Activation Of Wnt Signaling Research Articles

Balancing WNT signalling in early forebrain development: The role of LRP4 as a modulator of LRP6 function.

The specification of the forebrain relies on the precise regulation of WNT/ß-catenin signalling to support neuronal progenitor cell expansion, patterning, and morphogenesis. Imbalances in WNT signalling activity in the early neuroepithelium lead to congenital disorders, such as neural tube defects (NTDs). LDL receptor-related protein (LRP) family members, including the well-studied receptors LRP5 and LRP6, play critical roles in modulating WNT signalling capacity through tightly regulated interactions with their co-receptor Frizzled, WNT ligands, inhibitors and intracellular WNT pathway components. However, little is known about the function of LRP4 as a potential modulator of WNT signalling in the central nervous system. In this study, we investigated the role of LRP4 in the regulation of WNT signalling during early mouse forebrain development. Our results demonstrate that LRP4 can modulate LRP5- and LRP6-mediated WNT signalling in the developing forebrain prior to the onset of neurogenesis at embryonic stage 9.5 and is therefore essential for accurate neural tube morphogenesis. Specifically, LRP4 functions as a genetic modifier for impaired mitotic activity and forebrain hypoplasia, but not for NTDs in LRP6-deficient mutants. In vivo and in vitro data provide evidence that LRP4 is a key player in fine-tuning WNT signalling capacity and mitotic activity of mouse neuronal progenitors and of human retinal pigment epithelial (hTERT RPE-1) cells. Our data demonstrate the crucial roles of LRP4 and LRP6 in regulating WNT signalling and forebrain development and highlight the need to consider the interaction between different signalling pathways to understand the underlying mechanisms of disease. The findings have significant implications for our mechanistic understanding of how LRPs participate in controlling WNT signalling.

Abstract 4863: PMCA inhibition induces cytotoxic calcium stress and suppression of Wnt signaling in refractory molecular subtype of gastric cancer

Abstract Background: Gastric cancer (GC) is the fourth leading cause of cancer-related deaths worldwide, but its genetic heterogeneity retards the development of novel therapeutics. Recently, tumor molecular classification identified clinically refractory SEM (i.e., Stem-like, EMT and Mesenchymal) subtype GC which exhibited over-expression of the plasma membrane Ca2+ ATPase (PMCA) whose role is circumventing calcium overload causing ER stress and apoptosis. Here, we showed that PMCA4 is associated with Wnt signaling activity by regulating Secreted Frizzled-Related Protein 2 (SFRP2) expression and demonstrated that PMCA4 inhibition might achieve dual mechanism-specific anti-cancer effect by inducing calcium-mediated apoptosis and restraining oncogenic Wnt activity in SEM subtype GC. Materials and Methods: GC cell lines and patient-derived organoids (PDOs) were categorized either SEM-subtype (SNU484, SNU668, SK4, MKN1; GA077T, GA265T, GA333T, GA352T) or non-SEM subtype (MKN45, NCIN87, SNU16, SNU601; GA200T, GA247T, GA215T) based on transcriptome signatures or subtype-specific biomarker genes. PMCA4 expression was confirmed using western blot and its calcium clearance function was examined using Fluo-4 AM fluorescent Ca2+ dye. Biological and molecular characteristics of GC cell lines and PDOs upon PMCA4 inhibition were investigated using RT-qPCR, western blot, and TOPflash/FOPflash reporter assay. The target engagement of novel PMCA inhibitor PM058 (bis-indole maleimide derivative) was evaluated by cellular thermal shift assay (CETSA), and the efficacy was determined by CCK8 cell viability assay for cell lines and by Cell Titer-Glo for PDOs.Results: PMCA4 was highly expressed in SEM subtype GC cell lines and PDOs. The siRNA-mediated PMCA4 knockdown (KD) or pharmaceutical inhibition of PMCA4 suppressed the calcium clearance, the cell viability, and decreased cell invasion/migration. TCF/LEF-responsive reporter assay revealed that pharmaceutical inhibition of PMCA4 suppressed Wnt/beta-catenin signaling activity, proven by decrease in beta-catenin and Wnt target gene expression. PMCA inhibition was mechanistically associated with the increased expression of SFRP2, a Wnt antagonist. Importantly, PM058, a PMCA inhibitor, treatment resulted in highly efficacious anti-cancer effect in PDOs at nano molar range, which correlated with PMCA4 protein expression.Discussion and Conclusion: Our results provide the proof of concept that PMCA4 can be a promising therapeutic target for refractory subtype cancer. The proposed mechanism of PMCA4 inhibition is calcium overload - induced apoptosis and interfering with Wnt signaling activity via SFRP2 in GC. Importantly, we demonstrated a novel potent inhibitor PM058 that blocks PMCA function in clinically intractable SEM subtype GC cancer. Citation Format: Saeli Ban, Jungmin Kim, Jonghwan Bae, Ki Cheong Park, Suji Lee, Moon Hwan Kim, Jae-Ho Cheong. PMCA inhibition induces cytotoxic calcium stress and suppression of Wnt signaling in refractory molecular subtype of gastric cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4863.

