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Mammalian sex determination is controlled by the antagonistic interactions of two genetic pathways: The SRY-SOX9-FGF9 network promotes testis determination partly by opposing proovarian pathways, while RSPO1/WNT-β-catenin/FOXL2 signals control ovary development by inhibiting SRY-SOX9-FGF9. The molecular basis of this mutual antagonism is unclear. Here we show that ZNRF3, a WNT signaling antagonist and direct target of RSPO1-mediated inhibition, is required for sex determination in mice. XY mice lacking ZNRF3 exhibit complete or partial gonadal sex reversal, or related defects. These abnormalities are associated with ectopic WNT/β-catenin activity and reduced Sox9 expression during fetal sex determination. Using exome sequencing of individuals with 46, XY disorders of sex development, we identified three human ZNRF3 variants in very rare cases of XY female presentation. We tested two missense variants and show that these disrupt ZNRF3 activity in both human cell lines and zebrafish embryo assays. Our data identify a testis-determining function for ZNRF3 and indicate a mechanism of direct molecular interaction between two mutually antagonistic organogenetic pathways.

Wnt/β-catenin signaling, a highly conserved pathway through evolution, regulates key cellular functions including proliferation, differentiation, migration, genetic stability, apoptosis, and stem cell renewal. The Wnt pathway mediates biological processes by a canonical or noncanonical pathway, depending on the involvement of β-catenin in signal transduction. β-catenin is a core component of the cadherin protein complex, whose stabilization is essential for the activation of Wnt/β-catenin signaling. As multiple aberrations in this pathway occur in numerous cancers, WNT-directed therapy represents an area of significant developmental therapeutics focus. The recently described role of Wnt/β-catenin pathway in regulating immune cell infiltration of the tumor microenvironment renewed the interest, given its potential impact on responses to immunotherapy treatments. This article summarizes the role of Wnt/β-catenin pathway in cancer and ongoing therapeutic strategies involving this pathway.

A delicate equilibrium of WNT agonists and antagonists in the intestinal stem cell (ISC) niche is critical to maintaining the ISC compartment, as it accommodates the rapid renewal of the gut lining. Disruption of this balance by mutations in the tumour suppressor gene APC , which are found in approximately 80% of all human colon cancers, leads to unrestrained activation of the WNT pathway 1 , 2 . It has previously been established that Apc -mutant cells have a competitive advantage over wild-type ISCs 3 . Consequently, Apc -mutant ISCs frequently outcompete all wild-type stem cells within a crypt, thereby reaching clonal fixation in the tissue and initiating cancer formation. However, whether the increased relative fitness of Apc -mutant ISCs involves only cell-intrinsic features or whether Apc mutants are actively involved in the elimination of their wild-type neighbours remains unresolved. Here we show that Apc -mutant ISCs function as bona fide supercompetitors by secreting WNT antagonists, thereby inducing differentiation of neighbouring wild-type ISCs. Lithium chloride prevented the expansion of Apc -mutant clones and the formation of adenomas by rendering wild-type ISCs insensitive to WNT antagonists through downstream activation of WNT by inhibition of GSK3β. Our work suggests that boosting the fitness of healthy cells to limit the expansion of pre-malignant clones may be a powerful strategy to limit the formation of cancers in high-risk individuals. Using experiments in organoids and in vivo in mice, the authors show that Apc -mutant cells act as supercompetitors to initiate the formation of adenomas.

Bone homeostasis is tightly orchestrated and maintained by the balance between osteoblasts and osteoclasts. Recent studies have greatly expanded our understanding of the molecular mechanisms of cellular differentiation. However, the functional roles of non-coding RNAs particularly lncRNAs in remodeling bone architecture remain elusive. In our study, lncRNA H19 was found to be upregulated during osteogenesis in hMSCs. Stable expression of H19 significantly accelerated in vivo and in vitro osteoblast differentiation. Meanwhile, by using bioinformatic investigations and RIP assays combined with luciferase reporter assays, we demonstrated that H19 functioned as an miRNA sponge for miR-141 and miR-22, both of which were negative regulators of osteogenesis and Wnt/β-catenin pathway. Further investigations revealed that H19 antagonized the functions of these two miRNAs and led to de-repression of their shared target gene β-catenin, which eventually activated Wnt/β-catenin pathway and hence potentiated osteogenesis. In addition, we also identified a novel regulatory feedback loop between H19 and its encoded miR-675-5p. And miR-675-5p was found to directly target H19 and counteracted osteoblast differentiation. To sum up, these observations indicate that the lncRNA H19 modulates Wnt/β-catenin pathway by acting as a competing endogenous RNA, which may shed light on the functional role of lncRNAs in coordinating osteogenesis.

