Search

Background Circular RNAs (circRNAs) play crucial roles in multiple cancers, including colorectal cancer (CRC). Here, we explored the role of circRNA ArfGAP with coiled-coil, ankyrin repeat and PH domains 2 (circ-ACAP2) in the progression and radioresistance of CRC. Methods Quantitative real-time polymerase chain reaction (qPCR) and Western blot assay were used to detect RNA and protein expression, respectively. The proliferation, apoptosis, migration, invasion and radioresistance of CRC cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, transwell migration assay, transwell invasion assay and colony formation assay. The target interaction between microRNA-143-3p (miR-143-3p) and circ-ACAP2 or frizzled class receptor 4 (FZD4) was verified by dual-luciferase reporter assay. Murine xenograft model was established to explore the role of circ-ACAP2 in vivo. Results The expression of circ-ACAP2 was prominently enhanced in CRC tissues and cell lines. Circ-ACAP2 facilitated the proliferation, migration, invasion and radioresistance whereas inhibited the apoptosis of CRC cells. MiR-143-3p was a direct target of circ-ACAP2 in CRC cells. Circ-ACAP2 promoted the progression and radioresistance of CRC partly by sponging miR-143-3p. MiR-143-3p interacted with the 3' untranslated region (3'UTR) of FZD4 in CRC cells, and FZD4 overexpression partly reversed miR-143-3p-mediated effects in CRC cells. Wnt/β-catenin signalling was modulated by circ-ACAP2/miR-143-3p/FZD4 axis in CRC cells. Conclusion Circ-ACAP2 contributed to the development and radioresistance of CRC partly through targeting miR-143-3p/FZD4 axis, which provided novel potential diagnostic and therapeutic targets for CRC.

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families. While SMO relies on cholesterol binding to the 7TM core of the receptor to activate downstream signaling, underlying details of receptor activation remain obscure for FZDs. Here, we aimed to investigate the activation mechanisms of class F receptors utilizing a computational biology approach and mutational analysis of receptor function in combination with ligand binding and downstream signaling assays in living cells. Our results indicate that FZDs differ substantially from SMO in receptor activation-associated conformational changes. SMO manifests a preference for a straight TM6 in both ligand binding and functional readouts. Similar to the majority of GPCRs, FZDs present with a kinked TM6 upon activation owing to the presence of residue P6.43. Functional comparison of FZD and FZD P6.43F mutants in different assay formats monitoring ligand binding, G protein activation, DVL2 recruitment and TOPflash activity, however, underlines further the functional diversity among FZDs and not only between FZDs and SMO., The class Frizzled of G protein-coupled receptors (GPCRs) consist of ten Frizzled (FZD1-10) subtypes and Smoothened (SMO). Here the Schulte laboratory demonstrates that FZDs differ substantially from SMO in receptor activation-associated conformational changes, while SMO manifests a preference for a straight TM6, the TM6 of FZDs is kinked upon activation.

Treating postoperative (PO) pain is a clinical challenge. Inadequate PO pain management can lead to worse outcomes, for example chronic post-surgical pain. Therefore, acquiring new information on the PO pain mechanism would increase the therapeutic options available. In this paper, we evaluated the role of a natural substance, epigallocatechin-3-gallate (EGCG), on pain and neuroinflammation induced by a surgical procedure in an animal model of PO pain. We performed an incision of the hind paw and EGCG was administered for five days. Mechanical allodynia, thermal hyperalgesia, and motor dysfunction were assessed 24 h, and three and five days after surgery. At the same time points, animals were sacrificed, and sera and lumbar spinal cord tissues were harvested for molecular analysis. EGCG administration significantly alleviated hyperalgesia and allodynia, and reduced motor disfunction. From the molecular point of view, EGCG reduced the activation of the WNT pathway, reducing WNT3a, cysteine-rich domain frizzled (FZ)1 and FZ8 expressions, and both cytosolic and nuclear β-catenin expression, and the noncanonical β-catenin–independent signaling pathways, reducing the activation of the NMDA receptor subtype NR2B (pNR2B), pPKC and cAMP response element-binding protein (pCREB) expressions at all time points. Additionally, EGCG reduced spinal astrocytes and microglia activation, cytokines overexpression and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) pathway, downregulating inducible nitric oxide synthase (iNOS) activation, cyclooxygenase 2 (COX-2) expression, and prostaglandin E2 (PGE2) levels. Thus, EGCG administration managing the WNT/β-catenin signaling pathways modulates PO pain related neurochemical and inflammatory alterations.

BACKGROUND Although the promoting roles of Frizzled-7 (Fzd7) have been shown before, its effects in gastric cancer (GC) cell stemness are still unclear. The present study assessed the effects of Fzd7 on GC cell stemness and chemoresistance. MATERIAL AND METHODS Clinical samples were used to detect Fzd7 expression and online datasets were used to analyze the correlation between Fzd7 expression and GC patient prognosis. Quantitative real-time PCR (qPCR), Western blot, and spheroid formation were used to detect the stemness of cells and Fzd7-mediated effects on GC cell stemness. Cell viability was assessed to evaluate the role of Fzd7 in chemoresistance of GC cells. RESULTS We found that the expression of Frizzled-7 (Fzd7), a Wnt receptor, was increased in gastric cancer (GC) cells and tissues. Additionally, Fzd7 expression was correlated with shorter overall survival of GC patients. Knockdown of Fzd7 or using inhibitors of Wnt/Fzd (OMP-18R5/Vantictumad) decreased GC cell stemness, characterized as a decrease of spheroid formation ability and expression of stemness regulators. Notably, Fzd7 knockdown or inhibitors of Wnt/Fzd attenuated the chemoresistance of GC cells. Furthermore, elevation of Myc expression rescued the effects of Fzd7 inhibition on GC cell stemness and chemoresistance. CONCLUSIONS Our results suggest that inhibition of Fzd7 decreases the stemness and chemotherapeutic resistance of GC cells.

Glioblastoma (GBM) is a highly heterogeneous and aggressive brain tumor. Comprehensive genomic and transcriptomic analyses revealed that GBM segregates into three subgroups with characteristic signaling pathways. The Wnt pathway recently received increasing attention with the recognition of its importance in tumor development and recurrence through the promotion of glioma stem cells. As an extension of our previous translational studies, here we tested the possible interactions between key subgroup markers (IDH-1 R132H, EGFRvIII and both cytoplasmic and nuclear loss [c-/n-] of NF-1 expression) and the canonical (Wnt3a and beta-catenin) and non-canonical (Wnt5a and Fzd2) Wnt pathway markers by immunohistochemistry. These analyses revealed increased expression levels of both Wnt pathway markers with significant quantitative differences within, but not among subgroups. Wnt5a and Fzd2 levels were higher than the canonical marker levels in all subgroups. The strongest evidence for correlation between expression levels of the EGFRvIII subgroup marker and the Fzd2 Wnt marker was found, but weaker correlations also could be noted for IDH-1 R132H and NF-1 and some Wnt markers. The correlations detected between markers of the canonical and non-canonical pathways raised the possibility of cross-talk between the two pathways. Analyses of tumors with various NF-1 expression patterns (c+/n-; c-/n+ combined with c+/n+, and c-/n-) revealed that aberrant nuclear accumulation of NF-1 is accompanied by nuclear accumulation of beta-catenin, suggesting that they may act synergistically to define the tumor's biology. Altogether, our study presents expression characteristics of Wnt ligands and receptors, and suggests complex molecular interactions in subgroups of GBM.

