Expression Of Wnt Target Genes Research Articles

Irisin promotes intestinal epithelial cell proliferation via Wnt/β-catenin and focal adhesion kinase signaling pathways

The regeneration of epithelia is crucial for maintaining intestinal homeostasis. Irisin is an exercise-induced hormone originally found to be secreted by skeletal muscles, thereby regulating energy metabolism. Recent studies have revealed that irisin protected against gut inflammation. However, the direct effects of irisin on the intestinal epithelial cells remain to be elucidated. In this study, mouse intestinal organoids were used to assess the effects of irisin on the proliferation of the intestinal epithelial cells. At a concentration of 100 ng/mL irisin significantly increased the growth of the intestinal organoids and upregulated the Wnt/β-catenin and focal adhesion kinase (FAK) signaling pathway genes. Notably, a FAK inhibitor 14 blocked the effects of irisin on the proliferation of the intestinal epithelial cells by inhibiting FAK phosphorylation, as well as the expressions of Wnt target genes. Furthermore, irisin (100 ng/mL) improved the recovery of the intestinal organoids from cellular damages caused by TNF-α, and markedly increased the expression of Wnt target genes in the intestinal epithelial cells. Taken together, irisin activates Wnt/β-catenin and FAK signaling pathways in the intestinal epithelial cells, thereby promoting intestinal epithelial self-renewal under normal homeostatic conditions and intestinal epithelial regeneration upon damages.

Targeting CBP and p300: Emerging Anticancer Agents.

CBP and p300 are versatile transcriptional co-activators that play essential roles in regulating a wide range of signaling pathways, including Wnt/β-catenin, p53, and HIF-1α. These co-activators influence various cellular processes such as proliferation, differentiation, apoptosis, and response to hypoxia, making them pivotal in normal physiology and disease progression. The Wnt/β-catenin signaling pathway, in particular, is crucial for cellular proliferation, differentiation, tissue homeostasis, and embryogenesis. Aberrant activation of this pathway is often associated with several types of cancer, such as colorectal tumor, prostate cancer, pancreatic and hepatocellular carcinomas. In recent years, significant efforts have been directed toward identifying and developing small molecules as novel anticancer agents capable of specifically inhibiting the interaction between β-catenin and the transcriptional co-activators CBP and p300, which are required for Wnt target gene expression and are consequently involved in the regulation of tumor cell proliferation, migration, and invasion. This review summarizes the most significant and original research articles published from 2010 to date, found by means of a PubMed search, highlighting recent advancements in developing both specific and non-specific inhibitors of CBP/β-catenin and p300/β-catenin interactions. For a more comprehensive view, we have also explored the therapeutic potential of CBP/p300 bromodomain and histone acetyltransferase inhibitors in disrupting the transcriptional activation of genes involved in various signaling pathways related to cancer progression. By focusing on these therapeutic strategies, this review aims to offer a detailed overview of recent approaches in cancer treatment that selectively target CBP and p300, with particular emphasis on their roles in Wnt/β-catenin-driven oncogenesis.

Saroglitazar Enhances Memory Functions and Adult Neurogenesis via Up-Regulation of Wnt/β Catenin Signaling in the Rat Model of Dementia.

Peroxisome proliferator-activated receptors (PPARs) have emerged as a promising target for the treatment of various neurodegenerative disorders. Studies have shown that both PPAR α & γ individually modulate various pathophysiological events like neuroinflammation and insulin resistance, which are known to variedly affect neurogenesis. Our study aimed to evaluate the effect of saroglitazar (SGZR), a dual PPAR agonist, on adult neurogenesis and spatial learning and memory, in intracerebroventricular streptozotocin (ICV STZ)-induced dementia in rats. We have found that SGZR at the dose of 4 mg/kg per oral showed significant improvement in learning and memory compared to ICV STZ-treated rats. A substantial increase in neurogenesis was observed in the subventricular zone (SVZ) and the dentate gyrus (DG), as indicated by an increase in the number of 5-bromo-2'-deoxyuridine (BrdU)+ cells, BrdU+ nestin+ cells, and doublecortin (DCX)+cells. Treatment with SGZR also decreased the active form of glycogen synthase kinase 3β (GSK3β) and hence enhanced the nuclear translocation of the β-catenin. Enhanced expression of Wnt transcription factors and target genes indicates that the up-regulation of Wnt signaling might be involved in the observed increase in neurogenesis. Hence, it can be concluded that the SGZR enhances memory functions and adult neurogenesis via the upregulation of Wnt β-catenin signaling in ICV STZ-treated rats.