A decline in stem cell function impairs tissue regeneration during ageing, but the role of the stem-cell-supporting niche in ageing is not well understood. The small intestine is maintained by actively cycling intestinal stem cells that are regulated by the Paneth cell niche 1 , 2 . Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age owing to defects in both stem cells and their niche. The functional decline was caused by a decrease in stemness-maintaining Wnt signalling due to production of Notum, an extracellular Wnt inhibitor, in aged Paneth cells. Mechanistically, high activity of mammalian target of rapamycin complex 1 (mTORC1) in aged Paneth cells inhibits activity of peroxisome proliferator activated receptor α (PPAR-α) 3 , and lowered PPAR-α activity increased Notum expression. Genetic targeting of Notum or Wnt supplementation restored function of aged intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of aged stem cells and promoted recovery from chemotherapy-induced damage. Our results reveal a role of the stem cell niche in ageing and demonstrate that targeting of Notum can promote regeneration of aged tissues. Ageing-associated decline in intestinal stem cell function is mediated by increased Notum, a protein inhibitor of stemness-maintaining Wnt signalling, which is secreted by Paneth cells.

Key Points WNT signalling is a complex cascade and there is extensive crosstalk with other pathways. WNT signalling has crucial roles in both normal homeostasis and disease, and thereby there has been considerable difficulty in safely targeting the WNT pathway, thus hampering development to date. There are multiple points of intervention in the WNT signalling cascade that have been investigated; however, many of them may be limited from the standpoint of therapeutic efficacy owing to on-target side effects. WNT signalling has crucial roles in stem cells — both normal and cancer stem cells — in both the maintenance of potency and initiation of differentiation. Currently, several specific inhibitors and modulators of WNT signalling have entered clinical trials. Preliminary results and future prospects are discussed. The WNT pathway has a vast array of functions and aberrant WNT signalling is correspondingly implicated in numerous diseases, including cancer, fibrosis and nervous system disorders. Kahn discusses our understanding of this developmentally important pathway, the complexities associated with safely targeting it therapeutically and WNT-modulating agents that are currently being investigated. WNT–β-catenin signalling is involved in a multitude of developmental processes and the maintenance of adult tissue homeostasis by regulating cell proliferation, differentiation, migration, genetic stability and apoptosis, as well as by maintaining adult stem cells in a pluripotent state. Not surprisingly, aberrant regulation of this pathway is therefore associated with a variety of diseases, including cancer, fibrosis and neurodegeneration. Despite this knowledge, therapeutic agents specifically targeting the WNT pathway have only recently entered clinical trials and none has yet been approved. This Review examines the problems and potential solutions to this vexing situation and attempts to bring them into perspective.

Key Points WNTs are secreted glycoproteins that regulate multiple signalling pathways through both β-catenin (CTNNB1)-dependent and CTNNB1–independent mechanisms. The activation of WNT signalling pathways can be both positively and negatively correlated with patient outcomes in different types of cancer. WNT–CTNNB1 signalling can either promote or inhibit tumour initiation, growth, metastases and drug resistance in a cancer-stage-specific and a cancer-type-specific manner. CTNNB1-independent WNT signalling pathways also contribute to tumorigenesis and cancer progression in a context-dependent manner. Aberrations in WNT signalling pathways and alterations in other oncogene and tumour suppressor pathways cooperate to drive cancer initiation and progression. Multiple strategies for targeting WNT signalling — ranging from small molecules to blocking antibodies, and peptide agonists and antagonists — are now in development, thus paving the way for initial clinical trials using WNT modulators in cancer patients. This Review highlights the complexity and context-dependent roles of both β-catenin-dependent and β-catenin-independent WNT signalling pathways in cancer, as well as some of the ways in which WNT signalling might be targeted therapeutically. Since the initial discovery of the oncogenic activity of WNT1 in mouse mammary glands, our appreciation for the complex roles for WNT signalling pathways in cancer has increased dramatically. WNTs and their downstream effectors regulate various processes that are important for cancer progression, including tumour initiation, tumour growth, cell senescence, cell death, differentiation and metastasis. Although WNT signalling pathways have been difficult to target, improved drug-discovery platforms and new technologies have facilitated the discovery of agents that can alter WNT signalling in preclinical models, thus setting the stage for clinical trials in humans.

