Activation Of Wnt Pathway Research Articles

The effects of taraxasterol on liver fibrosis revealed by RNA sequencing.

Effective treatment of liver fibrosis remains a challenging medical problem. Taraxasterol (TAR) has anti-inflammatory, anti-tumor and hepatoprotective effects. Studies have shown that TAR has good biological activity against liver injury induced by various factors. However, the anti-fibrotic effect of TAR and its mechanism are never clarified. The purpose of this study was to investigate the effects of TAR in liver fibrosis and to reveal its possible mechanism by RNA sequencing. Our results suggested that TAR attenuated CCl4-induced hepatocyte necrosis, inflammatory infiltration and ECM deposition. TAR inhibited the levels of ALT, AST, ALP, γ-GT, LN, HA, PC III and IV-C in serum and TNF-α, IL-6, IL-1β and MDA in liver. In addition, TAR increased the activities of SOD and GSH-Px in liver. RNA sequencing analysis of liver tissues revealed that CCl4 and TAR significantly altered 4,155 genes and 2,675 genes, respectively. TAR reversed changes in ECM-related genes. More specifically, TAR mediated the expression of genes related to the activation of the Hippo pathway, while inhibiting the expression of genes related to the activation of HIF-1α, TGF-β/Smad, and Wnt pathways. In the validation experiments, the qRT-PCR results showed that the expression levels of Yap1, Tead3, Hif1α, Vegfa, Tgfβ1, Want3a, and Ctnnb1 mRNA were consistent with the RNA sequencing results. The Western blot results showed that TAR inhibited the levels of TGF-β1 and p-Smad2. In addition, the results in vitro were consistent with those in vivo. Therefore, we concluded that TAR improved CCl4-induced liver fibrosis by regulating Hippo, HIF-1α, TGF-β/Smad and Wnt pathways.

Macrophage regulation of Wnt pathway in canine and murine mammary cancer cells

Interactions between various cell types in a tumor microenvironment (TME) are important for cancer progression and metastasis. Tumor-associated macrophages (TAMs) play a key role in this intratumoral dialogue regulating Wnt signaling pathway in cancer cells. Co-culture of canine and murine tumor cells with TAMs lead to inhibition of the canonical Wnt pathway and activation of the non-canonical Wnt pathway in tumor cells resulting in change in tumor cell phenotype and subsequent higher potential to invasion and metastasis. These molecular changes in cancer cells that are induced by TAMs resemble the quercetin activity. This natural product is a plant flavonoid from the group of polyphenols. Its biological function is inhibition of the canonical Wnt pathway. As quercetin supplementation is broadly discussed in cancer patients, findings of this study show that this compound should be used with caution, as it may enhance cancer spread.

DKK1 Ameliorates Myofibroblast Differentiation in Urethral Fibrosis in Vivo and in Vitro by Regulating the Canonical Wnt Pathway.

Background: Urethral stricture is a common disorder of the lower urinary tract in men. A complex network of pathways and interactions are involved in the pathogenesis of urethral fibrosis. However, the mechanisms underlying urethral fibrosis remain poorly understood. Objectives: To investigate the critical role of the canonical Wnt pathway in development of urethral fibrosis and explore DKK1, the endogenous inhibitor of Wnt pathway, as a potential target to prevent urethral fibrosis in vitro and in vivo. Methods: Urethral fibrosis tissue derived from patients and rat models were harvested to assess the activation of the canonical Wnt pathway by using western blot, qRT-PCR and immunohistochemistryWe performed histological staining, western blot, qRT-PCR and immunohistochemistry to examine the effects of DKK1 treatment on in vivo rat urethral fibrosis models. In vitro, human urethral fibroblasts (HUFs) were cultured to examine the effects of DKK1 in TGFβ1-induced HUFs by CCK-8 assay, hydroxyproline assay, flow cytometry, cell migration assay, western blot, qRT-PCR and immunofluorescence. Results: The key components of Wnt signaling were upregulated in urethral fibrosis tissue derived from patients and rat models while DKK 1 was downregulated. DKK1 ameliorated TGFβ1-induced urethral fibrosis in rats. TGFβ1 induced myofibroblast differentiation by upregulating collagen I, collagen III, α-SMA, β-catenin and p-GSK-3β, while DKK1 was decreased. DKK1 significantly inhibited cell proliferation, collagen content, cell migration and promoted cell apoptosis in TGFβ1-induced HUFs. DKK1 significantly suppressed myofibroblast differentiation of TGFβ1-induced HUFs by downregulating collagen I, collagen III, α-SMA, β-catenin and p-GSK-3β with a mechanism independent of Smad2/3. Conclusions: Our study demonstrated that canonical Wnt pathway may be an essential mechanism underlying the pathogenesis of urethral fibrosis and explored the potential role of DKK1 participation in the development of urethral fibrosis.

