Wnt signaling regulates chemokine production and cell migration of circulating human monocytes.

The ß-catenin dependent canonical Wnt signaling pathway plays a crucial role in maintaining normal homeostasis. However, when dysregulated, Wnt signaling is closely associated with various pathological conditions, including inflammation and different types of cancer.Here, we show a new connection between the leukocyte inflammatory response and the Wnt signaling pathway. Specifically, we demonstrate that circulating human primary monocytes express distinct Wnt signaling components and are susceptible to stimulation by the classical Wnt ligand-Wnt-3a. Although this stimulation increased the levels of ß-catenin protein, the expression of the classical Wnt-target genes was not affected. Intriguingly, treating circulating human monocytes with Wnt-3a induces the secretion of cytokines and chemokines, enhancing monocyte migration. Mechanistically, the enhanced monocyte migration in response to Wnt stimuli is mediated through CCL2, a strong monocyte-chemoattractant.To further explore the physiological relevance of these findings, we conducted ex-vivo experiments using blood samples of patients with rheumatic joint diseases (RJD) - conditions where monocytes are known to be dysfunctional. Wnt-3a generated a unique cytokine expression profile, which was significantly distinct from that observed in monocytes obtained from healthy donors.Thus, our results provide the first evidence that Wnt-3a may serve as a potent stimulator of monocyte-driven immune processes. These findings contribute to our understanding of inflammatory diseases and, more importantly, shed light on the role of a core signaling pathway in the circulation.

RIPK4 downregulation impairs Wnt3A-stimulated invasiveness via Wnt/ß-catenin signaling in melanoma cells and tumor growth in vivo.

PURPOSE: The role of Wnt signaling in oncogenesis and drug resistance is well known. Receptor-interacting protein kinase (RIPK4) contributing to the increased activity of many signaling pathways, including Wnt/ß-catenin, may be an important target for designing new drugs for metastatic melanoma, but its role in melanoma is not fully understood. METHODS: We tested the effect of genetic manipulation of RIPK4 (CRISPR/Cas9) on xenograft growth. In addition, immunohistochemistry was used to detect active ß-catenin, Ki67 and necrosis in xenografts. Wnt signaling pathway activity was examined using Western blot and Top-Flash. The effect of RIPK4 knockout on melanoma cells in vitro stimulated Wnt3A on wound overgrowth, migration and invasion ability was then evaluated. RESULTS: Our study showed that CRISPR/Cas9-mediated RIPK4 knockout (KO) significantly reduced tumor growth in a mouse model of melanoma, particularly of WM266.4 cells. RIPK4 KO tumors exhibited lower percentages of Ki67+ cells as well as reduced necrotic area and decreased levels of active ß-catenin. In addition, we observed that RIPK4 knockout impaired Wnt3A-induced activation of LRP6 and ß-catenin, as manifested by a decrease in the transcriptional activity of ß-catenin in Top-Flash in both tested melanoma cell lines, A375 and WM266.4. Prolonged incubation (48 h) with Wnt3A showed reduced level of MMP9, C-myc, and increased SOX10, proteins whose transcription is also dependent on ß-catenin activity. Moreover, RIPK4 knockout led to the inhibition of scratch overgrowth, migration and invasion of these cells compared to their controls. CONCLUSION: RIPK4 knockdown inhibits melanoma tumor growth and Wnt3A stimulated migration and invasion indicating that RIPK4 might be a potential target for melanoma therapy.

The selenoprotein P-LRP5/6-WNT3A complex promotes tumorigenesis in sporadic colorectal cancer.

