Activation Of Wnt Pathway Research Articles

HCMV UL8 interaction with β-catenin and DVL2 regulates viral reactivation in CD34+ hematopoietic progenitor cells.

CD34+ hematopoietic progenitor cells (HPCs) are an important cellular reservoir for latent human cytomegalovirus (HCMV). Several HCMV genes are expressed during latency that are involved with the maintenance of the viral genome in CD34+ HPC. However, little is known about the process of viral reactivation in these cells. Here, we describe a viral protein, pUL8, and its interaction and stabilization with members of the Wnt/β-catenin pathway as an important component of viral reactivation. We further define that pUL8 and β-catenin interact with DVL2 via a PDZ-binding domain, and loss of UL8 interaction with β-catenin-DVL2 restricts viral reactivation. Our findings will be instrumental in understanding the molecular processes involved in HCMV reactivation in order to design new antiviral therapeutics.

Induction of epithelial-mesenchymal transition in thyroid follicular cells is associated with cell adhesion alterations and low-dose hyper-radiosensitivity.

Epithelial-mesenchymal transition (EMT) is associated with altered cellular adhesion. We previously demonstrated that cellular adhesion influences Low-dose Hyper-Radiosensitivity (HRS) in a variety of tumor cells. However, the relationship of low-dose HRS with the phenotypic plasticity incurred by EMT during the neoplastic transformation remains to be elucidated. To investigate whether acquisition of EMT phenotype during progressive neoplastic transformation may affect low-dose radiation sensitivity. Primary thyroid cells obtained from a human cystic thyroid nodule were first subjected to nutritional stress. This yielded immortalized INM-Thy1 cell strain, which was further treated with either multiple γ-radiation fractions (1.5 Gy each) or repetitive cycles of 3-methylcholanthrene and phorbol-12-myristate-13-acetate, yielding two progressive transformants, viz., INM-Thy1R and INM-Thy1C. Morphological alterations, chromosomal double-minutes, cell adhesion proteins, anchorage dependency, tumorigenicity in nude mice and cellular radiosensitivity were studied in these strains. Both transformants (INM-Thy1R, INM-Thy1C) displayed progressive tumorigenic features, viz., soft agar colony growth and solid tumor growth in nude mice, coupled with features of epithelial-mesenchymal transition and activated Wnt pathway. Incidentally, the chemical-induced transformant (INM-Thy1C) displayed a prominent HRS (αs/αr=29.35) which remained unaffected at high cell density. However, the parental (INM-Thy1) cell line as well as radiation-induced transformant (INM-Thy1R) failed to show this hypersensitivity. The study shows that induction of EMT in thyroid follicular cells may accompany increased susceptibility to low-dose ionizing radiation, which was attenuated by adaptive resistance acquired during radiation-induced transformation.

Cadherin-11 increases tumor cell proliferation and metastatic potential via Wnt pathway activation.

During epithelial-mesenchymal transition (EMT) in cancer progression, tumor cells switch cadherin profile from E-cadherin to cadherin-11 (CDH11), which is accompanied by increased invasiveness and metastatic activity. However, the mechanism through which CDH11 may affect tumor growth and metastasis remains elusive. Here, we report that CDH11 was highly expressed in multiple human tumors and was localized on the membrane, in the cytoplasm and, surprisingly, also in the nucleus. Interestingly, β-catenin remained bound to carboxy-terminal fragments (CTFs) of CDH11, the products of CDH11 cleavage, and co-localized with CTFs in the nucleus in the majority of breast cancer samples. Binding of β-catenin to CTFs preserved β-catenin activity, whereas inhibiting CDH11 cleavage led to β-catenin phosphorylation and diminished Wnt signaling, similar to CDH11 knockout. Our data elucidate a previously unknown role of CDH11, which serves to stabilize β-catenin in the cytoplasm and facilitates its translocation to the nucleus, resulting in activation of Wnt signaling, with subsequent increased proliferation, migration and invasion potential.

Upregulation of SOX9 promotes the self-renewal and tumorigenicity of cervical cancer through activating the Wnt/β-catenin signaling pathway.