A Feedback Loop of LINC00665 and the Wnt Signaling Pathway Expedites Osteosarcoma Cell Proliferation, Invasion, and Epithelial-Mesenchymal Transition.

Osteosarcoma (OS) is a malignant tumor with frequent occurrence among teenagers. Long non-coding RNAs (lncRNAs) play pro-cancer roles in many tumors. The purpose of this study was to figure out the functional role of a novel lncRNA long intergenic non-protein coding RNA 665 (LINC00665) in OS by observing the OS cell behaviors. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to analyze LINC00665 expression in OS cells. Cell function assays assessed the impacts of LINC00665 on OS cell phenotype. Immunofluorescence and western blot analyzed the function of LINC00665 on epithelial-mesenchymal transition (EMT) in OS. Moreover, mechanistic assays analyzed the downstream mechanism of LINC00665 in OS cells. LINC00665 was significantly up-regulated in OS cells. LINC00665 silence facilitated OS cell proliferation, migration, invasion, and EMT while inhibiting cell apoptosis. Mechanically, LINC00665 acted as a competing endogenous RNA (ceRNA) to sponge miR-1249-5p and thereby modulated Wnt family member 2B (WNT2B) to activate Wnt pathway. Wnt pathway activated LINC00665 expression transcriptionally. Our study uncovered the cancer-promoting role of LINC00665 in OS, and the feedback loop of LINC00665/miR-1249-5p/WNT2B/Wnt might be a potential target for OS treatment.

FFA Promoting Proliferation and Migration Via Classical Wnt Signaling Pathway in Peripheral T Cell Lymphoma

Background: Peripheral t cell lymphoma (PTCL) is an important component of NHL, the overall prognosis is worse than B Cell Lymphoma. In the hyperlipidaemic microenvironment, exploration of the relationship between de novo adipose synthesis and the proliferation and migration capacity of PTCL cells will help to discover therapeutic targets that interfere with tumor cell growth. This study attempts to explore that abnormally high levels of free fatty acids in the tumor microenvironment may promote PTCL migration and proliferation through the classical Wnt signaling pathway Methods: The proliferation activity of EL4 cells treated with high fatty acids（OA:OP=2:1） was detected by CCK8 method, and the proportion of cells with tumor-stemness was detected by flow cytometry and Hoechst33342 stain; A C57BL FGF21-/-mouse model of non-alcoholic steatohepatitis (Nash) was constructed with the MCD method (Cholesterol & Cholate Methionine/choline deficient); PTCL tumor-bearing mice model was established by injecting EL4 cell into spleen in situ; WB method was used to detect the activation of WNT signal pathway in vitro and vivo; migration was evaluated by transwell assay. Results: 1. In Vitro, in the OP+ EL4 group, Axin-1 and p-β-catenin were both significantly decreased (P<0.001, P=0.005), additionally treated with XAV939 (WNT inhibitor) or LiCl (WNT agonistor), Axin-1 and p-β-catenin both showed a significant increase (P<0.001, P=0.002) or degradation of GSK3 β/p-β-catenin (P=0.033, P<0.001). Suggesting that OP could activate the Wnt pathway in EL4 cells. 2. In Vivo, compared with the control group, the FGF21-/-MCD Group had the most number of nodules on the surface of the liver (FGF21-/-MCD vs WT-CD, P<0.001, vs WT-MCD, P=0.009, vs FGF21-/-CD, P<0.001), In FGF21-/-MCD Group, β-Catenin was increased in tumor tissues and adjacent tissues, especially in tumor tissues (P<0.05 in each group). Suggested that the migration enhancer in gross, and the mechanism may related with Wnt signaling pathway. 3. The co-culture assay showed that OP+El4 enhanced its migration ability, which could be decreased by Xav939 and enhanced by Licl(P<0.05 in each group). 4. in Vivo, After treatment with PPARA agonist, β-catenin EPCAM decreased in all MCD groups (P<0.05). It is suggested that fenofibrate can improve the tumor outcome by correcting abnormal lipid metabolism and weakening the activation of Wnt pathway. Conclusion: High free fatty acids are associated with a poor prognosis in PTCL. Possibly mechanism is the activation of the canonical Wnt pathway by free fatty acids, which promote enhanced tumor cell proliferation and migration capacity. Effective Hypolipidemic therapy may be a new combined therapeutic target for PTCL patients with hyperlipidemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TBL1 Inhibitor, Tegavivint, Supresses Cell Survival and Promotes Sensitivity to Chemotherapy in ALL and AML