Some studies suggest that the trace element selenium protects against colorectal cancer (CRC). However, the contribution of selenoprotein P (SELENOP), a unique selenocysteine-containing protein, to sporadic colorectal carcinogenesis challenges this paradigm. SELENOP is predominately secreted by the liver but is also expressed in various cells of the small intestine and colon in mice and humans. In this issue of the JCI, Pilat et al. demonstrate that increased SELENOP expression promoted the progression of conventional adenomas to carcinoma. SELENOP functioned as a modulator of canonical WNT signaling activity through interactions with WNT3A and its coreceptor LDL receptor-related protein 5/6 (LRP5/6). Secreted SELENOP formed a concentration gradient along the gut crypt axis, which might amplify WNT signaling activity by binding to LRPL5/6. The mechanism for WNT control via SELENOP may affect colorectal tumorigenesis and provide therapeutic targets for CRC.

hsa­miR­216a­3p regulates cell proliferation in oral cancer via the Wnt3a/ß­catenin pathway.

Oral cancer is one of the leading causes of death worldwide, with a reported 5­year survival rate of ~50% after treatment. The treatment measures for oral cancer are very expensive and affordability is low. Thus, it is necessary to develop more effective therapies to treat oral cancer. A number of studies have found that miRNAs are invasive biomarkers and have therapeutic potential in a variety of cancers. The present study included 30 oral patients and 30 healthy controls. Clinicopathological characteristic and miR­216a­3p/ß­catenin expression level of 30 oral cancer patients were analyzed. In addition, two oral cancer cell lines (HSC­6 and CAL­27) were used for mechanism­of­action study. The expression level of miR­216a­3p was higher in oral cancer patients compared with healthy controls and positively associated with tumor stage. Inhibition of miR­216a­3p potently suppressed cell viability and induced apoptosis of oral cancer cells. It was found that effects of miR­216a­3p on oral cancer were through Wnt3a signaling. It was also found that the expression level of ß­catenin was higher in oral cancer patients compared with healthy controls and positively associated with tumor stage; the effects of miR­216a­3p on oral cancer were through ß­catenin. In conclusion, miR­216a­3p and the Wnt­ß­catenin signaling pathway may be interesting candidates to develop effective therapies for oral cancers.

PDLIM1 inhibits cell migration and invasion in diabetic retinopathy via negatively regulating Wnt3a.

The injury of vascular endothelial cells is a crucial factor in the development of diabetic retinopathy (DR). PDLIM1 (a member of the PDZ and LIM protein family) has been reported to exert an essential function in vascular diseases. This study aimed to elucidate the role of PDLIM1 on retinal vascular endothelial cells in DR. Immunofluorescence staining was used to localize the expression of PDLIM1 in the mouse retina. In some tumor diseases, PDLIM1 has been reported to play a key role in regulating the Wnt pathway. However, no in-depth reports have been found in DR. Retinal capillary endothelial cells (RCECs) were treated with high-glucose and high-lipid (HG/HL) culture medium, and siRNA transfection to investigate the role of PDLIM1 in DR. PDLIM1 and Wnt3a expression was confirmed by qRT-PCR and western blotting. Flow cytometry, Transwell assay, and scratch assay were used to test the ability of cell apoptosis, migration, and invasion. PDLIM1 was mainly expressed in the retinal pigment epithelium (RPE), ganglion cell layer (GCL), inner plexus layer (IPL), and outer plexus layer (OPL). HG/HL increased Wnt3a levels and promoted cell's ability of apoptosis, migration, and invasion, which were reversed by the knockdown of PDLIM1. PDLIM1 was found to play a protective role in diabetic retinopathy by counter-regulating Wnt3a. PDLIM1 ameliorates cell apoptosis, migration, and invasion by negatively regulating Wnt3a in RCECs of DR, which suggests that PDLIM1 might be a promising therapeutic target for DR treatment.

WNT3a Signaling Inhibits Aromatase Expression in Breast Adipose Fibroblasts-A Possible Mechanism Supporting the Loss of Estrogen Responsiveness of Triple-Negative Breast Cancers.

