Wnt Pathway Genes Research Articles

MRNA coexpression patterns of Wnt pathway components and their clinicopathological associations in breast and colorectal cancer

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.

Aberrant Signaling Pathways in Sinonasal Intestinal-Type Adenocarcinoma.

Simple SummaryThe majority of patients with sinonasal intestinal-type adenocarcinoma are wood and leather workers. However, the genetic changes that lead to these tumors are not well known. We analyzed 50 tumors for mutations in a set of 120 genes that may be involved in causing this cancer type. We found that 72% of cases carried mutations in genes that are active in Wnt, DNA damage response, MAPK or PI3K signaling pathways. Pathway activation was not related to mutations of genes in these pathways, except for nuclear β-catenin expression to Wnt pathway mutation. No specific gene mutation, mutated pathway, nor pathway activity level was associated with histological subtype, clinical data or survival. Finally, none of the identified mutated genes occurred in such frequency as to be considered a characteristic genetic feature of sinonasal intestinal-type adenocarcinoma.Sinonasal intestinal-type adenocarcinoma (ITAC) is strongly related to occupational exposure to wood and leather dust, however, little is known on the genetic alterations involved in tumor development and progression. The aim of this study was to identify tumorigenic signaling pathways affected by gene mutations and their relation to clinical features. We applied whole exome sequencing of 120 cancer-related genes in 50 ITACs and analyzed the signaling activity of four specific pathways frequently affected by mutations. Genes involved in DNA damage response showed somatic mutations in 30% of cases, including four tumors that also harbored germline mutations. Genes in Wnt, MAPK and PI3K pathways harbored mutations in 20%, 20% and 24% of cases, respectively. Mutations and copy number gains in receptor tyrosine kinases possibly affecting MAPK and PI3K pathways occurred in 44% of cases. Expression of key pathway proteins showed no correlation to mutations in these pathways, except for nuclear β-catenin and APC/CTNNB1 mutation. No specific gene mutation, mutated pathway, nor pathway activity level showed correlation to clinical data or survival. In addition, a similar mutational profile was observed among histological subtypes. The wide spectrum of gene mutations suggests that ITAC is a genetically heterogeneous without specific characterizing gene mutations.

Genistein-induced Anticancer Effects on Acute Leukemia Cells Involve the Regulation of Wnt Signaling Pathway Through H4K20me1 Rather Than DNA Demethylation

ObjectiveTo investigate the effects and mechanisms of genistein on the gene expression in the Wnt pathway in acute leukemia (AL) cells.MethodsThe expression of Wnt pathway genes and cell cycle-related genes were analyzed in two AL cell lines. Pyrophosphate sequencing was performed to determine the methylation degree. Then, the enrichment of H4K20me1 and H3K9ac was determined using ChIP-qPCR. Flow cytometry was used to analyze the cell cycle.ResultsThe IC50 of genistein in the two AL cell lines was lower than that for the bone marrow mesenchymal stem cell line. Genistein upregulated H4K20me1, KMT5A and Wnt suppressor genes, including Wnt5a, and downregulated the downstream target genes of Wnt, such as c-myc and β-catenin. The methylation degree and H3K9ac enrichment in the Wnt5a promoter region remained unchanged. However, the enrichment of H4K20me1 in the Wnt5a promoter and coding regions increased. In addition, genistein upregulated Phospho-cdc2, Myt1, Cyclin A, Cyclin E2, p21 and Phospho-histone H3, but downregulated Phospho-wee1. Cell cycle arrest was induced in the G2/M phase.ConclusionGenistein inhibits the activation of the Wnt pathway by promoting the expression of Wnt5a through the activation of KMT5A and enrichment of H4K20me1 in the Wnt5a gene promoter and coding regions, rather than demethylation. Genistein also blocks the cell cycle in the G2/M phase. Therefore, genistein is a potential anti-leukemia drug.

The genetic polymorphisms of key genes in WNT pathway (LRP5 and AXIN1) was associated with osteoporosis susceptibility in Chinese Han population.