BACKGROUND Abnormal wnt signaling has been linked to different forms of cancer and has been described to be critical in acute myeloblastic leukemia (AML) and acute lymphoblastic leukemia (ALL). AML is a subtype of leukemia with a poor prognosis as long-term disease-free survival is only 30~40%. While B-ALL has a better prognosis, relapse of B-ALL still has a poor overall survival rate. Aberrant Wnt pathway activation is a common feature in AML and B-ALL as Wnt/catenin activation through β-catenin is involved in self- renewal of leukemia initiating cells. In the canonical Wnt pathway, the signaling of wnt inhibits β-catenin from being degraded. β-catenin regulates transcription of several genes by its translocation into the nucleus. Recently, it has been shown that transducin β-like protein 1 (TBL1) and its highly related family member TBLR1 are required for Wnt-β-catenin-mediated transcription. Overexpression of TBL1 or TBLR1 can lead to significantly enhanced β-catenin-mediated transcription. Conversely, depletion of TBL1 or TBLR1 abolishes the activation of β-catenin. It has been shown that Tegavivint, a novel small molecule inhibitor of Wnt-ß-catenin signaling, binds to transducin ß-like protein 1 (TBL1) preventing the binding of TBL1 to ß-catenin, which results in nuclear degradation of ß-catenin in MV4-11 cells. Here we focus on the effect of Tegavivint in combination with chemotherapy: We hypothesize that interrupting the binding of TBL1 to beta-catenin abrogates not only AML, but also B-ALL cell survival and tests if Tegavivint sensitizes AML cells to AraC and B-ALL cells to chemotherapeutic agents including Vincristine, Dexamethasone or the tyrosine kinase inhibitor Nilotinib as part of preclinical evaluation in acute leukemias. RESULTS As AML is a genetically heterogenous disease, we have tested Tegavivint in vitro in six AML cell lines (THP1, KG1, MOLM14, MOLM13m U937 and MV4-11). These six AML cell lines express β-catenin and TBL1 as determined by Western Blotting. Tegavivint decreases nuclear β-catenin without affecting cytoplasmic β-catenin and decreases non-phospho β-catenin, the active form of β-catenin. We also determined that the IC50 of Tegavivint after 24 hours of incubation ranged between 5-16 nM in these 6 cell lines using AnnexinV/ DAPI staining and flow cytometry. We observed that Tegavivint can sensitize all six AML cell lines to chemotherapy (Ara-C) within 24 hours of incubation. We have also tested Tegavivint on 5 ALL cell lines with different karyotypes (RCH-ACV, NALM6, REH, SUPB15, and RS4;11). We determined that the IC50 of Tegavivint ranged between 5-6.5nM in these 5 cell lines. We also found that Tegavivint can sensitize four ALL cells to chemotherapy using Vincristine and Dexamethasone and one B-ALL cell line (SUBP-B15: BCR-ABL+) to the tyrosine kinase inhibitor Nilotinib within 24 hours of incubation. Further in vitro evaluation of patient-derived B-ALL cells and in vivo evaluation of the sensitization of B-ALL and AML to chemotherapy are in progress. CONCLUSION Our preliminary data shows that AML and B-ALL are sensitive to TBL1- β-catenin inhibition using Tegavivint as a monotherapy and can be sensitized to chemotherapy when AraC or Vincristine, dexamethasone or Nilotinib treatment is combined with Tegavivint. As our data supports our hypothesis that TBL-1- β-catenin inhibition is a new target in B-ALL and AML therapy, further studies are in progress to preclinically evaluate this approach for clinical care.