Estrogen-dependent breast cancers rely on a constant supply of estrogens and expression of estrogen receptors. Local biosynthesis, by aromatase in breast adipose fibroblasts (BAFs), is their most important source for estrogens. Triple-negative breast cancers (TNBC) rely on other growth-promoting signals, including those from the Wnt pathway. In this study, we explored the hypothesis that Wnt signaling alters the proliferation of BAFs, and is involved in regulation of aromatase expression in BAFs. Conditioned medium (CM) from TNBC cells and WNT3a consistently increased BAF growth, and reduced aromatase activity up to 90%, by suppression of the aromatase promoter I.3/II region. Database searches identified three putative Wnt-responsive elements (WREs) in the aromatase promoter I.3/II. In luciferase reporter gene assays, promoter I.3/II activity was inhibited by overexpression of full-length T-cell factor (TCF)-4 in 3T3-L1 preadipocytes, which served as a model for BAFs. Full-length lymphoid enhancer-binding factor (LEF)-1 increased the transcriptional activity. However, TCF-4 binding to WRE1 in the aromatase promoter, was lost after WNT3a stimulation in immunoprecipitation-based in vitro DNA-binding assays, and in chromatin immunoprecipitation (ChIP). In vitro DNA-binding assays, ChIP, and Western blotting revealed a WNT3a-dependent switch of nuclear LEF-1 isoforms towards a truncated variant, whereas ß-catenin levels remained unchanged. This LEF-1 variant revealed dominant negative properties, and most likely recruited enzymes involved in heterochromatin formation. In addition, WNT3a induced the replacement of TCF-4 by the truncated LEF-1 variant, on WRE1 of the aromatase promoter I.3/II. The mechanism described here may be responsible for the loss of aromatase expression predominantly associated with TNBC. Tumors with (strong) expression of Wnt ligands actively suppress aromatase expression in BAFs. Consequently a reduced estrogen supply could favor the growth of estrogen-independent tumor cells, which consequently would make estrogen receptors dispensable. In summary, canonical Wnt signaling within (cancerous) breast tissue may be a major factor controlling local estrogen synthesis and action.

Klotho ameliorates angiotension-II-induced endothelial senescence via restoration of autophagy by inhibiting Wnt3a/GSK-3ß/mTOR signaling: A potential mechanism involved in prognostic performance of Klotho in coronary atherosclerotic disease.

OBJECTIVE: We aimed to evaluate the prognostic performance of circulating Klotho in coronary atherosclerotic disease (CAD), and to further explore the effect of Klotho on stress-mediated endothelial senescence and underlying mechanism. METHODS: A cohort of 295 patients had a 12-month follow-up for major adverse cardiovascular events (MACE). Serum Klotho was detected by enzyme linked immunosorbent assay. Cell viability, SA-ß-Gal staining, the expression of P53 and P16 were analyzed for endothelial senescence. Oxidative stress was evaluated by measurement of reactive oxygen species, superoxide dismutase and malondialdehyde. LC3, P62, Wnt3a, GSK-3ß and mTOR were analyzed by western blotting. Autophagosome formation was detected by adenovirus transfection. RESULTS: In epidemiological analysis, low Klotho (≤295.9 pg/ml) was significantly associated with MACE risk (HR=2.266, 95 %CI 1.229-4.176). In experimental analysis, Klotho alleviated endothelial senescence and oxidative stress caused by Ang-II exposure; Klotho restored impaired autophagic flux to ameliorate Ang-II induced endothelial senescence; Ang-II activated Wnt3a/GSK-3ß/mTOR signaling to inhibit autophagy, whereas Klotho restored autophagy through blockade of Wnt3a/GSK-3ß/mTOR signaling; Klotho ameliorated endothelial senescence by suppressing Wnt3a/GSK-3ß/mTOR pathway under Ang-II exposure. CONCLUSIONS: Prognostic significance of Klotho in CAD is potentially ascribed to its anti-endothelial senescence effect via autophagic flux restoration by inhibiting Wnt3a/ GSK-3ß/mTOR signaling.

Wnt3a is a promising target in colorectal cancer.

Most colorectal cancers (CRC) are associated with activated Wnt signaling, making it the fourth most prevalent type of cancer globally. To function properly, the Wnt signaling pathway requires secreted glycoproteins known as Wnt ligands (Wnts). Humans have 19 Wnts, which suggest a complicated signaling and biological process, and we still know little about their functions in developing CRC. This review aims to describe the canonical Wnt signaling in CRC, particularly the Wnt3a expression pattern, and their association with the angiogenesis and progression of CRC. This review also sheds light on the inhibition of Wnt3a signaling in CRC. Despite some obstacles, a thorough understanding of Wnts is essential for effectively managing CRC.

