Journal articles on the topic 'Wnt/b-Catenin pathway'
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1
Yu, Fujun, Yong Guo, Bicheng Chen, Liang Shi, Peihong Dong, Mengtao Zhou, and Jianjian Zheng. "LincRNA-p21 Inhibits the Wnt/β-Catenin Pathway in Activated Hepatic Stellate Cells via Sponging MicroRNA-17-5p." Cellular Physiology and Biochemistry 41, no. 5 (2017): 1970–80. http://dx.doi.org/10.1159/000472410.
Abstract:
Background/Aims: It is known that the activation of hepatic stellate cells (HSCs) is a pivotal step in the initiation and progression of liver fibrosis. Aberrant activated Wnt/β-catenin pathway is known to accelerate the development of liver fibrosis. microRNAs (miRNAs)-mediated Wnt/β-catenin pathway has been reported to be involved in HSC activation during liver fibrosis. However, whether long noncoding RNAs (lncRNAs) regulate Wnt/β-catenin pathway during HSC activation still remains unclear. Methods: Long intergenic noncoding RNA-p21 (lincRNA-p21) expression was detected in Salvianolic acid B (Sal B)-treated cells. Effects of lincRNA-p21 knockdown on HSC activation and Wnt/β-catenin pathway activity were analyzed in Sal B-treated cells. In lincRNA-p21-overexpressing cells, effects of miR-17-5p on HSC activation and Wnt/β-catenin pathway activity were examined. Results: LincRNA-p21 expression was up-regulated in HSCs after Sal B treatment. In primary HSCs, lincRNA-p21 expression was down-regulated at Day 5 relative to Day 2. Sal B-inhibited HSC activation including the reduction of cell proliferation, α-smooth muscle actin (α-SMA) and type I collagen was inhibited by lincRNA-p21 knockdown. Sal B-induced Wnt/β-catenin pathway inactivation was blocked down by loss of lincRNA-p21. Notably, lincRNA-p21, confirmed as a target of miR-17-5p, suppresses miR-17-5p level. Lack of the miR-17-5p binding site in lincRNA-p21 prevents the suppression of miR-17-5p expression. In addition, the suppression of HSC activation and Wnt/β-catenin pathway induced by lincRNA-p21 overexpression was almost inhibited by miR-17-5p. Conclusion: We demonstrate that lincRNA-p21-inhibited Wnt/β-catenin pathway is involved in the effects of Sal B on HSC activation and lincRNA-p21 suppresses HSC activation, at least in part, via miR-17-5p-mediated-Wnt/β-catenin pathway.
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Adjei-Fremah, Sarah, Emmanuel Kwaku Asiamah, Kingsley Ekwemalor, Louis Jackai, Keith Schimmel, and Mulumebet Worku. "Modulation of Bovine Wnt Signaling Pathway Genes by Cowpea Phenolic Extract." Journal of Agricultural Science 8, no. 3 (February 16, 2016): 21. http://dx.doi.org/10.5539/jas.v8n3p21.
Abstract:
<p class="ANMmaintext">The Wingless (Wnt) signaling pathway is a conserved pathway with essential roles in cellular and biological processes in mammals. Wnt signal transduction has been implicated in inflammation, innate immunity and homeostasis via Toll-like receptor and NF-kB pathways. Plant bioactive compounds are capable of modulating the Wnt signalling pathway, which can be either a canonical (B-Catenin dependent) or non-canonical (B-Catenin independent) mechanism. This study evaluated the effect of cowpea phenolic extract (CPE) on the expression and modulation of genes of the Wnt signaling pathway in cow blood. Whole blood collected from six Holstein-Friesian cows was treated with 10 ug/ml of the extract, and evaluated for packed cell volume (PCV), total count and viability of cells, and white blood cell differential count before and after treatment. Cowpea phenolic extract agonist activity in blood was measured using a Bovine toll-like receptor (TLR) 2, and TLR 4 ELISA kit. Total RNA was isolated from the blood cell pellet, reverse transcribed and used for real-time PCR to detect expression of 84 genes on the Cow Wnt signaling pathway array. The total cell-associated B-Catenin level was measured using a commercial ELISA kit. There was no treatment effect on PCV, total cell and viability (P > 0.05). The percentage of mononuclear cells were influenced by treatment, % monocytes (P = 0.0136) decreased and % lymphocytes (P = 0.0114) increased. Treatment with CPE activated cow blood cells, increased TLR2 release and total B-Catenin levels (6 ng/ml, P < 0.05), but TLR4 was not detected. Polyphenols from cowpea modulated the expression of Wnt signalling genes, especially canonical B-Catenin mediated pathway genes. Modulation of Wingless gene expression may be an important mechanism by which polyphenols in cowpea feed impact cellular immune response and homeostasis. Thus, further studies are needed to determine the association of CPE-mediated Wnt gene modulation on blood leucocytes subpopulations and animal health.</p>
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Li, Xinyu, Lingyu Geng, Xiangxiang Zhou, Kang Lu, Peipei Li, and Xin Wang. "5-Aza-2-Deoxycytidine Regulates Wnt Beta-Catenin Pathway in B Cell Lymphoma." Blood 126, no. 23 (December 3, 2015): 4810. http://dx.doi.org/10.1182/blood.v126.23.4810.4810.
Abstract:
Abstract Introduction: The Wnt/beta-catenin pathway is aberrantly activated in B cell lymphomas, unphosphorylated beta-catenin accumulates and translocates into the nucleus, regulates the expression of c-myc, cyclinD1 and many other target genes which govern fundamental cell functions, such as proliferation, cell cycle regulation and apoptosis. Methylation is a highlight of epigenetic regulation research which also occurred in lymphoma, but the concrete mechanism of how the demethylation drug 5-aza-2-deoxycytidine affect Wnt/beta-catenin pathway is still unknown. This study was designed to illuminate the implications on Wnt/beta-catenin pathway via demethylation 5-aza in B cell lymphoma. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from samples of 30 primary CLL patients. The PBMCs contained more than 90% of CD19+ B lymphocytes, which were detected by flow cytometry and were referred to as primary CLL cells. The activation of Wnt/beta-catenin pathway and DNMT-1 of B cell lymphoma cells lines (MEC-1, LY8, Jeko-1, Grant519, mino and sp53) and the 30 patients were detected by qPCR and western blot. The expressions of beta-catenin in 20 cases of B cell lymphoma tissues were measured by IHC. The B cell lines and PBMCs from 10 primary CLL patients were given 5-aza-2-deoxycytidine in different concentrations, the effects in the pathway and apoptosis were observed by WB and flow cytometry. Results: The expressions of beta-catenin, c-myc, cyclinD1 and DNMT-1 were aberrantly higher in all cell lines we used ( MEC-1,LY8, Jeko-1, Grant519, mino and sp53 Fig.1-A,B), most primary CLL patients (Fig.1-C), and B cell lymphoma tissues (Fig.1-D). The protein expressions of beta-catenin in MEC-1 were higer than primary CLL patients. 0, 0.5, 1.0, 2.0¦ÌM 5-aza-2-deoxycytidine were given to the B cells lines and PBMCs from primary CLL patients for 48h, beta-catenin were found accumulated, but c-myc and cyclinD1 in the downstream were reduced (Fig.2-A,B,C,D). For further understanding of aberrant accumulation ofbeta-catenin, we extracted the nuclear protein of MEC-1, nuclear beta-catenin protein expressions were found decreased and cytoplasmic were increased (Fig.2-E). After 5-aza treatment, the apoptosis rate increased and caspase pathway were activated (Fig.2-A,F). Conclusions: The enhanced expressions of beta-catenin, c-myc, cyclinD1 in the B cell lines and the B cell lymphoma samples indicated the Wnt/beta-catenin was aberrantly activated. After 5-aza treatment with the cell lines (MEC-1, Jeko-1, LY8) and primary CLL cells, the abnormal accumulation of beta-catenin protein was observed which was discrepancy with previous reports, but the decrease of c-myc and cyclinD1 suggested the pathway was inhibited, apoptosis also occurred. The increase of totalbeta-catenin protein was supposed to be an stress reaction of the 5-aza treatment, however, the redundant beta-catenin protein in B cell lymphoma was speculated to be combined with demethylated genes and resulted in dormancy of this pathway. Our results indicated that 5-aza played a demethylation role through Wnt/beta-catenin pathway in B cell lymphoma. The data are of interest in the context of epigenetic-based therapy in B cell lymphoma. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.