Genetic factors play a critical role in the pathogenesis of osteoporosis. The imbalance of WNT/β-catenin will cause the occurrence of osteoporosis. LRP5 and AXIN1 play an important role in the classical Wnt/β-catenin signaling pathway. Our study was aimed to determine the association between five candidate single nucleotide polymorphisms (SNPs) of LRP5 or AXIN1 and osteoporosis susceptibility in Chinese Han population. A total of 599 osteoporosis patients and 599 healthy individuals were recruited for this case-control study. Agena MassARRAY was used to genotype SNPs. The association between SNPs and osteoporosis susceptibility in different genetic models was analyzed by PLINK software. We used false-positive report probability (FPRP) analysis to detect whether the positive results were just chance or noteworthy observations. Multifactor dimension reduction (MDR) was used to analyze the interaction of SNP-SNP in the osteoporosis risk. Finally, haplotype analysis was performed by plink1.07 and Haploview software. We found that LRP5 rs11228240, AXIN1 rs2301522, and rs9921222 were significantly associated with the osteoporosis susceptibility. The results of subgroup analysis showed that LRP5 rs11228240 (protective factor) and AXIN1 rs2301522 (risk factor) were associated with the susceptibility of osteoporosis among participants who were age >60 years, female or BMI ≤ 24; AXIN1 rs9921222 significantly increased the risk of osteoporosis among participants with BMI ≤ 24. The genotype Ars2301522Crs9921222 could increase the susceptibility of osteoporosis (p = 0.026). The rs11228219LPR5, rs11228240 LPR5, rs2301522AXIN1, and rs9921222AXIN1 four-site model was the best model for predicting the osteoporosis risk (test accuracy = 0.541; CVC = 10/10). The LRP5-rs11228240, AXIN1-rs2301522, and AXIN1- rs9921222 were associated with osteoporosis susceptibility in Chinese Han population.

Cardiomyocyte-specific deletion of \u03b2-catenin protects mouse hearts from ventricular arrhythmias after myocardial infarction

Wnt/β-catenin signaling is activated in the heart after myocardial infarction (MI). This study aims to investigate if β-catenin deletion affects post-MI ion channel gene alterations and ventricular tachycardias (VT). MI was induced by permanent ligation of left anterior descending artery in wild-type (WT) and cardiomyocyte-specific β-catenin knockout (KO) mice. KO mice showed reduced susceptibility to VT (18% vs. 77% in WT) at 8 weeks after MI, associated with reduced scar size and attenuated chamber dilation. qPCR analyses of both myocardial tissues and purified cardiomyocytes demonstrated upregulation of Wnt pathway genes in border and infarct regions after MI, including Wnt ligands (such as Wnt4) and receptors (such as Fzd1 and Fzd2). At 1 week after MI, cardiac sodium channel gene (Scn5a) transcript was reduced in WT but not in KO hearts, consistent with previous studies showing Scn5a inhibition by Wnt/β-catenin signaling. At 8 weeks after MI when Wnt genes have declined, Scn5a returned to near sham levels and K+ channel gene downregulations were not different between WT and KO mice. This study demonstrated that VT susceptibility in the chronic phase after MI is reduced in mice with cardiomyocyte-specific β-catenin deletion primarily through attenuated structural remodeling, but not ion channel gene alterations.

Upgrading Hepatic Differentiation and Functions on 3D Printed Silk-Decellularized Liver Hybrid Scaffolds.