Sry-box9 (SOX9) maintains stem cell properties and plays crucial roles in many cancers. However, whether SOX9 is correlated with cervical cancer cell stemness and its detailed mechanism remains obscure. We studied the relationship between SOX9 and prognosis of cervical cancer through public database, and SOX9 was related to poor prognosis of cervical cancer. Elevated SOX9 expression enhanced the self-renewal properties and promotes tumorigenicity in cervical cancer. Overexpression of SOX9 could promote the expression of stem cell-related factors in cervical cancer cells and xenografts. Meanwhile, overexpression of SOX9 could also enhance the expressions of FZD10, β-catenin, and c-Myc in cervical cancer cells and xenografts, while inhibiting the expression of DDK1. The activation of Wnt pathway by chir-99 021 raised the tumor spheroid ability of SOX9 knockdown HeLa cells. In addition, SOX9 could transcriptional inhibit DKK1 and activate FZD10 and MYC by binding to their promoters to affect the Wnt/β-catenin pathway. These results demonstrated SOX9 regulated the self-renewal and tumorigenicity of cervical cancer through Wnt/β-catenin pathway by directly transcriptional activation of FZD10, MYC and transcriptional inhibition of DKK1.

Abstract 23 HLA-Homozygous iPSC from Cord Blood for the Generation of Cardiomyocytes for Allogeneic Cell Therapy to the Heart

Abstract Introduction Based on a previous grant on HLA-homozygous (HLAh) iPS-GMP (and ATMP conform) generation from allogeneic licensed cord blood (CB) units, the Düsseldorf CBB could provide the most frequent HLA-types for reprogramming (Liedtke et al. 2020). These HLAh iPSC provide the basis for the generation of cardiomyocytes from iPSC. Patients with acute heart failure or cardiomyopathies have limited options of treatment. iPSC technology allows for de novo generation of functional heart muscle in vitro, enabling the implementation of new therapy concepts for heart regeneration. Objectives The GMP-grade iPSC-lines are the backbone for the granted EU project (HEAL (101056712) on iPSC-cardiomyocyte aggregate manufacturing for allogeneic cell therapy. Besides GMP-compliant up-scaled cell production of cardiomyocytes, the project is designed to overcome many of the scientific, safety and regulatory hurdles necessary to initiate a first-in-man clinical HLAh-based heart regeneration study. Methods After CD34+ selection/short expansion of CD34, cells were reprogrammed by GMP-conform plasmids and stored in a master cell bank with most frequent HLA-haplotypes. All iPSC lines were tested extensively for genetic stability, mycoplasma, virus markers, endotoxins and microorganisms. The differentiation into cardiomyocytes was performed in correspondence to the Wnt modulation, i.e. first activating Wnt pathway and then inhibiting the pathway with the addition of small molecules (Halloin et al. 2019). For the up-scaled production, a 3D-cardiomyocyte aggregate culture in large stirred tank bioreactors (STBR) was applied. Cryopreservation using controlled rate freezing was adapted to tissue freezing. Results Selection of the most frequent homozygous haplotypes facilitated the production of HLAh iPSC lines for differentiation into cardiomyocytes. Efficient progress had been made for the improvement of STBR platform for sufficient production of cardiomyocytes, enabling the differentiation with a purity of up to 95%, measured via cardiac troponin T (cTnT). Discussion Although high yields of cardiomyocytes in a GMP-compliant process is getting more achievable by using STBRs, the safety and regulatory hurdles are still a major problem. For safety testing, toxicology, tumorigenicity assays will be performed in mice and in pig models. This will enable the prediction of arrhythmia-related risks. For a safe distribution of the finally released cell product optimization of cryopreservation of cardiomyocytes aggregates will be performed. Disclaimer: Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency (HADEA). Neither the European Union nor the granting authority can be held responsible for them.

SEPTIN3 Promotes Progression of Triple-Negative Breast Cancer via Activating Wnt Pathway.