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Langhammer, Tina-Susann, Catrin Roolf, Saskia Krohn, Christin Kretzschmar, Rayk Huebner, Arndt Rolfs, Mathias Freund, and Christian Junghanss. "PI3K/Akt Signaling Interacts With Wnt/β-Catenin Signaling But Does Not Induce An Accumulation Of β-Catenin In The Nucleus Of Acute Lymphoblastic Leukemia Cell Lines." Blood 122, no. 21 (November 15, 2013): 4886. http://dx.doi.org/10.1182/blood.v122.21.4886.4886.
Abstract:
Abstract Signaling pathways play essential roles in biological processes as development, cell proliferation and homeostasis. The accurate modulation of signaling pathways, their adapted interaction and their time- and tissue-specific adjusted regulation are required for normal cell development. PI3K/Akt and Wnt/β-Catenin signaling pathways act as key regulators in cell proliferation, differentiation and growth. Both signaling pathways include GSK3β as a common protein, which may mediate an interaction and cross-talk between the pathways. Aberrant activation of PI3K/Akt signaling has been linked to different types of leukemia while Wnt/β-Catenin signaling is known to be deregulated in some solid tumors. However, a potential role of Wnt/β-Catenin signaling for pathogenesis of acute lymphoblastic leukemia (ALL) has not yet been analyzed. In our study we analyzed both signaling pathways in different B- and T-ALL cell lines (RS4;11, SEM, REH, CEM, Jurkat, MOLT-4), thereby focusing mainly on their potential interaction via the protein GSK3β. Western Blot experiments were performed to evaluate the expression of specific PI3K/Akt and Wnt/β-Catenin key proteins. To evaluate the activation status of Wnt signaling immunofluorescence and protein fractionation experiments were performed, analyzing the activation linked nucleic localization of β-Catenin. The effect of pathway activation and inhibition on cell proliferation via chemical compounds was analyzed by WST-1 test. High pAkt levels were detected in B-ALL cell line SEM and T-ALL cell line CEM, indicating a hyperactive PI3K/Akt signaling, whereas other analyzed cell lines diplayed lower pAkt status. Among all cell lines analyzed SEM and CEM also showed the highest cytoplasmic β-Catenin levels, indicating a direct interaction of both signaling pathways. However, immunofluorescence and fractionation experiments revealed that a translocation of β-Catenin into the nucleus did not occur. To further investigate the role and interaction of PI3K/Akt and Wnt/β-Catenin signaling, pathway inhibiting and stimulating experiments were performed. Treatment of cells with Wnt3a led to activation of the Wnt/β-Catenin signaling cascade, characterized by nuclear β-Catenin accumulation. Inhibition of cell proliferation was detected after treatment with high concentrations Wnt3a (≥ 500 ng/ml). PI3K inhibition by LY294002 led to decreased phosphorylation of GSK3β at Ser9 and an increased decay of β-Catenin. Stimulation of PI3K/Akt signaling using activating ligand FLT3L induced GSK3β phosphorylation at Ser9 and accumulation of cytoplasmic β-Catenin. However a translocation of β-Catenin into the nucleus seems not to occur. In summary our results indicate that PI3K/Akt and Wnt/β-Catenin signaling can interact through their common protein GSK3β, but stimulation of the PI3K/Akt signaling pathway by addition of PI3K/Akt specific activators does not fully activate Wnt/β-Catenin signaling in ALL cells. Complete activation of the Wnt cascade characterized by translocation of β-Catenin into the nucleus can only be induced by use of specific Wnt effectors. Disclosures: No relevant conflicts of interest to declare.
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Fachel, Angela A., Ashlesha Shrikant Muley, Cem Meydan, Xabier Agirre, Leandro Cerchietti, Ari Melnick, and Kristy L. Richards. "Targeting ß-Catenin Signaling in DLBCL with ICG-001 and PAM-1 Suppresses Cell Proliferation and Induces Apoptosis." Blood 128, no. 22 (December 2, 2016): 5382. http://dx.doi.org/10.1182/blood.v128.22.5382.5382.
Abstract:
Abstract Introduction: DLBCL is a fast-growing, aggressive form of NHL and ~30% of the patients are not cured with the current treatment regimens. Recently Wnt/ ß-catenin signaling has been identified as a new target pathway for lymphoma. To specifically target B-catenin we picked two drugs that act downstream of the Wnt/B-catenin pathway. ICG-001 inhibits TCF/β-catenin mediated transcription by competing specifically with β-catenin for CREB binding protein (CBP), but not for p300. PAM-1 is a p21-activated kinase 4 (PAK4) allosteric modulator acting as a nucleo-cytoplasmic shuttling protein. PAK4 interacts with and phosphorylates β-catenin on Ser675, promoting TCF/B-catenin transcriptional activity and stabilizing β-catenin through inhibition of its degradation. We conducted a pre-clinical study with ICG-001 and PAM-1 ß-catenin inhibitors on 16 ABC and GCB DLBCL cell lines and provided a molecular rationale for Wnt/ß-catenin directed therapy. Material & Methods: RNAseq data from DLBCL cell lines, patient and normal B-cell samples were used to profile transcript abundance of the Wnt pathway in DLBCL. Western blotting was used to measure ß-catenin expression in 16 ABC and GCB DLBCL cell lines. Pharmacological inhibition of ß-catenin-signaling was tested using ICG-001 and PAM-1 compounds. A metabolically active cellular assay (CellTiter Glo) and flow cytometry with annexin V staining were used to determine cell viability and apoptosis. To address the specificity of the drug response, DLBCL cells were transduced with a ß-catenin/TCF pathway reporter, knocked down for ß-catenin, and canonical Wnt target expression was assessed using quantitative real-time (qRT-PCR) and Western blot. Results: ß-catenin is expressed on all DLBCL cell lines at different levels that do not correlate with the ABC/GCB classification. The ß-catenin inhibitors ICG-001 and PAM-1 significantly reduced viability in all ABC and GCB DLBCL cell lines, regardless of DLBCL subtype. The EC50 after 48h of treatment ranged from 1.4 μM to 6.3 μM for ICG001 and from 80 nM to 870 nM for PAM-1. Both compounds suppressed cell proliferation, induced apoptosis, and reduced Wnt/ß-catenin transcriptional activity. Conclusions: Our data indicate that targeting ß-catenin pathway with ICG-001 and PAM-1 could be therapeutically beneficial to DLBCL patients. Disclosures Melnick: Janssen: Research Funding.