We developed hybrid liver-specific three-dimensional (3D) printed scaffolds using a solubilized native decellularized liver (DCL) matrix and silk fibroin (SF) and investigated their ability to support functional cultures of hepatic cells. Rat livers were decellularized by perfusing detergents via the portal vein, solubilized using pepsin to form DCL, and characterized. SF blended with gelatin (8% w/v) was optimized with varying percentages of DCL to obtain silk gelatin-DCL bioink (SG-DCL). Different compositions of SG-DCL were studied by rheology for optimum versatility and print fidelity. 3D printed six-layered scaffolds were fabricated using a sophisticated direct-write 3D bioprinter. Huh7 cells were cultured on the 3D printed scaffolds for 3 weeks. 3D printed SG scaffolds without DCL along with 2D films (SG and SG-DCL) and 2D culture on tissue culture Petri dish control were used for comparative studies. The DCL matrix showed the absence of cells in histology and SEM. The combined SG-DCL ink at all of the studied DCL percentages (1-10%) revealed shear-thinning behavior in the printable range. The storage modulus value for the SG-DCL ink at all DCL percentages was higher than the loss modulus. In comparison to 2D controls, hepatic cells cultured on 3D SG-DCL revealed increased proliferation until 2 weeks and an upregulated expression of hepatocyte markers, including asialoglycoprotein receptor 1 (ASGR1). The Wnt pathway gene β-catenin was upregulated by more than 4-fold in 3D SG-DCL on day 3, while it showed a decline on day 7 as compared to 3D SG and also 2D controls. The expression of the epithelial cell adhesion molecule (EpCAM) was however lower in both 2D SG-DCL (2-fold) and 3D SG-DCL (2.5-fold) as compared to that in 2D controls. Immunofluorescence studies validated the protein expression of ASGR1 in 3D SG-DCL. Albumin (ALB) was not identified on SG scaffolds but prominently expressed in 3D SG-DCL constructs. In comparison to 2D SG, both ALB (1.8-fold) and urea (5-fold) were enhanced in cells cultured on 3D SG-DCL on day 7 of culture. Hence, the SG-DCL 3D printed scaffolds provide a conducive microenvironment for elevating differentiation and functions of hepatic cells possibly through an involvement of the Wnt/β-catenin signaling pathway.

Gastrointestinal symptoms in patients with isolated oligodontia and a Wnt gene mutation.

Since Wnt signaling plays an important role in both tooth agenesis and altered intestine homeostasis, the aim was to compare gastrointestinal symptoms in patients with isolated oligodontia caused by a Wnt pathway gene mutation and controls. A case-control study was designed to compare self-reported gastrointestinal symptoms among patients with isolated oligodontia, caused by a Wnt signaling gene mutation, and fully dentate controls. The Gastrointestinal Symptom Rating Scale (GSRS) was used to assess gastrointestinal symptoms. Prevalence and severity of gastrointestinal symptoms among patients and age- and gender-matched controls were evaluated. Twenty patients with isolated oligodontia and a pathogenic variant in the wnt pathway genes WNT10A, LRP6, or PAX9 participated. The prevalence of gastrointestinal symptoms was higher in the oligodontia patients compared to their controls (Χ2 (1)=87.33, p=.008). Mean GSRS total scores (p=.011) and domain scores for "abdominal pain" (p=.022), "reflux" (p=.003) and constipation (p=.030) were higher for these oligodontia patients compared to their controls. Gastrointestinal symptoms are more prevalent and more severe in patients with isolated oligodontia and a deficiency in a Wnt pathway-related gene, when compared to controls without tooth agenesis.

Braf mutation induces rapid neoplastic transformation in the aged and aberrantly methylated intestinal epithelium

ObjectiveSessile serrated lesions (SSLs) are common across the age spectrum, but the BRAF mutant cancers arising occur predominantly in the elderly. Aberrant DNA methylation is uncommon in SSL from young...