Abstract 1338: Subpopulations of cancer associated fibroblasts serve as cancer stem cell niche in ovarian cancer via activation of non-canonical Wnt signaling

Abstract Ovarian cancer (OC) is the most lethal gynecologic cancer and the 5th leading cause of cancer-related deaths among women in the US. One of the main reasons for the poor outcome is a high recurrence rate and development of chemoresistance. The standard care involves debulking surgery and carbo-taxol chemotherapy. While most patients respond well initially, a small fraction of OC cells survive - these persister cells are enriched in cancer stem cells (CSCs), and are capable of tumor initiation and cause relapse. Most studies on OC stem cells (OCSCs) have focused on their cell-intrinsic properties and not much is known about the role of the tumor microenvironment (TME) in regulating their growth, fate, and sustenance. Cancer-associated fibroblasts (CAFs) are a key constituent of the OC TME and have been reported to play important roles in regulating tumorigenesis, metastasis, and immune evasion. Moreover, the residual tumors following neo-adjuvant chemotherapy are highly fibrotic. Our studies using matched pre- and post-chemotherapy OC patient tumors revealed an enrichment in OCSCs and CAFs following chemotherapy. Using heterotypic 3D cocultures of patient-derived CAFs and OC cells and in vivo limiting dilution assay, we found that CAFs could induce OCSCs. CAFs stimulated symmetric division of OCSCs as well as dedifferentiation of some non-OCSCs. Using a novel interface interaction assay that we developed, we found that CAFs could influence only those OC cells that are in their immediate neighborhood. We have identified the underlying mechanism involving a non-canonical Wnt signaling pathway in the OCSCs induced by Wnt5a secreted by CAFs. Using functional rescue experiments, we determined that Wnt5a acted via its coreceptor ROR2 on the OCSCs that activated PKC and CREB1 to induce stemness. Inhibiting the CAF-OCSC crosstalk using a Wnt5a-specific inhibitor, Box5 abrogated the effects of CAFs on OCSCs both in vitro and in vivo. While Box5 treatment alone was sufficient to inhibit tumor growth in vivo, its combination with carboplatin sensitized tumors to carboplatin by eradicating residual OCSCs. CAFs are heterogenous and not all CAFs are capable of inducing OCSCs, similarly, only a subpopulation of OC cells can respond to CAFs and dedifferentiate into OCSCs. We performed single-cell RNA-seq using heterotypic 3D cocultures of patient-derived CAFs and OC cells, and characterized the pathways activated in Wnt5ahigh CAFs. Using trajectory analysis, we identified OC subpopulations that respond to CAF signals and become OCSCs. In summary, we have identified a novel mechanism by which a subpopulation of CAFs in the OC TME act as OCSC niche. Our research provides compelling evidence in support of targeting Wnt5a, in combination with the standard of care carbo-taxol chemotherapy, to prevent OC relapse and chemoresistance. Citation Format: Xue Xiao, Yiming Fang, David Pepin, Dmitriy Zamarin, Anirban K. Mitra. Subpopulations of cancer associated fibroblasts serve as cancer stem cell niche in ovarian cancer via activation of non-canonical Wnt signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1338.

Dexmedetomidine-mediated neuroprotection against sevoflurane-induced brain development abnormality in fetal mice brain.

Brain development is susceptible to external influences during the gestation period so the neurotoxicity of anesthetics has gained a lot of attention. We aimed to investigate the neurotoxicity of sevoflurane to fetal mice brain as well as the neuroprotective effects of dexmedetomidine. Pregnant mice were treated with 2.5% sevoflurane for 6 hours. The changes in fetal brain development were assayed with immunofluorescence and western blot. The pregnant mice were intraperitoneally injected with dexmedetomidine or vehicle from gestation day (G) 12.5 to G15.5. Our results showed maternal sevoflurane exposure could not only inhibit neurogenesis but also lead to precocious generation of astrocytes in fetal mice brains. The fetal mice brain of sevoflurane group exhibited a significant inhibition in the activity of Wnt signaling and the expression of CyclinD1, Ngn2. Chronic dexmedetomidine administration could minimize the negative effects caused by sevoflurane by activating the Wnt signaling pathway. This study has uncovered a Wnt signaling-related mechanism of the neurotoxicity of sevoflurane and confirmed the neuroprotective effect of dexmedetomidine, which could provide pre-clinical evidence for clinical decision-making.