CSIG-40. STEAROYL-COA DESATURASE 1 (SCD1) IS REQUIRED FOR WNT SIGNALING TO INDUCE AN APOPTOSIS IN IDH MUTANT GLIOMA

Abstract BACKGROUND AND HYPOTHESES SCD1, a major enzyme of saturated fatty acids, has been implicated to be important for tumor metabolic reprograming. Our previous study show that a high level of SCD1 mRNA is associated with IDH1mut lower grade gliomas. IDH1mut glioma cells are more sensitive to SFA induced apoptosis. However, the underlying mechanism remains unclear. In this study, we investigate the functions of SCD1 in IDHmut glioma and the potential contribution of SCD1 for cancer therapy of glioma. STUDY DESIGN AND METHODS The genetically engineered IDH wild-type, IDHmut and patient derived glioma cell lines were used to evaluate SCD1 functions. The expression of proteins were checked by Western-blotting assay. SCD1 was silenced by CRISPR or siRNA. The transcriptome change after SCD1 knockdown was profiled by RNA-seq or single cell RNA-seq (scRNA-seq). RESULTS AND CONCLUSIONS SCD1 transient silencing slowed down the cell growth, suggesting that SCD1 may possess an oncogenic property. RNA-seq analysis revealed that SCD1 inhibition decreased the expression of wnt-signaling pathway genes in IDH-1mut cells. scRNA confirmed that CRISPR SCD1 significantly decreased wnt signaling in the patient cell line Ts603. Therefore, we activated wnt pathway using a small chemical compound, BML-2838. Consistent with recent studies, wnt pathway induction led to a dramatically suppression of glioma cells growth. However, SCD1 silencing reversed this inhibitory effect. Further investigation revealed that SCD1 inhibition reduced the nucleus translocation of phosphorylated beta-catenin. Overall, the results suggest that SCD1 is vital for the onset of wnt pathway in glioma cells. High level of SCD1 expression may render the IDHmut glioma cells more sensitive to wnt pathway induced apoptosis. RELEVANCE AND IMPORTANCE In clinical, the 5-years survival rate of glioma remains low. SCD1 have been considered as a target for glioma therapy, recently. Our data provides a new insight on the strategy to target SCD1 in clinical.

CSIG-37. MERLIN S13 DEPHOSPHORYLATION DRIVES MENINGIOMA WNT SIGNALLING AND CELL PROLIFERATION

Abstract How Merlin-intact meningiomas arise in the absence of NF2/Merlin inactivation is incompletely understood. Here, we integrate single-cell RNA sequencing of 86,000 cells from meningioma xenografts with APEX2 proteomic proximity-labelling mass spectrometry and functional biochemical approaches to discover Merlin Serine 13 (S13) dephosphorylation drives meningioma Wnt signalling and cell proliferation. Cell biology, molecular biology, and biochemical techniques were used to validate Merlin functions in meningioma cells or xenografts using wildtype Merlin constructs or Merlin constructs encoding S13A, phosphomimetic S13D, or cancer-associated missense substitutions (L46R, A211D). Single-cell RNA sequencing of meningioma xenografts showed Merlin rescue activated the Wnt pathway in Merlin-deficient meningiomas. Proteomic proximity-labelling mass spectrometry revealed b-catenin, PKC, and PP1A interactions with wildtype Merlin, but not with Merlin L46R or A211D. b-catenin does not interact with other FERM family members, and Merlin contains a unique N-terminal domain (NTD) with a PKC phosphorylation motif overlapping with a PP1A dephosphorylation motif at S13. Thus, we hypothesized the Merlin S13, PKC, and PP1A may be important for Wnt signalling in Merlin-intact meningiomas. In support of this hypothesis, over-expression of wildtype Merlin or Merlin S13A but not Merlin DNTD, S13D, L46R, A211D, or other FERM family members drove meningioma Wnt signalling and sustained meningioma cell proliferation in vivo. Moreover, b-catenin was detected in proximity to Merlin S13D but not Merlin S13A in meningioma cells. Meningioma cell fractionation and immunofluorescence showed Merlin S13D over-expression stabilized b-catenin at the plasma membrane and inhibited Wnt signalling. Phospho-proteomic mass spectrometry and custom phospho-specific antibodies integrated with shRNA or siRNA gene suppression demonstrated PKC phosphorylated Merlin S13, but meningioma Wnt pathway activation induced PP1A to dephosphorylate Merlin S13 and drive cell proliferation. In summary, Merlin S13 dephosphorylation drives meningioma Wnt signalling and cell proliferation. These data reveal a novel tumor-promoting function of NF2/Merlin in Merlin-intact meningiomas.

Sarcomatoid Adrenal Cortical Carcinoma: Report of a Rare Case

Abstract Introduction/Objective Adrenal cortical carcinoma (ACC) is a rare neoplasm with oncocytic, myxoid, and sarcomatoid variants. Sarcomatoid is the least common variant. To our knowledge, there are only 23 cases of sarcomatoid ACC reported in the English literature. Typically, ACC’s express SF1, Melan A, inhibin, and synaptophysin. It has been reported that the sarcomatoid component does not express the aforementioned immunomarkers; however, it can display mutational expression of p53 and β-catenin. Methods/Case Report A 77-year-old man was incidentally found to have a left adrenal gland mass. Hormone levels were unremarkable, and adrenalectomy was performed. Gross examination of the specimen revealed a 4.5 cm well-circumscribed tan and focally hemorrhagic mass confined to the adrenal gland. Histologically, the mass was composed of pleomorphic round to polygonal cells with clear to eosinophilic cytoplasm and spindle cells. The mitotic count exceeded 20 in 50 high power fields. Lymphovascular invasion and extensive necrosis were present. Immunohistochemical studies revealed that the sarcomatoid component expressed SF1 and synaptophysin focally. The non-sarcomatoid component exhibited focal expression of synaptophysin and diffuse expression of SF1 and Melan A. Both components displayed null expression of p53, and neither expressed β-catenin. Ki67 proliferation index was &amp;gt; 95% in the sarcomatoid component. Results (if a Case Study enter NA) NA Conclusion In sarcomatoid ACC, both sarcomatoid and non-sarcomatoid components have been shown to harbor TP53 and CTNNB1 mutations, with the latter leading to activation of Wnt pathway and causing diffuse and strong nuclear expression of β-catenin. In the present case, tumor cells lacked expression of p53, which might have played a role in the evolution of the tumor.