Background Adult stem cells and progenitors are responsible for breast tissue regeneration. Human breast epithelial progenitors are organized in a lineage hierarchy consisting of bipotent progenitors (BPs), myoepithelial- and luminal-restricted progenitors (LRPs) where the LRP differentiation into mature luminal cells requires estrogen receptor (ER) signaling. However, the experimental evidence exploring the relationship between the BPs and LRPs has remained elusive. In this study, we report the presence of a basal-like luminal progenitor (BLP) in human breast epithelial cells. Methods Breast reduction samples were used to obtain different subsets of human breast epithelial cell based on cell surface marker expression using flow cytometry. Loss of function and gain of function studies were employed to demonstrate the role of NOTCH3 (NR3)-FRIZZLED7 (FZD7) signaling in luminal cell fate commitment. Results Our results suggest that, NR3-FZD7 signaling axis was necessary for luminal cell fate commitment. Similar to LRPs, BLPs (NR3highFZD7highCD90+MUC1−ER−) differentiate to generate NR3medFZD7medCD90−MUC1+ER+ luminal cells. Unlike LRPs however, BLP’s proliferation and differentiation potentials depend on NR3 and regulated in part by FZD7 signaling. Lastly, we show that BLPs have a higher colony-forming potential than LRPs and that they are continuously generated from the NOTCH3−FZD7low subset of the bipotent progenitors. Conclusion Our data indicate that BPs differentiate to generate basal-like luminal progenitors that in turn differentiate into LRPs. These results provide new insights into the hierarchical organization of human breast epithelial cell and how cooperation between the Notch and Wnt signaling pathways define a new progenitor cell type. Electronic supplementary material The online version of this article (10.1186/s13287-019-1361-3) contains supplementary material, which is available to authorized users.

Wnt ligands signal through canonical or non-canonical signaling pathways. Although both routes share common elements, such as the Fz2 receptor, they differ in the co-receptor and in many of the final responses; for instance, whereas canonical Wnts increase β-catenin stability, non-canonical ligands downregulate it. However, both types of ligands stimulate tumor cell invasion. We show here that both the canonical Wnt3a and the non-canonical Wnt5a stimulate Fz2 tyrosine phosphorylation, Fyn binding to Fz2, Fyn activation and Fyn-dependent Stat3 phosphorylation. Wnt3a and Wnt5a require Src for Fz2 tyrosine phosphorylation; Src binds to canonical and non-canonical co-receptors (LRP5/6 and Ror2, respectively) and is activated by Wnt3a and Wnt5a. This Fz2/Fyn/Stat3 branch is incompatible with the classical Fz2/Dvl2 pathway as shown by experiments of over-expression or depletion. Fyn is necessary for transcription of genes associated with invasiveness, such as Snail1, and for activation of cell invasion by both Wnt ligands. Our results extend the knowledge about canonical Wnt pathways, demonstrating additional roles for Fyn in this pathway and describing how this protein kinase is activated by both canonical and non-canonical Wnts.

In autism spectrum disorder (ASD), disrupted functional and structural connectivity in the social brain has been suggested as the core biological mechanism underlying the social recognition deficits of this neurodevelopmental disorder. In this study, we aimed to identify genetic and neurostructural abnormalities at birth in a non-human primate model of ASD, the common marmoset with maternal exposure to valproic acid (VPA), which has been reported to display social recognition deficit in adulthood. Using a comprehensive gene expression analysis, we found that 20 genes were significantly downregulated in VPA-exposed neonates. Of these, Frizzled3 (FZD3) and PIK3CA were identified in an axon guidance signaling pathway. FZD3 is essential for the normal development of the anterior commissure (AC) and corpus callosum (CC); hence, we performed diffusion tensor magnetic resonance imaging with a 7-Tesla scanner to measure the midsagittal sizes of these structures. We found that the AC size in VPA-exposed neonates was significantly smaller than that in age-matched controls, while the CC size did not differ. These results suggest that downregulation of the genes related to axon guidance and decreased AC size in neonatal primates may be linked to social brain dysfunctions that can happen later in life.

Wnts are secreted signaling molecules that are implicated in a variety of growth-related processes. Frizzled proteins have been identified as receptors for Wnt ligands in vertebrates and invertebrates, but a functional role for dioecious flatworm Frizzleds has not been determined. To evaluate the endogenous role of Frizzled proteins during development, we have identified and characterized a Schistosoma japonicum frizzled gene (Sjfz7). We found that Sjfz7 encodes a 698 amino acid protein with typical characteristics of Frizzled proteins. The immunohistochemical localization pattern showed that Sjfz7 protein was extensively distributed in almost all tissues of S. japonicum, including subtegumental muscle cells, parenchymal cells, intestinal epithelial cells and male and female germ cells. This indicated that Sjfz7-mediated Wnt signaling might be associated with the development of musculature, intestinal tract and reproductive organs in schistosome. Comparing mRNA levels between frizzled family members showed that Sjfz7 mRNA was consistently higher in the developmental stages analyzed, suggesting that Sjfz7 may be responsible for more functional tasks than other frizzled family members. Comparing frizzled mRNA levels between not fully developed and normal worms suggested that Wnt signaling might be abnormal in not fully developed worms.

To discriminate between closely related members of a protein family that differ at a limited number of spatially distant positions is a challenge for drug discovery. We describe a combined computational design and experimental selection approach for generating binders targeting functional sites with large, shape complementary interfaces to read out subtle sequence differences for subtype-specific antagonism. Repeat proteins are computationally docked against a functionally relevant region of the target protein surface that varies in the different subtypes, and the interface sequences are optimized for affinity and specificity first computationally and then experimentally. We used this approach to generate a series of human Frizzled (Fz) subtype-selective antagonists with extensive shape complementary interaction surfaces considerably larger than those of repeat proteins selected from random libraries. In vivo administration revealed that Wnt-dependent pericentral liver gene expression involves multiple Fz subtypes, while maintenance of the intestinal crypt stem cell compartment involves only a limited subset. Chris Garcia, David Baker and colleagues use a computational approach to develop designed repeat protein binders (DRPBs), which function as human Frizzled (Fz) subtype-selective antagonists and enable identification of Fz subtypes active in different organs.

Recently, molecular dynamics simulations, from all atom and coarse grained to hybrid methods bridging the two scales, have provided exciting functional insights into class F (Frizzled and Taste2) GPCRs (about 40 members in humans). Findings include: (i) The activation of one member of the Frizzled receptors (FZD4) involves a bending of transmembrane helix TM7 far larger than that in class A GPCRs. (ii) The affinity of an anticancer drug targeting another member (Smoothened receptor) decreases in a specific drug-resistant variant, because the mutation ultimately disrupts the binding cavity and affects TM6. (iii) A novel two-state recognition mechanism explains the very large agonist diversity for at least one member of the Taste2 GPCRs, hTAS2R46.