Oncogenic Wnt3a is a promising sensitive biomarker for monitoring hepatocarcinogenesis.

BACKGROUND: The effective treatment for hepatocellular carcinoma (HCC) depends on early diagnosis. Previously, the abnormal expression of Wnt3a as the key signaling molecule in the Wnt/ß-catenin pathway was found in HCC cells and could be released into the circulation. In this study, we used rat model of hepatocarcinogenesis to dynamically investigate the alteration of oncogenic Wnt3a and to explore its early monitor value for HCC. METHODS: Sprague-Dawley rats (SD) were fed with diet 2-fluorenylacetamide (2-FAA, 0.05%) for inducing hepatocarcinogenesis, and grouped based on liver morphological alteration by Hematoxylin & Eosin (H&E) staining; rats fed with normal chow were used as normal control (NC). Total RNA and protein were purified from rat livers. Differently expressed genes (DEGs) or Wnt3a mRNA, cellular distribution, and Wnt3a protein levels were analyzed by whole genome microarray with signal logarithm ratio (SLR log2cy5/cy3), immunohistochemistry, and enzyme-linked immunosorbent assay, respectively. RESULTS: Models of rat hepatocarcinogenesis were successfully established based on liver histopathological H&E staining. Rats were divided into the cell degeneration (rDeg), precancerosis (rPre-C) and HCC (rHCC) groups. Total numbers of the up- and down-regulated DEGs with SLR ≥ 8 were 55 and 48 in the rDeg group, 268 and 57 in the rPre-C group, and 312 and 201 in the rHCC group, respectively. Significantly altered genes were involved in cell proliferation, signal transduction, tumor metastasis, and apoptosis. Compared with the NC group, Wnt3a mRNA was increased by 4.6 folds (P < 0.001) in the rDeg group, 7.4 folds (P < 0.001) in the rPre-C group, and 10.4 folds (P < 0.001) in the rHCC group; the positive rates of liver Wnt3a were 66.7% (P = 0.001) in the rDeg group, 100% (P < 0.001) in the rPre-C group, and 100% (P < 0.001) in the rHCC group, respectively. Also, there were significant differences of liver Wnt3a (P < 0.001) or serum Wnt3a (P < 0.001) among different groups. CONCLUSIONS: Overexpression of Wnt3a was associated with rat hepatocarcinogenesis and it should be expected to be a promising monitoring biomarker for HCC occurrence at early stage.

Tumor Suppressor miRNA-503 Inhibits Cell Invasion in Head and Neck Cancer through the Wnt Signaling Pathway via the WNT3A/MMP Molecular Axis.

Head and neck cancer (HNC) is the fifth most common cancer worldwide, and its incidence and death rates have been consistently high throughout the past decades. MicroRNAs (miRNAs) have recently gained significant attention because of their role in the regulation of a variety of biological processes via post-transcriptional silencing mechanisms. Previously, we determined a specific profile of miRNAs associated with HNC using a miRNA microarray analysis. Of the 23 miRNAs with highly altered expression in HNC cells, miR-503 was the most significantly downregulated miRNA. In this study, we confirmed that miR-503 acts as a tumor suppressor, as our results showed decreased levels of miR-503 in cancer cells and patients with HNC. We further characterized the role of miR-503 in the malignant functions of HNC. Although there was a minimal effect on cell growth, miR-503 was found to inhibit cellular invasion significantly. Algorithm-based studies identified multiple potential target genes and pathways associated with oncogenic mechanisms. The candidate target gene, WNT3A, was confirmed to be downregulated by miR-503 at both the mRNA and protein levels and validated by a reporter assay. Furthermore, miR-503 modulated multiple invasion-associated genes, including matrix metalloproteinases (MMPs), through the Wnt downstream signaling pathway. Overall, this study demonstrates that miR-503 suppresses HNC malignancy by inhibiting cell invasion through the Wnt signaling pathway via the WNT3A/MMP molecular axis. The modulation of miR-503 may be a novel therapeutic approach to intervene in cancer invasion.