Stimulasi Pulsed Electromagnetic Field (PEMF) pada Osteogenesis Pergerakan Gigi Ortodonti

The prevalence of malocclusion and the need for orthodontic treatment in Indonesia is relatively very high. Orthodontic treatment that transmits mechanical forces to the periodontal ligament and alveolar bone cells can stimulate the periodontal ligament and alveolar bone remodeling. While this time, orthodontic treatment requires a long time, resulting in several adverse effects and discomfort. For this reason, a method that can accelerate orthodontic treatment is needed which aims to increase stability after active orthodontic treatment, to reduce the potential for relapse, and to accelerate retention time. Pulsed Electromagnetic Field (PEMF) stimulation is one of the methods that have this goal. This aim to study the stimulation mechanism of PEMF on the osteogenesis process of orthodontic tooth movement. Alveolar bone remodeling played an essential role in orthodontic tooth movement, is an active and dynamic process. The dynamic process depends on a balance between resorption and bone formation (osteogenesis). PEMF stimulation is a nonsurgical method that can promote osteogenesis through activation of Wnt signaling. Several studies showed that this method could accelerate retention time and orthodontic treatment. PEMF stimulated osteogenesis through activation of Wnt signaling in orthodontic tooth movement.

C1orf109 promotes malignant phenotype of liver cancer via wnt signaling pathway in a CK2-dependent manner

Chromosome 1 open reading frame 109 (C1orf09) is a protein whose expression pattern and biological function in humans, particularly in malignant tumors, have not been explored. In this study, both bioinformatics and immunohistochemical staining revealed that C1orf109 was overexpressed in the cytoplasm of liver cancer cells, and the positive ratio of C1orf109 in liver cancer samples (42.5%, 37/87) was significantly higher than that in normal liver tissues (10%, 3/30, P = 0.0012). C1orf109 expression was correlated with an advanced TNM stage (P = 0.017) and vascular invasion (P = 0.023) and predicted the poor overall survival of patients with liver cancer (P = 0.001). C1orf109 facilitated tumor growth, colony formation, migration, and invasion by activating Wnt signaling by upregulating non-phosphorylated β-catenin and its downstream target genes such as CyclinD1, c-myc, and MMP7. Our results also suggest that C1orf109 interacts and co-localizes with casein kinase II (CK2) to activate Wnt signaling. Treatment with a CK2-specific inhibitor markedly counteracted the increased expression of CyclinD1, c-Myc, and MMP7, as well as the upregulation of tumor proliferation and invasion caused by C1orf109 overexpression. Taken together, our results indicate that C1orf109 accelerates liver cancer cell proliferation and invasion by strengthening the Wnt signaling pathway in a CK2-dependent manner.

The DNA demethylation-regulated SFRP2 dictates the progression of endometriosis via activation of the Wnt/β-catenin signaling pathway

BackgroundEndometriosis cause decreases in life quality and pelvic pain in reproductive-age women. Methylation abnormalities played a functional role in the progression of endometriosis, this study aimed to explore the mechanisms mediated by abnormal methylation in the development of EMS.Materials and methodsNext-generation sequencing dataset and methylation profiling dataset were used to screen out the key gene SFRP2. Western bolt, Real-time PCR, Aza-2?deoxycytidine treatment, luciferase reporter assay, Methylation-specific PCR , Bisulfite sequencing PCR and lentivirus infection were carried out to detect the methylation status and signaling pathway with the primary epithelial cells. Transwell assay and wound scratch assay were implemented to observe the differences of migration ability with the intervening with the expression of SFRP2.ResultsTo define the role of the DNA methylation-regulated genes in the pathogenesis of EMS, we performed both DNA methylomic and expression analyses of ectopic endometrium and ectopic endometrium epithelial cells(EEECs) and found that SFRP2 is demethylated/upregulated in ectopic endometrium and EEECs. The expression of lentivirus carrying SFRP2 cDNA up-regulates the activity of Wnt signaling and the protein expression of ?-catenin in EEECs. SFRP2 impact on the invasion and migration of ectopic endometrium by modulating the activities of the Wnt/?-catenin signaling pathway. The invasion and migration ability of EEECs were significantly strengthened after demethylation treatment including 5-Aza and the knockdown of DNMT1.ConclusionIn summary, the increased SFRP2 expression-induced Wnt/?-catenin signaling due to the demethylation of the SFRP2 promoter plays an important role in the pathogenesis of EMS, suggesting that SFRP2 might be a therapeutic target for EMS treatment.

The mechanisms underlying the enrichment and action of glypican-1-positive exosomes in colorectal cancer cells