Aberrant activation of Wnt/β-catenin signaling occurs frequently in cancer. However, therapeutic targeting of this pathway is complicated by the role of Wnt in stem cell maintenance and tissue homeostasis. Here, we evaluated antibodies blocking 6 of the 10 human Wnt/Frizzled (FZD) receptors as potential therapeutics. Crystal structures revealed a common binding site for these monoclonal antibodies (mAbs) on FZD, blocking the interaction with the Wnt palmitoleic acid moiety. However, these mAbs displayed gastrointestinal toxicity or poor plasma exposure in vivo. Structure-guided engineering was used to refine the binding of each mAb for FZD receptors, resulting in antibody variants with improved in vivo tolerability and developability. Importantly, the lead variant mAb significantly inhibited tumor growth in the HPAF-II pancreatic tumor xenograft model. Taken together, our data demonstrate that anti-FZD cancer therapeutic antibodies with broad specificity can be fine-tuned to navigate in vivo exposure and tolerability while driving therapeutic efficacy.

Wnt ligand binding initiates the interaction between Frizzled and Dvl proteins. However, the regulation of Frizzled–Dvl proteins interaction remains largely unknown. The present study aims to elucidate the regulation of Frizzled–Dvl interaction by WDR34. The protein levels of WDR34 in hepatocellular carcinoma (HCC) tissues were examined by western blot and immunohistochemistry. The effects of WDR34 on the growth and migration of HCC cells were examined using MTT assay and Boyden chamber assay. The interaction between Frizzled and Dvl was evaluated by immunoprecipitation and GST pull-down assay. In this study, we have shown that WDR34, the binding protein of Frizzled (Fz) activated beta-catenin/TCF signaling by enhancing the interaction between Fz and Dvl2. WDR34 was found to up-regulate in HCC tissues, and its expression was negatively correlated with the survival of HCC patients. WDR34 promoted the growth, colony formation and migration of HCC cells. However, knocking down the expression of WDR34 inhibited the growth, colony formation and migration of HCC cells. Taken together, this study demonstrated the oncogenic roles of WDR34 in the progression of HCC and suggested that WDR34 might be a therapeutic target for HCC.

Environmental factors such as diet and endocrine-disrupting chemicals have individually been shown to mediate metabolic function. However, the underlying mechanism by which the combination disrupts adipocyte morphology and fat storage remains unknown. The current study evaluated early-life programming by diet and phthalate exposure. During gestation and lactation, pregnant Long-Evans hooded rat dams were fed either a control (C) or high-fat (HF) diet and were orally administered one of three phthalate dosages (0, 200 or 1000 μg/kg/day), yielding six groups of offspring: C-0, C-200, C-1000, HF-0, HF-200 and HF-1000. On postnatal day (PND) 90, gonadal fat pads were collected and analyzed for histology, gene expression and DNA methylation. Differences in body weight were observed only in males. Hematoxylin and eosin staining revealed larger adipocyte size in HF-0 vs. C-0 females. Exposure to 200 or 1000 μg/kg/day phthalates modulated diet-induced changes in adipose morphology. Compared to C-0 females, HF-0 females also had higher expression of the adipogenesis gene Wnt receptor, frizzled 1 (Fzd1) and the triglyceride cleaving enzyme lipoprotein lipase (Lpl). These increases in gene expression were accompanied by lower DNA methylation surrounding the transcription start sites of the two genes. Diet-driven effects were observed in unexposed females but not in phthalate-treated rats. Results suggest a sex-specific association between perinatal HF diet and body weight, adipocyte size and DNA methylation. Perinatal phthalate exposure appears to produce a phenotype that more closely resembles HF-fed animals.

Significance In malignant melanoma, one of the most aggressive cancers, the components of the WNT pathway are frequently deregulated and have been shown to drive self-renewal and metastatic progression of melanoma cells. Identifying tumorigenic properties of key members of the WNT signaling network in the pathogenesis of melanoma represents a pivotal task in the field and underlies successful design of targeted therapeutic approaches. In the present study, we characterized Frizzled 3 receptor (FZD3) as a critical factor underlying tumorigenic properties of aggressive human melanoma. Using in vivo and in vitro tumorigenic assays, combined with functional transcriptomics analysis of FZD3 knockdown in human patient-derived cells, we determined key signaling nodes regulated by FZD3 activity during malignant transformation., Frizzled 3 receptor (FZD3) plays an important role in the homeostasis of the neural crest and its derivatives, which give rise to pigment-synthesizing cells, melanocytes. While the role for FZD3 in specification of the melanocytic lineage from neural crest is well established, its significance in the formation of melanoma, its associated malignancy, is less understood. In this study we identified FZD3 as a critical regulator of human melanoma tumorigenesis. Down-regulation of FZD3 abrogated growth, colony-forming potential, and invasive capacity of patient-derived melanoma cells. Xenotransplantation of tumor cells with down-regulated FZD3 levels originating from melanomas carrying the BRAF(V600) mutation uniformly suppressed their capacity for tumor and metastasis formation. FZD3 knockdown leads to the down-regulation of the core cell cycle protein components (cyclins D1, E2, B1, and CDKs 1, 2, and 4) in melanomas with a hyperactive BRAF oncogene, indicating a dominant role of this receptor during melanoma pathogenesis. Enriched pathway analysis revealed that FZD3 inhibits transcriptional networks controlled by CREB5, FOXD1, and ATF3, which suppress the activity of MAPK-mediated signaling. Thus, FZD3 establishes a positive-feedback mechanism that activates MAPK signal transduction network, critical to melanoma carcinogenesis. Importantly, high levels of FZD3 mRNA were found to be correlated with melanoma advancement to metastatic stages and limited patient survival. Changes in gene-expression patterns mediated by FZD3 activity occur in the absence of nuclear β-catenin function, thus representing an important therapeutic target for the melanoma patients whose disease progresses independent of canonical WNT signaling.

Endometriosis is a chronic inflammatory syndrome and nearly 6%‐10% of women are affected by it during the reproductive period. Previous studies have proved that microRNAs (miRNAs) are implicated in the pathogenesis of ovarian endometriosis. In this study, we aimed to investigate that restored miR‐488 would effectively inhibit the development of endometriosis. The microarray‐based data analysis was performed to screen endometriosis‐related differentially expressed genes (DEGs). The mouse model in endometriosis syndrome was established by being subcutaneously injected with Estradiol benzoate, and the ectopic endometrial tissues and normal endometrial tissues were collected. Additionally, the endometrial glandular epithelial cells were extracted from the endometrial glandular epithelial tissues from normal and endometriosis mice. In order to examine the role of miR‐488 in mice with endometriosis, we measured miR‐488 expression and expression levels of Frizzled‐7 (FZD7), cyclinD1, β‐catenin, and c‐Myc in vivo and in vitro. Finally, we detected the effect of miR‐488 on cell proliferation, apoptosis, migration and invasion in vitro. FZD7 was upregulated in human endometriosis. The data showed higher expression levels of FZD7, β‐catenin, c‐Myc and cyclinD1, and lower miR‐488 expression in mouse endometrial tissues. FZD7 was the target gene of miR‐488. Furthermore, elevated miR‐488 in isolated mouse endometrial glandular endometrial cells inhibited FZD7, the translocation of β‐catenin to nucleus, the activation of Wnt pathway, and the cell proliferation, migration and invasion. Collectively, these findings indicated that up‐regulated miR‐488 may reduce the proliferation, migration and invasion of endometrial glandular epithelial cells through inhibiting the activation of Wnt pathway by down‐regulating FZD7.