There is a lack of targeted therapies for triple-negative breast cancer (TNBC), necessitating the search for novel targets. Patients with TNBC exhibit elevated expression of neuron-specific septin-3 (SEPTIN3), leading to poor prognosis. This study aimed to investigate the modulation of SEPTIN3 expression in TNBC cells. The relative expression levels of SEPTIN3 in TNBC tissues and cell lines were determined using Western blotting and qRT-PCR. We generated lentivirally transduced TNBC cell lines so such that SEPTIN3 was overexpressed or knocked down. Next, the effect of SEPTIN3 on the biological behavior of TNBC cells was detected using a series of functional assays, including CCK8, colony formation, scratch, and transwell assays. We monitored the tumorigenicity of SEPTIN3 overexpressed cells and performed Ki-67 immunostaining in mice. The mechanism mediated by SEPTIN3 was studied using functional enrichment analysis and Western blotting. Protein and mRNA expression levels of SEPTIN3 were observed to be increased in TNBC tissues and cell lines. SEPTIN3 knockdown reduced cell growth, invasion, and migration, whereas SEPTIN3 overexpression exerted the opposite effects. SEPTIN3 was observed to favor cell growth and tumorigenicity in vivo. In addition, SEPTIN3 promoted TNBC cell aggressiveness and proliferation via activation of the Wnt signaling pathway. SEPTIN3 emerged as an oncogene that accelerates tumor progression by regulating the Wnt signaling pathway.

SMAD4 and KCNQ3 alterations are associated with lymph node metastases in oesophageal adenocarcinoma

Metastasis in oesophageal adenocarcinoma (OAC) is an important predictor of survival. Radiological staging is used to stage metastases in patients, and guide treatment selection, but is limited by the accuracy of the approach. Improvements in staging will lead to improved clinical decision making and patient outcomes. Sequencing studies on primary tumours and pre-cancerous tissue have revealed the mutational landscape of OAC, and increasingly cheap and widespread sequencing approaches offer the potential to improve staging assessment. In this work we present an analysis of lymph node metastases found by radiological and pathological sampling, identifying new roles of the genes SMAD4 and KCNQ3 in metastasis. Through transcriptomic analysis we find that both genes are associated with canonical Wnt pathway activity, but KCNQ3 is uniquely associated with changes in planar cell polaritiy associated with non-canonical Wnt signalling. We go on to validate our observations in KCNQ3 in cell line and xenograph systems, showing that overexpression of KCNQ3 reduces wound closure and the number of metastases observed. Our results suggest both genes as novel biomarkers of metastatic risk and offer new potential routes to drug targeting.

Different expression of DACT1, DACT2, and CYCLIN D1 genes in human colorectal cancer tissues and its association with clinicopathological characteristics

Aberrant activation of Wnt pathway is linked to dysregulation of several genes. DACT1 and DACT2 are members of the DACT family that participate in antagonizing of the Wnt signaling cascade. Thus in this study, we assessed the mRNA levels of DACT1, DACT2, and CYCLIN D1 in 70 pairs of CRC tissues compared to the adjacent tissues. Determination of the mRNA levels of DACT1, DACT2, and CYCLIN D1 was done by Quantitative Real-Time PCR (qRT-PCR). The correlation between DACT1, DACT2, and CYCLIN D1 genes was also examined. Receiver operating characteristic (ROC) curves was plotted to assess the diagnostic power. The association between histopathological parameters and the DACT1, DACT2, and CYCLIN D1 genes was investigated. The expression levels of DACT1 and CYCLIN D1 were remarkably higher in CRC tissues compared to the adjacent tissues (p < 0.0001). However, the expression of DACT2 was decreased (p < 0.001). Our results showed a significant correlation between the expression of DACT1 and CYCLIN D1 (p < 0.0001). DACT1 (AUC = 0.74, p < 0.0001), DACT2 (AUC = 0.69, p < 0.0003), and CYCLIN D1 (AUC = 0.75, p < 0.0001) had good effectiveness in separation between CRC samples and adjacent tissues. We found a significant association between DACT1 expression with tumor site (p < 0.01). Also, a significant association was detected between DACT2 and CYCLIN D1 with tumor stage (p < 0.005 and p < 0.038, respectively). The findings suggested that DACT1 could function as an oncogene, whereas DACT2 was downregulated and can be considered as a tumor suppressor in CRC.

