WNT5B drives osteosarcoma stemness, chemoresistance and metastasis.

BACKGROUND: Treatment for osteosarcoma, a paediatric bone cancer with no therapeutic advances in over three decades, is limited by a lack of targeted therapies. Osteosarcoma frequently metastasises to the lungs, and only 20% of patients survive 5 years after the diagnosis of metastatic disease. We found that WNT5B is the most abundant WNT expressed in osteosarcoma tumours and its expression correlates with metastasis, histologic subtype and reduced survival. METHODS: Using tumor-spheroids to model cancer stem-like cells, we performed qPCR, immunoblotting, and immunofluorescence to monitor changes in gene and protein expression. Additionally, we measured sphere size, migration and forming efficiency to monitor phenotypic changes. Therefore, we characterised WNT5B's relevance to cancer stem-like cells, metastasis, and chemoresistance and evaluated its potential as a therapeutic target. RESULTS: In osteosarcoma cell lines and patient-derived spheres, WNT5B is enriched in stem cells and induces the expression of the stemness gene SOX2. WNT5B promotes sphere size, sphere-forming efficiency, and cell proliferation, migration, and chemoresistance to methotrexate (but not cisplatin or doxorubicin) in spheres formed from conventional cell lines and patient-derived xenografts. In vivo, WNT5B increased osteosarcoma lung and liver metastasis and inhibited the glycosaminoglycan hyaluronic acid via upregulation of hyaluronidase 1 (HYAL1), leading to changes in the tumour microenvironment. Further, we identified that WNT5B mRNA and protein correlate with the receptor ROR1 in primary tumours. Targeting WNT5B through inhibition of WNT/ROR1 signalling with an antibody to ROR1 reduced stemness properties, including chemoresistance, sphere size and SOX2 expression. CONCLUSIONS: Together, these data define WNT5B's role in driving osteosarcoma cancer stem cell expansion and methotrexate resistance and provide evidence that the WNT5B pathway is a promising candidate for treating osteosarcoma patients. KEY POINTS: WNT5B expression is high in osteosarcoma stem cells leading to increased stem cell proliferation and migration through SOX2. WNT5B expression in stem cells increases rates of osteosarcoma metastasis to the lungs and liver in vivo. The hyaluronic acid degradation enzyme HYAL1 is regulated by WNT5B in osteosarcoma contributing to metastasis. Inhibition of WNT5B with a ROR1 antibody decreases osteosarcoma stemness.

Mechanisms and Physiological Roles of Polymorphisms in Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a significant pregnancy complication linked to perinatal complications and an elevated risk of future metabolic disorders for both mothers and their children. GDM is diagnosed when women without prior diabetes develop chronic hyperglycemia due to ß-cell dysfunction during gestation. Global research focuses on the association between GDM and single nucleotide polymorphisms (SNPs) and aims to enhance our understanding of GDM's pathogenesis, predict its risk, and guide patient management. This review offers a summary of various SNPs linked to a heightened risk of GDM and explores their biological mechanisms within the tissues implicated in the development of the condition.

Regulation and Function of FOXC1 in Osteoblasts.

Estrogens, which bind to estrogen receptor alpha (ERα), are important for proper bone mineral density. When women go through menopause, estrogen levels decrease, and there is a decrease in bone quality, along with an increased risk for fractures. We previously identified an enhancer near FOXC1 as the most significantly enriched binding site for estrogen receptor alpha (ERα) in osteoblasts. FOXC1 is a transcription factor belonging to a large group of proteins known as forkhead box genes and is an important regulator of bone formation. Here, we demonstrate that 17ß-estradiol (E2) increases the mRNA and protein levels of FOXC1 in primary mouse and human osteoblasts. GATA4 is a pioneer factor for ERα and it is also recruited to enhancers near Foxc1. Knockdown of Gata4 in mouse osteoblasts in vitro decreases Foxc1 expression as does knockout of Gata4 in vivo. Functionally, GATA4 and FOXC1 interact and regulate osteoblast proteins such as RUNX2, as demonstrated by ChIP-reChIP and luciferase assays. The most enriched motif in GATA4 binding sites from ChIP-seq is for FOXC1, supporting the notion that GATA4 and FOXC1 cooperate in regulating osteoblast differentiation. Together, these data demonstrate the interactions of the transcription factors ERα, GATA4, and FOXC1 to regulate each other's expression and other osteoblast differentiation genes.