Abstract 13973: Human iPSC-Derived Multi-Cell-Type Engineered Heart Tissues as an in vitro Model to Study Pathological Cardiac Hypertrophy

Introduction Pathological cardiac hypertrophy has not been fully recapitulated in the in vitro settings. Currently used in vitro models are presumably oversimplified since the vast majority are based on iPSC-derived cardiomyocytes (CMs) exclusively and do not include any cardiac non-myocytes. The aim of this study was to generate engineered heart tissues (EHTs) containing five hiPSC-derived cardiac cell types, i.e., CMs, endothelial cells (ECs), macrophages (Mϕs), fibroblasts (FBs), and smooth muscle cells (SMCs) and treat them with prohypertrophic compounds. Methods: Mesoderm induction preceded all cell-type-specific differentiation steps. CMs were then generated by Wnt signaling inhibition and ECs by VEGF stimulation. Mϕ differentiation required treatment with IL-3+M-CSF. FBs and SMCs were generated by Wnt pathway activation followed by bFGF or TGFβ+bFGF stimulation. The characterization of different iPSC-derivatives and EHTs was based on the MICS (MACSima Imaging Cycling Staining) technology and profiling of hallmark genes and proteins. To study the response to prohypertrophic stimuli, EHTs were treated with 20 μM phenylephrine (PE) and 50 nM endothelin-1 (ET-1) for 9-10 days. Results: The expression of cell-type-specific markers and functional assays were the determinants of successful differentiation. For instance, CMs were identified based on cTnT expression and contractile capabilities, Mϕs by expression of CD68 and phagocytic activity, and ECs by CD31 expression and tube formation abilities. Multi-cell-type EHTs were assembled from 60% CMs, 20% ECs, 10% Mϕs, 5% FBs and 5% SMCs. These EHTs displayed accelerated development, i.e., faster remodeling and earlier onset of beating compared to CM-only EHTs. Preliminary results show that multi-cell-type EHTs exhibited higher maximal forces and increased stiffness compared to CM-only controls. The exposure to PE+ET-1 led to a decrease in force, activation of the hypertrophic gene program, and elevation of NT-pro BNP production. Conclusions: We successfully generated functional multi-cell-type EHTs containing five hiPSC-derived cardiac cell types. Upon hypertrophic stimulation, these EHTs developed a pathological phenotype that resembles the hallmarks of a failing human heart.

Pygo1 Regulates the Behavior of Human Non-Small-Cell Lung Cancer via the Wnt/β-Catenin Pathway.

Abnormal activation of the classical Wnt pathway has been reported in non-small-cell lung cancer (NSCLC) previously. Pygo family genes, the core regulators of Wnt/β-catenin signaling, were also reported to be involved in tumorigenesis. However, the role of the homolog Pygo1 in human lung cancer remains unclear. In the current study, we demonstrated an association of increased Pygo1 expression with consistent high nuclear β-catenin signals across pathological tissue samples of early-stage human NSCLC. Overexpression of Pygo1 in lung cancer cells resulted in enhanced G1/S cell phase transformation, reduced apoptosis, and increased cell proliferation. These changes were accompanied by the downregulation of cell cycle-related proteins, such as RB, p16, p53, and p27Kip1, and increased expression of CyclinE1. Migration, wound healing, and colony formation assays revealed that Pygo1 overexpression enhanced the invasion and migration of lung cancer cells, increased the formation of clones, and suppressed E-cadherin expression. In addition, overexpression of Pygo1 in lung cancer cells led to an increase of β-catenin/TCF4 complex, as well as upregulated expression of target genes of β-catenin. In vivo experiments also revealed that Pygo1 overexpression promoted the tumorigenicity of a xenograft tumor model, while Wnt inhibition partially blocked the effect of Pygo1 overexpression. In conclusion, Pygo1 affects human NSCLC via the canonical Wnt/β-catenin pathway, which provides new clues for lung cancer pathology.