AXIN1 bi-allelic variants disrupting the C-terminal DIX domain cause craniometadiaphyseal osteosclerosis with hip dysplasia

Sclerosing skeletal dysplasias result from an imbalance between bone formation and resorption. We identified three homozygous, C-terminally truncating AXIN1 variants in seven individuals from four families affected by macrocephaly, cranial hyperostosis, and vertebral endplate sclerosis. Other frequent findings included hip dysplasia, heart malformations, variable developmental delay, and hematological anomalies. In line with AXIN1 being a central component of the β-catenin destruction complex, analyses of primary and genome-edited cells harboring the truncating variants revealed enhanced basal canonical Wnt pathway activity. All three AXIN1-truncating variants resulted in reduced protein levels and impaired AXIN1 polymerization mediated by its C-terminal DIX domain but partially retained Wnt-inhibitory function upon overexpression. Addition of a tankyrase inhibitor attenuated Wnt overactivity in the AXIN1-mutant model systems. Our data suggest that AXIN1 coordinates the action of osteoblasts and osteoclasts and that tankyrase inhibitors can attenuate the effects of AXIN1 hypomorphic variants.

Am80, a retinoic acid receptor agonist, activates the cardiomyocyte cell cycle and enhances engraftment in the heart.

Human induced pluripotent stem cell-derived (hiPSC) cardiomyocytes are a promising source for regenerative therapy. To realize this therapy, however, their engraftment potential after their injection into the host heart should be improved. Here, we established an efficient method to analyze the cell cycle activity of hiPSC cardiomyocytes using a fluorescence ubiquitination-based cell cycle indicator (FUCCI) system. Invitro high-throughput screening using FUCCI identified a retinoic acid receptor (RAR) agonist, Am80, as an effective cell cycle activator in hiPSC cardiomyocytes. The transplantation of hiPSC cardiomyocytes treated with Am80 before the injection significantly enhanced the engraftment in damaged mouse heart for 6months. Finally, we revealed that the activation of endogenous Wnt pathways through both RARA and RARB underlies the Am80-mediated cell cycle activation. Collectively, this study highlights an efficient method to activate cell cycle in hiPSC cardiomyocytes by Am80 as a means to increase the graft size after cell transplantation into a damaged heart.

SHH PATHWAY ACTIVATION DRIVES MALIGNANT TRANSFORMATION IN A SUBSET OF MPNSTS AND IS A POTENTIAL THERAPEUTIC TARGET

Abstract BTFC travel award recipient Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive Schwann-cell derived sarcomas and the leading cause of mortality in Neurofibromatosis Type 1. The molecular pathways driving malignant transformation is not well understood. In this study, we leveraged multi-platform genomic and epigenomic profiling of human neurofibromas and MPNSTs to identify targetable molecular pathways that lead to malignant transformation. Fresh-frozen tumors (N = 108) were studied including methylation profiling, RNA sequencing, and whole exome sequencing. Unsupervised consensus clustering of methylome and transcriptome data identified 2 distinct MPNST subgroups. Pathway analysis showed that MPNST-G1 tumors are characterized by SHH pathway activation (NES=1.9952, p&amp;lt;0. 0001, FDR&amp;lt;0.001), while MPNST-G2 tumors are characterized by WNT pathway activation (NES=1.7630, p&amp;lt;0.0001, FDR=0.0058). We observed significantly higher rates of PTCH1 deletions, a negative regulator of SHH pathway, in MPNST-G1 (62.5% vs 0%, Fisher Exact Test=0.0065, p&amp;lt;0.05) and PTCH1 promoter hypermethylation (mean beta value: 0.2899 vs. 0.06119, p&amp;lt;0.05). A computational drug screen identified sonedigib, a SHH pathway inhibitor, as a potential treatment option for MPSNT-G1. We validated the importance of SHH pathway in malignant transformation by knocking out PTCH1 in neurofibroma cell lines and demonstrated increased cellular proliferation, cellular migration and invasion. In addition, treatment with sonedigib decreased cellular viability in MPNST cell lines and improved survival in mouse xenograft models. These results suggest a targeted approach should be taken for treating MPNSTs, and SHH and WNT pathway inhibition may be promising avenue for therapeutic development.

Procoagulant genes may affect angiogenesis, epithelial-mesenchymal transition, survival prognosis and tumor immune microenvironment in patients with urothelial carcinoma.