Inhibition of WHSC1 As a Promising Therapeutic Strategy in Childhood ALL

Childhood acute lymphoblastic leukemia (cALL) is the most frequent pediatric cancer, accounting for ~25% of all pediatric tumors. Nearly 20% of patients do not respond to current treatments, making cALL one of the leading cause of disease-related mortality amongst children. Moreover, sizable portion of childhood leukemia survivors develop significant long-term or late effects of treatment. This rate of refractory/relapse cases and the increased risk of chronic morbidity and early mortality among survivors highlight the pressing need for new treatment strategies in cALL. These can be developed through in-depth investigation of the genetic architecture underlying cALL. For this purpose, we performed whole exome sequencing (WES) of matched tumor-normal pair genomic DNA from 200 cALL patients to identify putative somatic driving mutations. A repertoire of 132 candidate drivers were further validated by performing a targeted knockdown functional screen in 3 pre-B ALL cell lines (NALM6, REH and 697). Through this screen, we identified WHSC1 as putative driver gene. WHSC1 is a methyltransferase that dimethylates lysine 36 on histone H3 (H3K36me2). Eight of the 200 (4%) cALL patients harboured a mutation in WHSC1 (7 pre-B and 1 pre-T ALL). In all 8 cases, the mutation led to a change at amino acid 1099 from a glutamic acid to lysine (E1099K). It has been shown that this activating mutation is associated with global increase in H3K36me2, which is accompanied by substantial changes in gene expression. Transcriptome analysis, using RNA-seq technique, of 6 cALL patients (2 with the mutation and 4 without) revealed distinctive differential expression pattern. We observed a specific enrichment in Wnt pathway genes in presence of the WHSC1 E1099K mutation (6.52 fold, P= 0.05), suggesting a stem cell signature. MCTP39 has been identified as an inhibitor of WHSC1, but has never been tested in ALL. Thus, we treated 4 pre-B ALL cell lines, 3 wild-type for WHSC1 (697, REH and Nalm6) and 1 carrying the mutation E1099K (RS411), as well as the non-leukemic human B cell precursor GM12878 cell line with variable concentrations of MCTP39 and calculated IC50. The IC50 in the leukemic cell lines 697, REH, Nalm6 and RS411 were 2.5µM, 1.7µM, 0.88µM and 0.89µM respectively, whereas it reached 25µM in the non-leukemic GM12878, suggesting a specificity for transformed cells. Although Nalm6 and RS411 had almost the same IC50 at 48h, cell growth curves showed differences in late effect (beyond 3 days), with RS411 being more sensitive to MCTP39. The latter differential effect has been confirmed by colony assay with reduction in plating efficiency of 69% and 45% in RS411 and Nalm6, respectively, when treated 48h with MCTP39 at 0.25µM. This result suggests that MCTP39 targets specifically stem/progenitor cells, particularly those that harbor the mutation WHSC1 E1099K. To confirm that the cellular effect of MCTP39 is due to WHSC1 inhibition, we assessed the impact of MCTP39 on H3K36me2 expression, using fluorometric assay and western blot. In both Nalm6 and RS411cell lines, MCTP39 led to significant decrease in H3K36me2. Then, we analyzed whether MCTP39 synergizes with doxorubicin, an anthracycline that is commonly used in cALL treatment. In both Nalm6 and RS411 cell lines, MCTP39 showed a clear synergistic effect at all concentration tested, permitting 40% reduction in doxorubicin concentration to obtain equal effect. Altogether, this study indicates that inhibition of WHSC1 is a promising treatment strategy in cALL patients, particularly in those carrying WHSC1 mutation. Further investigations are needed to recapitulate these results in vivo and to elucidate the pathways affected as result of the inhibition of H3K36me2 by MCTP39. DisclosuresNo relevant conflicts of interest to declare.

P085 Expression profiling of Wnt pathway genes in colon biopsies of patients with Ulcerative Colitis

Abstract Background There is an increasing demand of agents that can promote mucosal healing in Inflammatory Bowel Disease (IBD). Wnt/β-catenin signaling plays a critical role in epithelial regeneration and repair, and stimulating regeneration in the damaged epithelium by modulating Wnt signaling has been suggested as a potential treatment option for IBD. To guide development of Wnt modulating therapeutic molecules for IBD, an understanding of how Wnt signaling may be altered in IBD tissues is required. While earlier work showed altered Wnt pathway gene expression in UC tissues, these studies failed to consider disease conditions (moderate vs severe) and patient treatment history on expression of the Wnt family genes. These previous studies utilized RT-qPCR or microarray and did not reveal how Wnt pathway gene expression might be affected specifically in the epithelium and in the adjacent stromal stem cell niche. Here we report our work investigating expression patterns of Wnt pathway genes in UC biopsies from 12 patients with moderate and severe disease. Patients had either received no anti-TNF treatment or had gone through anti-TNF treatment and partially responded to the treatment. Methods Expression of a set of Wnt pathway genes was assessed in UC colon and rectum biopsies by RNAscope in situ hybridization and compared to expression patterns in normal control colon. The genes included the Wnt target genes AXIN2, LGR5 and RNF43, Wnt ligands and the FZD5 and LRP6 receptors enriched in the intestinal epithelium as well as key Wnt signal modulators RSPO1-4. Results Expression of Wnt target genes were mildly reduced in the UC colon epithelium, while their expression in some crypts appeared much lower. Overall expression levels of Wnt pathway genes did not differ between moderate and severe UC colon and Wnt target gene expression was more affected in the anti-TNF treated colons, which may reflect more refractory disease. Expression of FZD5, LRP6 and the key niche factor RSPO2, was reduced in the UC colon. RSPOs are normally expressed in the stromal cells next to the crypt bottom stem cell compartment but this expression pattern was disrupted in the UC colon as a result of immune cell infiltration. Although expression of Wnts was induced in the UC colon tissues, the location of expression was altered due to tissue damage, potentially making the Wnts less accessible to the intestinal stem cells. Conclusion Reduced expression of Wnt receptors, RSPOs and Wnt target genes indicate insufficient Wnt signal induction in the damaged colon epithelium of UC patients. This suggests that repair of the damaged epithelium by Wnt agonist treatment may constitute a new mechanism of action and benefit patients with UC.