Brasenia-inspired hydrogel with sustained and sequential release of BMP and WNT activators for improved bone regeneration

Although bone morphogenetic protein (BMP) and WNT signaling play pivotal roles in bone development, homeostasis, and regeneration, the applications of proteins to stimulate corresponding signaling pathways showed limited outcomes in the repair and regeneration of bone defects that might be attributed to the reciprocal interventions of these pathways. In order to satisfy the combinational and sequential activation of BMP and WNT pathways, inspired by the heterogeneous hydrogel-liked structures of Brasenia, heterogeneous alginate/chitosan hydrogels were fabricated and spatially loaded with FK506 and BIO to achieve sustained and sequential release of the activators. Alkaline phosphatase staining, alizarin red staining and qRT-PCR results suggested that FK506 and BIO enhanced osteoblastic differentiation in vitro when used separately. Besides, by mixing and matching the activators and the hydrogel layers, a superior releasing mode that a combination of early FK506 release and following BIO release was identified via both in vitro and in vivo explorations for most efficient bone regeneration. These results suggested that drug-loaded heterogeneous hydrogels possess great potentials in treating bone loss defects for future clinical practice.

Gallic acid alleviates gastric precancerous lesions through inhibition of epithelial mesenchymal transition via Wnt/β-catenin signaling pathway

Ethnopharmacological relevanceGallic acid (GA) is a natural polyphenolic compound derived from Rhus chinensis Mill. with a variety of biological activities such as astringent sweat, cough, dysentery, hemostasis, and detoxification, and is widely used in China as a treatment for cough, bleeding, and gastrointestinal disorders. In recent years, the anticancer activity of GA has been demonstrated in a variety of cancers, affecting multiple cellular pathways associated with cancer onset, development and progression. Aim of the studyTo investigate the role and potential mechanism of GA on gastric precancerous lesions (GPL), the key turning point of gastritis to gastric cancer, with the aim of delaying, blocking or reversing the dynamic overall process of “inflammation-cancer transformation” and thus blocking GPL to prevent the development of gastric cancer. Materials and methodsIn this study, we established N-Nitroso-N-methylurea (MNU)-induced GPL mice model and induced precancerous lesions of gastric cancer cells (MC), i.e. epithelial mesenchymal transition (EMT), in human gastric mucosal epithelial cells (GES-1) with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). We used conventional pathology, immunohistochemistry, RNA sequencing, Western blot and other techniques to study the therapeutic effect of GA on GPL and its possiblemechanism in vitro and in vivo. ResultsThe results showed that compared with normal GES-1 cells, MC cells had the characteristics of malignant cells such as abnormal proliferation, invasion and metastasis, accompanied by decreased expression of EMT-related protein E-cadherin and increased expression of N-cadherin and Vimentin. GA can inhibit the malignant behavior of MC cell proliferation and induce its G0/G1 phase arrest, which is achieved by downregulating the Wnt/β-catenin signaling pathway and thereby inhibiting the EMT process. However, when we incubated with the Wnt pathway activator (Wnt agonist 1), the effect of GA was reversed. Furthermore, analysis of human gastric specimens showed that activation of the Wnt/β-catenin pathway was significantly associated with GPL pathological changes. Meanwhile, GA reversed MNU-induced intestinal metaplasia and partial dysplasia in GPL mice. ConclusionTaken together, these results indicate that GA prevents the occurrence and development of GPL by inhibiting the Wnt/β-catenin signaling pathway and then inhibiting the EMT process, which may become potential candidates for the treatment of GPL.

HDAC2 Induces DNA Methyltransferase DNMT3B Expression to Regulate the Wnt Signaling Pathway and Thus Promotes Glioma Development and Progression.

Purpose To investigate the role and molecular mechanism of HDAC2 in glioma. Methods GSE16011, GSE31262, and GSE90598 datasets were used to identify co-expressed genes, GO analysis, and KEGG analysis to identify gene enrichment pathways, and PPI networks were constructed to identify gene interrelationships. HDAC2 enrichment on DNMT3B promoter and DNMT3B enrichment on Bcl2 CpG island was detected by a ChIP assay. The expression, prognosis, and hierarchical distribution of HDAC2, DNMT3B, and Bcl2 were examined in the CGGA database, and the correlation between HDAC2 and DNMT3B, Bcl2, and DNMT3B and Bcl2 was assessed. Results The HDAC2-DNMT3B-Bcl2 axis is differentially expressed and interacts in gliomas. HDAC2 activates the transcriptional activity of DNMT3B, and DNMT3B inhibits the expression of Bcl2. HDAC2 and DNMT3B are highly expressed in gliomas and have a poor prognosis, while Bcl2 is lowly expressed in gliomas and has a good prognosis. Conclusion HDAC2 promotes DNMT3B transcriptional repression of Bcl2 expression and Wnt pathway activity, thereby activating glioma cell activity in vitro and in vivo.