Synergistic effect of anticancer drug resistance and Wnt3a on primary ciliogenesis in A549 cell-derived anticancer drug-resistant subcell lines.

Primary cilia, antenna-like cellular sensor structures, are generated from the mother centriole in the G0/G1 cell-cycle phase under control by cellular signaling pathways involving Wnt, hedgehog, and platelet-derived growth factor. Although primary ciliary dynamics have been reported to be closely related to ciliopathy and tumorigenesis, the molecular basis for the role of primary cilia in human disease is lacking. To clarify how Wnt3a affects primary ciliogenesis in anticancer drug-resistant cells, we derived specific drug-resistant subcell lines from A549 human lung cancer cells using anticancer drugs doxorubicin, dasatinib, and paclitaxel (A549/Dox, A549/Das, and A549/Pac, respectively). The primary cilia-containing cell population and primary cilia length increased in the A549/Dox and A549/Pac subcell lines under increased MDR1 expression, when compared to those in the parental A549 cells. In the A549/Das subcell line, primary cilia length increased but the cell population was not affected. In addition, Wnt3a increased primary cilia-containing cell population and primary cilia length in A549/Dox, A549/Das, and A549/Pac cells, without change of cell growth. Abnormal shapes of primary cilia were frequently observed by anticancer drug resistance and Wnt3a stimulation. Taken together, our results indicate that anticancer drug resistance and Wnt3a affect primary ciliogenesis synergistically, suggesting a potential new strategy for overcoming anticancer drug resistance.

Mechanisms and inhibition of Porcupine-mediated Wnt acylation.

Wnt signalling is essential for regulation of embryonic development and adult tissue homeostasis1-3, and aberrant Wnt signalling is frequently associated with cancers4. Wnt signalling requires palmitoleoylation on a hairpin 2 motif by the endoplasmic reticulum-resident membrane-bound O-acyltransferase Porcupine5-7 (PORCN). This modification is indispensable for Wnt binding to its receptor Frizzled, which triggers signalling8,9. Here we report four cryo-electron microscopy structures of human PORCN: the complex with the palmitoleoyl-coenzyme A (palmitoleoyl-CoA) substrate; the complex with the PORCN inhibitor LGK974, an anti-cancer drug currently in clinical trials10; the complex with LGK974 and WNT3A hairpin 2 (WNT3Ap); and the complex with a synthetic palmitoleoylated WNT3Ap analogue. The structures reveal that hairpin 2 of WNT3A, which is well conserved in all Wnt ligands, inserts into PORCN from the lumenal side, and the palmitoleoyl-CoA accesses the enzyme from the cytosolic side. The catalytic histidine triggers the transfer of the unsaturated palmitoleoyl group to the target serine on the Wnt hairpin 2, facilitated by the proximity of the two substrates. The inhibitor-bound structure shows that LGK974 occupies the palmitoleoyl-CoA binding site to prevent the reaction. Thus, this work provides a mechanism for Wnt acylation and advances the development of PORCN inhibitors for cancer treatment.

Pulsed electromagnetic fields inhibit mandibular bone deterioration depending on the Wnt3a/ß-catenin signaling activation in type 2 diabetic db/db mice.