More severe phenotype of early-onset osteoporosis associated with recessive form of LRP5 and combination with DKK1 or WNT3A.

BackgroundEarly‐onset osteoporosis (EOOP) is defined by low bone mineral density (BMD), which increases the risk of fracture. Although the prevalence of osteoporosis at a young age is unknown, low BMD is highly linked to genetic background. Heterozygous pathogenic variants in low‐density lipoprotein receptor‐related protein 5 (LRP5) are associated with EOOP. This study aimed to investigate the genetic profile in patients with EOOP to better understand the variation in phenotype severity by using a targeted gene sequencing panel associated with bone fragility.Method and ResultsWe used a sequencing panel with 17 genes reported to be related to bone fragility for analysis of 68 patients with EOOP. We found a high positivity rate of EOOP with LRP5 variants (14 patients, 20.6%). The remaining 79.4% of patients with EOOP but without LRP5 variants showed variable disease severity, as observed in patients with at least one variant in this gene. One patient, with multiple fractures and spine L1‐L4 BMD Z‐score −2.9, carried a novel pathogenic homozygous variant, c.2918T>C, p.(Leu973Pro), without any pseudoglioma. In addition to carrying the LRP5 variant, 2 other patients carried a heterozygous variant in Wnt signaling pathway genes: dickkopf WNT signaling pathway inhibitor 1 (DKK1) [NM_012242.4: c.359G>T, p.(Arg120Leu)] and Wnt family member 3A (WNT3A) [NM_033131.3: c.377G>A, p. (Arg126His)]. As compared with single‐variant LRP5 carriers, double‐variant carriers had a significantly lower BMD Z‐score (−4.1 ± 0.8) and higher mean number of fractures (6.0 ± 2.8 vs. 2.2 ± 1.9). Analysis of the family segregation suggests the inheritance of BMD trait.ConclusionSevere forms of EOOP may occur with carriage of 2 pathogenic variants in genes encoding regulators of the Wnt signaling pathway. Two‐variant carriers of Wnt pathway genes had severe EOOP. Moreover, DKK1 and WNT3A genes should be included in next‐generation sequence analyses of bone fragility.

Brief multi‐drug treatment via a wearable silk‐hydrogel bioreactor induces long‐term limb re‐patterning and regeneration in adult Xenopus Laevis

Limb regeneration requires the temporal and spatial patterning of molecular signals within a suitable microenvironment such that differentiation and growth can be orchestrated. Despite advances in limb prostheses, ideal solutions to limb loss will involve re-structuring native tissues to regain biological function. Our novel biomedical engineering approach was designed to induce patterning by controlling the microenvironment at the wound site in amputated hindlimbs of Xenopus laevis, without having to micromanage every molecular cascade. Post-metamorphic, non-regenerative frogs which had undergone hindlimb amputation were treated with an innovative wearable bioreactor composed of a silk-hydrogel infused with 5 pro-regenerative compounds for only 24 hours. Animals were maintained for 18 months where morphometric and histological analyses were performed at regular intervals to monitor neural, vascular, and osseous repatterning. The 24-hr. brief exposure to the bioreactor induced remarkable growth with patterned structures not observed in untreated controls. Bone remodelling, re-vascularization, and digit patterning were observed 18 months after the initial 24 hour bioreactor treatment. 18-month post treatment regenerates also regained sensorimotor abilities within the regenerated hindlimb. Whole transcriptomic analysis at early regenerative periods confirmed changes to blastemal profiles related only to drug-reactor exposures, which included changes in differential gene expression profiles related to crucial pro-regenerative mechanisms such as sox2, KLf4, WNT pathway genes and upregulation of pro-regenerative inflammatory and neural pathways. Our data provide strong evidence that a non-regenerative animal can be induced to re-pattern limbs when exposed to a controlled bioreactor device. This first induction of an advanced limb regenerative response in fully adult organisms suggests using a biomedical engineering approach with silk-hydrogel as a bioreactor for simultaneous and compound delivery of multiple drug compounds is a promising avenue for induction of regeneration in adult, non- regenerative species.