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Tran, Bang Manh, Dustin James Flanagan, Gregor Ebert, Nadia Warner, Hoanh Tran, Theodora Fifis, Georgios Kastrappis, et al. "The Hepatitis B Virus Pre-Core Protein p22 Activates Wnt Signaling." Cancers 12, no. 6 (May 31, 2020): 1435. http://dx.doi.org/10.3390/cancers12061435.
Abstract:
An emerging theme for Wnt-addicted cancers is that the pathway is regulated at multiple steps via various mechanisms. Infection with hepatitis B virus (HBV) is a major risk factor for liver cancer, as is deregulated Wnt signaling, however, the interaction between these two causes is poorly understood. To investigate this interaction, we screened the effect of the various HBV proteins for their effect on Wnt/β-catenin signaling and identified the pre-core protein p22 as a novel and potent activator of TCF/β-catenin transcription. The effect of p22 on TCF/β-catenin transcription was dose dependent and inhibited by dominant-negative TCF4. HBV p22 activated synthetic and native Wnt target gene promoter reporters, and TCF/β-catenin target gene expression in vivo. Importantly, HBV p22 activated Wnt signaling on its own and in addition to Wnt or β-catenin induced Wnt signaling. Furthermore, HBV p22 elevated TCF/β-catenin transcription above constitutive activation in colon cancer cells due to mutations in downstream genes of the Wnt pathway, namely APC and CTNNB1. Collectively, our data identifies a previously unappreciated role for the HBV pre-core protein p22 in elevating Wnt signaling. Understanding the molecular mechanisms of p22 activity will provide insight into how Wnt signaling is fine-tuned in cancer.
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Rogaczewski, Patryk, Michał Janiak, Kamil Borysewicz, Tadeusz Głos, Navid Ahmadi, and Łukasz Szylberg. "Clinical significancy of WNT pathway inhibition in various cancers." Journal of Education, Health and Sport 12, no. 11 (November 3, 2022): 183–91. http://dx.doi.org/10.12775/jehs.2022.12.11.024.
Abstract:
The tumor microenvironment (TME) plays an important role in the cell cycle. There is a correlation between the Wnt/B–catenin signaling pathway and TME. This article reviews methods of inhibiting Wnt Pathway, a useful process in the treatment of various cancers. Compounds of Wnt/β–Catenin Signaling Pathway, such as TCF–1, have an impact on the differentiation and migration of CD8+ T cells. CCL4 expression is regulated by the beta–catenin protein to recruit CD103+ dendritic cells, which enables CD8+ T cell activation. Inhibition of the Wnt/β–catenin pathway has an impact on ovarian cancer patients’ prognosis, reducing the development of ovarian cancer. Research shows that inhibition of the pathway with the use of the LGK974 inhibitor may boost immunity, especially when applied with a Paclitaxel mix. After treatment, expression of the inhibitory receptors CTLA–4, TIM3, PD–1 on CD8+ T cells decreased. The combination of LGK974 and Paclitaxel can cause the death of tumor cells and significantly inhibit their proliferation. The application of dose–dense Paclitaxel avoids toxicity related to the maximum dose needed to protect the patient's immune system by increasing CD8+ TILs. There are concerns regarding toxicity of the LGK 974, especially for cells dependent on the Wnt/β–catenin pathway to maintain homeostasis. Many Wnt/β–catenin pathway inhibitors are tested against colorectal cancer (CRC) with successful results. These include NSAIDs, porcupine inhibitors, tankyrase inhibitors, Wnt5A inhibitors, and disheveled protein inhibitors.The Wnt/β–catenin pathway, when expressed in Triple Negative Breast Cancer (TNBC), leads to the transition of epithelial to mesenchymal cells. In early clinical development, there are multiple inhibitors (ex. KYA1797K) targeting the Wnt/β–catenin pathway in TNBC cells, which could become a viable anticancer strategy.
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Thanendrarajan, S., Y. Kim, and I. G. H. Schmidt-Wolf. "Understanding and Targeting the Wnt/β-Catenin Signaling Pathway in Chronic Leukemia." Leukemia Research and Treatment 2011 (December 4, 2011): 1–7. http://dx.doi.org/10.4061/2011/329572.
Abstract:
It has been revealed that the Wnt/β-catenin signaling pathway plays an important role in the development of solid tumors and hematological malignancies, particularly in B-cell neoplasia and leukemia. In the last decade there have been made experimental approaches targeting the Wnt pathway in chronic leukemia. In this paper we provide an overview about the current state of knowledge regarding the Wnt/β-catenin signaling pathway in chronic leukemia with special focus on therapeutic options and strategies.
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Shi, Chong-Shan, Ning-Na Huang, Kathleen Harrison, Sang-Bae Han, and John H. Kehrl. "The Mitogen-Activated Protein Kinase Kinase Kinase Kinase GCKR Positively Regulates Canonical and Noncanonical Wnt Signaling in B Lymphocytes." Molecular and Cellular Biology 26, no. 17 (September 1, 2006): 6511–21. http://dx.doi.org/10.1128/mcb.00209-06.
Abstract:
ABSTRACT Wnt ligands bind receptors of the Frizzled (Fz) family to control cell fate, proliferation, and polarity. Canonical Wnt/Fz signaling stabilizes β-catenin by inactivating GSK3β, leading to the translocation of β-catenin to the nucleus and the activation of Wnt target genes. Noncanonical Wnt/Fz signaling activates RhoA and Rac, and the latter triggers the activation of c-Jun N-terminal kinase (JNK). Here, we show that exposure of B-lymphocytes to Wnt3a-conditioned media activates JNK and raises cytosolic β-catenin levels. Both the Rac guanine nucleotide exchange factor Asef and the mitogen-activated protein kinase kinase kinase kinase germinal center kinase-related enzyme (GCKR) are required for Wnt-mediated JNK activation in B cells. In addition, we show that GCKR positively affects the β-catenin pathway in B cells. Reduction of GCKR expression inhibits Wnt3a-induced phosphorylation of GSK3β at serine 9 and decreases the accumulation of cytosolic β-catenin. Furthermore, Wnt signaling induces an interaction between GCKR and GSK3β. Our findings demonstrate that GCKR facilitates both canonical and noncanonical Wnt signaling in B lymphocytes.
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Qiang, Ya-Wei, Bo Hu, Yu Chen, Wei Qiang, Christoph Heuck, Bart Barlogie, and John D. Shaughnessy. "Bortezomib Induces Activation of b-Catenin/TCF Signaling Pathway in Multiple Myeloma." Blood 118, no. 21 (November 18, 2011): 1851. http://dx.doi.org/10.1182/blood.v118.21.1851.1851.
