WNT signalling control by KDM5C during development affects cognition.

Although KDM5C is one of the most frequently mutated genes in X-linked intellectual disability1, the exact mechanisms that lead to cognitive impairment remain unknown. Here we use human patient-derived induced pluripotent stem cells and Kdm5c knockout mice to conduct cellular, transcriptomic, chromatin and behavioural studies. KDM5C is identified as a safeguard to ensure that neurodevelopment occurs at an appropriate timescale, the disruption of which leads to intellectual disability. Specifically, there is a developmental window during which KDM5C directly controls WNT output to regulate the timely transition of primary to intermediate progenitor cells and consequently neurogenesis. Treatment with WNT signalling modulators at specific times reveal that only a transient alteration of the canonical WNT signalling pathway is sufficient to rescue the transcriptomic and chromatin landscapes in patient-derived cells and to induce these changes in wild-type cells. Notably, WNT inhibition during this developmental period also rescues behavioural changes of Kdm5c knockout mice. Conversely, a single injection of WNT3A into the brains of wild-type embryonic mice cause anxiety and memory alterations. Our work identifies KDM5C as a crucial sentinel for neurodevelopment and sheds new light on KDM5C mutation-associated intellectual disability. The results also increase our general understanding of memory and anxiety formation, with the identification of WNT functioning in a transient nature to affect long-lasting cognitive function.

DKK1 is a strong candidate for mesiodens and taurodontism.

A mutation in DKK1 gene leads to inhibitory DKK1 function, over-activation of WNT/ß-catenin signaling, disruptive development of dental epithelium, and subsequent mesiodens formation.

The Flavonol Quercitrin Hinders GSK3 Activity and Potentiates the Wnt/ß-Catenin Signaling Pathway.

The Wnt/ß-catenin signaling pathway dictates cell proliferation and differentiation during embryonic development and tissue homeostasis. Its deregulation is associated with many pathological conditions, including neurodegenerative disease, frequently downregulated. The lack of efficient treatment for these diseases, including Alzheimer's disease (AD), makes Wnt signaling an attractive target for therapies. Interestingly, novel Wnt signaling activating compounds are less frequently described than inhibitors, turning the quest for novel positive modulators even more appealing. In that sense, natural compounds are an outstanding source of potential drug leads. Here, we combine different experimental models, cell-based approaches, neuronal culture assays, and rodent behavior tests with Xenopus laevis phenotypic analysis to characterize quercitrin, a natural compound, as a novel Wnt signaling potentiator. We find that quercitrin potentiates the signaling in a concentration-dependent manner and increases the occurrence of the Xenopus secondary axis phenotype mediated by Xwnt8 injection. Using a GSK3 biosensor, we describe that quercitrin impairs GSK3 activity and increases phosphorylated GSK3ß S9 levels. Treatment with XAV939, an inhibitor downstream of GSK3, impairs the quercitrin-mediated effect. Next, we show that quercitrin potentiates the Wnt3a-synaptogenic effect in hippocampal neurons in culture, which is blocked by XAV939. Quercitrin treatment also rescues the hippocampal synapse loss induced by intracerebroventricular injection of amyloid-ß oligomers (AßO) in mice. Finally, quercitrin rescues AßO-mediated memory impairment, which is prevented by XAV939. Thus, our study uncovers a novel function for quercitrin as a Wnt/ß-catenin signaling potentiator, describes its mechanism of action, and opens new avenues for AD treatments.

Therapeutic Potential of Naturally Occurring Small Molecules to Target the Wnt/ß-Catenin Signaling Pathway in Colorectal Cancer.

Colorectal cancer (CRC) ranks second in the number of cancer deaths worldwide, mainly due to late diagnoses, which restrict treatment in the potentially curable stages and decrease patient survival. The treatment of CRC involves surgery to remove the tumor tissue, in addition to radiotherapy and systemic chemotherapy sessions. However, almost half of patients are resistant to these treatments, especially in metastatic cases, where the 5-year survival rate is only 12%. This factor may be related to the intratumoral heterogeneity, tumor microenvironment (TME), and the presence of cancer stem cells (CSCs), which is impossible to resolve with the standard approaches currently available in clinical practice. CSCs are APC-deficient, and the search for alternative therapeutic agents such as small molecules from natural sources is a promising strategy, as these substances have several antitumor properties. Many of those interfere with the regulation of signaling pathways at the central core of CRC development, such as the Wnt/ß-catenin, which plays a crucial role in the cell proliferation and stemness in the tumor. This review will discuss the use of naturally occurring small molecules inhibiting the Wnt/ß-catenin pathway in experimental CRC models over the past decade, highlighting the molecular targets in the Wnt/ß-catenin pathway and the mechanisms through which these molecules perform their antitumor activities.