Purpose: To identify novel mutations in FZD4 and to investigate their pathogenicity in a cohort of Chinese patients with familial exudative vitreoretinopathy (FEVR). Methods: Next-generation sequencing was performed in patients with a clinical diagnosis of FEVR. Wide-field angiography was performed in probands and family members if available. Clinical data were collected from patient charts. The effect of the mutations in FZD4 on its biologic activity in the Norrin/β-catenin signaling pathway was analyzed with the luciferase reporter assay. Results: Four novel mutations in FZD4 (c.1188_1192del/p.F396fs, c.1220delC/p.A407Vfs*24, c.905G>A/p.C302Y, c.1325T>A/p.V442E) were identified in four unrelated families. The mutations were not detected in 200 healthy individuals. The variability of the ocular phenotypes was not only observed in the probands and parents harboring the same mutation but also between two eyes in one individual. All four novel mutations introduced reduction in luciferase activity. Compared with the wild-type, the FZD4 level of the four mutants also decreased variably. Conclusions: Four novel mutations in FZD4 were identified in Chinese patients with FEVR. No correlation in the reduced luciferase activity and the ocular phenotype was observed in this study. This study further emphasized the complexity of the FEVR-causing machinery.

There is a need for novel strategies to treat aggressive breast cancer subtypes and overcome drug resistance. ZnO nanoparticles (NPs) have potential in cancer therapy due to their ability to potently and selectively induce cancer cell apoptosis. Here, we tested the in vitro chemotherapeutic efficacy of ZnONPs loaded via a mesoporous silica nanolayer (MSN) towards drug-sensitive breast cancer cells (MCF-7: estrogen receptor-positive, CAL51: triple-negative) and their drug-resistant counterparts (MCF-7TX, CALDOX). ZnO-MSNs were coated on to gold nanostars (AuNSs) for future imaging capabilities in the NIR-II range. Electron and confocal microscopy showed that MSN-ZnO-AuNSs accumulated close to the plasma membrane and were internalized by cells. High-resolution electron microscopy showed that MSN coating degraded outside the cells, releasing ZnONPs that interacted with cell membranes. MSN-ZnO-AuNSs efficiently reduced the viability of all cell lines, and CAL51/CALDOX cells were more susceptible than MCF7/MCF-7-TX cells. MSN-ZnO-AuNSs were then conjugated with the antibody to Frizzled-7 (FZD-7), the receptor upregulated by several breast cancer cells. We used the disulphide (S-S) linker that could be cleaved with a high concentration of glutathione normally observed within cancer cells, releasing Zn2+ into the cytoplasm. FZD-7 targeting resulted in approximately three-fold amplified toxicity of MSN-ZnO-AuNSs towards the MCF-7TX drug-resistant cell line with the highest FZD-7 expression. This study shows that ZnO-MSs are promising tools to treat triple-negative and drug-resistant breast cancers and highlights the potential clinical utility of FZD-7 for delivery of nanomedicines and imaging probes specifically to these cancer types.

Frizzled (Fzd) proteins are Wnt receptors and play essential roles in development, homeostasis, and oncogenesis. How Wnt/Fzd signaling is coupled to physiological regulation remains unknown. Cholesterol is reported as a signaling molecule regulating morphogen such as Hedgehog signaling. Despite the elusiveness of the in-depth mechanism, it is well-established that pancreatic cancer specially requires abnormal cholesterol metabolism levels for growth. In this study, it is unexpectedly found that among ten Fzds, Fzd5 has a unique capacity to bind cholesterol specifically through its conserved extracellular linker region. Cholesterol-binding enables Fzd5 palmitoylation, which is indispensable for receptor maturation and trafficking to the plasma membrane. In Wnt-addicted pancreatic ductal adenocarcinoma (PDAC), cholesterol stimulates tumor growth via Fzd5-mediated Wnt/β-catenin signaling. A natural oxysterol, 25-hydroxylsterol competes with cholesterol and inhibits Fzd5 maturation and Wnt signaling, thereby alleviating PDAC growth. This cholesterol-receptor interaction and ensuing receptor lipidation uncover a novel mechanism by which Fzd5 acts as a cholesterol sensor and pivotal connection coupling lipid metabolism to morphogen signaling. These findings further suggest that cholesterol-targeting may provide new therapeutic opportunities for treating Wnt-dependent cancers.

Developing synapses mature through the recruitment of specific proteins that stabilize presynaptic and postsynaptic structure and function. Wnt ligands signaling via Frizzled (Fz) receptors play many crucial roles in neuronal and synaptic development, but whether and how Wnt and Fz influence synaptic maturation is incompletely understood. Here, we show that Fz2 receptor cleavage via the γ-secretase complex is required for postsynaptic development and maturation. In the absence of γ-secretase, Drosophila neuromuscular synapses fail to recruit postsynaptic scaffolding and cytoskeletal proteins, leading to behavioral deficits. Introducing presenilin mutations linked to familial early-onset Alzheimer's disease into flies leads to synaptic maturation phenotypes that are identical to those seen in null alleles. This conserved role for γ-secretase in synaptic maturation and postsynaptic development highlights the importance of Fz2 cleavage and suggests that receptor processing by proteins linked to neurodegeneration may be a shared mechanism with aspects of synaptic development.

Wnt signaling regulates physiological processes ranging from cell differentiation to bone formation. Dysregulation of Wnt signaling is linked to several human ailments, including colorectal, pancreatic, and breast cancers. As such, modulation of this pathway has been an attractive strategy for therapeutic development of anticancer agents. Since the discovery of Wnt proteins more than 35 years ago, research efforts continue to focus on understanding the biochemistry of their molecular interactions and their biological functions. Wnt is a secreted glycoprotein covalently modified with a cis-unsaturated fatty acyl group at a conserved serine residue, and this modification is required for Wnt secretion and activity. To initiate signaling, Wnt proteins bind to cell-surface Frizzled (FZD) receptors, but the molecular basis for recognition of Wnt's fatty acyl moiety by the extracellular cysteine-rich domain of FZD has become clear only very recently. Here, we review the most recent developments in the field, focusing on structural and biochemical studies of the FZD receptor family and highlighting new insights into their molecular arrangement and mode of regulation by cis-unsaturated fatty acids. Additionally, we examine how other lipid-binding proteins recognize fatty acyl chains on Wnt proteins in the regulation of Wnt secretion and activities. Altogether, this perspective expands our understanding of fatty acid–protein interactions in the FZD system and provides a basis for guiding future research in the field.

Background Recurrence and metastasis are the major problems of bladder urothelial carcinoma, which mainly attribute to tumor cell stemness, epithelial-mesenchymal transition (EMT) and chemoresistance. Methods TCGA database was interrogated for gene mRNA expression in bladder urothelial carcinoma samples. CCLE database was interrogated for gene mRNA expression in bladder cancer cell lines. The correlation between two genes was analyzed by Pearson statistics. 37 human bladder urothelial carcinoma specimens were adopted for immunohistochemistry. Bladder cancer cells RT4, J82, and UM-UC-3 were used to carry out loss and gain of function studies. Kaplan-Meier method was performed to analyze the overall survival. Findings WNT7B is downregulated in high-grade bladder urothelial carcinomas. Low WNT7B expression is associated with unfavorable prognosis. Loss and gain of function studies showed that WNT7B inhibits bladder urothelial carcinoma cell EMT, stem-like properties and chemoresistance. FZD5, a specific receptor for WNT7B, mediates WNT7B signaling. ELF3 is a downstream component of WNT7B signaling, which transcriptionally modulates NOTCH1, a tumor suppressor in bladder urothelial carcinoma. Interpretation These data demonstrate that WNT7B/FZD5-ELF3-NOTCH1 signaling functions as a tumor-suppressing pathway in bladder urothelial carcinoma.