GATA4 and estrogen receptor alpha bind at SNPs rs9921222 and rs10794639 to regulate AXIN1 expression in osteoblasts.

Osteoporosis is a serious public health problem that affects 200 million people worldwide. Genome-wide association studies have revealed the association between several single nucleotide polymorphisms (SNPs) near WNT/ß-catenin signaling genes and bone mineral density (BMD). The activation of ß-catenin by WNT ligands is required for osteoblast differentiation. SNP rs9921222 is an intronic variant of AXIN1 (a scaffold protein in the destruction complex that regulates ß-catenin signaling) that correlates with BMD. However, the biological mechanism of SNP rs9921222 has never been reported. Here, we show that the genotype of SNP rs9921222 correlates with the expression of AXIN1 in human osteoblasts. RNA and genomic DNA were analyzed from primary osteoblasts from 111 different individuals. Homozygous TT at rs9921222 correlates with a higher expression of AXIN1 than homozygous CC. Regional association analysis showed that rs9921222 is in high linkage disequilibrium (LD) with SNP rs10794639. In silico transcription factor analysis predicted that rs9921222 is within a GATA4 motif and rs10794639 is adjacent to an estrogen receptor alpha (ERα) motif. Mechanistically, GATA4 and ERα bind at SNPs rs9921222 and rs10794639 as detected by ChIP-qPCR. Luciferase assays demonstrate that rs9921222 is the causal SNP to alter ERα and GATA4 binding. GATA4 promoted the expression, and in contrast, ERα suppressed the expression of AXIN1 via the histone deacetylase complex member SIN3A. Functionally, the level of AXIN1 negatively correlates with the level of transcriptionally active ß-catenin. In summary, we have discovered a molecular mechanism of the SNP rs9921222 to regulate AXIN1 through GATA4 and ERα binding in human osteoblasts.

WNT5B in cellular signaling pathways.

The Wnt signaling ligand WNT5B is implicated in various developmental pathways, both in normal and pathological physiology. Most of the research on WNT5B has been associated with expression analysis and disease states, leaving the signaling pathways underexplored. Here, we review the current understandings of WNT5B's regulation of signal transduction, from receptors to downstream mediators and transcription factors. We also describe its roles in ß-catenin-dependent and ß-catenin-independent (Planar Cell Polarity and Wnt/Ca2+) Wnt signaling.

Estrogen receptor alpha and NFATc1 bind to a bone mineral density-associated SNP to repress WNT5B in osteoblasts.

Genetic factors and estrogen deficiency contribute to the development of osteoporosis. The single-nucleotide polymorphism (SNP) rs2887571 is predicted from genome-wide association studies (GWASs) to associate with osteoporosis but has had an unknown mechanism. Analysis of osteoblasts from 110 different individuals who underwent joint replacement revealed that the genotype of rs2887571 correlates with WNT5B expression. Analysis of our ChIP-sequencing data revealed that SNP rs2887571 overlaps with an estrogen receptor alpha (ERα) binding site. Here we show that 17ß-estradiol (E2) suppresses WNT5B expression and further demonstrate the mechanism of ERα binding at the enhancer containing rs2887571 to suppress WNT5B expression differentially in each genotype. ERα interacts with NFATc1, which is predicted to bind directly at rs2887571. CRISPR-Cas9 and ChIP-qPCR experiments confirm differential regulation of WNT5B between each allele. Homozygous GG has a higher binding affinity for ERα than homozygous AA and results in greater suppression of WNT5B expression. Functionally, WNT5B represses alkaline phosphatase expression and activity, decreasing osteoblast differentiation and mineralization. Furthermore, WNT5B increases interleukin-6 expression and suppresses E2-induced expression of alkaline phosphatase during osteoblast differentiation. We show that WNT5B suppresses the differentiation of osteoblasts via receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2), which activates DVL2/3/RAC1/CDC42/JNK/SIN3A signaling and inhibits ß-catenin activity. Together, our data provide mechanistic insight into how ERα and NFATc1 regulate the non-coding SNP rs2887571, as well as the function of WNT5B on osteoblasts, which could provide alternative therapeutic targets for osteoporosis.

