond that of transcriptional cofactors.

Wnt Ligands Secreted by Subepithelial Mesenchymal Cells Are Essential for the Survival of Intestinal Stem Cells and Gut Homeostasis.

Targeting of Wnt signaling represents a promising anti-cancer therapy. However, the consequences of systemically attenuating the Wnt pathway in an adult organism are unknown. Here, we globally prevent Wnt secretion by genetically ablating Wntless. We find that preventing Wnt signaling in the entire body causes mortality due to impaired intestinal homeostasis. This is caused by the loss of intestinal stem cells. Reconstitution of Wnt/ß-catenin signaling via delivery of external Wnt ligands prolongs the survival of intestinal stem cells and reveals the essential role of extra-epithelial Wnt ligands for the renewal of the intestinal epithelium. Wnt2b is a key extra-epithelial Wnt ligand capable of promoting Wnt/ß-catenin signaling and intestinal homeostasis. Wnt2b is secreted by subepithelial mesenchymal cells that co-express either Gli1 or Acta2. Subepithelial mesenchymal cells expressing high levels of Wnt2b are predominantly Gli1 positive.