Diabetes mellitus (DM) influence induces poor osseointegration. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is a critical factor in successful dental implants. Certain microRNAs play important roles during bone development, and others are deregulated in diabetes. This study investigated the roles of miR-129-5p in the osteoblast differentiation regulation. Exosomes containing miR-129-5p inhibited the osteoblast differentiation and was found in the blood of DM rats. The BMSCs isolated from the jaw of rats were used to detect the miR-129-5p expression. Frizzled (FZD) proteins function as receptors for WNT ligands. The FZD4 was the target of miR-129-5p in dual luciferase assay and Western blot. The miR-129-5p inhibited osteoblast differentiation and decreased the osteoblast markers. The exosomes isolated from the blood of DM rats showed more miR-129-5p level. Results suggested that the exosomes containing miR-129-5p maybe regulators of BMSCs in jaw. The collected exosomes containing miR-129-5p showed the inhibition effect in osteoblast differentiation and decreased the expression osteoblastic markers by targeting FZD4/β-catenin signaling pathway. Therefore, the exosomes containing miR-129-5p in DM rats inhibits osteoblast differentiation by targeting FZD4/β-catenin pathway.

Aberrant Wnt signaling is implicated in carcinogenesis triggering efforts for the development of new therapeutic agents, many of which have entered clinical trials. We extend our previous analysis of WNT3, FZD7, LEF1 expression levels in breast and colorectal cancer including WNT2, FZD4 and β-catenin expression, in an effort to delineate their relative expression levels along with concurrent expression patterns and possible prognostic value. We analyzed 82 breast and 102 colorectal carcinomas for relative mRNA expression levels of the investigated genes by RT-PCR relative quantification with the ΔΔCt method. Statistical analysis was performed in order to determine associations of relative mRNA expression and linear correlations. β-catenin expression was determined by immunochemistry. Regarding breast carcinomas, decreased relative mRNA expression levels of WNT2, FZD4 were found frequently and WNT2 expression was correlated with ER/ PR status (p = 0.045/p = 0.028), whereas β-catenin with grade (p = 0.026). In colorectal carcinomas, increased relative mRNA expression levels of WNT2 and FZD4 were found in 59% and 32% of cases respectively, whereas β-catenin showed decreased mRNA expression levels in 57% of cases and a correlation with pN-category (p = 0.037). Linear correlations were observed between WNT2/FZD4 (R=0.542, p 0.001), WNT2/β-catenin (R=0.254, p = 0.010), FZD4/β-catenin (R=0.406, p 0.001) expression and a correlation between mRNA expression and membranous/cytoplasmic β-catenin emerged (p = 0.039/0.046). Our results suggest a possible clinical significance for Wnt pathway gene expression levels in both tumour types. The concurrent expression of the investigated genes as well as the different expression profiles, underlines the complexity of this pathway and the necessity of patient selection in order to maximize the efficacy of drugs targeting Wnt pathway.

SUMMARYThe orientation of epithelial cells in the plane of the tissue, known as planar cell polarity (PCP), is regulated by interactions of asymmetrically localized PCP protein complexes. In the Xenopus neural plate, Van Gogh-like2 (Vangl2) and Prickle3 (Pk3) proteins form a complex at the anterior cell boundaries, but how this complex is regulated in vivo remains largely unknown. Here we show that Vangl2-Pk3 association is inhibited by Frizzled3 (Fz3), a core PCP protein that is specifically expressed in the neuroectoderm and is essential for the establishment of PCP in this tissue. Proximity biotinylation and crosslinking studies revealed that the Vangl2-Pk3 interaction is suppressed by overexpressed Fz3, but enhanced in Fz3 morphants. In addition, Fz3 induced Vangl2 phosphorylation on T76 and T78, and this phosphorylation was required for Fz3-mediated inhibition of Vangl2-Pk3 complex formation. Consistent with this observation, the complex of Pk3 with nonphosphorylatable Vangl2 was not polarized in the neural plate. These findings provide evidence for in vivo regulation of the Vangl2-Pk3 complex formation and localization by a Frizzled receptor.

As the leading cause of worldwide hospital-acquired infection, Clostridioides difficile (C. difficile) infection has caused heavy economic and hospitalized burden, while its pathogenesis is not fully understood. Toxin B (TcdB) is one of the major virulent factors of C. difficile. Recently, CSPG4 and FZD2 were reported to be the receptors that mediate TcdB cellular entry. However, genetic ablation of genes encoding these receptors failed to completely block TcdB entry, implicating the existence of alternative receptor(s) for this toxin. Here, by employing the CRISPR-Cas9 screen in CSPG4-deficient HeLa cells, we identified LDL receptor-related protein-1 (LRP1) as a novel receptor for TcdB. Knockout of LRP1 in both CSPG4-deficient HeLa cells and colonic epithelium Caco2 cells conferred cells with increased TcdB resistance, while LRP1 overexpression sensitized cells to TcdB at a low concentration. Co-immunoprecipitation assay showed that LRP1 interacts with full-length TcdB. Moreover, CROPs domain, which is dispensable for TcdB's interaction with CSPG4 and FZD2, is sufficient for binding to LRP1. As such, our study provided evidence for a novel mechanism of TcdB entry and suggested potential therapeutic targets for treating C. difficile infection.

Intrahepatic cholangiocarcinoma (ICC) is a primary liver cancer with high aggressiveness and extremely poor prognosis. The role of circular RNAs (circRNAs) in ICC carcinogenesis and progression remains to be determined.CircRNA microarray was performed to screen significantly upregulated circRNAs in paired ICC and non-tumor tissues. Colony formation, transwell, and xenograft models were used to examine the role of circRNAs in ICC proliferation and metastasis. RNA pulldown, mass spectrometry, chromatin immunoprecipitation, RNA-binding protein immunoprecipitation, chromatin isolation by RNA purification, electrophoretic mobility shift assay, and luciferase reporter assays were used to explore the molecular sponge role of the circRNA (via miRNA binding), and the interaction between circRNA and RNA-binding proteins.Hsa_circ_0050898, which originated from exon 1 to exon 20 of the ACTN4 gene (named circACTN4), was significantly upregulated in ICC. High circACTN4 expression was associated with enhanced tumor proliferation and metastasis in vitro and in vivo, as well as a worse prognosis following ICC resection. In addition, circACTN4 upregulated Yes-associated protein 1 (YAP1) expression by sponging miR-424-5p. More importantly, circACTN4 also recruited Y-box binding protein 1 (YBX1) to stimulate Frizzled-7 (FZD7) transcription. Furthermore, circACTN4 overexpression in ICC cells enhanced the interaction between YAP1 and β-catenin, which are the core components of the Hippo and Wnt signaling pathways, respectively.CircACTN4 was upregulated in ICC and promoted ICC proliferation and metastasis by acting as a molecular sponge of miR-424-5p, as well as by interacting with YBX1 to transcriptionally activate FZD7. These results suggest that circACTN4 is a potential prognostic marker and therapeutic target for ICC.Intrahepatic cholangiocarcinoma is a primary liver cancer associated with aggressiveness and extremely poor prognosis. It is essential for therapeutic development that we uncover relevant pathogenic pathways. Herein, we showed that a circular RNA (circACTN4) was highly expressed in intrahepatic cholangiocarcinoma and was positively associated with tumor growth and metastasis through key developmental signaling pathways. Thus, circACTN4 could be a prognostic biomarker and therapeutic target for intrahepatic cholangiocarcinoma.