BackgroundGlypican-1 (GPC1) is overexpressed in several tumors, and GPC1+ exosomes have shown the potential to predict early colorectal cancer (CRC). However, the mechanisms underlying the enrichment and action of GPC1+ exosomes in CRC remain unknown. MethodsThe expression of slit guidance ligand 2 (SLIT2), hypoxia-inducible factor (HIF)-1α/2α, and GPC1 in clinical CRC tissues was detected using immunohistochemistry and western blot. Exosomes were isolated from the supernatants of CRC cell cultures. The effects of SLIT2, hypoxia, heparin, and phospholipase C (PLC) on exosomal GPC1 expression and GPC1+ exosome enrichment in CRC cells were analyzed with western blot and flow cytometry. CRC cell proliferation was assessed with MTT and colony formation assays. Co-immunoprecipitation was used to detect the binding of GPC1 and SLIT2 in SW480 cells. Nude mice were subcutaneously inoculated with SW480 cells with different treatments. The Wnt signaling was detected. ResultsSLIT2 was poorly expressed and GPC1, HIF-1α, and HIF-2α were highly expressed in human CRC tissues. SLIT2 in CRC cells inhibited GPC1+ exosome enrichment and exosomal GPC1 expression. PLC and heparin increased GPC1+ exosome enrichment in CRC cells in a concentration-dependent manner. Hypoxia increased the enrichment of GPC1+ exosomes in CRC cells depending on HIF-2α expression. GPC1+ exosomes stimulated CRC cell proliferation and xenograft tumor growth through activation of Wnt signaling. ConclusionsGPC1+ exosome enrichment is related to PLC and heparin. Hypoxia increases the enrichment of GPC1+ exosomes in CRC cells by activating HIF-2α and downregulating SLIT2. GPC1+ exosomes further drive CRC progression by activating Wnt signaling.

ANKRD1 activates the Wnt signaling pathway by modulating CAV3 expression and thus promotes BMSC osteogenic differentiation and bone formation in ovariectomized mice

Bone marrow-derived mesenchymal stem cells (BMSCs) are considered promising materials for treating bone diseases such as osteoporosis (OP). This research explored the functions and molecular mechanism of ankyrin repeat domain 1 (ANKRD1) in BMSC osteogenesis. An OP model in mice was established by bilateral ovariectomy. Manipulation of ANKRD1 expression in BMSCs or femurs was achieved by lentivirus infection. Increased ANKRD1 expression was observed in BMSCs during osteogenic induction. Silencing of ANKRD1 impaired the osteogenesis of BMSCs, as shown by the decreased alkaline phosphatase (ALP) activity, osteogenic gene (Runx2, Col1a1, Bglap, and Spp1) expression, and mineralized formation. ANKRD1-mediated promotion of osteogenesis was also reproduced in mouse MC3T3-E1 preosteoblastic cells. Activation of Wnt/β-catenin signaling, a well-known osteogenic stimulus, was also impaired in ANKRD1-silenced BMSCs. Overexpression of ANKRD1 resulted in the opposite effects on osteogenesis and Wnt/β-catenin signaling. Mechanistic studies revealed that ANKRD1 modulated caveolin-3 (CAV3) expression by reducing CAV3 ubiquitination, and the knockdown of CAV3 impaired the functions of ANKRD1. Additionally, a very low level of ANKRD1 was observed in the BMSCs from OP mice. Rescue of ANKRD1 significantly restored osteogenic differentiation and Wnt signaling activation in BMSCs from ovariectomized mice. The results of micro-CT, H&E staining, and IHC staining showed that ANKRD1 also promoted bone formation and Wnt activation and ameliorated pathological alterations in the femurs of OP mice. Collectively, this study demonstrated that ANKRD1 plays an important role in regulating the osteogenic differentiation of BMSCs and is a promising target for the treatment of OP and other bone diseases.

TGM2 accelerates migration and differentiation of BMSCs by activating Wnt/β-catenin signaling

BackgroundTransglutaminase 2 (TGM2) is a gene previously reported to be associated with the differentiation of bone marrow mesenchymal stem cells (BMSCs). The study was developed to reveal the impact of TGM2 on the migration and differentiation of BMSCs.MethodsCells were isolated from bone marrow of mice and then the surface antigens were identified by flow cytometry. Wound healing assays were conducted to assess the migratory ability of BMSCs. The mRNA levels of TGM2 and osteoblast-associated genes (ALP, OCN, and RUNX2) were subjected to RT-qPCR analysis, and protein levels of these genes as well as β-catenin were quantitated by western blotting. Alizarin red staining was conducted for detection of osteogenic ability. The activation of Wnt signaling was assessed by TOP/FOP flash assays.ResultsSurface antigens were positively identified in MSCs, indicating good multidirectional differentiation ability of cells. TGM2 silencing suppressed BMSC migration while weakening mRNA and protein levels of osteoblast-associated genes. While TGM2 overexpression exerts the opposite impact on cell migration and expression levels of osteoblast-associated genes. Additionally, overexpressed TGM2 promotes the mineralization of BMSCs according to results of Alizarin red staining. Moreover, TGM2 activated the Wnt/β-catenin signaling, and DKK1 (an inhibitor of Wnt signaling) reversed the promoting influence of TGM2 on cell migration and differentiation.ConclusionTGM2 promotes the migration and differentiation of BMSCs via activation of the Wnt/β-catenin signaling.

Ndst1,aheparan sulfatemodificationenzyme,regulates neuroectodermal patterningbyenhancingWntsignalingin Xenopus.