A comprehensive view on the fisetin impact on colorectal cancer in animal models: Focusing on cellular and molecular mechanisms.

Flavonoids, including fisetin, have been linked to a reduced risk of colorectal cancer (CRC) and have potential therapeutic applications for the condition. Fisetin, a natural flavonoid found in various fruits and vegetables, has shown promise in managing CRC due to its diverse biological activities. It has been found to influence key cell signaling pathways related to inflammation, angiogenesis, apoptosis, and transcription factors. The results of this study demonstrate that fisetin induces colon cancer cell apoptosis through multiple mechanisms. It impacts the p53 pathway, leading to increased levels of p53 and decreased levels of murine double minute 2, contributing to apoptosis induction. Fisetin also triggers the release of important components in the apoptotic process, such as second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI and cytochrome c. Furthermore, fisetin inhibits the cyclooxygenase-2 and wingless-related integration site (Wnt)/epidermal growth factor receptor/nuclear factor kappa B signaling pathways, reducing Wnt target gene expression and hindering colony formation. It achieves this by regulating the activities of cyclin-dependent kinase 2 and cyclin-dependent kinase 4, reducing retinoblastoma protein phosphorylation, decreasing cyclin E levels, and increasing p21 levels, ultimately influencing E2 promoter binding factor 1 and cell division cycle 2 (CDC2) protein levels. Additionally, fisetin exhibits various effects on CRC cells, including inhibiting the phosphorylation of Y-box binding protein 1 and ribosomal S6 kinase, promoting the phosphorylation of extracellular signal-regulated kinase 1/2, and disrupting the repair process of DNA double-strand breaks. Moreover, fisetin serves as an adjunct therapy for the prevention and treatment of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA)-mutant CRC, resulting in a reduction in phosphatidylinositol-3 kinase (PI3K) expression, Ak strain transforming phosphorylation, mTOR activity, and downstream target proteins in CRC cells with a PIK3CA mutation. These findings highlight the multifaceted potential of fisetin in managing CRC and position it as a promising candidate for future therapy development.

Inhibition of GSK3α,β rescues cognitive phenotypes in a preclinical mouse model of CTNNB1 syndrome

CTNNB1 syndrome is a rare monogenetic disorder caused by CTNNB1 de novo pathogenic heterozygous loss-of-function variants that result in cognitive and motor disabilities. Treatment is currently lacking; our study addresses this critical need. CTNNB1 encodes β-catenin which is essential for normal brain function via its dual roles in cadherin-based synaptic adhesion complexes and canonical Wnt signal transduction. We have generated a Ctnnb1 germline heterozygous mouse line that displays cognitive and motor deficits, resembling key features of CTNNB1 syndrome in humans. Compared with wild-type littermates, Ctnnb1 heterozygous mice also exhibit decreases in brain β-catenin, β-catenin association with N-cadherin, Wnt target gene expression, and Na/K ATPases, key regulators of changes in ion gradients during high activity. Consistently, hippocampal neuron functional properties and excitability are altered. Most important, we identify a highly selective inhibitor of glycogen synthase kinase (GSK)3α,β that significantly normalizes the phenotypes to closely meet wild-type littermate levels. Our data provide new insights into brain molecular and functional changes, and the first evidence for an efficacious treatment with therapeutic potential for individuals with CTNNB1 syndrome.

Proteomic insights into the regulatory function of ARID1A in colon cancer cells.