Factors related to coagulation regulation are closely related to angiogenesis, epithelial-mesenchymal transition, tumor proliferation and metastasis, and tumor immune microenvironment remodeling in tumors. To date, there are no quantitative indicators of coagulation associated with urothelial cancer. We classified urothelial cancer into high coagulation and low coagulation subtypes by screening for procoagulant-related molecular features and screened out relevant genes representing the coagulation state of urothelial carcinoma. Tumors with increased procoagulant gene expression were consistently associated with higher T-staging (p < 0.001), lymph node metastasis (p < 0.001), stage (p < 0.001), and grade (p = 0.046). Furthermore, high expression of procoagulant genes predicts a worse prognosis, a higher tumor proliferation rate and increased angiogenesis within the tumor. In addition, according to cibersort algorithm, the increased expression of procoagulant gene was negatively correlated with the degree of T-lymphocyte infiltration and positively correlated with the degree of M2 macrophage infiltration. Increased expression of procoagulant genes in data sets treated with immune checkpoints also predicted worse response and worse prognosis. At the same time, the expression of procoagulant genes in bladder cancer promoted the activation of coagulation, EMT, TGF-β and WNT pathways.

Transcriptome analysis of primary sporadic neuroendocrine tumours of the intestine identified three different molecular subgroups

BackgroundIntestinal neuroendocrine tumours (I-NETs) represent a non-negligible entity among intestinal neoplasms, with metastatic spreading usually present at the time of diagnosis. In this context, effective molecular actionable targets are still lacking. Through transcriptome analysis, we aim at refining the molecular taxonomy of I-NETs, also providing insights towards the identification of new therapeutic vulnerabilities. Materials and MethodsA retrospective series of 38 primary sporadic, surgically-resected I-NETs were assessed for transcriptome profiling of 20,815 genes. ResultsTranscriptome analysis detected 643 highly expressed genes. Unsupervised hierarchical clustering, differential expression analysis and gene set enriched analysis identified three different tumour clusters (CL): CL-A, CL-B, CL-C. CL-A showed the overexpression of ARGFX, BIRC8, NANOS2, and SSTR4 genes. Its most characterizing signatures were those related to cell-junctions, and activation of mTOR and WNT pathway. CL-A was also enriched in T CD8 + lymphocytes. CL-B showed the overexpression of PCSK1, QPCT, ST18, and TPH1 genes. Its most characterizing signatures were those related to adipogenesis, neuroendocrine metabolism, and splice site machinery-related processes. CL-B was also enriched in T CD4 + lymphocytes. CL-C showed the overexpression of ALB, ANG, ARG1, and HP genes. Its most characterizing signatures were complement/coagulation and xenobiotic metabolism. CL-C was also enriched in M1/2 macrophages. These CL-based differences may have therapeutic implications in refining the management of I-NET patients. At last, we described a specific gene-set for differentiating I-NET from pancreatic NET. DiscussionOur data represent an additional step for refining the molecular taxonomy of I-NET, identifying novel transcriptome subgroups with different biology and therapeutic opportunities.

MiR-455-3p inhibits gastric cancer progression by repressing Wnt/β-catenin signaling through binding to ARMC8

BackgroundGlobally, gastric cancer (GC) is one of the world’s most widespread malignancies, with persistent high mortality and morbidity rates. Increasing evidence now suggests that microRNAs (miRNAs) participate in many biological processes, with miR-455-3p having key roles in the progression of diverse cancers. Nevertheless, miR-455-3p function and expression in GC remain unclear.MethodsWe explored miR-455-3p expression in GC using quantitative polymerase chain reaction (qPCR). To further examine the effect of miR-455-3p in GC, after transfecting miR-455-3p mimics or inhibitors into GC cells, 5-ethynyl-2’-deoxyuridine (EdU) incorporation and colony formation assays were performed to examine cell proliferation. Flow cytometry was used to detect apoptosis, and expression levels of Bax, Bcl-2, Snail, N-cadherin, E-cadherin, and Caspase-3 were assessed by western blotting (WB). Using online databases and luciferase assays, we identified armadillo repeat-containing protein 8 (ARMC8) as a promising target of miR-455-3p. A mouse tumor model was established to investigate actions of miR-455-3p in vivo. Expression levels of C-myc, cyclinD1, and β-catenin were examined using WB and immunofluorescence.ResultsMiR-455-3p expression was attenuated in GC tissue and cell lines. MiR-455-3p overexpression inhibited GC cell proliferation, epithelial-mesenchymal transition (EMT), as well as facilitated apoptosis, while suppression of miR-455-3p had the opposite effects. From luciferase assays, we confirmed that ARMC8 was a novel and direct downstream target gene of miR-455-3p, and that the tumor suppressive role of miR-455-3p was in part reversed due to ARMC8 overexpression. Moreover, miR-455-3p inhibited GC growth in vivo via ARMC8. We also observed that miR-455-3p repressed canonical Wnt pathway activation by binding to ARMC8.ConclusionsMiR-455-3p exerted tumor inhibitory effects in GC by targeting ARMC8. Therefore, intervening in the miR-455-3p/ARMC8/Wnt/βcatenin axis could be a promising novel treatment strategy for GC.