Abstract:
Abstract Abstract 1851 Background: The proteasome inhibitor Bortezomib (Bz) shows significant activity in Multiple Myeloma (MM) by acting on MM cell directly as well as by augmenting bone formation in vitro and in vivo. Its effect on the bone could be traced to promoting differentiation of mesenchymal stem cells into osteoblast cells by regulating BMP2 and canonical Wnt signaling. However, the molecular mechanism mediating the direct anti-MM activity of Bz remains to be fully understood. Initially the rationale for the use of Bz in MM was inhibition of NF-kB signaling, yet subsequent studies showed that Bz actually induces activation of this pathway. In this study, we examined whether Bz regulates the activity of canonical Wnt signaling pathway in MM and whether the growth-inhibition effect of Bz was associated with activation of this pathway by using multiple MM cell lines including EJM, H929, INA6, KMS28BM, JJN3, L363, OPM1, OPM2, RPMI8226, UTMC, XG2 and XG6 as well as primary plasma cells (PC) from six patients with newly diagnosed MM. Methods/Results: Immunoblotting demonstrated that Bz induces stabilization of b-catenin protein in three MM cell lines (H929, OPM2 and UTMC) in a time- and dose-dependent manner. These changes were not seen when the same cell lysate were immunoblotted for other catenin family members, a-catenin and g-catenin. Increased levels of b-catenin protein response to Bz treatment were observed in other 9 MM cell lines (EJM, INA6, KMS28BM, JJN3, L363, OPM1, RPMI8226, XG2 and XG6) and in the 6 CD138+ sorted bone marrow PC from patients with MM. To determine if Bz regulation of b-catenin level is a specific effect of the inhibition of 26S proteasome subunit we treated the same MM cell lines with another proteasome inhibitor, MG132. Similar results were observed in response to MG132 for all four MM cell lines, suggesting the effect of Bz on b-catenin protein is 26S proteosome inhibitor specific. Increases in b-catenin protein levels in MM cells were not due to increased Ctnnb1/CTNNB (b-catenin) gene transcription as b-catenin mRNA did not change in these cells treated with Bz. These results indicate that proteasome inhibition increases b-catenin is independent of transcriptional upregulation. To determine whether Bz induces the nuclear localization and transcriptional activity of b-catenin, cells were incubated with Bz for 6 hours and then fractionated to separate the nuclear and cytoplasmic fractions. Treatment with Bz resulted an increase in nuclear b-catenin as well as b-catenin in cytoplasm in four cell lines including H929, INA6, OPM1 and MM144. Increase in cytoplasmic and nuclear b-catenin was further confirmed by immunofluorescence with antibodies specific for active form of b-catenin. To determine whether Bz affects b-catenin-mediated transcriptional activity, we used a TCF/LEF luciferase reporter construct cloned in lentiviral vector. OPM2 cells were infected with lentiviral particle containing the TCF reporter or containing empty vector and were then treated with serial concentrations of Bz. The luciferase activity exhibited a dose-dependent response to Bz analogous to the stabilization of b-catenin. Similar results were observed in 7 out of 8 MM cell lines compared with untreated control. Stimulation of TCF transcriptional activity by Bz was independent of modifiers of extracellular Wnt ligands, such as Frizzled receptors, LRP5/6 co-receptors and sFRPs or the activation of intracellular GSK3b. Conclusion: These results indicate that Bz augments activation of canonical Wnt signaling by preventing b-catenin protein from proteosome-mediated degradation in MM cells. Concentrations of Bz for stimulating TCF transcriptional activity are comparable to those being used to induce inhibition of MM proliferation. Experiments modulating cytoplasmic as well as the nuclear players and interactions of the Wnt-pathway are ongoing to determine if Bz mediated activation of b-catenin signaling is responsible for its direct anti-MM effect. Disclosures: Barlogie: Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.
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Xu, Leilei, Zhicheng Dai, Chao Xia, Zhichong Wu, Zhenhua Feng, Xu Sun, Zhen Liu, Yong Qiu, Jack Chun-Yiu Cheng, and Zezhang Zhu. "Asymmetric Expression of Wnt/B-catenin Pathway in AIS." Spine 45, no. 12 (February 10, 2020): E677—E683. http://dx.doi.org/10.1097/brs.0000000000003409.
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Gonzalez, Maria E., Eric R. Fearon, and Celina Kleer. "Abstract PD5-06: PD5-06 CCN6 suppresses spindle metaplastic breast carcinoma in part via antagonizing Wnt/β-catenin signaling." Cancer Research 83, no. 5_Supplement (March 1, 2023): PD5–06—PD5–06. http://dx.doi.org/10.1158/1538-7445.sabcs22-pd5-06.
Abstract:
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.
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Morrison, J. A., A. J. Klingelhutz, and N. Raab-Traub. "Epstein-Barr Virus Latent Membrane Protein 2A Activates β-Catenin Signaling in Epithelial Cells." Journal of Virology 77, no. 22 (November 15, 2003): 12276–84. http://dx.doi.org/10.1128/jvi.77.22.12276-12284.2003.
Abstract:
ABSTRACT The Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) functions to maintain latency in EBV-infected B lymphocytes. Although LMP2A is nonessential for the immortalization of B lymphocytes by EBV, its expression in B lymphocytes prevents viral reactivation by blocking B-cell receptor activation and signaling. LMP2A also provides an antiapoptotic signal in transgenic mice that express LMP2A in B lymphocytes. LMP2A activates phosphatidylinositol 3-kinase (PI3K) and the serine/threonine kinase Akt in lymphocytes and epithelial cells. Here we show that EBV LMP2A activates the PI3K and β-catenin signaling pathways in telomerase-immortalized human foreskin keratinocytes (HFK). LMP2A activated Akt in a PI3K-dependent manner, and the downstream Akt targets glycogen synthase kinase 3β (GSK3β) and the Forkhead transcription factor FKHR were phosphorylated and inactivated in LMP2A-expressing HFK cells. GSK3β is a negative regulator of the Wnt signaling pathway, and inactivation of GSK3β by LMP2A signaling led to stabilization of β-catenin, the central oncoprotein of Wnt signaling. In LMP2A-expressing cells, β-catenin accumulated in the cytoplasm and translocated into the nucleus via a two-step mechanism. The cytoplasmic accumulation of β-catenin downstream of LMP2A was independent of PI3K signaling, whereas its nuclear translocation was dependent on PI3K signaling. In the nucleus, β-catenin activated a reporter responsive to T-cell factor, and this activation was augmented by LMP2A coexpression. The Wnt pathway is inappropriately activated in 90% of colon cancers and is dysregulated in several other cancers, and these data suggest that activation of this pathway by LMP2A may contribute to the generation of EBV-associated cancers.
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Fagotto, F., K. Guger, and B. M. Gumbiner. "Induction of the primary dorsalizing center in Xenopus by the Wnt/GSK/beta-catenin signaling pathway, but not by Vg1, Activin or Noggin." Development 124, no. 2 (January 15, 1997): 453–60. http://dx.doi.org/10.1242/dev.124.2.453.