The AT-rich interacting domain-containing protein 1A (ARID1A) is a tumor suppressor gene that has been implicated in several cancers, including colorectal cancer (CRC). The present study used a proteomic approach to elucidate the molecular mechanisms of ARID1A in CRC carcinogenesis. Stable ARID1A-overexpressing SW48 colon cancer cells were established using lentivirus transduction and the successful overexpression of ARID1A was confirmed by western blotting. Label-free quantitative proteomic analysis using liquid chromatography-tandem mass spectrometry identified 705 differentially altered proteins in the ARID1A-overexpressing cells, with 310 proteins significantly increased and 395 significantly decreased compared with empty vector control cells. Gene Ontology enrichment analysis highlighted the involvement of the altered proteins mainly in the Wnt signaling pathway. Western blotting supported these findings, as a decreased protein expression of Wnt target genes, including c-Myc, transcription factor T cell factor-1/7 and cyclin D1, were observed in ARID1A-overexpressing cells. Among the altered proteins involved in the Wnt signaling pathway, the interaction network analysis revealed that ARID1A exhibited a direct interaction with E3 ubiquitin-protein ligase zinc and ring finger 3 (ZNRF3), a negative regulator of the Wnt signaling pathway. Further analyses using the The Cancer Genome Atlas colon adenocarcinoma public dataset revealed that ZNRF3 expression significantly impacted the overall survival of patients with CRC and was positively correlated with ARID1A expression. Finally, an increased level of ZNRF3 in ARID1A-overexpressing cells was confirmed by western blotting. In conclusion, the findings of the present study suggest that ARID1A negatively regulates the Wnt signaling pathway through ZNRF3, which may contribute to CRC carcinogenesis.

A phase 1/2 study of the TBL1 inhibitor, tegavivint (BC2059), in patients (pts) with advanced hepatocellular carcinoma (aHCC) with β-catenin activating mutations.

TPS4192 Background: β-catenin mutations are present in up to 40% of aHCC pts. Elevated nuclear β-catenin expression levels correlate with poor responses to standard of care and β-catenin regulates both tumor metabolism and the immune microenvironment. Tegavivint is a first-in-class small molecule inhibitor of TBL1, a novel downstream Wnt-signaling pathway target. Tegavivint binds to TBL1 in the β-catenin pocket, disrupting the formation of the activation complex necessary for oncogenic activity, and enabling degradation of free nuclear β-catenin. The safety, clinical activity, and PK/PD of tegavivint was demonstrated through a proof-of-concept study in pts with desmoid tumors and is being studied through multiple Investigator Initiated Trials in AML (NCT04874480), pediatric solid tumors (NCT04851119), NSCLC (NCT04780568), and lymphoma (NCT05755087). Tegavivint was also studied in preclinical mouse models of β-cateninexon3 mutant HCC and the H22 syngeneic HCC model. In these models tegavivint decreased Wnt target gene expression and enhanced CD3+ T-cell infiltration in liver tumors. Tegavivint treatment of established β-cateninexon3 activated tumors resulted in reduced tumor growth and burden. Based on these promising preclinical results, the following phase 1/2 exploratory study was designed. Methods: This is a phase 1/2 study of tegavivint in pts with aHCC to characterize safety, PK/PD, and preliminary antitumor activity (NCT05797805). Eligibility includes aHCC pts ≥18 years old, with AXIN1or CTNNB1 mutation for all pts, except those in the single agent dose escalation; have BCLC Stage C or Stage B disease not amendable to local therapy or curative approaches, have Child-Pugh class A or B7 liver score, and must have received at least one prior line of systemic therapy. This study will be conducted in 2 parts. First, tegavivint will be administered as a single agent in a dose escalation/optimization and subsequent dose expansion cohort. Upon completion of the dose escalation via a 3+3 design, two dose levels will be selected for the dose selection optimization to determine the recommended phase 2 dose (RP2D) for use in the dose expansion. If sufficient clinical benefit is observed, the combination of tegavivint plus pembrolizumab will be explored in the second part of the study in aHCC pts previously treated with a PD-1/PD-L1 inhibitor. Tegavivint will be administered intravenously weekly. Primary objective is safety and secondary objectives are preliminary efficacy and PK/PD. The first patient began treatment in October of 2023 and the first dose escalation cohort was completed in January. Three institutions are open for enrollment; 5 other sites are pending site activation; all sites are in the United States and Canada. Clinical trial information: NCT05797805 .