Coordinated changes in the expression of Wnt pathway genes following human and rat peripheral nerve injury.

A human neuroma-in continuity (NIC), formed following a peripheral nerve lesion, impedes functional recovery. The molecular mechanisms that underlie the formation of a NIC are poorly understood. Here we show that the expression of multiple genes of the Wnt family, including Wnt5a, is changed in NIC tissue from patients that underwent reconstructive surgery. The role of Wnt ligands in NIC pathology and nerve regeneration is of interest because Wnt ligands are implicated in tissue regeneration, fibrosis, axon repulsion and guidance. The observations in NIC prompted us to investigate the expression of Wnt ligands in the injured rat sciatic nerve and in the dorsal root ganglia (DRG). In the injured nerve, four gene clusters were identified with temporal expression profiles corresponding to particular phases of the regeneration process. In the DRG up- and down regulation of certain Wnt receptors suggests that nerve injury has an impact on the responsiveness of injured sensory neurons to Wnt ligands in the nerve. Immunohistochemistry showed that Schwann cells in the NIC and in the injured nerve are the source of Wnt5a, whereas the Wnt5a receptor Ryk is expressed by axons traversing the NIC. Taken together, these observations suggest a central role for Wnt signalling in peripheral nerve regeneration.

Generic injuries are sufficient to induce ectopic Wnt organizers in Hydra.

During whole-body regeneration, a bisection injury can trigger two different types of regeneration. To understand the transcriptional regulation underlying this adaptive response, we characterized transcript abundance and chromatin accessibility during oral and aboral regeneration in the cnidarian Hydra vulgaris. We found that the initial response to amputation at both wound sites is identical and includes widespread apoptosis and the activation of the oral-specifying Wnt signaling pathway. By 8 hr post amputation, Wnt signaling became restricted to oral regeneration. Wnt pathway genes were also upregulated in puncture wounds, and these wounds induced the formation of ectopic oral structures if pre-existing organizers were simultaneously amputated. Our work suggests that oral patterning is activated as part of a generic injury response in Hydra, and that alternative injury outcomes are dependent on signals from the surrounding tissue. Furthermore, Wnt signaling is likely part of a conserved wound response predating the split of cnidarians and bilaterians.

CeRNA Network Regulation of TGF-β, WNT, FOXO, Hedgehog Pathways in the Pharynx of Ciona robusta.

The transforming growth factor-β (TGF-β) family of cytokines performs a multifunctional signaling, which is integrated and coordinated in a signaling network that involves other pathways, such as Wintless, Forkhead box-O (FOXO) and Hedgehog and regulates pivotal functions related to cell fate in all tissues. In the hematopoietic system, TGF-β signaling controls a wide spectrum of biological processes, from immune system homeostasis to the quiescence and self-renewal of hematopoietic stem cells (HSCs). Recently an important role in post-transcription regulation has been attributed to two type of ncRNAs: microRNAs and pseudogenes. Ciona robusta, due to its philogenetic position close to vertebrates, is an excellent model to investigate mechanisms of post-transcriptional regulation evolutionarily highly conserved in immune homeostasis. The combined use of NGS and bioinformatic analyses suggests that in the pharynx, the hematopoietic organ of Ciona robusta, the Tgf-β, Wnt, Hedgehog and FoxO pathways are involved in tissue homeostasis, as they are in human. Furthermore, ceRNA network interactions and 3′UTR elements analyses of Tgf-β, Wnt, Hedgehog and FoxO pathways genes suggest that different miRNAs conserved (cin-let-7d, cin-mir-92c, cin-mir-153), species-specific (cin-mir-4187, cin-mir-4011a, cin-mir-4056, cin-mir-4150, cin-mir-4189, cin-mir-4053, cin-mir-4016, cin-mir-4075), pseudogenes (ENSCING00000011392, ENSCING00000018651, ENSCING00000007698) and mRNA 3′UTR elements are involved in post-transcriptional regulation in an integrated way in C. robusta.