Corrigendum: WNT5B in Physiology and Disease.

[This corrects the article DOI: 10.3389/fcell.2021.667581.].

WNT5B in Physiology and Disease.

WNT5B, a member of the WNT family of proteins that is closely related to WNT5A, is required for cell migration, cell proliferation, or cell differentiation in many cell types. WNT5B signals through the non-canonical ß-catenin-independent signaling pathway and often functions as an antagonist of canonical WNT signaling. Although WNT5B has a high amino acid identity with WNT5A and is often assumed to have similar activities, WNT5B often exhibits unique expression patterns and functions. Here, we describe the distinct effects and mechanisms of WNT5B on development, bone, adipose tissue, cardiac tissue, the nervous system, the mammary gland, the lung and hematopoietic cells, compared to WNT5A. We also highlight aberrances in non-canonical WNT5B signaling contributing to diseases such as osteoarthritis, osteoporosis, obesity, type 2 diabetes mellitus, neuropathology, and chronic diseases associated with aging, as well as various cancers.

GATA4 regulates mesenchymal stem cells via direct transcriptional regulation of the WNT signalosome.

GATA4 is a transcription factor that regulates osteoblast differentiation. However, GATA4 is expressed at a higher level in mesenchymal stem cells (MSCs) than in osteoblasts. Therefore, the role of GATA4 in limb bud mesenchyme differentiation was investigated in mice by knocking out Gata4 using Cre-recombinase controlled by the Prx1 promoter (herein called Gata4 Prx-cKO mice). µCT analysis of the Gata4 Prx-cKO mice showed a decrease in trabecular bone properties compared with wildtype (Gata4fl/fl) littermates. Gata4 Prx-cKO mice have fewer MSCs as measured by CFU-F assays, mesenchymal progenitor cells (MPC2) (flow cytometry of Sca1+/CD45-/CD34-/CD44hi) and nestin immunofluorescence. Gata4 Prx-cKO bone marrow-derived MSCs have a significant reduction in WNT ligands, including WNT10B, and WNT signalosome components compared to control cells. Chromatin immunoprecipitation demonstrates that GATA4 is recruited to enhancers near Wnt3a, Wnt10b, Fzd6 and Dkk1. GATA4 also directly represses YAP in wildtype cells, and the absence of Gata4 leads to increased YAP expression. Together, we show that the decrease in MSCs is due to loss of Gata4 and a WNT10B-dependent positive autoregulatory loop. This leads to a concurrent increase of YAP and less activated ß-catenin. These results explain the decreased trabecular bone in Gata4 Prx-cKO mice. We suggest that WNT signalosome activity in MSCs requires Gata4 and Wnt10b expression for lineage specification.

Anti-osteoporotic effects of Pueraria candollei var. mirifica on bone mineral density and histomorphometry in estrogen-deficient rats.

Although it has been clearly shown that Pueraria mirifica and its phytoestrogens can mimic estrogen in preventing bone loss, as osteoporosis is an asymptomatic disease, the therapeutic effects of P. mirifica should be acknowledged. In this study, 6-month-old female rats were ovariectomized, kept for 4 weeks to induce bone loss, divided into five groups, and treated with P. mirifica at doses of 0, 5, 25, and 50 mg/kg BW/day (PM0, PM5, PM25, and PM50 groups, respectively) or 7 mg/kg BW/day of puerarin (PU group) for 12 weeks. Only the trabecular bone mineral densities (BMDs) of tibia metaphysis (at the 12th, 14th, and 16th week) and total and trabecular BMDs of L4 (at the 16th week) of the PM50 group were significantly higher than those of the PM0 group. However, the BMDs of tibia metaphysis and L4 at the 16th week of the study period were kept significantly lower than those of the 0 week, and the BMD was also significantly lower than that of the 4th week for tibia metaphysis. The trabecular bone area (BV/TV), trabecular number (Tb.N), and osteoblast surface (Ob.S/BS) were significantly higher, and trabecular space (Tb.Sp) was significantly lower in the PM50 group, as compared with those of the PM0 group. This study indicates that P. mirifica could be used as an anti-osteoporotic agent for postmenopausal women. Since P. mirifica could mainly retain bone mass at the levels before bone loss is initiated, the use of other anabolic agents in combination with P. mirifica is recommended for osteoporotic patients.