cWnt-signalling plays a crucial role in stem cell maintenance and tissue homeostasis. Secreted frizzled-related proteins(SFRP), Wnt inhibitors consist of the N-terminal cysteine rich domain(CRD) and the C-terminal netrin(NTR) domain. SFRP1 binds to the Wnt ligands and frizzled receptors(FZ) either through its SFRP1CRD or through its SFRP1Netrin domains; however, very little is known on these binding affinities. Here, we attempted to understand the interactions and binding affinities of SFRP1-Wnt5B, SFRP1-FZ(2, 37) and Wnt5B-FZ(2, 37) that are mainly expressed in murine hair follicle stem cells. SFRP1CRD, SFRP1Netrin, Wnt5B and FZ(2, 37) structures were built using homology modelling, followed by their molecular dynamics simulations. SFRP1CRD showed lower fluctuation when in complex with FZ2, FZ3 and FZ7 and Wnt5B as compared to SFRP1Netrin using RMSF and RMSD. However, free energy showed SFRP1Netrin was energetically more stable than SFRP1CRD. SFRP1Netrin formed more number of interactions with FZ as compared to SFRP1CRD. Importantly, SFRP1Netrin favoured binding to the FZ receptors(FZ3 FZ7 FZ2) as compared to Wnt5B ligand. Conversely, the SFRP1CRD showed more affinity towards the Wnt5B ligand as compared to FZ receptors. Wnt5B showed the best binding affinity with FZ3 followed by SFRP1CRD and SFRP1Netrin. Therefore, SFRP1Netrin can bind to the FZ3 with higher binding affinity and may inhibit non-canonical Wnt-signalling pathway. Our study provides the comprehensive information on the binding affinities among the Wnt5B, SFRP1CRD/Netrin and FZ(2, 37). Thus, this information might also help in designing novel strategies to inhibit aberrant Wnt-signalling.Communicated by Ramaswamy H. Sarma.

Recently, a new genetically autosomal recessive color phenotype emerged in the red pied bovine Montbeliarde breed. It is characterized by a dilution of the red areas of the coat and was denominated 'milca'. A genome-wide homozygosity scan of 106 cases followed by haplotype analysis revealed a candidate region within BTA2 between positions 89.95 and 91.63 Mb. Analysis of whole-genome sequence data generated from milca animals identified a strong candidate variant within the coding region of the Frizzled-7 gene (FZD7). This gene encodes for a G-protein coupled receptor for Wnt signaling proteins. The variant induces a glycine to alanine substitution in the second extracellular loop, p.(Gly414Ala). Cross-species amino acid alignments revealed that this glycine is conserved among orthologs and most paralogs, suggesting that it plays an important role in FZD function. In addition, genotyping data revealed that the mutant allele is restricted to the Montbeliarde breed, at a 3.7% frequency. All homozygous cows for the mutant allele exhibited the milca phenotype whereas all heterozygotes had no coat color defects. In conclusion, this study strongly suggests that, in cattle, a mutation of FZD7 alone is sufficient to cause a coat color phenotype without any strong other adverse effect.

Glioma is a devastating cancer with a rich vascular network. No anti-angiogenic treatment is available for prolonging the overall survival of glioma patients. Recent studies have demonstrated that the endothelial differentiation of glioma stem cells (GSCs) into glioma-derived endothelial cells (GDECs) may be a novel target for anti-angiogenic therapy in glioma; however, the underlying mechanisms of this process remain unknown. Here, we report that wingless-related integration site (WNT) family member 5A (WNT5A) plays significant roles in GSC endothelial differentiation and GDECs angiogenesis. WNT5A is preferentially secreted by GDECs, and inhibition of WNT5A suppresses angiogenesis and tumorigenesis in GDECs. Silencing of WNT5A in GDECs also disrupts the impact of GDECs on stimulating GSC endothelial differentiation. Frizzled-4 is a receptor that mediates the effect of WNT5A on GSC endothelial differentiation and angiogenesis of GDECs via GSK3β/β-catenin/epithelial-mesenchymal transition signalling. The shWNT5A@cRGD-DDD liposomes, targeting WNT5A, exert anti-angiogenic effects in vivo. In this study, we identified that WNT5A has a dual functional role in modulating the endothelial differentiation of GSCs and angiogenesis of GDECs, indicating that WNT5A is a potential target for anti-angiogenesis-based therapeutics in glioma.

To orchestrate collective polarization across tissues, planar cell polarity (PCP) proteins localize asymmetrically to cell junctions, a conserved feature of PCP that requires the atypical cadherin Celsr1. We report that mouse Celsr1 engages in bothtrans- andcis-interactions, and organizes into dense and highly stable punctate assemblies. We provide evidence suggesting that PCP-mutant variant of Celsr1, Celsr1Crsh, selectively impairs lateralcis-interactions. Although Celsr1Crshmediates cell adhesion in trans, it displays increased mobility, diminishes junctional enrichment, and fails to engage in homophilic adhesion with the wild-type protein, phenotypes that can be rescued by ectopiccis-dimerization. Using biochemical and super-resolution microscopy approaches, we show that although Celsr1Crshphysically interacts with PCP proteins Frizzled6 and Vangl2, it fails to organize these proteins into asymmetric junctional complexes. Our results suggest mammalian Celsr1 functions not only as atrans-adhesive homodimeric bridge, but also as an organizer of intercellular Frizzled6 and Vangl2 asymmetry through lateral,cis-interactions.

Background Frizzled (FZD) proteins function as receptors for WNT ligands. Members in FZD family including FZD2, FZD4, FZD7, FZD8 and FZD10 have been demonstrated to mediate cancer cell epithelial-mesenchymal transition (EMT). Methods CCLE and TCGA databases were interrogated to reveal the association of FZD5 with EMT. EMT was analyzed by investigating the alterations in CDH1 (E-cadherin), VIM (Vimentin) and ZEB1 expression, cell migration and cell morphology. Transcriptional modulation was determined by ChIP in combination with Real-time PCR. Survival was analyzed by Kaplan–Meier method. Results In contrast to other FZDs, FZD5 was identified to prevent EMT in gastric cancer. FZD5 maintains epithelial-like phenotype and is negatively modulated by transcription factors SNAI2 and TEAD1. Epithelial-specific factor ELF3 is a downstream effecter, and protein kinase C (PKC) links FZD5 to ELF3. ELF3 represses ZEB1 expression, further guarding against EMT. Moreover, FZD5 signaling requires its co-receptor LRP5 and WNT7B is a putative ligand for FZD5. FZD5 and ELF3 are associated with longer survival, whereas SNAI2 and TEAD1 are associated with shorter survival. Conclusions Taken together, FZD5-ELF3 signaling blocks EMT, and plays a potential tumor-suppressing role in gastric cancer.