Metabolic control by the Bithorax Complex-Wnt signaling crosstalk in Drosophila.

Adipocytes distributed throughout the body play crucial roles in lipid metabolism and energy homeostasis. Regional differences among adipocytes influence normal function and disease susceptibility, but the mechanisms driving this regional heterogeneity remain poorly understood. Here, we report a genetic crosstalk between the Bithorax Complex ( BX-C ) genes and Wnt/Wingless signaling that orchestrates regional differences among adipocytes in Drosophila larvae. Abdominal adipocytes, characterized by the exclusive expression of abdominal A ( abd-A ) and Abdominal B ( Abd-B ), exhibit distinct features compared to thoracic adipocytes, with Wnt signaling further amplifying these disparities. Depletion of BX-C genes in adipocytes reduces fat accumulation, delays larval-pupal transition, and eventually leads to pupal lethality. Depleting Abd-A or Abd-B reduces Wnt target gene expression, thereby attenuating Wnt signaling-induced lipid mobilization. Conversely, Wnt signaling stimulated abd-A transcription, suggesting a feedforward loop that amplifies the interplay between Wnt signaling and BX-C in adipocytes. These findings elucidate how the crosstalk between cell-autonomous BX-C gene expression and Wnt signaling define unique metabolic behaviors in adipocytes in different anatomical regions of fat body, delineating larval adipose tissue domains.

Inhibition of TBL1 cleavage alleviates doxorubicin-induced cardiomyocytes death by regulating the Wnt/β-catenin signal pathway.

Doxorubicin (DOX) is a widely used anthracycline anticancer agent; however, its irreversible effects on the heart can result in DOX-induced cardiotoxicity (DICT) after cancer treatment. Unfortunately, the pathophysiology of DICT has not yet been fully elucidated, and there are no effective strategies for its prevention or treatment. In this investigation, the novel role of transducin beta-like protein 1 (TBL1) in developing and regulating DICT was explored. We observed a reduction in TBL1 protein expression levels as well as cleavage events in the transplanted cardiac tissues of patients diagnosed with Dilated Cardiomyopathy and DICT. It was revealed that DOX selectively induces TBL1 cleavage at caspase-3 preferred sites-D125, D136, and D215. Interestingly, overexpression of the uncleaved TBL1 mutant (TBL1uclv) variant reduced apoptosis, effectively preventing DOX-induced cell death. We confirmed that cleaved TBL1 cannot form a complex with β-catenin. As a result, Wnt reporter activity and Wnt target gene expression collectively indicate a decrease in Wnt/β-catenin signalling, leading to DICT progression. Furthermore, the cleaved TBL1 triggered DOX-induced abnormal electrophysiological features and disrupted calcium homeostasis. However, these effects were improved in TBL1uclv-overexpressing human-induced pluripotent stem cell-derived cardiomyocytes. Finally, in a DICT mouse model, TBL1uclv overexpression inhibited the DICT-induced reduction of cardiac contractility and collagen accumulation, ultimately protecting cardiomyocytes from cell death. Our findings reveal that the inhibition of TBL1 cleavage not only mitigates apoptosis but also enhances cardiomyocyte function, even in the context of DOX administration. Consequently, this study's results suggest that inhibiting TBL1 cleavage may be a novel strategy to ameliorate DICT.