Enhanced sensitivity to Wnts is an emerging hallmark of a subset of cancers, defined in part by mutations regulating the abundance of their receptors. Whether these mutations identify a clinical opportunity is an important question. Inhibition of Wnt secretion by blocking an essential post-translational modification, palmitoleation, provides a useful therapeutic intervention. We developed a novel potent, orally available PORCN inhibitor, ETC-1922159 (henceforth called ETC-159) that blocks the secretion and activity of all Wnts. ETC-159 is remarkably effective in treating RSPO-translocation bearing colorectal cancer (CRC) patient-derived xenografts. This is the first example of effective targeted therapy for this subset of CRC. Consistent with a central role of Wnt signaling in regulation of gene expression, inhibition of PORCN in RSPO3-translocated cancers causes a marked remodeling of the transcriptome, with loss of cell cycle, stem cell and proliferation genes, and an increase in differentiation markers. Inhibition of Wnt signaling by PORCN inhibition holds promise as differentiation therapy in genetically defined human cancers.

Basal cell carcinoma (BCC) is the most frequent cancer in humans and results from constitutive activation of the Hedgehog pathway 1 . Several Smoothened inhibitors are used to treat Hedgehog-mediated malignancies, including BCC and medulloblastoma 2 . Vismodegib, a Smoothened inhibitor, leads to BCC shrinkage in the majority of patients with BCC 3 , but the mechanism by which it mediates BCC regression is unknown. Here we used two genetically engineered mouse models of BCC 4 to investigate the mechanisms by which inhibition of Smoothened mediates tumour regression. We found that vismodegib mediates BCC regression by inhibiting a hair follicle-like fate and promoting the differentiation of tumour cells. However, a small population of tumour cells persists and is responsible for tumour relapse following treatment discontinuation, mimicking the situation found in humans 5 . In both mouse and human BCC, this persisting, slow-cycling tumour population expresses LGR5 and is characterized by active Wnt signalling. Combining Lgr5 lineage ablation or inhibition of Wnt signalling with vismodegib treatment leads to eradication of BCC. Our results show that vismodegib induces tumour regression by promoting tumour differentiation, and demonstrates that the synergy between Wnt and Smoothened inhibitors is a clinically relevant strategy for overcoming tumour relapse in BCC. Treatment of basal cell carcinoma with Smoothened inhibitors leaves a small population of quiescent cells that can drive relapse but can be eliminated by additional treatment with a Wnt signalling inhibitor.

The stability of the Wnt pathway transcription factor β-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits β-catenin-mediated transcription. XAV939 stimulates β-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies. Target for Wnt inhibitors Deregulation of the Wnt pathway, a signalling system involved in embryogenesis and in many other processes in living cells, has been implicated in many cancers, making it an attractive target for anticancer therapies. But while inhibitors of Notch and Hedgehog pathways have reached the clinical trial stage, 'drugable' targets for Wnt inhibitors have proved elusive. Now, using a chemical genetics approach, a small molecule inhibitor of the Wnt pathway has been identified and its direct target and mechanism of action characterized. XAV939 inhibits Wnt signalling with high specificity via the stabilization of axin, a concentration-limiting factor of the β-catenin degradation complex. As well as suggesting new drug targets, this work provides insights into how the Wnt pathway is physiologically regulated. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. Here, a small molecule inhibitor of the Wnt pathway is identified and its direct target and mechanism of action are characterized, providing new insights into the physiological regulation of the Wnt pathway and new possibilities for targeted Wnt pathway therapeutics.