Abstract:
The molecular nature of the primary dorsalizing inducing event in Xenopus is controversial and several secreted factors have been proposed as potential candidates: Wnts, Vg1, Activin and Noggin. Recent studies, however, have provided new insight into the activity of the dorsalizing region, called the Nieuwkoop Center. (1) The activity of this dorsalizing center involves an entire signal transduction pathway that requires maternal beta-catenin (Heasman, J., Crawford, A., Goldstone, K., Garner-Hamrick, P., Gumbiner, B., McCrea, P., Kintner, C., Noro, C. Y. and Wylie, C. (1994) Cell 79, 791–803). (2) A transcription factor with potent dorsalizing activity, Siamois, is expressed within the Nieuwkoop Center (Lemaire, P., Garrett, N. and Gurdon, J. B. (1995) Cell 81, 85–94). We have used these two properties of the Nieuwkoop Center to evaluate the dorsalizing activity of the four secreted factors Wnt8, Vg1, Activin and Noggin. The requirement for beta-catenin was tested by coexpressing a cadherin, which sequesters beta-catenin at the cell membrane and specifically blocks its intracellular signaling activity (Fagotto, F., Funayama, N., Gluck, U. and Gumbiner, B. M. (1996) J. Cell Biol. 132, 1105–1114). Induction of Siamois expression was detected by RT-PCR. Of the four growth factors, only Wnt was sensitive to inhibition of beta-catenin activity and only Wnt could induce Siamois expression. Therefore, Wnt is able to induce a bonafide Nieuwkoop Center, while Vg1, Activin and Noggin probably induce dorsal structures by a different mechanism. To order the steps in the Nieuwkoop Center signaling cascade, we have tested the relationship between beta-catenin and GSK, a serine-threonine kinase that has been implicated in axis formation in a step downstream of Wnt. We found that GSK acts upstream of beta-catenin, similar to the order of these components in the Wingless pathway in Drosophila. We have also examined the relationship between the Wnt/beta-catenin pathway and Siamois. We show that beta-catenin induces expression of Siamois and that the free signaling pool of beta-catenin is required for normal expression of endogenous Siamois. We conclude that the sequence of steps in the signaling pathway is Wnt-->GSK-->beta-catenin-->Siamois.
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Behrouj, Hamid, Atefeh Seghatoleslam, Pooneh Mokarram, and Saeid Ghavami. "Effect of casein kinase 1α inhibition on autophagy flux and the AKT/phospho-β-catenin (S552) axis in HCT116, a RAS-mutated colorectal cancer cell line." Canadian Journal of Physiology and Pharmacology 99, no. 3 (March 2021): 284–93. http://dx.doi.org/10.1139/cjpp-2020-0449.
Abstract:
The Wnt/β-catenin pathway, which interferes with cell proliferation, differentiation, and autophagy, is commonly dysregulated in colorectal cancer (CRC). Mutation of the RAS oncogene is the most prevalent genetic alteration in CRC and has been linked to activation of protein kinase B (AKT) signaling. Phosphorylation of β-catenin at Ser 552 by AKT contributes to β-catenin stability, transcriptional activity, and increase of cell proliferation. Casein kinase 1 alpha (CK1α) is an enzyme that simultaneously regulates Wnt/β-catenin and AKT. The link of the AKT and Wnt pathway to autophagy in RAS-mutated CRC cells has not well identified. Therefore, we investigated how pharmacological CK1α inhibition (D4476) is involved in regulation of autophagy, Wnt/β-catenin, and AKT pathways in RAS-mutated CRC cell lines. qRT-PCR and immunoblotting experiments revealed that phospho-AKT (S473) and phospho-β-catenin (S552) are constitutively increased in RAS-mutated CRC cell lines, in parallel with augmented CK1α expression. The results also showed that D4476 significantly reduced the AKT/phospho-β-catenin (S552) axis concomitantly with autophagy flux inhibition in RAS-mutated CRC cells. Furthermore, D4476 significantly induced apoptosis in RAS-mutated CRC cells. In conclusion, our results indicate that CK1α inhibition reduces autophagy flux and promotes apoptosis by interfering with the AKT/phospho-β-catenin (S552) axis in RAS-mutated CRC cells.
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Ge, Xueling, Xiao Lv, Peipei Li, and Xin Wang. "Overexpression of Metadherin in the Pathogenesis of Diffuse Large B-Cell Lymphoma,." Blood 118, no. 21 (November 18, 2011): 3653. http://dx.doi.org/10.1182/blood.v118.21.3653.3653.
Abstract:
Abstract Abstract 3653 Introduction: Diffuse large-B-cell lymphoma (DLBCL) is an aggressive malignancy of mature B lymphocytes with variable biological and cytogenetic features, as well as clinical outcomes. Further investigating specific biomarkers and cellular signaling pathways, understanding molecular pathogenesis of DLBCL and developing more targeted and effective treatments are indispensable for significantly increasing the survival and alleviating the suffering of patients. Metadherin (MTDH) has been demonstrated as a potentially crucial mediator of various types of human malignancies. It promotes tumor progression by modulating multiple oncogenic signaling pathways, such as NF-kB, PI3K/Akt and Wnt/b-catenin pathways. However, the expression and role of MTDH in DLBCL have not been reported yet. This study focuses on illuminating the role of MTDH and the relationship between MTDH and Wnt/b-catenin pathway in the pathogenesis of DLBCL. Methods: Sixteen fresh and thirty araffin-embedded tissues from DLBCL patients were collected. The tissues from patients of reactive hyperplasia of lymph node were used as control group. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers served as normal control compared with human DLBCL cell lines LY1 and LY8. LY1 and LY8 cells were treated with tumor necrosis factor-a(TNF-a,250pg/ml) and cultured for 48 hours to induce the upregulation of MTDH protein. The expressions of MTDH and b-catenin mRNA in DLBCL tissues and controls were determined by quantitative PCR. MTDH and b-catenin protein expressions and localizations were examined by Western-blot analysis and immunohistochemical staining. The impacts of MTDH overexpression induced by TNF-a on LY1 and LY8 cells!&hibar; proliferation and apoptosis were assessed by 3H-TdR incorporation method and flow cytometry. The effects of MTDH upregulation on expression of total b-catenin and its nuclear translocation were analyzed by nuclear protein extraction and Western-blot analysis. Results: A remarkable elevation of MTDH and b-catenin on mRNA level was detected in DLBCL tissues (Figure 1A, P<0.001). The expression of MTDH and b-catenin protein, with b-catenin nuclear localization, was also significantly increased in DLBCL cell lines and DLBCL tissues in comparison to their counterparts (Figure 1B, P<0.001). Immunohistochemical analysis showed high expression of MTDH in 23 of 30(76.67%) paraffin-embedded archival DLBCL specimens (Figure 1C). Statistical analysis suggested that the over expression of MTDH was strongly correlated to the clinical staging of patients with DLBCL (Table 1). The expression of MTDH protein in LY1 and LY8 cells was upregulated after treated with TNF- a(MTDH/b-actin: 0.98\!À0.15 v. 1.18\!À0.18, P<0.05; 0.95\!À0.13 v. 1.19\!À0.19, P<0.05). Furthermore, we determined that the increase of MTDH in DLBCL cells could distinctly enhance cell proliferation and inhibit cell apoptosis (Figure 1D, P<0.05). Upregulation of MTDH elevated the protein level of total b-catenin and translocation of b-catenin to the nucleus (Figure 1E, P<0.05). Conclusion: Our study indicated that MTDH and b-catenin were markedly overexpressed, with b-catenin nuclear localization, in DLBCL. Overexpression of MTDH was correlated with the clinical staging of patients with DLBCL. The effect of MTDH promoting growth and survival of DLBCL cells may be mediated through regulation of Wnt/b-catenin signaling pathway. These results suggest that MTDH contributes to the pathogenesis of DLBCL mediated by activation of Wnt/b-catenin pathway. This novel study may contribute to further investigation on the useful biomarkers and potential therapeutic target in the DLBCL patients. Disclosures: No relevant conflicts of interest to declare.