Establishment of Anoxia/cytokine-induced Injury and Regeneration in Colonoids

Intestinal epithelial cells (IECs) play key roles in nutrient absorption and fluid secretion, providing a barrier to protect cells. IECs, derived from intestinal stem cells (ISCs), are crucial in maintaining intestinal homeostasis and regeneration, which is crucial in gastrointestinal diseases such as inflammatory bowel disease (IBD). To study epithelial injury/regeneration, primary cultured colonoids were utilized as a model system to mimic injury. They retain the composition and differentiation of the tissue, making it a suitable model for studying homeostasis and regeneration of ISCs in vitro. Anoxia and proinflammatory cytokines frequently coexist and exacerbate one another during intestinal injury. However, anoxic conditions have been mainly ignored in previous research. Thus, we developed a regeneration model using cytokines/anoxia injury to better recapitulate intestinal inflammation. Colonoids were treated with cytokines TNF-α and IFN-γ (5 ng/μL) in anoxic conditions for 24 hours to provoke an injury. After 24 hours of injury, the colonoids were exposed to normal oxygen and media conditions for regeneration. Regeneration was observed for 24, 48, and 72 hours and documented using photomicrographs. Morphological analysis detailed that injury was most severe 24 hours post-injury with the combined induction of cytokines and anoxia resulting in decreased colonoid area and number of buds compared to control groups. RT-PCR analysis detailed mRNA expression of stem cell markers ( Lgr5, Bmi1, and Hopx), Wnt ( Axin2, Myc2, and Ascl2), and YAP target genes ( Sca1 and Ctgf) were increased at peak injury and returned to pre-injury levels at 72 hours of recovery. Regeneration of the colonoids is consistent with in vivo observations, thus confirming the validity of the model. This versatile model can be used to study responses to injury and regeneration of intestinal epithelium and the function of ISCs with applications in intestinal disease therapeutics. University of Maryland College of Agriculture and Natural Resources and the Maryland Agricultural Experiment Station Undergraduate Research Grant Grant #4307572 from the Maryland Stem Cell Research Fund. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nkd1 functions downstream of Axin2 to attenuate Wnt signaling.

Wnt signaling is a crucial developmental pathway involved in early development as well as stem-cell maintenance in adults and its misregulation leads to numerous diseases. Thus, understanding the regulation of this pathway becomes vitally important. Axin2 and Nkd1 are widely utilized negative feedback regulators in Wnt signaling where Axin2 functions to destabilize cytoplasmic β-catenin, and Nkd1 functions to inhibit the nuclear localization of β-catenin. Here, we set out to further understand how Axin2 and Nkd1 regulate Wnt signaling by creating axin2gh1/gh1, nkd1gh2/gh2 single mutants and axin2gh1/gh1;nkd1gh2/gh2 double mutant zebrafish using sgRNA/Cas9. All three Wnt regulator mutants were viable and had impaired heart looping, neuromast migration defects, and behavior abnormalities in common, but there were no signs of synergy in the axin2gh1/gh1;nkd1gh2/gh2 double mutants. Further, Wnt target gene expression by qRT-PCR and RNA-seq, and protein expression by mass spectrometry demonstrated that the double axin2gh1/gh1;nkd1gh2/gh2 mutant resembled the nkd1gh2/gh2 phenotype demonstrating that Nkd1 functions downstream of Axin2. In support of this, the data further demonstrates that Axin2 uniquely alters the properties of β-catenin-dependent transcription having novel readouts of Wnt activity compared with nkd1gh2/gh2 or the axin2gh1/gh1;nkd1gh2/gh2 double mutant. We also investigated the sensitivity of the Wnt regulator mutants to exacerbated Wnt signaling, where the single mutants displayed characteristic heightened Wnt sensitivity, resulting in an eyeless phenotype. Surprisingly, this phenotype was rescued in the double mutant, where we speculate that cross-talk between Wnt/β-catenin and Wnt/Planar Cell Polarity pathways could lead to altered Wnt signaling in some scenarios. Collectively, the data emphasizes both the commonality and the complexity in the feedback regulation of Wnt signaling.

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

The β-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 β-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.

Abstract LB301: BRAFV600E drives WNT signalling in colorectal cancer via aberrant DNA methylation