Diet-Associated Inflammation Modulates Inflammation and WNT Signaling in the Rectal Mucosa, and the Response to Supplementation with Dietary Fiber.

Inflammation drives colorectal cancer development, and colorectal cancer risk is influenced by dietary factors, including dietary fiber. Hyperactive WNT signaling occurs in colorectal cancer and may regulate inflammation. This study investigated (i) relationships between the inflammatory potential of diet, assessed using the Energy-adjusted Dietary Inflammatory Index (E-DII), and markers of WNT signaling, and (ii) whether DII status modulated the response to supplementation with two types of dietary fiber. Seventy-five healthy participants were supplemented with resistant starch and/or polydextrose (PD) or placebo for 50 days. Rectal biopsies were collected before and after intervention and used to assess WNT pathway gene expression and crypt cell proliferation. E-DII scores were calculated from food frequency questionnaire data. High-sensitivity C-reactive protein (hsCRP) and fecal calprotectin concentrations were quantified. hsCRP concentration was significantly greater in participants with higher E-DII scores [least square means (LSM) 4.7 vs. 2.4 mg/L, P = 0.03]. Baseline E-DII score correlated with FOSL1 (β = 0.503, P = 0.003) and WNT11 (β = 0.472, P = 0.006) expression, after adjusting for age, gender, body mass index, endoscopy procedure, and smoking status. WNT11 expression was more than 2-fold greater in individuals with higher E-DII scores (LSM 0.131 vs. 0.059, P = 0.002). Baseline E-DII modulated the effects of PD supplementation on FOSL1 expression (P = 0.04). More proinflammatory diets were associated with altered WNT signaling and appeared to modulate the effects of PD supplementation on expression of FOSL1 This is the first study to investigate relationships between the E-DII and molecular markers of WNT signaling in rectal tissue of healthy individuals.Prevention Relevance: Our finding that more inflammatory dietary components may impact large bowel health through effects on a well-recognized pathway involved in cancer development will strengthen the evidence base for dietary advice to help prevent bowel cancer.

Clinical-grade whole-genome sequencing and 3\u2032 transcriptome analysis of colorectal cancer patients

BackgroundClinical-grade whole-genome sequencing (cWGS) has the potential to become the standard of care within the clinic because of its breadth of coverage and lack of bias towards certain regions of the genome. Colorectal cancer presents a difficult treatment paradigm, with over 40% of patients presenting at diagnosis with metastatic disease. We hypothesised that cWGS coupled with 3′ transcriptome analysis would give new insights into colorectal cancer.MethodsPatients underwent PCR-free whole-genome sequencing and alignment and variant calling using a standardised pipeline to output SNVs, indels, SVs and CNAs. Additional insights into the mutational signatures and tumour biology were gained by the use of 3′ RNA-seq.ResultsFifty-four patients were studied in total. Driver analysis identified the Wnt pathway gene APC as the only consistently mutated driver in colorectal cancer. Alterations in the PI3K/mTOR pathways were seen as previously observed in CRC. Multiple private CNAs, SVs and gene fusions were unique to individual tumours. Approximately 30% of patients had a tumour mutational burden of > 10 mutations/Mb of DNA, suggesting suitability for immunotherapy.ConclusionsClinical whole-genome sequencing offers a potential avenue for the identification of private genomic variation that may confer sensitivity to targeted agents and offer patients new options for targeted therapies.

Wnt/β-Catenin Signaling Regulates CXCR4 Expression and [68Ga] Pentixafor Internalization in Neuroendocrine Tumor Cells.