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Ko, Alex Y., Claudia Zierold-Fairman, and Erik A. Ranheim. "The beta-catenin pathway is involved in normal B cell development and function (83.3)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S112. http://dx.doi.org/10.4049/jimmunol.178.supp.83.3.
Abstract:
Abstract Wnt/Fzd signaling plays a key role in development, stem cell maintenance, and tumorigenesis, particularly via stabilization of ß-catenin protein levels. TCF-1 and LEF-1 knockout mice suggested a role for this pathway in lymphoid development as well. We have previously shown that Fzd9−/− mice exhibit a decrease in pre-B cells at a stage when self-renewing division is occurring in preference over further differentiation, prior to light chain Ig recombination (Hardy C-D). We now report further evidence for a role of ß-catenin in normal B cell function and development. By flow cytometric analysis, we find that cellular ß-catenin levels rise at precisely the stage of B cell development that shows diminished numbers in Fzd9−/− mice. Expression of LEF-1, the transcriptional partner of ß-catenin, also is tightly regulated during this developmental sequence by differential transcription of full length and dominant negative forms, further suggesting that Wnt/Fzd signals impact on B-cell lymphopoiesis. In mature B-cells, activation through BCR, Toll-like receptors, and CD40 all induce an increase in beta-catenin protein levels, while changes in the expression of genes involved in both positive and negative regulation of canonical Wnt signaling are differentially regulated by these stimuli, highlighting a potential novel role for this pathway in B-lymphocyte proliferation, differentiation, and survival.
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Mani, Mala, Jui Dutta, Yunyu Zhang, Daniel E. Carrasco, Yiming Zhou, Moshe Gatt, Bart Barlogie, John D. Shaughnessy, Kenneth C. Anderson, and Daniel Ruben Carrasco. "The BCL9 Oncogene Promotes Tumor Progression by Conferring Enhanced Proliferative, Metastatic and Angiogenic Properties to Myeloma Cells." Blood 112, no. 11 (November 16, 2008): 648. http://dx.doi.org/10.1182/blood.v112.11.648.648.
Abstract:
Abstract Wnt signaling plays an important role in tissue development and maintenance during embryogenesis, cell differentiation, and stem cell growth. Several components of the Wnt signaling cascade have been shown to function as either tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis. Deregulation of the canonical Wnt/b-catenin pathway has been implicated in numerous human epithelial malignancies as well as hematologic malignancies including multiple myeloma (MM), generating immense interest in these molecules as targets for cancer therapy. Activation of Wnt/b-catenin in cancer has been associated with mutations that enable b-catenin to escape degradation by the proteasome, thereby allowing its accumulation in the nucleus where it functions as a transcriptional regulator in conjunction with coactivators by constitutively activating target genes such as c-Myc and Cyclin D1. To date, however, no mutations in Wnt pathway have been documented in MM, suggesting that mechanisms other than gene mutation may contribute to Wnt pathway deregulation. BCL9, a key component of the Wnt pathway, is required for b-catenin transcriptional activity and resides on chromosome 1q21, a region frequently involved in secondary chromosomal aberrations associated with MM tumor progression. Here we provide evidence that dysregulation of BCL9 expression is a novel oncogenic mechanism of Wnt pathway activation in MM. Using in vitro and in vivo functional analyses, we demonstrate that BCL9 is a bonafide oncogene that is aberrantly expressed in MM and associated with survival. Using the TCF- specific luciferase reporter, we show that enforced expression of BCL9 in MM cells enhanced b-catenin mediated transcription by >12 fold, suggesting a possible role of BCL9 overexpression in the pathogenesis of MM. BCL9 enhanced proliferation (1.5 fold, P<0.02), migration (3.5 fold, P<0.0001) and the metastatic potential of MM cells. We also showed that BCL9 plays an important role in tumor progression by regulating Cyclin D1 and c-Myc mediated cell proliferation, CD44 mediated tumor metastasis, as well as VEGF mediated host angiogenesis. Importantly, BCL9 knockdown significantly increased the survival in a xenograft mouse model of human MM (P=0.001), associated with decreased tumor burden and host angiogenesis. In summary, we have demonstrated that BCL9 is a novel and potent oncogene of the Wnt pathway in MM, playing fundamental roles in tumor progression by regulating proliferation, migration, invasion, angiogenesis and the metastatic potential of tumor cells. The pleiotropic roles of BCL9 and its aberrant expression highlight its importance as an attractive and novel therapeutic target in the treatment of MM.
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Calaminus, Simon D., Gareth Inman, Cedric Ghaevert, Owen Sansom, Steve P. Watson, Tessa L. Holyoake, and Laura Machesky. "Alterations In Wnt Signalling In the Megakaryocytic Lineage Leads to Bone Marrow Failure and Myelofibrosis." Blood 116, no. 21 (November 19, 2010): 628. http://dx.doi.org/10.1182/blood.v116.21.628.628.
Abstract:
Abstract Abstract 628 Introduction: Wnt signalling is fundamental in controlling stem cell self-renewal, cell proliferation and development in multicellular organisms. Stabilization of beta catenin or loss of the scaffold protein adenomatis polyposis coli (APC) causes aberrant activation of wnt signalling and often leads to cancer. Mutations to wnt pathway members in haematopoietic stem cells leads to haematopoietic failure and rapid lethality. In this study, we demonstrate that aberrant wnt signalling in the megakaryocyte lineage underlies myelofibrosis. Methods: We created a series of mice with altered wnt pathway signalling in their megakaryocytic lineage using PF4-Cre (platelet factor 4 cre) as follows: Ctnnb1fx(ex3)/wt_ Expresses stabilized active beta catenin henceforth termed PF4bcat+; APCfx/fx_ Loss of APC stabilises beta catenin termed PF4APC-KO; Ctnnb1fx/fx b-catenin knockout termed PF4bcat-KO. Results: By day 40, PF4bcat+ and PF4APC-KO mice are severely underweight, anaemic (wt 6.8+0.2×106/ml v. PF4bcat+ 4.2+0.3×106/ml, PF4APC-KO 5.5+0.5×106/ml), and have a significant reduction in platelet number (wt 1033+37×106/ml v. PF4bcat+ 717+57×106/ml, PF4APC-KO 747+68×106/ml). Furthermore PF4bcat+ and PF4APC-KO mice develop bone marrow fibrosis and consistently die within 50 days of birth. Both populations of mice have splenic extramedullary haemopoiesis with hyperplasia of splenic megakaryocytes, leading to a dramatic increase in spleen to body size ratio. In addition, both PF4bcat+ and PF4APC-KO mice have increased peripheral blood levels of active TGFb, providing a likely molecular basis of the induction in bone marrow fibrosis. PF4APC-KO and PF4bcat+ mutant mice show a dramatic (>10-fold) increase in platelet b-catenin protein levels over wt samples. By comparison, human myelofibrosis patients (n=16) show a 2.7+0.6-fold increase in platelet b-catenin expression over controls. Moreover, overexpression of b-catenin within human patient samples is correlated with a worsening Primary Myelofibrosis prognostic score (Blood, 2009 vol 113 p. 2895), with those patients with a low score (n=7) having a 1.3+/−0.37-fold increase over control, intermediate score (n=4) 4.52+/−1.23-fold, and high score (n=1) 4.56-fold. In contrast PF4bcat-KO mice show no changes in whole blood counts, weight, or evidence of splenic extramedullary haematopoiesis, indicating that b-catenin removal does not adversely affect megakaryocyte development or function. Conclusions: Stabilisation of b-catenin within mouse megakaryocytes leads to a myelodysplastic disorder and myelofibrosis. This finding demonstrates a defined role for aberrant activation of the wnt signalling pathway and marks the wnt pathway and the megakaryocyte lineage as important potential drug targets for the treatment of myelodysplastic disorders. Disclosures: No relevant conflicts of interest to declare.