Abstract Objective: The BRAF V600E mutation drives the generation of an aggressive subtype of colorectal cancer (CRC). Although activation of WNT signalling is a generic hallmark of CRC, mutations in APC are uncommon in BRAF V600E-mutant CRC. Instead, mutations in RNF43 are the suspected WNT signalling driving event in these tumours. Here, we aimed to uncover how the WNT pathway is activated in BRAF V600E-mutant colon cancer. Design: CRISPR-LbCpf1-corrected BRAF (V600E) and RNF43 (P441fs) organoids were analysed for their growth potential in the presence or absence of growth factors, for their ability to form tumours, and by immunoblotting, RNA sequencing, and DNA methylation assays. Results: BRAF E600V organoids regained dependency on epidermal growth factor (EGF) receptor signalling for ERK pathway activation and proliferation, and completely lost tumorigenic potential. Correction of BRAF V600E, but not RNF43 P441fs, caused suppression of WNT target genes, while genes reflecting epithelial differentiation and negative regulators of WNT signalling were upregulated in BRAF E600V organoids. DNA methylation analysis revealed promoter hypermethylation of WNT antagonist genes in BRAF V600E organoids. Gene body hypermethylation, associated with transcriptional upregulation, was observed in key WNT-driven intestinal stem cell marker genes LGR5 and EPHB2, and in the WNT-effector TCF4. Treatment of BRAF V600E organoids with the DNA demethylating agent 5-aza-2′-deoxycytidine resulted in upregulation of WNT antagonist genes and a marked decrease in WNT target gene expression. Conclusions: BRAFV600E, rather than mutant RNF43, is a prominent force behind WNT pathway activation, and is essential for maintaining tumorigenic capacity. BRAFV600E causes widespread dysregulation of DNA methylation leading to activation of WNT signalling. Citation Format: Layla El Bouazzaoui, Jeroen M. Bugter, Emre Küçükköse, André Verheem, Jasmin B. Post, Nicola Fenderico, Inne H. Borel Rinkes, Hugo J. Snippert, Madelon M. Maurice, Onno Kranenburg. BRAFV600E drives WNT signalling in colorectal cancer via aberrant DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB301.

Transport of CLCA2 to the nucleus by extracellular vesicles controls keratinocyte survival and migration.

Chloride channel accessory 2 (CLCA2) is a transmembrane protein, which promotes adhesion of keratinocytes and their survival in response to hyperosmotic stress. Here we show that CLCA2 is transported to the nucleus of keratinocytes via extracellular vesicles. The nuclear localization is functionally relevant, since wild-type CLCA2, but not a mutant lacking the nuclear localization signal, suppressed migration of keratinocytes and protected them from hyperosmotic stress-induced cell death. In the nucleus, CLCA2 bound to and activated β-catenin, resulting in enhanced expression of Wnt target genes. Mass-spectrometry-based interaction screening and functional rescue studies identified RNA binding protein 3 as a key effector of nuclear CLCA2. This is of likely relevance in vivo because both proteins co-localize in the human epidermis. Together, these results identify an unexpected nuclear function of CLCA2 in keratinocytes under homeostatic and stress conditions and suggest a role of extracellular vesicles and their nuclear transport in the control of key cellular activities.

Perturbed liver gene zonation in a mouse model of non-alcoholic steatohepatitis

Liver zonation characterizes the separation of metabolic pathways along the lobules and is required for optimal hepatic function. Wnt signaling is a master regulator of spatial liver zonation. A perivenous-periportal Wnt activity gradient orchestrates metabolic zonation by activating gene expression in perivenous hepatocytes, while suppressing gene expression in their periportal counterparts. However, the understanding as to the liver gene zonation and zonation regulators in diseases is limited. Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fat accumulation, inflammation, and fibrosis. Here, we investigated the perturbation of liver gene zonation in a mouse NASH model by combining spatial transcriptomics, bulk RNAseq and in situ hybridization. Wnt-target genes represented a major subset of genes showing altered spatial expression in the NASH liver. The altered Wnt-target gene expression levels and zonation spatial patterns were in line with the up regulation of Wnt regulators and the augmentation of Wnt signaling. Particularly, we found that the Wnt activator Rspo3 expression was restricted to the perivenous zone in control liver but expanded to the periportal zone in NASH liver. AAV8-mediated RSPO3 overexpression in controls resulted in zonation changes, and further amplified the disturbed zonation of Wnt-target genes in NASH, similarly Rspo3 knockdown in Rspo3+/− mice resulted in zonation changes of Wnt-target genes in both chow and HFD mouse. Interestingly, there were no impacts on steatosis, inflammation, or fibrosis NASH pathology from RSPO3 overexpression nor Rspo3 knockdown. In summary, our study demonstrated the alteration of Wnt signaling in a mouse NASH model, leading to perturbed liver zonation.