Enhancement of TKI sensitivity in lung adenocarcinoma through m6A-dependent translational repression of Wnt signaling by circ-FBXW7

BackgroundTyrosine kinase inhibitors (TKIs) that specifically target mutational points in the EGFR gene have significantly reduced suffering and provided greater relief to patients with lung adenocarcinoma (LUAD). The third-generation EGFR-TKI, Osimertinib, has been successfully employed in clinical treatments to overcome resistance to both original and acquired T790M and L858R mutational points. Nevertheless, the issue of treatment failure response has emerged as an insurmountable problem.MethodsBy employing a combination of multiple and integrated approaches, we successfully identified a distinct population within the tumor group that plays a significant role in carcinogenesis, resistance, and recurrence. Our research suggests that addressing TKI resistance may involve targeting the renewal and repopulation of stem-like cells. To investigate the underlying mechanisms, we conducted RNA Microarray and m6A Epi-Transcriptomic Microarray analyses, followed by assessment of transcription factors. Additionally, we specifically designed a tag to detect the polypeptide circRNA-AA, and its expression was confirmed through m6A regulations.ResultsWe initially identified unique molecular signatures present in cancer stem cells that contributed to poor therapeutic responses. Activation of the alternative Wnt pathway was found to sustain the renewal and resistant status of these cells. Through bioinformatics analysis and array studies, we observed a significant decrease in the expression of circFBXW7 in Osimertinib-resistant cell lines. Notably, the abnormal expression pattern of circFBXW7 determined the cellular response to Osimertinib. Functional investigations revealed that circFBXW7 inhibits the renewal of cancer stem cells and resensitizes both resistant LUAD cells and stem cells to Osimertinib. In terms of the underlying mechanism, we discovered that circFBXW7 can be translated into short polypeptides known as circFBXW7-185AA. These polypeptides interact with β-catenin in an m6A-dependent manner. This interaction leads to reduced stability of β-catenin by inducing subsequent ubiquitination, thereby suppressing the activation of canonical Wnt signaling. Additionally, we predicted that the m6A reader, YTHDF3, shares common binding sites with hsa-Let-7d-5p. Enforced expression of Let-7d post-transcriptionally decreases the levels of YTHDF3. The repression of Let-7d by Wnt signaling releases the stimulation of m6A modification by YTHDF3, promoting the translation of circFBXW7-185AA. This creates a positive feedback loop contributing to the cascade of cancer initiation and promotion.ConclusionsOur bench study, in vivo experiments, and clinical validation have unequivocally shown that circFBXW7 effectively inhibits the abilities of LUAD stem cells and reverses resistance to TKIs by modulating Wnt pathway functions through the action of circFBXW7-185AA on β-catenin ubiquitination and inhibition. The regulatory role of circRNA in Osimertinib treatment has been rarely reported, and our findings reveal that this process operates under the influence of m6A modification. These results highlight the tremendous potential of this approach in enhancing therapeutic strategies and overcoming resistance to multiple TKI treatments.

RNA-binding Protein QKI Inhibits Osteogenic Differentiation Via Suppressing Wnt Pathway