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Xu, Na, Liu Xiaoli, Qingfeng Du, Zhi Liu, Rong Li, Jun Yang, Shuang Wang, Min Zhong, and Yu Wang. "Artesunate,a Antimalarial Agent,Exhibits Potent Anti-Leukemia Activities in Vitro and Inbibit Wnt/b-Catenin Pathway." Blood 114, no. 22 (November 20, 2009): 4822. http://dx.doi.org/10.1182/blood.v114.22.4822.4822.
Abstract:
Abstract Abstract 4822 Artesunate (ART), a remarkable antimalarial agent, its antitumor effect was widely studied recently. But whether the antitumor mechanisms of ART involves in Wnt/b-catenin signalling, a major oncogenic pathway have yet been poorly understood. In address this question,we treated human leukemia cell lines,such as K562 cells, HL-60 cells,U937cells and KG1a cells with 12.5ug/ml,50ug/ml, 100 ug/ml and 200ug/ml ART for 24h, 48h and 72h respectively. Determined by the Cell Counting Kit-8 and flow cytometry analysis on apoptosis, we found that ART suppressed the leukemia cells proliferation and promoted the apoptosis of leukemia cells in a time- and concentration-dependent manner. Furthermore, we detected the mRNA level and protein expression of b-catenin and Wnt/b-catenin target genes c-myc and cyclinD1 by QRT-PCR and western blotting when the above leukemia cell lines treated with ART for 48h. The results demonstrated that the the mRNA level and protein expression of c-myc and cyclinD1 was lower in ART groups than in the contorl groups,and in concentration-dependent manner. on the other hand, there was no significant difference in the mRNA level and protein expression of b-catenin in the ART groups compare to the contorl groups. Moreover, we observed b-catenin by immunofluorescence technology, the Bioluminescent imaging demonstrated that ART translocated b-catenin from nucleus to adherent junctions of membrane,and binding to E-cadherin on the cell membrane. Our results provide the vitro evidence for the anti-luekemia mechanism of ART correlated to the inhibition of hyperactive Wnt/b-catenin signaling pathway. Combination With the present studies, the antitumor mechanism of ART is invoved in multiple signaling pathways, and the well known low toxicity. We thus speculate that ART might be a promising candidate drug for the treatment of leukemia. Disclosures: No relevant conflicts of interest to declare.
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Mannino, Federica, and Post Doc. "ODP599 Modulation of Wnt/b-catenin and Autophagy in an in vitro Model of Glucocorticoid-induced Osteoporosis." Journal of the Endocrine Society 6, Supplement_1 (November 1, 2022): A186. http://dx.doi.org/10.1210/jendso/bvac150.384.
Abstract:
Abstract Background During bone aging and osteoporosis, formation and resorption are not tightly coupled; the main cells involved in bone remodeling are osteoblasts and osteoclast but also osteocytes play a pivotal role in this process. Osteocytes are the most abundant cell type in bone and are mainly responsible for sensing mechanical signals on the bones, controlling osteoblast and osteoclast activities through cell-to-cell communication and via secreted factors. In particular, osteocytes regulate bone resorption, thanks to the production of RANKL, reducing osteoclast activity. Ellagic acid, a natural polyphenolic compound derived from pomegranate, could modulate cell function via specific estrogen receptor b; activation of ERb plays a critical role in bone remodeling suppressing osteoclast differentiation and function, promoting osteoblast proliferation through the Wnt/b-catenin pathway, increasing osteoprotegerin levels. In addition, stimulation of ERb plays an anti-inflammatory effect, increasing the expression of IL-10 and reducing the expression of proinflammatory cytokines, such as IL-1β and TNF-α, and can regulate the expression of autophagy inhibiting the PI3K/Akt/mTOR pathway. The hypothesis here tested is that ellagic acid, through ERb modulation, could induce Wnt/b-catenin and autophagy pathways in osteocytes challenged with dexamethasone to mimic glucocorticoid-induced osteoporosis. Materials and methods The osteocyte cells MLO-A5 were differentiated into osteocytes under appropriate culturing conditions. Cells were treated with Ellagic acid (1µM) following dexamethasone (1µM) challenge for 24h to induce an in vitro model of osteoporosis. At the end of the treatment period, cell viability was evaluated by MTT assay; qPCR was performed to evaluate the expression of the genes involved in osteocyte function (SOST, RANKL, Destrin and Dmp1) and Wnt/B-catenin signaling pathway (Wnt5a, Wnt10b, B-Catenin and DKK1); Western Blot was performed to evaluate the expression of proteins involved in apoptosis (cleaved-Caspase3) and autophagy (Beclin-1, LC3 and p62). In addition, the expression of Sclerostin and nuclear translocation of B-Catenin was evaluated by immunofluorescence. Results Ellagic acid reduced the gene expression of SOST, RANKL, Dmp1 and increased the expression of Destrin compared to untreated cells stimulated with dexamethasone; caused a significant increase of Wnt5a, Wnt10b and B-Catenin expression and reduced significantly the expression of DKK1. CGS21680 inhibited dexamethasone-induced apoptosis, reducing the expression of Caspase3, and increased the expression of Beclin-1 and LC3 compared to cell treated with dexamethasone. Finally, treatment with Ellagic acid stimulated the nuclear translocation of B-Catenin, promoting the transcription of genes involved in osteogenesis. Conclusion These preliminary data suggest that stimulation of ERb through Ellagic acid could modulate bone remodelling through activation of Wnt/b-catenin pathway and autophagy, providing evidence on the possible use of this natural compound as a new therapeutic approach for osteoporosis. Presentation: No date and time listed
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Jiang, Jiahao, Shuaihua Feng, Zexiang Li, Yangqian Luo, Zhenyuan Wang, Mingyang Li, and Guanbao Wu. "The Expression of MDM2 Gene Promoted Chondrocyte Proliferation in Rats with Osteoarthritis via the Wnt/β-Catenin Pathway." Cellular and Molecular Biology 67, no. 6 (February 27, 2022): 236–41. http://dx.doi.org/10.14715/cmb/2021.67.6.31.