ILKAP Promotes the Metastasis of Hepatocellular Carcinoma Cells by Inhibiting β-Catenin Degradation and Enhancing the WNT Signaling Pathway.

The incidence of Hepatocellular carcinoma (HCC) and HCC-related deaths have remarkably increased over the recent decades. It has been reported that β-catenin activation can be frequently observed in HCC cases. This study identified the integrin-linked kinase-associated phosphatase (ILKAP) as a novel β-catenin-interacting protein. ILKAP is localized both in the nucleus and cytoplasm and regulates the WNT pathway in different ways. First, it is demonstrated that ILKAP activates the WNT pathway in HCC cells by increasing the protein level of β-catenin and other proteins associated with the WNT signaling, such as c-Myc and CyclinD1. Next, it is shown that ILKAP promotes the metastasis of HCC both in vitro and in vivo in a zebrafish xenograft model. It is also found that ILKAP dephosphorylates the GSK3β and CK1, contributing to the reduced ubiquitination of β-catenin. Furthermore, it is identified that ILKAP functions by mediating binding between TCF4 and β-catenin to enhance expression of WNT target genes. Taken together, the study demonstrates a critical function of ILKAP in metastasis of HCC, since ILKAP is crucial for the activation of the WNT pathway via stabilization of β-catenin and increased binding between TCF4 and β-catenin.

The Warburg Trap: A Novel Therapeutic Approach for Targeting Osteosarcoma.

Although urgently needed, no significant improvements in osteosarcoma (OS) therapy have been achieved within the last decades. Here, we present a new therapeutic approach based on drug combinations consisting of mitochondrial complex I (MCI) inhibitors and ionophores that induce cancer cell-specific cell death based on a modulation of cellular energy metabolism and intracellular pH (pHi) named the Warburg Trap (WT). The effects of several drug combinations on intracellular pH, cell viability, colony-forming capacity and expression of WNT-target genes were analysed using OS cell lines and primary human osteoblasts (HOB). Tumour take rates and tumour volumes were analysed in vivo using a chicken chorioallantoic membrane assay (CAM). Several WT drug combinations induced the intracellular acidification and apoptotic cell death in OS cells, whereas HOBs tolerated the treatment. A significant inhibition of the colony-forming ability of OS cells and downregulation of WNT-target genes suggest that cancer stem cells (CSCs) are also targeted by the WT approach. In vivo, we observed a significant reduction in the tumour take rates in response to WT drug treatment. Our data suggest that the Warburg Trap is a promising approach for the development of a novel and effective OS therapy to replace or supplement the current OS chemotherapy.

HIF signaling overactivation inhibits lateral line neuromast development through Wnt in zebrafish

The lateral line is critical for prey detection, predator avoidance, schooling, and rheotaxis behavior in fish. As similar to hair cells in the mammalian inner ear, the lateral line sensory organ called neuromasts is a popular model for hair cell regeneration. However, the mechanism of lateral line development has not been fully understood. In this study, we showed for the first time that hypoxia-inducible factor (HIF) signaling is involved in lateral line development in zebrafish. hif1ab and epas1b were highly expressed in neuromasts during lateral line development. Hypoxia response induced by a prolyl hydroxylase domain-containing proteins (PHD) inhibitor treatment or vhl gene knockout significantly reduced hair cells and support cells in neuromast during lateral line development. In addition, inhibition of Hif-1α or Epas1 could partially rescue hair cells in the larvae with increased HIF activity, respectively. Moreover, the support cell proliferation and the expression of Wnt target genes decreased in vhl mutants which suggests that Wnt signaling mediated the role of HIF signaling in lateral line development. Collectively, our results demonstrate that HIF signaling overactivation inhibits lateral line development in zebrafish and suggest that inhibition of HIF signaling might be a potential therapeutic method for hair cell death.