Loss of Somatostatin Receptor 2 (SSTR2) expression and rising CXC Chemokine Receptor Type 4 (CXCR4) expression are associated with dedifferentiation in neuroendocrine tumors (NET). In NET, CXCR4 expression is associated with enhanced metastatic and invasive potential and worse prognosis but might be a theragnostic target. Likewise, activation of Wnt/β-catenin signaling may promote a more aggressive phenotype in NET. We hypothesized an interaction of the Wnt/β-catenin pathway with CXCR4 expression and function in NET. The NET cell lines BON-1, QGP-1, and MS-18 were exposed to Wnt inhibitors (5-aza-CdR, quercetin, and niclosamide) or the Wnt activator LiCl. The expressions of Wnt pathway genes and of CXCR4 were studied by qRT-PCR, Western blot, and immunohistochemistry. The effects of Wnt modulators on uptake of the CXCR4 ligand [68Ga] Pentixafor were measured. The Wnt activator LiCl induced upregulation of CXCR4 and Wnt target gene expression. Treatment with the Wnt inhibitors had opposite effects. LiCl significantly increased [68Ga] Pentixafor uptake, while treatment with Wnt inhibitors decreased radiopeptide uptake. Wnt pathway modulation influences CXCR4 expression and function in NET cell lines. Wnt modulation might be a tool to enhance the efficacy of CXCR4-directed therapies in NET or to inhibit CXCR4-dependent proliferative signaling. The underlying mechanisms for the interaction of the Wnt pathway with CXCR4 expression and function have yet to be clarified.

Colorectal Cancer-Associated Smad4 R361 Hotspot Mutations Boost Wnt/β-Catenin Signaling through Enhanced Smad4-LEF1 Binding.

About 10% to 30% of patients with colorectal cancer harbor either loss of or missense mutations in SMAD4, a critical component of the TGFβ signaling pathway. The pathophysiologic function of missense mutations in Smad4 is not fully understood. They usually map to the MH2 domain, specifically to residues that are involved in heterodimeric complex formation with regulatory Smads (such as Smad2/3) and ensuing transcriptional activation. These detrimental effects suggest that SMAD4 missense mutations can be categorized as loss-of-function. However, they tend to cluster in a few hotspots, which is more consistent with them acting by a gain-of-function mechanism. In this study, we investigated the functional role of Smad4 R361 mutants by re-expressing two R361 Smad4 variants in several Smad4-null colorectal cancer cell lines. As predicted, R361 mutations disrupted Smad2/3-Smad4 heteromeric complex formation and abolished canonical TGFβ signaling. In that, they were similar to SMAD4 loss. However, RNA sequencing and subsequent RT-PCR assays revealed that Smad4mut cells acquired a gene signature associated with enhanced Lef1 protein function and increased Wnt signaling. Mechanistically, Smad4 mutant proteins retained binding to Lef1 protein and drove a commensurate increase in downstream Wnt signaling as measured by TOP/FOP luciferase assay and Wnt-dependent cell motility. Consistent with these findings, human colorectal cancers with SMAD4 missense mutations were less likely to acquire activating mutations in the key Wnt pathway gene CTNNB1 (encoding β-catenin) than colorectal cancers with truncating SMAD4 nonsense mutations. IMPLICATIONS: Our studies suggest that in colorectal cancer hotspot mutations in Smad4 confer enhanced Wnt signaling and possibly heightened sensitivity to Wnt pathway inhibitors. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/5/823/F1.large.jpg.

Canine DVL2 variant contributes to brachycephalic phenotype and caudal vertebral anomalies

A frameshift deletion variant in the Wnt pathway gene dishevelled 2 (DVL2) is associated with a truncated, kinked tail (“screw tail”) in English Bulldogs, French Bulldogs and Boston Terriers. These breeds are also characterized by distinctive morphological traits, including a wide head, flat face and short-limbed dwarfism, which are characteristic of Robinow syndrome in humans, caused by defects in genes such as DVL1 and DVL3. Based on these phenotypic and genetic similarities, it has previously been hypothesized that the canine DVL2 variant results in a syndromic phenotype called the Robinow-like syndrome. In our study, we investigated the distribution of the DVL2 variant in 1954 dogs from 15 breeds, identifying breeds with allele variation and enabling the dissection of the genotype–phenotype correlation for the first time. With CT examinations in American Staffordshire Terriers, we confirmed that the DVL2 allele is associated with caudal vertebral malformations and a brachycephalic phenotype. We also hypothesize that the variant may be linked to additional health conditions, including brachycephalic obstructive airway syndrome and congenital heart defects. Altogether, our study strengthens the role of DVL2 as one of the contributors to the “bulldog type” morphology and features on the spectrum of human Robinow syndrome.