Type 2 diabetes mellitus (T2DM) patients have compromised mandibular bone architecture/quality, which markedly increase the risks of tooth loosening, tooth loss, and failure of dental implantation. However, it remains lacks effective and safe countermeasures against T2DM-related mandibular bone deterioration. Herein, we studied the effects of pulsed electromagnetic fields (PEMF) on mandibular bone microstructure/quality and relevant regulatory mechanisms in T2DM db/db mice. PEMF exposure (20 Gs, 15 Hz) for 12 weeks preserved trabecular bone architecture, increased cortical bone thickness, improved material properties and stimulated bone anabolism in mandibles of db/db mice. PEMF also upregulated the expression of canonical Wnt3a ligand (but not Wnt1 or Wnt5a) and its downstream ß-catenin. PEMF improved the viability and differentiation of primary osteoblasts isolated from the db/db mouse mandible, and stimulated the specific activation of Wnt3a/ß-catenin signaling. These positive effects of PEMF on mandibular osteoblasts of db/db mice were almost totally abolished after Wnt3a silencing in vitro, which were equivalent to the effects following blockade of canonical Wnt signaling using the broad-spectrum antagonist DKK1. Injection with Wnt3a siRNA abrogated the therapeutic effects of PEMF on mandibular bone quantity/quality and bone anabolism in db/db mice. Our study indicates that PEMF might become a non-invasive and safe treatment alternative resisting mandibular bone deterioration in T2DM patients, which is helpful for protecting teeth from loosening/loss and securing the dental implant stability.

Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/ß-catenin signaling in obstructed kidneys in vivo.

Follistatin (FS)-like 1 (FSTL1) is a member of the FS-SPARC (secreted protein, acidic and rich in cysteine) family of secreted and extracellular matrix proteins. The functions of FSTL1 have been studied in heart and lung injury as well as in wound healing; however, the role of FSTL1 in the kidney is largely unknown. Here, we show using single-cell RNA-Seq that Fstl1 was enriched in stromal cells in obstructed mouse kidneys. In addition, immunofluorescence demonstrated that FSTL1 expression was induced in fibroblasts during kidney fibrogenesis in mice and human patients. We demonstrate that FSTL1 overexpression increased renal fibrosis and activated the Wnt/ß-catenin signaling pathway, known to promote kidney fibrosis, but not the transforming growth factor ß (TGF-ß), Notch, Hedgehog, or Yes-associated protein (YAP) signaling pathways in obstructed mouse kidneys, whereas inhibition of FSTL1 lowered Wnt/ß-catenin signaling. Importantly, we show that FSTL1 interacted with Wnt ligands and the Frizzled (FZD) receptors but not the coreceptor lipoprotein receptor-related protein 6 (LRP6). Specifically, we found FSTL1 interacted with Wnt3a through its extracellular calcium-binding (EC) domain and von Willebrand factor type C-like (VWC) domain, and with FZD4 through its EC domain. Furthermore, we show that FSTL1 increased the association of Wnt3a with FZD4 and promoted Wnt/ß-catenin signaling and fibrogenesis. The EC domain interacting with both Wnt3a and FZD4 also enhanced Wnt3a signaling. Therefore, we conclude that FSTL1 is a novel extracellular enhancer of the Wnt/ß-catenin pathway.

Wnt3a but not CDX-2 expression is associated with differentiated thyroid cancer.

OBJECTIVE: Thyroid neoplasm incidence has increased worldwide, mostly due to the advancements in medical imaging and screening rates. The aberrant Wnt/ß-catenin pathway has been identified as a key mechanism, and it has also been related to the metastatic activity of differentiated thyroid cancer. We aimed to verify the difference in the expression of Wnt3a, a canonical activator of the ß-catenin signaling, and CDX-2, a transcription factor upregulated by Wnt/ß-catenin pathway, in multinodular goiter and differentiated thyroid cancer and to determine their prognostic value. METHODS: We included 194 thyroid tissue surgical specimen and their clinicopathological data: study group (differentiated thyroid cancer, n=154) and control group (multinodular goiter, n=40). Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tissue by the primary antibodies Wnt3a and CDX-2. RESULTS: High Wnt3a expression was significantly associated with differentiated thyroid cancer (p=0.031). CDX-2 was negative in all differentiated thyroid cancer cases (100%) and also in multinodular goiter. Wnt3a expression was significantly associated with tumors ≤20 mm (p=0.044) and with the absence of capsule invasion (p=0.031). The multivariate analyses suggested that older age (≥55), independent of capsular invasion and tumor size, was an independent prognostic factor for Wnt3a expression (p=0.058). CONCLUSIONS: Wnt3a expression but not CDX-2 is correlated with differentiated thyroid cancer samples in comparison to multinodular goiter. Although its prognostic value was limited to tumor size and capsule invasion, a combined model in a panel of immune markers can add accuracy in the classification of challenging thyroid follicular-derived lesions.