Oral squamous cell carcinoma (OSCC) accounts for approximately 90% of malignant epithelial tumors of the oral and maxillofacial region. OSCC has high rate of metastasis and poor prognosis. Tobacco and/or alcohol consumption and human papillomavirus infection are relatively exact susceptibility factors for OSCC, but the specific process of oral mucosal carcinogenesis and progression is very complicated. microRNA-302b (miR-302b) could regulate various characteristics of many tumor cells, such as proliferation and apoptosis, but its role and mechanism in OSCC have not been reported. This research aims to study the effect of miR-302b on the invasion and migration ability of OSCC and the mechanism by which it functions as well as to identify new prognostic indicators and therapeutic targets for OSCC patients. Functional studies showed that the miR-302b level was negatively correlated with the invasion and migration ability of OSCC. The studies also showed that the overexpression of miR-302b could attenuate the invasion and migration ability of OSCC cells and reduce lymphangiogenesis and the lung metastasis rate of OSCC cells in a mouse model. Mechanistic studies were performed by quantitative polymerase chain reactions, luciferase assays, and RNA pull-down experiments. The results verified that frizzled class receptor 6 (FZD6) is a target gene of miR-302b in OSCC that could promote cell invasion and migration. Clinical studies demonstrate that the protein expression level of FZD6 was higher in OSCC and metastatic lymph nodes than in normal oral mucosa epithelium. Taken together, these data showed that miR-302b could inhibit the invasion and migration ability of OSCC cells by targeting and downregulating FZD6, thereby inhibiting OSCC metastasis. As a new target gene of miR-302b, FZD6 has the potential to become a prognostic and therapeutic target for OSCC patients.

The endoplasmic reticulum (ER) membrane protein complex (EMC) regulates the synthesis and quality control of membrane proteins with multiple transmembrane domains. One of the membrane spanning subunits, EMC3, is a core member of the EMC complex that provides essential hydrophilic vestibule for substrate insertion. Here, we show that the EMC subunit Emc3 plays critical roles in the retinal vascular angiogenesis by regulating Norrin/Wnt signaling. Postnatal endothelial cell (EC)-specific deletion of Emc3 led to retarded retinal vascular development with a hyperpruned vascular network, the appearance of blunt-ended, aneurysm-like tip endothelial cells (ECs) with reduced numbers of filopodia and leakage of erythrocytes at the vascular front. Diminished tube formation and cell proliferation were also observed in EMC3 depleted human retinal endothelial cells (HRECs). We then discovered a critical role for EMC3 in expression of FZD4 receptor of β-catenin signaling using RNA sequencing, real-time quantitative PCR (RT-qPCR) and luciferase reporter assay. Moreover, augmentation of Wnt activity via lithium chloride (LiCl) treatment remarkably enhanced β-catenin signaling and cell proliferation of HRECs. Additionally, LiCl partially reversed the angiogenesis defects in Emc3-cKO mice. Our data reveal that Emc3 plays essential roles in angiogenesis through direct control of FZD4 expression and Norrin/β-catenin signaling.

Dysregulation of Wnt signaling is implicated in multiple ocular disorders. The roles of Wnt co-receptors LRP5 and LRP6 in Wnt signaling regulation remain elusive, as most retinal cells express both of the co-receptors. To address this question, LRP5 and LRP6 were individually knocked-out in a human retinal pigment epithelium cell line using the CRISPR-Cas9 technology. Wnt signaling activity induced by various Wnt ligands was measured using wild-type and the KO cell lines. The results identified three groups of Wnt ligands based on their co-receptor specificity: 1) activation of Wnt signaling only through LRP6, 2) through both LRP5 and LRP6 and 3) predominantly through LRP5. These results indicate that LRP5 and LRP6 have differential roles in Wnt signaling regulation.

Familial exudative vitreoretinopathy (FEVR) is a hereditary vitreoretinal disorder characterized by incomplete vascular development in the peripheral retina. The secondary pathological processes le...

An organizer is defined as a group of cells that secrete extracellular proteins that specify the fate of surrounding cells according to their concentration. Their function during embryogenesis is key in patterning new growing tissues. Although organizers should also participate in adult development when new structures are regenerated, their presence in adults has only been identified in a few species with striking regenerative abilities, such as planarians. Planarians provide a unique model to understand the function of adult organizers, since the presence of adult pluripotent stem cells provides them with the ability to regenerate any body part. Previous studies have shown that the differential activation of the WNT/&beta, catenin signal in each wound is fundamental to establish an anterior or a posterior organizer in the corresponding wound. Here, we identify the receptors that mediate the WNT/&beta, catenin signal in posterior-facing wounds. We found that Wnt1-Fzd1-LRP5/6 signaling is evolutionarily conserved in executing a WNT/&beta, catenin signal to specify cell fate and to trigger a proliferative response. Our data allow a better understanding of the mechanism through which organizers signal to a &ldquo, competent&rdquo, field of cells and integrate the patterning and growth required during de novo formation of organs and tissues.

Zacarías-Fluck, et al identify and validate the Wnt receptor Fzd9 as a key effector of Myc-Wnt signaling cross-talk in a mouse model of Myc-driven pancreatic insulinomas., The huge cadre of genes regulated by Myc has obstructed the identification of critical effectors that are essential for Myc-driven tumorigenesis. Here, we describe how only the lack of the receptor Fzd9, previously identified as a Myc transcriptional target, impairs sustained tumor expansion and β-cell dedifferentiation in a mouse model of Myc-driven insulinoma, allows pancreatic islets to maintain their physiological structure and affects Myc-related global gene expression. Importantly, Wnt signaling inhibition in Fzd9-competent mice largely recapitulates the suppression of proliferation caused by Fzd9 deficiency upon Myc activation. Together, our results indicate that the Wnt signaling receptor Fzd9 is essential for Myc-induced tumorigenesis in pancreatic islets.

WNT proteins are secreted symmetry breaking signals that interact with cell surface receptors of the FZD family to regulate a multitude of developmental processes. Studying selectivity between WNTs and FZDs has been hampered by the paucity of purified WNT proteins and by their apparent non-selective interactions with the FZD receptors. Here, we describe an engineered protein, called F7L6, comprised of antibody-derived single-chain variable fragments, that selectively binds to human FZD7 and the co-receptor LRP6. F7L6 potently activates WNT/β-catenin signaling in a manner similar to Wnt3a. In contrast to Wnt3a, F7L6 engages only FZD7 and none of the other FZD proteins. Treatment of human pluripotent stem (hPS) cells with F7L6 initiates transcriptional programs similar to those observed during primitive streak formation and subsequent gastrulation in the mammalian embryo. This demonstrates that selective engagement and activation of FZD7 signaling is sufficient to promote mesendodermal differentiation of hPS cells.