Piperine suppresses the Wnt/ß-catenin pathway and has anti-cancer effects on colorectal cancer cells.

More than 94% of colorectal cancer cases have mutations in one or more Wnt/ß-catenin signaling pathway components. Inactivating mutations in APC or activating mutations in ß-catenin (CTNNB1) lead to signaling overactivation and subsequent intestinal hyperplasia. Numerous classes of medicines derived from synthetic or natural small molecules, including alkaloids, have benefited the treatment of different diseases, including cancer, Piperine is a true alkaloid, derived from lysine, responsible for the spicy taste of black pepper (Piper nigrum) and long pepper (Piper longum). Studies have shown that piperine has a wide range of pharmacological properties; however, piperine molecular mechanisms of action are still not fully understood. By using Wnt/ß-catenin pathway epistasis experiment we show that piperine inhibits the canonical Wnt pathway induced by overexpression of ß-catenin, ß-catenin S33A or dnTCF4 VP16, while also suppressing ß-catenin nuclear localization in HCT116 cell line. Additionally, piperine impairs cell proliferation and migration in HCT116, SW480 and DLD-1 colorectal tumor cell lines, while not affecting the non-tumoral cell line IEC-6. In summary, piperine inhibits the canonical Wnt signaling pathway and displays anti-cancer effects on colorectal cancer cell lines.

The Chalcone Lonchocarpin Inhibits Wnt/ß-Catenin Signaling and Suppresses Colorectal Cancer Proliferation.

The deregulation of the Wnt/ß-catenin signaling pathway is a central event in colorectal cancer progression, thus a promising target for drug development. Many natural compounds, such as flavonoids, have been described as Wnt/ß-catenin inhibitors and consequently modulate important biological processes like inflammation, redox balance, cancer promotion and progress, as well as cancer cell death. In this context, we identified the chalcone lonchocarpin isolated from Lonchocarpus sericeus as a Wnt/ß-catenin pathway inhibitor, both in vitro and in vivo. Lonchocarpin impairs ß-catenin nuclear localization and also inhibits the constitutively active form of TCF4, dnTCF4-VP16. Xenopus laevis embryology assays suggest that lonchocarpin acts at the transcriptional level. Additionally, we described lonchocarpin inhibitory effects on cell migration and cell proliferation on HCT116, SW480, and DLD-1 colorectal cancer cell lines, without any detectable effects on the non-tumoral intestinal cell line IEC-6. Moreover, lonchocarpin reduces tumor proliferation on the colorectal cancer AOM/DSS mice model. Taken together, our results support lonchocarpin as a novel Wnt/ß-catenin inhibitor compound that impairs colorectal cancer cell growth in vitro and in vivo.

Toxoplasma gondii Impairs Myogenesis in vitro, With Changes in Myogenic Regulatory Factors, Altered Host Cell Proliferation and Secretory Profile.

Toxoplasma gondii is the causative agent of toxoplasmosis, a parasitic disease with a wide global prevalence. The parasite forms cysts in skeletal muscle cells and neurons, although no evident association with inflammatory infiltrates has been typically found. We studied the impact of T. gondii infection on the myogenic program of mouse skeletal muscle cells (SkMC). The C2C12 murine myoblast cell line was infected with T. gondii tachyzoites (ME49 strain) for 24 h followed by myogenic differentiation induction. T. gondii infection caused a general decrease in myotube differentiation, fusion and maturation, along with decreased expression of myosin heavy chain. The expression of Myogenic Regulatory Factors Myf5, MyoD, Mrf4 and myogenin was modulated by the infection. Infected cultures presented increased proliferation rates, as assessed by Ki67 immunostaining, whereas neither host cell lysis nor apoptosis were significantly augmented in infected dishes. Cytokine Bead Array indicated that IL-6 and MCP-1 were highly increased in the medium from infected cultures, whereas TGF-ß1 was consistently decreased. Inhibition of the IL-6 receptor or supplementation with recombinant TGF-ß failed to reverse the deleterious effects caused by the infection. However, conditioned medium from infected cultures inhibited myogenesis in C2C12 cells. Activation of the Wnt/ß-catenin pathway was impaired in T. gondii-infected cultures. Our data indicate that T. gondii leads SkMCs to a pro-inflammatory phenotype, leaving cells unresponsive to ß-catenin activation, and inhibition of the myogenic differentiation program. Such deregulation may suggest muscle atrophy and molecular mechanisms similar to those involved in myositis observed in human patients.