Neural tissue is derived from three precursor regions: neural plate, neural crest, and preplacodal ectoderm. These regions are determined by morphogen-mediated signaling. Morphogen distribution is generally regulated by binding to an extracellular matrix component, heparan sulfate (HS) proteoglycan. HS is modified by many enzymes, such as N-deacetyl sulfotransferase 1 (Ndst1), which is highly expressed in early development. However, functions of HS modifications in ectodermal patterning are largely unknown. In this study, we analyzed the role of Ndst1 using Xenopus embryos. We found that ndst1 was expressed in anterior neural plate and the trigeminal region at the neurula stage. ndst1 overexpression expanded the neural crest (NC) region, whereas translational inhibition reduced not only the trigeminal region, but also the adjacent NC region, especially the anterior part. At a later stage, ndst1 knocked-down embryos showed defects in cranial ganglion formation. We also found that Ndst1 activates Wnt signaling pathway at the neurula stage. Taken together, our results suggest that N-sulfonated HS accumulates Wnt ligand and activates Wnt signaling in ndst1-expressing cells, but that it inhibits signaling in non-ndst1-expressing cells, leading to proper neuroectodermal patterning.

The method of sinus node-like pacemaker cells from human induced pluripotent stem cells by BMP and Wnt signaling.

The embryonic development of sinus nodes (SAN) is co-regulated by multiple signaling pathways. Among these, the bone morphogenetic protein (BMP) and Wnt signaling pathways are involved in the development of SAN. In this study, the effects of BMP and Wnt signaling on the differentiation of SAN-like pacemaker cells (SANLPCs) were investigated. Human induced pluripotent stem cells (hiPSCs) were divided into four groups: control, BMP4, CHIR-3, and BMP4 + CHIR (CHIR: a Wnt signaling activator). The samples were tested at day (D) 15 of differentiation. The final protocol for the activation of BMP signaling at D0-D3 and reactivation of Wnt signaling at D5-D7 in the differentiation of hiPSCs were determined. The results showed that the mRNA levels of pacemaker markers (TBX18, SHOX2, TBX3, HCN4, and HCN1) were higher in the BMP4 + CHIR group than in the control group, and working myocardial genes were downregulated. The immunofluorescence assay revealed that the expression of SHOX2 and HCN4 increased in the BMP4 + CHIR group compared to that in the other groups. In addition, the results of patch clamps revealed that a funny current of higher density and typical SAN action potentials were recorded, except in the control group, in which the L-type calcium current was higher in the BMP4 + CHIR group than in the other groups. Finally, the proportion of SANLPCs (cTnT+ NKX2.5-) was further enhanced by the combination of BMP4 and CHIR treatment. In summary, the combination of BMP and Wnt signaling promotes the differentiation of SANLPCs from hiPSCs.

Abstract PD5-06: PD5-06 CCN6 suppresses spindle metaplastic breast carcinoma in part via antagonizing Wnt/β-catenin signaling

Abstract Background: Metaplastic breast carcinomas (mBrCAs) are a rare and highly aggressive subtype of triple negative breast cancer, with histological evidence of non-glandular differentiation and frequent activation of the canonical (-catenin-dependent) Wnt pathway. Our laboratory has reported that CCN6 is expressed in normal mammary epithelium, but CCN6 expression is lost in 68% of spindle mBrCAs. We found mice with mammary epithelial cell-specific conditional deletion of Ccn6 (MMTV-Cre; Ccn6fl/fl mice) develop mammary tumors that recapitulate human spindle mBrCAs, including upregulation of Wnt pathway genes. We investigated if and how secreted CCN6 protein functions in tumor suppression in spindle mBrCA via effects on the canonical Wnt pathway. Methods: To investigate CCN6 binding to the Wnt co-receptors LRP6 and FZD8 proteins, we performed Flag-IPs on MDA-MB231 mesenchymal-like breast cancer cells expressing Flag-CCN6 or vector. Effects of CCN6 on b-catenin subcellular localization and gene and protein expression were studied by IHC, IF, qRT-PCR and immunoblot in human cell lines and MMTV-Cre;Ccn6fl/fl tumors. To test effects of recombinant CCN6 on canonical Wnt signaling, we used the Leading-Light Wnt Reporter Assay and also tested CCN6 effects in WNT3A- and WNT10B-mediated Wnt signaling activation and on MDA-MB231 cell invasion. To study b-catenin/TCF function in invasive growth of CCN6-deficient cancer cells, we employed two independent approaches: i) expression of a dominant-negative Tcf4 (dnTcf4) versus control vector in MMTV-Cre; Ccn6fl/fl tumor-derived cells; and ii) expression of a constitutively active mutant (S33Y) b-catenin in concert with treatment with recombinant human CCN6 (rhCCN6; 500 ug/ml) versus BSA control. Syngeneic orthotopic mammary tumor transplants of MMTV-Cre;Ccn6fl/fl were used in vivo for rescue experiments with i.p. injections of rhCCN6 or BSA. We monitored tumor growth and morphology, and performed IHC to determine b-catenin localization and expression. Results: We found in co-IPs that CCN6 interacts with LRP6 and FZD8 to form a complex that antagonizes canonical Wnt signaling. CCN6 ectopic expression in MDA-MB231 cells led to reduced nuclear and increased membrane localization of b-catenin and decreased invasive growth in vitro. In vivo, CCN6 protein administration to MMTV-Cre; Ccn6fl/fl mice reduced tumor growth and was linked to decreased nuclear b-catenin in the tumors. Conclusion: CCN6 antagonizes canonical Wnt/b-catenin in part by binding Wnt ligands, leading to reduced active b-catenin in the cytoplasm and nucleus. Our data indicate a critical role for b-catenin activation for CCN6-deficient mBrCA tumor phenotypes. In vivo, rhCCN6 protein reduces tumorigenesis in MMTV-Cre; Ccn6fl/fl mBrCA tumors, highlighting how CCN6 restoration or b-catenin inhibition could be new therapeutic approaches for mBrCAs. Citation Format: Maria E. Gonzalez, Eric R. Fearon, Celina Kleer. PD5-06 CCN6 suppresses spindle metaplastic breast carcinoma in part via antagonizing Wnt/β-catenin signaling [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-06.