Dysregulation of MSCs differentiation is associated with many pathophysiological processes. Genetically modified MSCs transplantation helps restore bone loss efficiently. BMSCs-specific QKI overexpressing and knockdown mice were built to explore QKI's role in bone formation and fat accumulation. Primary BMSCs with QKI overexpression and knockout were subjected to osteogenic and adipogenic differentiation. ALP staining and oil red O staining were performed to evaluate the differences between the groups. RNA immunoprecipitation was performed to identify the QKI-related pathway. QKI deficient BMSCs were transplanted into mice with glucocorticoid-induced osteoporosis to evaluate its therapeutic potential. Mice harboring BMSC-specific transgenic QKI exhibited reduced bone mass, while BMSC-specific QKI-deficient mice showed an increase in bone mass. Osteogenic differentiation of QKI deficient BMSCs was promoted and adipogenic differentiation was inhibited, while QKI overexpression in BMSCs displayed the opposite effects. To define the underlying mechanisms, RIP sequencing was performed. Wnt pathway-related genes were the putative direct target mRNAs of QKI, Canonical Wnt pathway activation was involved in QKI's effects on osteogenic differentiation. RNA immunoprecipitation quantitative real-time Polymerase Chain Reaction (PCR) and RNA fluorescence in situ hybridization experiments further validated that QKI repressed the expressions of Wnt5b, Fzd7, Dvl3 and β-catenin via direct binding to their putative mRNA specific sites. Glucocorticoid-induced osteoporotic mice transplanted with QKI deficient BMSCs exhibited less bone loss compared with mice transplanted with control BMSCs. QKI suppressed BMSCs osteogenic differentiation by downregulating the expressions of Wnt5b, Fzd7, Dvl3 and β-catenin. Loss of QKI in BMSCs transplantation may provide a new strategy for the treatment of orthopedic diseases such as osteoporosis.

Differential sensitivity to Wnt signaling gradients in human gastric organoids derived from corpus and antrum.

Wnt signaling regulates gastric stem cell proliferation and differentiation. Although similar Wnt gradients exist within the corpus and antrum of the human stomach, there are striking differences in gland architecture and disease manifestation that suggest Wnt may differentially regulate progenitor cell function in each compartment. In this study, we tested sensitivities to Wnt activation in human gastric corpus and antral organoids to determine whether progenitor cells have region-specific differences in Wnt responsiveness. Human patient-matched corpus and antral organoids were grown in the presence of varying concentrations of the Wnt pathway activator CHIR99021 to assess regional sensitivity to Wnt signaling on growth and proliferation. Corpus organoids were further studied to understand how high Wnt affected cellular differentiation and progenitor cell function. A lower concentration of CHIR99021 stimulated peak growth in corpus organoids compared with patient-matched antral organoids. Supramaximal Wnt signaling levels in corpus organoids suppressed proliferation, altered morphology, reduced surface cell differentiation, and increased differentiation of deep glandular neck and chief cells. Surprisingly, corpus organoids grown in high CHIR99021 had enhanced organoid forming potential, indicating that progenitor cell function was maintained in these nonproliferative, deep glandular cell-enriched organoids. Passaging high-Wnt quiescent organoids into low Wnt rescued normal growth, morphology, and surface cell differentiation. Our findings suggest that human corpus progenitor cells have a lower threshold for optimal Wnt signaling than antral progenitor cells. We demonstrate that Wnt signaling in the corpus regulates a bimodal axis of differentiation, with high Wnt promoting deep glandular cell differentiation and suppressing proliferation while simultaneously promoting progenitor cell function.NEW & NOTEWORTHY This study demonstrates that human gastric corpus organoids have a lower Wnt signaling threshold to drive optimal growth relative to patient-matched antral organoids. Paradoxically, supramaximal Wnt levels suppress corpus organoid proliferation, yet promote differentiation toward deep glandular cell types while simultaneously enhancing progenitor cell function. These findings provide novel insights into how Wnt signaling differentially regulates homeostasis in the human gastric corpus and antrum and contextualizes patterns of Wnt activation diseases.

Overcoming the Challenges of Phytochemicals in Triple Negative Breast Cancer Therapy: The Path Forward.

Triple negative breast cancer (TNBC) is a very aggressive subtype of breast cancer that lacks estrogen, progesterone, and HER2 receptor expression. TNBC is thought to be produced by Wnt, Notch, TGF-beta, and VEGF pathway activation, which leads to cell invasion and metastasis. To address this, the use of phytochemicals as a therapeutic option for TNBC has been researched. Plants contain natural compounds known as phytochemicals. Curcumin, resveratrol, and EGCG are phytochemicals that have been found to inhibit the pathways that cause TNBC, but their limited bioavailability and lack of clinical evidence for their use as single therapies pose challenges to the use of these phytochemical therapies. More research is required to better understand the role of phytochemicals in TNBC therapy, or to advance the development of more effective delivery mechanisms for these phytochemicals to the site where they are required. This review will discuss the promise shown by phytochemicals as a treatment option for TNBC.