Background Anti-angiogenic therapy has been widely applied to the clinical treatment of malignant tumors. However, the efficacy of such treatments has been called into question, especially in triple-negative breast cancer (TNBC). Bevacizumab, the first anti-angiogenic agent approved by FDA, actually increases invasive and metastatic properties of TNBC cells, resulting from the activation of Wnt/β-catenin signaling in response to hypoxia. As a critical receptor of Wnt/β-catenin signaling, Frizzled-7 (Fzd7) is aberrantly expressed in TNBC, indicating Fzd7 a potential target for developing drugs to be combined with anti-angiogenic agents. Methods Hybridoma technique and antibody humanization technique were utilized to generate a Fzd7-targeting antibody (SHH002-hu1). Biolayer interferometry (BLI) assay and near infrared (NIR) imaging were conducted to detect the affinity and targeting ability of SHH002-hu1. Next, whether SHH002-hu1 could suppress the invasion and migration of TNBC cells induced by Bevacizumab were validated, and the underlying molecular mechanisms were elucidated by luciferase reporter and western blot assays. The nude-mice transplanted TNBC models were established to assess the anti-TNBC activities of SHH002-hu1 when combined with Bevacizumab. Then, the effects on putative TNBC stem-like cells and Wnt/β-catenin signaling were evaluated by immunofluorescence (IF). Further, the tumor-initiating and self-renew capacity of TNBC cells were studied by secondary nude mouse xenograft model and sphere formation assay. In addition, the effects of SHH002-hu1 on the adaptation of TNBC cells to hypoxia were evaluated by the detection of vasculogenic mimicry (VM) and hypoxia-inducible factor-1α (HIF-1α) transcriptional activity. Results The novel humanized antibody targeting Fzd7 (SHH002-hu1) exhibited extremely high affinity with Fzd7, and specifically targeted to Fzd7+ cells and tumor tissues. SHH002-hu1 repressed invasion, migration and epithelial-mesenchymal cell transformation (EMT) of TNBC cells induced by Bevacizumab through abating Wnt/β-catenin signaling. SHH002-hu1 significantly enhanced the capacity of Bevacizumab to inhibit the growth of TNBC via reducing the subpopulation of putative TNBC stem-like cells, further attenuating Bevacizumab-enhanced tumor-initiating and self-renew capacity of TNBC cells. Moreover, SHH002-hu1 effectively restrained the adaptation of TNBC cells to hypoxia via disrupting Wnt/β-catenin signaling. Conclusion SHH002-hu1 significantly enhances the anti-TNBC capacity of Bevacizumab, and shows the potential of preventing TNBC recurrence, suggesting SHH002-hu1 a good candidate for the synergistic therapy together with Bevacizumab.

Nuclear paraspeckles assembly transcript 1 (NEAT1) is a well-known long noncoding RNA (lncRNA) with various functions in different physiological and pathological processes. Notably, aberrant NEAT1 expression is implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease (AD). However, the molecular mechanism of NEAT1 in AD remains poorly understood. In this study, we investigated that NEAT1 regulated microtubules (MTs) polymerization via FZD3/GSK3β/p-tau pathway. Downregulation of NEAT1 inhibited Frizzled Class Receptor 3 (FZD3) transcription activity by suppressing H3K27 acetylation (H3K27Ac) at the FZD3 promoter. Our data also demonstrated that P300, an important histone acetyltransferases (HAT), recruited by NEAT1 to bind to FZD3 promoter and mediated its transcription via regulating histone acetylation. In addition, according to immunofluorescence staining of MTs, metformin, a medicine for the treatment of diabetes mellitus, rescued the reduced length of neurites detected in NEAT1 silencing cells. We suspected that metformin may play a neuroprotective role in early AD by increasing NEAT1 expression and through FZD3/GSK3β/p-tau pathway. Collectively, NEAT1 regulates microtubule stabilization via FZD3/GSK3β/P-tau pathway and influences FZD3 transcription activity in the epigenetic way.

Spag6 encodes an axoneme central apparatus protein that is required for normal flagellar and cilia motility. Recent findings suggest that Spag6 also plays a role in ciliogenesis, orientation of cilia basal feet, and planar polarity. Sensory cells of the inner ear display unique structural features that underlie their mechanosensitivity. They represent a distinctive form of cellular polarity, known as planar cell polarity (PCP). However, a role for Spag6 in the inner ear has not yet been explored. In the present study, the function of Spag6 in the inner ear was examined using Spag6-deficient mice. Our results demonstrate hearing loss in the Spag6 mutants, associated with abnormalities in cellular patterning, cell shape, stereocilia bundles, and basal bodies, as well as abnormally distributed Frizzled class receptor 6 (FZD6), suggesting that Spag6 participates in PCP regulation. Moreover, we found that the subapical microtubule meshwork was disrupted. Our observations suggest new functions for Spag6 in hearing and PCP in the inner ear.

The G protein-coupled receptors Frizzled1-10 (FZD1-10) act as molecular checkpoints mediating intracellular signaling induced by 19 mammalian, secreted Wingless/Int-1 lipoglycoproteins (WNTs). Despite the vital roles of these signaling components in health and disease, our knowledge about WNT/FZD selectivity, and the mechanisms of receptor activation and intracellular signal propagation by individual ligand/receptor pairs is limited due to the current lack of suitable biophysical techniques. Here, we developed fluorescence-based biosensors that detect WNT-induced FZD conformational changes in living cells in order to assess WNT action via FZDs at the most proximal level, i.e. the receptor conformation. By testing a panel of recombinant ligands on conformational biosensors representing all four homology clusters of FZDs, we discover yet unappreciated selectivities of WNTs to their receptors and, surprisingly, identify distinct ligand-induced receptor conformations. Furthermore, we demonstrate that FZDs can undergo conformational changes upon WNT binding without being dependent on the WNT co-receptors LRP5/6. This sensor toolbox provides an advanced platform for a thorough investigation of the 190 possible WNT/FZD pairings and for future screening campaigns targeting synthetic FZD ligands. Furthermore, our findings shed new light on the complexity of the WNT/FZD signaling system and have substantial implications for our understanding of fundamental biological processes including embryonal development and tumorigenesis.

Toxin B (TcdB) produced by Clostridioides difficile is a main pathogenicity factor that affects a variety of different cell types within the colonic mucosa. TcdB is known to utilize frizzled-1,2,7 and chondroitin sulfate proteoglycan-4 (CSPG4) as protein receptors. By using human cervical cancer cell line HeLa CSPG4 knockout (CSPG4&minus, /&minus, ) cells as well as TcdB mutants which do not bind to either CSPG4 or frizzled-1,2,7, or both, we evaluated the impact of the individual receptors for cytopathic and cytotoxic effects of TcdB. We compared TcdB from the reference strain VPI10463 (TcdBVPI) and the endemic strain R20291 (TcdBR20) which does not interact with frizzled-1,2,7. TcdBVPI devoid of CSPG4 binding (TcdBVPI &Delta, CROP) shows identical cytopathic potency as full-length TcdB in HeLa CSPG4&minus, cells, indicating that interaction with frizzled proteins is not affected in the presence of the C-terminal CROP domain. We validated CSPG4 as cellular receptor for both TcdB toxinotypes in HeLa and HEp-2 cells. By exchange of a single phenylalanine residue, 1597 with serine, we generated a mutated TcdBVPI variant (TcdBVPI F1597S) that in accordance with TcdBR20 lacks binding to frizzled-1,2,7 and showed identical potency as TcdBR20 on HeLa cells. This enabled us to estimate the respective share of CSPG4 and frizzled-1,2,7 in the cytotoxic and cytopathic effect induced by TcdB. Our data reveal that binding to frizzled-1,2,7 and to CSPG4 occurs independently and in an additive manner.