CUB domain-containing protein 1 (CDCP1) binds transforming growth factor beta family members and increase TGF-ß1 signaling pathway.

CUB domains are most exclusively found in secreted proteins and in a few transmembrane proteins. These domains are approximately 110 amino acids long and have four conserved cysteines that form a ß-sandwich fold. CUB domains proteins are involved in a wide range of biological functions. We have shown that CUB domains from Tolloid/BMP1 can bind BMP4 and block BMP signaling in the developing frog embryo. CUB domain-containing protein 1 (CDCP1) is one of the few transmembrane glycoprotein that contains three extracellular CUB domains and regulates anchorage-independent growth and cancer cell migration through activation of Src kinases. In the extracellular space, only a few proteins were found to interact with CDCP1 and at the moment no ligand was found. We demonstrate by using real time protein interaction on BIAcore chip that CDCP1 CUB domains bind directly to TGF-ß1 and BMP4. CDCP1 enhances TGF-ß1 signaling reporter activity and phosphorylated Smad2 levels but does not modulate BMP signaling pathway. CDCP1 actions on TGF-ß/Smad2 signaling are dependent on Smad2 and TGFRI and do not require Src or PKCδ binding. Our findings uncover a new co-receptor for TGF-ß1 and bring up new questions on whether CDCP1 cooperates with TGF-ß1 to promote cancer progression.

Telocinobufagin and Marinobufagin Produce Different Effects in LLC-PK1 Cells: A Case of Functional Selectivity of Bufadienolides.

Bufadienolides are cardiotonic steroids (CTS) identified in mammals. Besides Na⁺/K⁺-ATPase inhibition, they activate signal transduction via protein⁻protein interactions. Diversity of endogenous bufadienolides and mechanisms of action may indicate the presence of functional selectivity and unique cellular outcomes. We evaluated whether the bufadienolides telocinobufagin and marinobufagin induce changes in proliferation or viability of pig kidney (LLC-PK1) cells and the mechanisms involved in these changes. In some experiments, ouabain was used as a positive control. CTS exhibited an inhibitory IC50 of 0.20 (telocinobufagin), 0.14 (ouabain), and 3.40 µM (marinobufagin) for pig kidney Na⁺/K⁺-ATPase activity and concentrations that barely inhibited it were tested in LLC-PK1 cells. CTS induced rapid ERK1/2 phosphorylation, but corresponding proliferative response was observed for marinobufagin and ouabain instead of telocinobufagin. Telocinobufagin increased Bax:Bcl-2 expression ratio, sub-G0 cell cycle phase and pyknotic nuclei, indicating apoptosis. Src and MEK1/2 inhibitors blunted marinobufagin but not telocinobufagin effect, which was also not mediated by p38, JNK1/2, and PI3K. However, BIO, a GSK-3ß inhibitor, reduced proliferation and, as telocinobufagin, phosphorylated GSK-3ß at inhibitory Ser9. Combination of both drugs resulted in synergistic antiproliferative effect. Wnt reporter activity assay showed that telocinobufagin impaired Wnt/ß-catenin pathway by acting upstream to ß-catenin stabilization. Our findings support that mammalian endogenous bufadienolides may exhibit functional selectivity.

N-myc regulates growth and fiber cell differentiation in lens development.