Induction of Pro-Fibrotic CLIC4 in Dermal Fibroblasts by TGF-ß/Wnt3a Is Mediated by GLI2 Upregulation.

Chloride intracellular channel 4 (CLIC4) is a recently discovered driver of fibroblast activation in Scleroderma (SSc) and cancer-associated fibroblasts (CAF). CLIC4 expression and activity are regulated by TGF-ß signalling through the SMAD3 transcription factor. In view of the aberrant activation of canonical Wnt-3a and Hedgehog (Hh) signalling in fibrosis, we investigated their role in CLIC4 upregulation. Here, we show that TGF-ß/SMAD3 co-operates with Wnt3a/ß-catenin and Smoothened/GLI signalling to drive CLIC4 expression in normal dermal fibroblasts, and that the inhibition of ß-catenin and GLI expression or activity abolishes TGF-ß/SMAD3-dependent CLIC4 induction. We further show that the expression of the pro-fibrotic marker α-smooth muscle actin strongly correlates with CLIC4 expression in dermal fibroblasts. Further investigations revealed that the inhibition of CLIC4 reverses morphogen-dependent fibroblast activation. Our data highlights that CLIC4 is a common downstream target of TGF-ß, Hh, and Wnt-3a through signalling crosstalk and we propose a potential therapeutic avenue using CLIC4 inhibitors.

WNT/beta-catenin signalling interrupts a senescence-induction cascade in human mesenchymal stem cells that restricts their expansion.

Senescence, the irreversible cell cycle arrest of damaged cells, is accompanied by a deleterious pro-inflammatory senescence-associated secretory phenotype (SASP). Senescence and the SASP are major factors in aging, cancer, and degenerative diseases, and interfere with the expansion of adult cells in vitro, yet little is known about how to counteract their induction and deleterious effects. Paracrine signals are increasingly recognized as important senescence triggers and understanding their regulation and mode of action may provide novel opportunities to reduce senescence-induced inflammation and improve cell-based therapies. Here, we show that the signalling protein WNT3A counteracts the induction of paracrine senescence in cultured human adult mesenchymal stem cells (MSCs). We find that entry into senescence in a small subpopulation of MSCs triggers a secretome that causes a feed-forward signalling cascade that with increasing speed induces healthy cells into senescence. WNT signals interrupt this cascade by repressing cytokines that mediate this induction of senescence. Inhibition of those mediators by interference with NF-κB or interleukin 6 signalling reduced paracrine senescence in absence of WNT3A and promoted the expansion of MSCs. Our work reveals how WNT signals can antagonize senescence and has relevance not only for expansion of adult cells but can also provide new insights into senescence-associated inflammatory and degenerative diseases.

Common genetic variation in alcohol-related hepatocellular carcinoma: a case-control genome-wide association study.