Regulation of Cx43 and its role in trichloroethylene-induced cardiac toxicity in H9C2 rat cardiomyocytes

Trichloroethylene (TCE), a widespread environmental contaminant, has been linked to congenital heart defects. Abnormal regulation of Connexin 43 is closely associated with various cardiac diseases. However, it is yet to be established how Cx43 responds to environmental pollutants. Here, we aim to explore the role of Cx43 in TCE-induced cardiac toxicity using H9C2 cardiomyocytes. EdU incorporation assay and cell cycle analysis revealed that increased number of TCE-treated cells entered into the S stage, indicating that TCE exposure provoked cell proliferation. Additionally, compromised mitochondrial function was observed in TCE-treated cells, and inhibition of mitochondrial permeability transition pore (mPTP) with Cyclosporin A or eliminating mitochondrial ROS by MitoQ alleviated the TCE-induced cardiac toxicity. Importantly, TCE exposure increased the protein expression levels of Cx43 and stimulated the recruitment of Cx43 to the mitochondria. TCE exposure disrupted canonical Wnt signal pathway, resulting in downregulation of antioxidant genes and β-catenin. The adverse effects of TCE on Wnt signal pathway activation, mitochondrial function and cell proliferation were efficiently counteracted by either Cx43 knockdown or pharmaceutical activator of Wnt signaling, CHIR-99021. Taken together, our results for the first time revealed that dysregulation of Cx43 mediates TCE-induced heart defects via mitochondrial dysfunction and Wnt signaling inhibition, suggesting that Cx43 can be a potential molecular marker or therapeutic target for cardiac diseases caused by environmental pollutants.

The Relationship between LRP6 and Wnt/β-Catenin Pathway in Colorectal and Esophageal Cancer

High expression of low-density lipoprotein receptor-related protein 6 (LRP6), a key component of the Wnt/β-catenin signaling pathway, is reported to be associated with malignant potential in some solid tumors including breast cancer and hepatocellular carcinoma. Few reports, however, have examined its function and clinical significance in colorectal cancers (CRC) demonstrating constitutive activation of Wnt signaling. Here, we compared the expression level and function of LRP6 in CRC with that of esophageal squamous cell carcinoma (ESCC) bearing few Wnt/β-catenin pathway mutations. On immunohistochemical staining, high LRP6 expression was noted in three of 68 cases (4.4%), and high β-catenin in 38 of 67 cases (56.7%) of CRC. High LRP6 expression was found in 21 of 82 cases (25.6%), and high β-catenin expression in 29 of 73 cases (39.7%) of ESCC. In our in vitro studies, LRP6 knockdown hardly changed Wnt signaling activity in CRC cell lines with mutations in Wnt signaling downstream genes. In contrast, in ESCC cell lines without Wnt signaling-related mutations, LRP6 knockdown significantly decreased Wnt signaling activity. LRP6 function may depend on constitutive activation of Wnt signaling.

Wnt/β-catenin modulating drugs regulate somatostatin receptor expression and internalization of radiolabelled octreotide in neuroendocrine tumor cells.