CNSC-02. INVESTIGATING THE ROLE OF THE WNT SIGNALING TRANSDUCTION PATHWAY IN GLIOMA BLOOD-BRAIN-BARRIER INTEGRITY

Abstract The WNT signaling transduction pathway has been linked to cancer stem cell self-renewal, differentiation, and blood brain barrier (BBB) development. Previous studies have shown that WNT pathway inhibition increased BBB permeability, specifically in WNT medulloblastoma. Glioblastoma, an aggressive malignant tumor with poor prognosis, has also been shown to have high WNT expression and variable BBB permeability. We hypothesize that activating WNT pathway inhibition in glioblastoma stem cells (GSCs) will inhibit glioma progression, increase BBB permeability within the tumor microenvironment, and enhance overall treatment responsiveness. We transduced primary pediatric derived glioma stem cells with overexpressing vectors for WNT inhibitors (DKK1 or WIF1). To characterize effects of DKK1 or WIF1 overexpression, we evaluated cell migration patterns via RTCA xCelligence, cell cycle progression via Annexin 5 assays, and cell viability via cell-titer glo. Brain endothelial cell integrity was evaluated with the treatment of CHIR99021, WNT pathway activator, or GSC condition media with immunoblotting for junctional protein expression (Claudin-5, Claudin-3, Occludin, ZO-1, VE-Cadherin). Verification of WNT inhibitory overexpressing GSCs for DKK1 and WIF1 was performed using RNAseq, proteomics, and immunoblotting. DKK1-GSCs demonstrated a statistically significant impairment in the migratory slope compared to empty vector GSCs. Cell cycle analysis also revealed that DKK1-GSCs were arrested in G0/1 phase while WIF1-GSCs were arrested in S/G2M phase. CHIR99021 endothelial treatment resulted in increased secretion of WNT inhibitors DKK1 and SFRP1 and resultantly, there was a decrease in junctional protein expression. Overall, WNT inhibitor overexpressing GSCs exhibited unique cellular patterns and indirectly disrupted endothelial cell integrity. Future studies will evaluate orthotopic WNT inhibitor overexpressing transplant rodent models to determine BBB integrity, drug permeability, model survival, and chemotherapeutic treatment response. By enhancing our understanding of the WNT pathway in GSCs, we anticipate understanding the use of future therapeutic WNT pathway targeting to increase chemotherapeutic responsiveness and improve glioblastoma prognosis.

Arjunolic acid modulate pancreatic dysfunction by ameliorating pattern recognition receptor and canonical Wnt pathway activation in type 2 diabetic rats

BackgroundArjunolic acid (AA) is a potent phytochemical with multiple therapeutics effects. In this study, AA is evaluated on type 2 diabetic (T2DM) rats to understand the mechanism of β-cell linkage with Toll-like receptor 4 (TLR-4) and canonical Wnt signaling. However, its role in modulating TLR-4 and canonical Wnt/β-catenin crosstalk on insulin signaling remains unclear during T2DM.AimThe current study is aimed to examine the potential role of AA on insulin signaling and TLR-4-Wnt crosstalk in the pancreas of type 2 diabetic rats. MethodMultiple methods were used to determine molecular cognizance of AA in T2DM rats, when treated with different dosage levels. Histopathological and histomorphometry analysis was conducted using masson trichrome and H&E stains. While, protein and mRNA expressions of TLR-4/Wnt and insulin signaling were assessed using automated Western blotting (jess), immunohistochemistry, and RT-PCR. ResultsHistopathological findings revealed that AA had reversed back the T2DM-induced apoptosis and necrosis caused to rats pancreas. Molecular findings exhibited prominent effects of AA in downregulating the elevated level of TLR-4, MyD88, NF-κB, p-JNK, and Wnt/β-catenin by blocking TLR-4/MyD88 and canonical Wnt signaling in diabetic pancreas, while IRS-1, PI3K, and pAkt were all upregulated by altering the NF-κB and β-catenin crosstalk during T2DM. ConclusionOverall results, indicate that AA has potential to develop as an effective therapeutic in the treatment of T2DM associated meta-inflammation. However, future preclinical research at multiple dose level in a long-term chronic T2DM disease model is warranted to understand its clinical relevance in cardiometabolic disease.