BACKGROUND: Hepatocellular carcinoma is a frequent consequence of alcohol-related liver disease, with variable incidence among heavy drinkers. We did a genome-wide association study (GWAS) to identify common genetic variants for alcohol-related hepatocellular carcinoma. METHODS: We conducted a two-stage case-control GWAS in a discovery cohort of 2107 unrelated European patients with alcohol-related liver disease aged 20-92 years recruited between Oct 22, 1993, and March 12, 2017. Cases were patients with alcohol-related hepatocellular carcinoma diagnosed by imaging or histology. Controls were patients with alcohol-related liver disease without hepatocellular carcinoma. We used an additive logistic regression model adjusted for the first ten principal components to assess genetic variants associated with alcohol-related hepatocellular carcinoma. We did another analysis with adjustment for age, sex, and liver fibrosis. New candidate associations (p<1 × 10-6) and variants previously associated with alcohol-related hepatocellular carcinoma were evaluated in a validation cohort of 1933 patients with alcohol-related liver disease aged 29-92 years recruited between July 21, 1995, and May 2, 2019. We did a meta-analysis of the two case-control cohorts. FINDINGS: The discovery cohort included 775 cases and 1332 controls. Of 7 962 325 variants assessed, we identified WNT3A-WNT9A (rs708113; p=1·11 × 10-8) and found support for previously reported regions associated with alcohol-related hepatocellular carcinoma risk at TM6SF2 (rs58542926; p=6·02 × 10-10), PNPLA3 (rs738409; p=9·29 × 10-7), and HSD17B13 (rs72613567; p=2·49 × 10-4). The validation cohort included 874 cases and 1059 controls and three variants were replicated: WNT3A-WNT9A (rs708113; p=1·17 × 10-3), TM6SF2 (rs58542926; p=4·06 × 10-5), and PNPLA3 (rs738409; p=1·17 × 10-4). All three variants reached GWAS significance in the meta-analysis: WNT3A-WNT9A (odds ratio 0·73, 95% CI 0·66-0·81; p=3·93 × 10-10), TM6SF2 (1·77, 1·52-2·07; p=3·84×10-13), PNPLA3 (1·34, 1·22-1·47; p=7·30 × 10-10). Adjustment for clinical covariates yielded similar results. We observed an additive effect of at-risk alleles on alcohol-related hepatocellular carcinoma. WNT3A-WNT9A rs708113 was not associated with liver fibrosis. INTERPRETATION: WNT3A-WNT9A is a susceptibility locus for alcohol-related hepatocellular carcinoma, suggesting an early role of the Wnt-ß-catenin pathway in alcohol-related hepatocellular carcinoma carcinogenesis. FUNDING: Ligue Nationale contre le Cancer, Bpifrance, INSERM, AFEF, CARPEM, Labex OncoImmunology, and Agence Nationale de la Recherche.

Rhizoma drynariae total flavonoids combined with calcium carbonate ameliorates bone loss in experimentally induced Osteoporosis in rats via the regulation of Wnt3a/ß-catenin pathway.

BACKGROUND: Rhizoma drynariae, a traditional Chinese herb, is commonly used in treatment of bone healing in osteoporotic fractures. However, whether the Rhizoma drynariae total flavonoids (RDTF) can promote the absorption of calcium and enhance the bone formation is unclear. The aim of the present study was to investigate the preventive effects of RDTF combined with calcium carbonate (CaCO3) on estrogen deficiency-induced bone loss. METHODS: Three-month-old Sprague-Dawley rats were ovariectomized (OVX) and then treated with CaCO3, RDTF, and their admixtures for ten weeks, respectively. The bone trabecular microstructure, bone histopathological examination, and serum biomarkers of bone formation and resorption were determined in the rat femur tissue. The contents of osteoprotegerin (OPG), receptor activator of the NF-κB (RANK), and its ligand (RANKL) in marrow were analyzed by ELISA, and the protein expressions of Wnt3a, ß-catenin, and phosphorylated ß-catenin (p-ß-catenin) were analyzed by Western blot. Statistical analysis was conducted by using one-way analysis of variance (ANOVA) followed by LSD post hoc analysis or independent samples t test using the scientific statistic software SPSS version 20.0 RESULTS: RDTF combined with CaCO3 could promote osteosis and ameliorate bone loss to improve the repair of cracked bone trabeculae of OVX rats. Furthermore, RDTF combined with CaCO3 also could prevent OVX-induced decrease in collagen fibers in the femoral tissue of ovariectomized rats and promote the regeneration of new bone or cartilage tissue, while CaCO3 supplementation promoted the increase in bone mineral content. Nevertheless, there was no difference in the expression of Wnt3a, ß-catenin and p-ß-catenin between osteopenic rats and RDTF treated rats, but RDTF combined with CaCO3 could activate the Wnt3a/ß-catenin pathway. CONCLUSIONS: RDTF combined with CaCO3 could ameliorate estrogen deficiency-induced bone loss via the regulation of Wnt3a/ß-catenin pathway.