Knockdown of SOX9 alleviates tracheal fibrosis through the Wnt/β-catenin signaling pathway.

Trachealfibrosis is an important cause of tracheal stenosis without effective treatments, and new drug targets need to be developed. The role of SOX9 in the injury and repair of the trachea is unknown; this study aims to investigate the role of SOX9 in the regulation of tracheal fibrosis based on clinical samples from patients with tracheal injury and a model of tracheal fibrosis produced by tracheal brushing in rats. The results showed that the expressions of SOX9 and mesenchymal and ECM-related indicators were increased in the injury and fibrosis of the trachea in patients and rats. Serum SOX9 levels exhibited a sensitivity of 83.87% and specificity of 90% in distinguishing patients with tracheal fibrosis from healthy volunteers when the cut‑off value was 13.24ng/ml. Knockdown SOX9 can markedly inhibit granulation tissue proliferation, reduce inflammation and ECM deposition, promote epithelial regeneration and granulation tissue apoptosis, and attenuate the tracheal fibrosis after injury. Additionally, RNA sequencing showed that the proliferation, migration, and ECM deposition of tracheal granulation tissue were related to the activation of Wnt pathway, activation of the β-catenin, and p-GSK3β after injury can be inhibited by the knockdown of SOX9. In summary, SOX9 is upregulated in tracheas fibrosis and may be a novel factor to promote tracheal fibrosis progression. Inhibiting SOX9 may be used to prevent and treat tracheal fibrosis in the future. KEY MESSAGE : The expression of SOX9 is upregulated the process of injury and repair of the tracheal fibrosis. Knocking down SOX9 can attenuate tracheal fibrosis after injury by inhibiting inflammation response, granulation tissue proliferation, ECM deposition, and promoting granulation tissue apoptosis. The Wnt/β-catenin-SOX9 axis is activated during tracheal injury and fibrosis, and inhibition of SOX9 can partially alleviate tracheal fibrosis. SOX9 may act as a new diagnostic and therapeutic target in patients with tracheal fibrosis in the future.

324 (PB104) - Quantitative analysis of EGFR expression and distribution upon drug treatment in colorectal organoid models

Background: Human organoids are patient-derived tissues that can be expanded in vitro by stimulating the (cancer) stem cells with WNT pathway activators, treatment with differentiation inhibitors and by providing growth factors in an extracellular matrix environment. These organoids form three-dimensional (3D) multicellular structures that polarize and self-organize and express relevant disease-related markers, which makes an organoid biobank ideal for testing the efficacy and mode of action of compounds and antibodies across a diverse patient population. Here, our colorectal organoid biobank and high content imaging platform were used to characterize EGFR dynamics upon drug treatment. Methods: An established biobank of normal colon and colorectal cancer organoid models was characterized for its sensitivity to the growth factor EGF, EGFR-inhibitors (Cetuximab), and standard-of-care compounds. To study the distribution of the EGF receptor, antibodies followed by immunofluorescence staining were used to detect the localization and distribution upon growth factor stimulation. (Inducible) knockdown organoid models with shRNA constructs targeting EGFR were generated to validate the findings. Results: The 3D organoid screening and our proprietary image analysis platform allows the in-depth detection and distinction of growth factor responses that not necessarily only relate to proliferation. By making use of this system, the sensitivity of organoid models to EGF stimulation that are independent of EGF for their outgrowth could be determined. By combining the detection of the target, EGFR, with the detection of targeting EGFR-inhibitory antibodies by immunofluorescence followed by image analysis, the redistribution of the receptor upon EGF-stimulation and in response to antibody exposure could be visualized and quantified. This revealed that EGFR is internalized in the first hours upon EGF stimulation. Along with making use of (inducible) knockdown organoids, it also revealed that different EGFR-targeting antibodies have different effects on the redistribution, internalization, and degradation of EGFR. Conclusions: The high content imaging platform in combination with high throughput screening in 3D-cultured organoid models allows compound and antibody testing in physiologically relevant patient-derived models to not only study the sensitivity to novel compounds, but also for patient stratification and to study the mode of action or understand the underlying relevant biology. The outcome of this work demonstrated that organoids are versatile, multicellular structures that are highly suitable for studying the expression and redistribution of disease-relevant targets. No conflict of interest.