ELTD1 Suppression Enhances Human Embryonic Stem Cell Hematopoiesis By Facilitating Hemogenic Endothelial Progenitor Induction Via Wnt/β-Catenin Signaling Inhibition

Objectives Human embryonic stem cells (hESCs) serve as an attractive cell source for generating functional blood cells and hold great potential for cell-based therapy in future clinical applications. However, the underlying molecular mechanisms that regulate hESC differentiation into hematopoietic cells remain elusive. Therefore, understanding hESC differentiation is essential for the acquisition of sufficient and functional cells in vitro for successful clinical translation. Materials and Methods ELTD1 was identified as a potential mediator of hESC hematopoietic differentiation by genome-wide gene profiling. ELTD1-deleted human embryonic stem cell (hESC) lines were constructed by the iCRISPR/Cas9 technology and were used to undergo hematopoietic differentiation. Flow cytometry, quantitative RT-PCR, immunofluorescence, and RNA sequencing were used for phenotype and molecular mechanism assessment. Results Real-time PCR analysis and bioinformatic studies demonstrated that the dynamic expression of ELTD1 parallels the hemogenic endothelial progenitors-related genes expression and subsequently decreased as they differentiate into hematopoietic cells ( Figure A, C), leading us to speculate that it might play a role in hESC early hematopoietic differentiation. To further confirm these results, stage-specific cell populations at different time points were sorted to document the cell-type specificity of ELTD1. As shown in Figure B, ELTD1 was exclusively expressed in CD31 +CD34 + HEP populations. Loss-of-function studies revealed that ELTD1 deletion promoting the induction of CD31 +CD34 + HEPs at day 6, thereby promoting endothelial-to-hematopoietic transition to generate more CD43 +/CD45 + hematopoietic cells, and the results were confirmed by flow cytometry and immunofluorescence analyses ( Figure D, E and F). To dissect the molecular mechanism by which ELTD1 regulates hESC hematopoietic differentiation, the differentiated cells at day 6 were used to perform the RNA-Seq. KEGG analysis showed that hematopoietic cell lineage-related genes and Wnt signaling pathway were prominently enriched in ELTD1-deleted cells compared to WT-H1, consistent with the enhancement of HEPs and hematopoietic cells generation caused by ELTD1 deletion ( Figure G). Moreover, decreased expression of Wnt target genes: SOX17, TCF7L1, MYC, CCND1, BIRC5, and AXIN2 was further validated by quantitative RT-PCR analysis ( Figure H). To determine whether the downregulation of Wnt/β-catenin signaling is required for ELTD1 deletion-mediated enhancement of early hematopoietic differentiation from hESCs, we treated ELTD1-KO1# and ELTD1-KO2# hESCs with the Wnt agonist CHIR99021. As expected, treatment with CHIR99021 inhibited the generation of CD31 +CD34 + HEPs compared to ELTD1 deleted alone at day 6, which was validated by flow cytometry ( Figure I). Conclusions We identified ELTD1 as a novel regulator of hematopoietic differentiation, which may act via Wnt/β-catenin signaling, and provided new insights into understanding the mechanism of human hematopoiesis.

Region-specific Wnt signaling responses promote gastric polyp formation in patients with familial adenomatous polyposis.

Germline adenomatous polyposis coli (APC) mutation in patients with familial adenomatous polyposis (FAP) promotes gastrointestinal polyposis, including the formation of frequent gastric fundic gland polyps (FGPs). In this study, we investigated how dysregulated Wnt signaling promotes FGPs and why they localize to the corpus region of the stomach. We developed a biobank of FGP and surrounding nonpolyp corpus biopsies and organoids from patients with FAP for comparative studies. Polyp biopsies and polyp-derived organoids exhibited enhanced Wnt target gene expression. Polyp-derived organoids with intrinsically upregulated Wnt signaling showed poor tolerance to further induction, suggesting that high Wnt restricts growth. Targeted genomic sequencing revealed that most gastric polyps did not arise via APC loss of heterozygosity. Studies in genetic mouse models demonstrated that heterozygous Apc loss increased epithelial cell proliferation in the corpus but not the antrum, while homozygous Apc loss was not maintained in the corpus yet induced hyperproliferation in the antrum. Our findings suggest that heterozygous APC mutation in patients with FAP may be sufficient to drive polyp formation in the corpus region while subsequent loss of heterozygosity to further enhance Wnt signaling is not tolerated. This finding contextualizes the abundant yet benign nature of gastric polyps in FAP patient corpus compared with the rare, yet adenomatous polyps in the antrum.