Differentiated neuroendocrine tumors (NETs) express somatostatin receptors (SSTRs), targets for therapy with either unlabeled or radioactively labeled somatostatin analogs (SSA). Associated with worse prognosis, dedifferentiated NET loose SSTR expression, which may be linked to deregulation of Wnt/β-catenin signaling on an intracellular level. The aim of the present study was to investigate the effect of Wnt/β-catenin signaling pathway alterations on SSTR expression and its function in NET. The NET cell lines BON-1 and QGP-1 were incubated with the Wnt-inhibitors 5-aza-2'-deoxycytidine (5-aza-CdR), Quercetin, or Niclosamide, or the Wnt activator lithium chloride (LiCl). Expression of SSTR1, SSTR2, and SSTR5 was determined by quantitative RT-PCR (qRT-PCR), immunocytomicroscopy and western blot. Changes in the Wnt pathway were analyzed by qRT-PCR of selected target genes and the TaqMan Array Human WNT Pathway. Receptor-associated function was determined by measuring the cellular uptake of [125I-Tyr3] octreotide. The mRNAs of SSTRs 1-5 were expressed in both cell lines. Wnt inhibitors caused downregulation of Wnt target genes, while 5-aza-CdR had the highest inhibitory effect. LiCl lead to an upregulation of Wnt genes, which was more marked in QGP-1 cells. SSTR expression increased in both cell lines upon Wnt inhibition. All three Wnt inhibitors lead to a marked increase in the specific uptake of [125I-Tyr3]octreotide, with 5-aza-CdR showing the greatest effect (increase by more than 50% in BON-1 cells), while a decreased uptake of [125I-Tyr3]octreotide was seen upon activation of Wnt signaling by LiCl. We demonstrate here that Wnt signaling orchestrates SSTR expression and function in a preclinical NET model. Wnt inhibition increases [125I-Tyr3]octreotide uptake offering an opportunity to enhance the efficacy of SSTR-targeted theranostic approaches.

Characterization and impact of Wnt5A signaling on outcomes of urothelial carcinoma.

560 Background: Active Wnt signaling via WNT5A through ROR1 and its partner, ROR2, or WNT5A/frizzled 2 (FZD2) is linked to processes driving tumorigenesis, disease progression and therapy resistance. The role of this pathway in the pathogenesis of urothelial carcinoma (UC) is not fully elucidated. In adult tissue, ROR1 is largely absent, which makes it ideal for targeted therapies, with several ROR1 targeting agents in early clinical development. We utilized a large dataset of molecularly characterized UC tumors to investigate the significance of Wnt5a/ ROR1, ROR2 or FZD2 transcriptional expression. Methods: NextGen Sequencing of DNA (592 genes or WES)/RNA (WTS) was performed for 4743 UC tumors submitted to Caris Life Sciences (Phoenix, AZ). PD-L1 expression (SP142; Positive (+): ³2+, ³%5) was tested by IHC. Gene expression profiles were analyzed for a transcriptional signature predictive of response to immunotherapy (T cell-inflamed; Bao, 2020). WNT5a, ROR1, ROR2, and FZD2-high and -low expression were defined as ³ top and < bottom quartile of transcripts per million (TPM), respectively. Mann-Whitney U and X2/Fisher-Exact tests were applied where appropriate, with P-values adjusted for multiple comparisons ( q < .05). Real-world overall survival (OS) information was obtained from insurance claims data and Kaplan Meier estimates were calculated for molecularly defined cohorts. Results: We observed similar expression of these WNT5A signaling pathway genes between upper (N=795) and lower tract UC (N=3,204): WNT5A (22.7 v. 22.2 median TMP (mTPM), q = .18), FZD2 (3.4 v 3.5, q = .93), ROR1 (2.0 v. 1.7, q = .05), and ROR2 (2.1 v 2.5, q < .01). WNT family gene expression varied significantly between primary (N=2,756) and metastatic sites (N=1,361): WTN5A (25.2 v 16.8 mTPM), FZD2 (3.2 v 4.05), ROR1 (1.7 v 2.1), and ROR2 (2.4 v 2.6) for primary vs. metastatic sites respectively ( q < .05 for all). Comparison of high- and low-expression subgroups revealed variation in the prevalence of TP53, FGFR3 and RB1 mutations, as well as PDL1+ staining and T cell-inflamed scores (Table). High gene expression for ROR2 (HR 0.66, 95% CI 0.56-0.78, p < .001) and FZD2 (HR 0.75, 95% CI 0.63-0.89, p < .001) was associated with worse OS compared to low gene expression. No significant difference in OS was observed for WNT5A (HR 0.97, 95% CI 0.82-1.15, p < .76) and ROR1 (HR 0.86, 95% CI 0.72-1.01, p < .068). Conclusions: Distinct genomic and immune landscapes for the four investigated WNT pathway components were observed and should be leveraged to identify combination therapies that complement the current pipeline of WNT pathway-targeting drugs. [Table: see text]

Identification of miR-1-3p, miR-143–3p and miR-145–5p association with bone metastasis of Gleason 3+4 prostate cancer and involvement of LASP1 regulation