The oncogenic transcription factor FOXQ1 is a differential regulator of Wnt target genes

The forkhead box transcription factor FOXQ1 contributes to the pathogenesis of carcinomas. In colorectal cancers, FOXQ1 promotes tumour metastasis by inducing epithelial-to-mesenchymal transition (EMT) of cancer cells. FOXQ1 may exacerbate cancer by activating the oncogenic Wnt/β-catenin signalling pathway. However, the role of FOXQ1 in the Wnt pathway remains to be resolved. Here, we report that FOXQ1 is an activator of Wnt-induced transcription and regulator of β-catenin target gene expression. Upon Wnt pathway activation, FOXQ1 synergises with the β-catenin nuclear complex to boost the expression of major Wnt targets. In parallel, we find that FOXQ1 controls the differential expression of various Wnt target genes in a β-catenin-independent manner. Using RNA sequencing of colorectal cancer cell lines, we show that Wnt signalling and FOXQ1 converge on a transcriptional programme linked to EMT and cell migration. Additionally, we demonstrate that FOXQ1 occupies Wnt-responsive elements in β-catenin target gene promoters and recruits a similar set of co-factors to the β-catenin-associated transcription factor Tcf7l1. Taken together, our results indicate a multifaceted role of FOXQ1 in Wnt/β-catenin signalling, which may drive the metastasis of colorectal cancers.

142 (PB022) - Loss of SALL2 in colorectal cancer correlates with Wnt pathway activation, a new mechanism involving SALL2-dependent AXIN2 transcriptional regulation.

Background: SALL2 is a developmental transcription factor involved in the regulation of cell proliferation, migration, and survival. Massive data analyses of cancer versus normal tissues indicate that SALL2 mRNA is significantly decreased in colorectal cancer (CRC), a cancer type characterized by hyperactivation of the Wnt pathway. Interestingly, our previous ChIP-seq analyses suggested that SALL2 regulates genes associated with the Wnt pathway, including the negative regulator AXIN2. Currently, SALL2 function and its relationship with the Wnt/β-catenin pathway in colorectal cancer is unknown.

CYP24A1 Involvement in Inflammatory Factor Regulation Occurs via the Wnt Signaling Pathway

ObjectiveWhile the upregulation of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) gene expression has been reported in colon cancer, its role in tumorigenesis remains largely unknown. In this study, we aimed to investigate the involvement of CYP24A1 in Wnt pathway regulation via the nuclear factor kappa B (NF-κB) pathway.MethodsThe human colon cancer cell lines HCT-116 and Caco-2 were subjected to stimulation with interleukin-6 (IL-6) as well as tumor necrosis factor alpha (TNF-α), with subsequent treatment using the NF-κB pathway-specific inhibitor ammonium pyrrolidinedithiocarbamate (PDTC). Furthermore, CYP24A1 expression was subjected to knockdown via the use of small interfering RNA (siRNA). Subsequently, NF-κB pathway activation was determined by an electrophoretic mobility shift assay, and the transcriptional activity of β-catenin was determined by a dual-luciferase reporter assay. A mouse ulcerative colitis (UC)-associated carcinogenesis model was established, wherein TNF-α and the NF-κB pathway were blocked by anti-TNF-α monoclonal antibody and NF-κB antisense oligonucleotides, respectively. Then the tumor size and protein level of CYP24A1 were determined.ResultsIL-6 and TNF-α upregulated CYP24A1 expression and activated the NF-κB pathway in colon cancer cells. PDTC significantly inhibited this increase in CYP24A1 expression. Additionally, knockdown of CYP24A1 expression by siRNA could partially antagonize Wnt pathway activation. Upregulated CYP24A1 expression was observed in the colonic epithelial cells of UC-associated carcinoma mouse models. Anti-TNF-α monoclonal antibody and NF-κB antisense oligonucleotides decreased the tumor size and suppressed CYP24A1 expression.ConclusionTaken together, this study suggests that inflammatory factors may increase CYP24A1 expression via NF-κB pathway activation, which in turn stimulates Wnt signaling.

Discovery of Novel Pyrazole-Based KDM5B Inhibitor TK-129 and Its Protective Effects on Myocardial Remodeling and Fibrosis

Lysine-specific demethylase 5B (KDM5B) has been recognized as a potential drug target for cardiovascular diseases. In this work, we first found that the KDM5B level was increased in mouse hearts after transverse aortic constriction (TAC) and in Ang II-induced activated cardiac fibroblasts. Structure-based design and further optimizations led to the discovery of highly potent pyrazole-based KDM5B inhibitor TK-129 (IC50 = 0.044 μM). TK-129 reduced Ang II-induced activation of cardiac fibroblasts in vitro, exhibited good PK profile (F = 42.37%), and reduced isoprenaline-induced myocardial remodeling and fibrosis in vivo. Mechanistically, we found that KDM5B up-regulation in cardiac fibroblast activation was associated with the activation of Wnt-related pathway. The protective effects of TK-129 were associated with its KDM5B inhibition and blocking KDM5B-related Wnt pathway activation. Taken together, TK-129 may represent a novel KDM5-targeting lead compound for cardiac remodeling and fibrosis.