Myc proto-oncogenes regulate diverse cellular processes during development, but their roles during morphogenesis of specific tissues are not fully understood. We found that c-myc regulates cell proliferation in mouse lens development and previous genome-wide studies suggested functional roles for N-myc in developing lens. Here, we examined the role of N-myc in mouse lens development. Genetic inactivation of N-myc in the surface ectoderm or lens vesicle impaired eye and lens growth, while "late" inactivation in lens fibers had no effect. Unexpectedly, defective growth of N-myc-deficient lenses was not associated with alterations in lens progenitor cell proliferation or survival. Notably, N-myc-deficient lens exhibited a delay in degradation of DNA in terminally differentiating lens fiber cells. RNA-sequencing analysis of N-myc-deficient lenses identified a cohort of down-regulated genes associated with fiber cell differentiation that included DNaseIIß. Further, an integrated analysis of differentially expressed genes in N-myc-deficient lens using normal lens expression patterns of iSyTE, N-myc-binding motif analysis and molecular interaction data from the String database led to the derivation of an N-myc-based gene regulatory network in the lens. Finally, analysis of N-myc and c-myc double-deficient lens demonstrated that these Myc genes cooperate to drive lens growth prior to lens vesicle stage. Together, these findings provide evidence for exclusive and cooperative functions of Myc transcription factors in mouse lens development and identify novel mechanisms by which N-myc regulates cell differentiation during eye morphogenesis.

Derricin and derricidin inhibit Wnt/ß-catenin signaling and suppress colon cancer cell growth in vitro.

Overactivation of the Wnt/ß-catenin pathway in adult tissues has been implicated in many diseases, such as colorectal cancer. Finding chemical substances that can prevent this phenomenon is an emerging problem. Recently, several natural compounds have been described as Wnt/ß-catenin inhibitors and might be promising agents for the control of carcinogenesis. Here, we describe two natural substances, derricin and derricidin, belonging to the chalcone subclass, that show potent transcriptional inhibition of the Wnt/ß-catenin pathway. Both chalcones are able to affect the cell distribution of ß-catenin, and inhibit Wnt-specific reporter activity in HCT116 cells and in Xenopus embryos. Derricin and derricidin also strongly inhibited canonical Wnt activity in vitro, and rescued the Wnt-induced double axis phenotype in Xenopus embryos. As a consequence of Wnt/ß-catenin inhibition, derricin and derricidin treatments reduce cell viability and lead to cell cycle arrest in colorectal cancer cell lines. Taken together, our results strongly support these chalcones as novel negative modulators of the Wnt/ß-catenin pathway and colon cancer cell growth in vitro.

Isoquercitrin suppresses colon cancer cell growth in vitro by targeting the Wnt/ß-catenin signaling pathway.

Flavonoids are plant-derived polyphenolic molecules that have potential biological effects including anti-oxidative, anti-inflammatory, anti-viral, and anti-tumoral effects. These effects are related to the ability of flavonoids to modulate signaling pathways, such as the canonical Wnt signaling pathway. This pathway controls many aspects of embryonic development and tissue maintenance and has been found to be deregulated in a range of human cancers. We performed several in vivo assays in Xenopus embryos, a functional model of canonical Wnt signaling studies, and also used in vitro models, to investigate whether isoquercitrin affects Wnt/ß-catenin signaling. Our data provide strong support for an inhibitory effect of isoquercitrin on Wnt/ß-catenin, where the flavonoid acts downstream of ß-catenin translocation to the nuclei. Isoquercitrin affects Xenopus axis establishment, reverses double axes and the LiCl hyperdorsalization phenotype, and reduces Xnr3 expression. In addition, this flavonoid shows anti-tumoral effects on colon cancer cells (SW480, DLD-1, and HCT116), whereas exerting no significant effect on non-tumor colon cell (IEC-18), suggesting a specific effect in tumor cells in vitro. Taken together, our data indicate that isoquercitrin is an inhibitor of Wnt/ß-catenin and should be further investigated as a potential novel anti-tumoral agent.

Flavonoids and Wnt/ß-catenin signaling: potential role in colorectal cancer therapies.

It is now well documented that natural products have played an important role in anticancer therapy. Many studies focus on the ability of these natural compounds to modulate tumor-related signaling pathways and the relationship of these properties to an anticancer effect. According to the World Health Organization (WHO), colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer death among men and women. Therefore, finding strategies to fight against CRC is an emergent health problem. CRC has a strong association with deregulation of Wnt/ß-catenin signaling pathway. As some types of natural compounds are capable of modulating the Wnt/ß-catenin signaling, one important question is whether they could counteract CRC. In this review, we discuss the role of flavonoids, a class of natural compounds, on Wnt/ß-catenin regulation and its possible potential for therapeutic usage on colorectal cancer.