Gleason Score (GS) 3 + 4 prostate cancer (PCa) is heterogeneous in clinical course and molecular features. Risk stratification of indolent and aggressive PCa with GS 3 + 4 is critical, especially those with bone metastasis (BM) potential. Microarray-based microRNA(miRNA) profiling with eight PCa cases with or without BM was used to screen the candidate miRNAs associated with BM. Transwell and MTS assays were used to characterize the function of miRNAs and target gene LASP1. RT-qPCR and immunohistochemistry assays were utilized to illustrate the clinical significance of miRNAs and target gene in a cohort of 309 Chinese PCa cases. In the current study, we identified that miR-1-3p, miR-143–3p and miR-145–5p are associated with BM of GS 3 + 4 PCa. Through functional experiments, we show that miR-1-3p/143–3p/145–5p promotes proliferation and migration of PCa in vitro. LASP1 was predicted as the common target of these three miRNAs which was further confirmed by a luciferase assay. Overexpression of LASP1 was correlated with higher GS, higher pathological stage, and the presence of metastasis by immunohistochemistry. siRNA knockdown of LASP1 significantly suppressed proliferation and migration, whereas overexpression of LASP1 promoted it. Bioinformatics analysis revealed the involvement of Wnt signaling pathway in LASP1 mediated function. LASP1 may activate Wnt signaling by interacting with β-catenin. In all, we suggest that miR-1-3p/143–3p/145–5p are associated with BM of Gleason 3 + 4 PCa. LASP1 is the common target of these miRNAs and may active Wnt signaling by interacting with β-catenin.

Oncogenic SIRT7 inhibits GATA4 transcriptional activity and activates the Wnt signaling pathway in ovarian cancer

ObjectiveSirtuin-7 (SIRT7) is a class III histone deacetylase that plays an important role in cancer development and frequently overexpressed in carcinomas. In this study, the tumor-supporting role and underlying mechanisms of SIRT7 were characterized in ovarian cancer (OC) aggressiveness. MethodsSIRT7 expression was examined in OC tissues and cells. Interactions among SIRT7, GATA4, Wnt signaling pathway were explored by bioinformatics tools and experimental validations. The effect of SIRT7 and GATA4 on malignant phenotypes of OC cells were examined with gain- and loss-of-function experiments. A nude mouse model of OC was developed to verify the in vitro findings. ResultsIt was noted that SIRT7 was highly expressed in OC tissues and cells. Cell lines with higher SIRT7 expression (OVCAR-3 and OVCAR-8) were used for subsequent in vitro experiments. The experimental data indicated that silencing of SIRT7 suppressed the OC cell proliferation, colony formation, migration, and invasion, and promoted cell senescence, which could be abolished by GATA4 knockdown. Mechanistically, SIRT7 promoted deacetylation of GATA4 and consequently inhibited the transcriptional activity of GATA4. In addition, GATA4 induced OC cell senescence by inhibiting Wnt signaling pathway. Further in vivo experiments substantiated that SIRT7 knockdown or overexpressed GATA4 could effectively inhibit tumor growth of nude mice. ConclusionTaken together, our findings indicated that SIRT7 enhanced development of OC by suppressing GATA4 and activating Wnt signaling pathway, suggesting the potential of SIRT7/GATA4/Wnt axis as a therapeutic target for OC.

Canonical Wnt signaling activation by chimeric antigen receptors for efficient cardiac differentiation from mouse embryonic stem cells

BackgroundCanonical Wnt signaling is involved in a variety of biological processes including stem cell renewal and differentiation, embryonic development, and tissue regeneration. Previous studies reported the stage-specific roles of the Wnt signaling in heart development. Canonical Wnt signal activation by recombinant Wnt3a in the early phase of differentiation enhances the efficiency of myocardial cell production from pluripotent stem cells. However, the hydrophobicity of Wnt proteins results in high cost to produce the recombinant proteins and presents an obstacle to their preparation and application for therapeutics, cell therapy, or molecular analysis of Wnt signaling.MethodsTo solve this problem, we generated an inexpensive molecule-responsive differentiation-inducing chimeric antigen receptor (designated as diCAR) that can activate Wnt3a signaling. The extracellular domains of low-density-lipoprotein receptor-related protein 6 (LRP6) and frizzeled-8 (FZD8) were replaced with single-chain Fv of anti-fluorescein (FL) antibody, which can respond to FL-conjugated bovine serum albumin (BSA-FL) as a cognate ligand. We then analyzed the effect of this diCAR on Wnt signal activation and cardiomyocyte differentiation of mouse embryonic stem cells in response to BSA-FL treatment.ResultsEmbryonic stem cell lines stably expressing this paired diCAR, named Wnt3a-diCAR, showed TCF/β-catenin-dependent transactivation by BSA-FL in a dose-dependent manner. Treatment with either Wnt3a recombinant protein or BSA-FL in the early phase of differentiation revealed similar changes of global gene expressions and resulted in efficient myocardial cell differentiation. Furthermore, BSA-FL-mediated signal activation was not affected by a Wnt3a antagonist, Dkk1, suggesting that the signal transduction via Wnt3a-diCAR is independent of endogenous LRP6 or FZD8.ConclusionWe anticipate that Wnt3a-diCAR enables target-specific signal activation, and could be an economical and powerful tool for stem cell-based regeneration therapy.