Abstract:
This study aimed to investigate the regulatory mechanism of MDM2 gene expression on cartilage cell proliferation in Osteoarthritis (OA) rats. For this purpose, 22 SD rats were randomly divided into normal control (10 cases) and treated (12 cases) groups. Treated group was used for OA modelling with the modified Hulth method. After a week, RT-PCR was used to detect MDM2 in cartilage tissue of rats, Wnt 1, Wnt 3 a, Wnt 10 b and β-catenin genes mRNA expression. Rat chondrocytes were isolated and cultured, and the recombinant eukaryotic expression vector pcDNA3.1 myc-siRNA-MDM2-β-catenin and co-expression plasmid pcDNA3.1 myc-siRNA-MDM2-β-catenin was used to transfect chondrocytes and the proliferation and related gene expression levels of the transfected chondrocytes were detected by MTT method and RT-PCR. The results showed that compared with the control group, MDM2, Wnt 1, Wnt 3 a, Wnt 10b and β-catenin genes in OA rat cartilage constructed by Hulth method were increased (p<0.05). The pcDNA3.1 myc-beta-catenin transfection slowed down the proliferation of OA chondrocytes, different from the non-transfected OA group (p<0.001), and increased Wnt 1, Wnt 3a, Wnt 10b and β-catenin genes expression compared with the Control group (p<0.05), but did not affect the expression of MDM2. The transfection of siRNA-MDM2 was opposite to pcDNA3.1 myc-β-catenin. The co-expression plasmid pcDNA3.1 myc-siRNA-MDM2-beta-catenin transfection did not affect the proliferation of OA chondrocytes. In general, the high expression of MDM2 in OA rats restricts the proliferation of chondrocytes, which may be related to the main pathogenesis of the occurrence and development of OA in vivo, and the regulation of MDM2 on the proliferation of chondrocytes may be achieved through the Wnt/ β-catenin pathway.
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Siapati, Elena K., Magda Papadaki, Zoi Kozaou, Erasmia Rouka, Evridiki Michali, Dimitrios Gogos, Ioanna Savvidou, Despoina S. Kyriakou, Nikolaos I. Anagnostopoulos, and George Vassilopoulos. "AML Survival and In Vivo Progression Is Dependent On β-Catenin Signaling." Blood 114, no. 22 (November 20, 2009): 2398. http://dx.doi.org/10.1182/blood.v114.22.2398.2398.
Abstract:
Abstract Abstract 2398 Poster Board II-375 B-catenin is the central effector molecule of the canonical wnt signaling pathway which governs cell fate and differentiation during embryogenesis as well as self-renewal of hematopoietic stem cells. Deregulation of the pathway has been observed in various malignancies including myeloid leukemias where over-expression of β-catenin is an independent adverse prognostic factor. In the present study we examined the functional outcome of stable β-catenin down-regulation through lentivirus-mediated expression of short hairpin RNA (shRNA). Reduction of the β-catenin levels in AML cell lines and patient samples diminished their in vitro proliferation ability without significantly affecting cell viability. In order to study the role of β-catenin in vivo, we transplanted leukemic cell lines with control or reduced levels of β-catenin in NOD/SCID animals and analyzed the engraftment levels in the bone marrow. We observed that while the immediate homing of the cells was not affected by the β-catenin levels, the bone marrow engraftment was directly dependent on its levels. Subsequent examination of bone marrow sections revealed that the reduced engraftment was partly due to the inability of the cells with lower β-catenin levels to dock to the endosteal niches, a finding that was confirmed in competitive repopulation assays with untransduced cells. When we examined the expression levels of adhesion molecules and integrins in engrafted cells in vivo, we observed a significant down-regulation of CD44 expression, a molecule that participates in the interaction of HSCs with the niche. Gene expression analysis of the components of the wnt signaling pathway showed that the pathway is subject to tight transcriptional regulation with minor expression deviations. We did, however, observe an up-regulation in components that participate in the non-canonical wnt signaling pathways such as the WNT5B ligand. Ongoing experiments in normal cord blood CD34+ cells will determine the in vivo role of β-catenin signaling in normal hematopoietic progenitors. In conclusion, our study showed that β-catenin comprises an integral part in the development and progression of AML in vivo, indicating that manipulation of the wnt pathway may hold a therapeutic potential in the management of AML. Disclosures: No relevant conflicts of interest to declare.
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Reis, Marco, Cathrin J. Czupalla, Nicole Ziegler, Kavi Devraj, Jenny Zinke, Sascha Seidel, Rosario Heck, et al. "Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression." Journal of Experimental Medicine 209, no. 9 (August 20, 2012): 1611–27. http://dx.doi.org/10.1084/jem.20111580.
Abstract:
Endothelial Wnt/β-catenin signaling is necessary for angiogenesis of the central nervous system and blood–brain barrier (BBB) differentiation, but its relevance for glioma vascularization is unknown. In this study, we show that doxycycline-dependent Wnt1 expression in subcutaneous and intracranial mouse glioma models induced endothelial Wnt/β-catenin signaling and led to diminished tumor growth, reduced vascular density, and normalized vessels with increased mural cell attachment. These findings were corroborated in GL261 glioma cells intracranially transplanted in mice expressing dominant-active β-catenin specifically in the endothelium. Enforced endothelial β-catenin signaling restored BBB characteristics, whereas inhibition by Dkk1 (Dickkopf-1) had opposing effects. By overactivating the Wnt pathway, we induced the Wnt/β-catenin–Dll4/Notch signaling cascade in tumor endothelia, blocking an angiogenic and favoring a quiescent vascular phenotype, indicated by induction of stalk cell genes. We show that β-catenin transcriptional activity directly regulated endothelial expression of platelet-derived growth factor B (PDGF-B), leading to mural cell recruitment thereby contributing to vascular quiescence and barrier function. We propose that reinforced Wnt/β-catenin signaling leads to inhibition of angiogenesis with normalized and less permeable vessels, which might prove to be a valuable therapeutic target for antiangiogenic and edema glioma therapy.
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Rieger, Megan E., Andrew H. Sims, Ebony R. Coats, Robert B. Clarke, and Karoline J. Briegel. "The Embryonic Transcription Cofactor LBH Is a Direct Target of the Wnt Signaling Pathway in Epithelial Development and in Aggressive Basal Subtype Breast Cancers." Molecular and Cellular Biology 30, no. 17 (July 6, 2010): 4267–79. http://dx.doi.org/10.1128/mcb.01418-09.
Abstract:
ABSTRACT L imb- b ud and h eart (LBH) is a novel key transcriptional regulator of vertebrate development. However, the molecular mechanisms upstream of LBH and its role in adult development are unknown. Here we show that in epithelial development, LBH expression is tightly controlled by Wnt signaling. LBH is transcriptionally induced by the canonical Wnt pathway, as evident by the presence of conserved functional T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) binding sites in the LBH locus and rapid β-catenin-dependent upregulation of endogenous LBH by Wnt3a. In contrast, LBH induction by Wnt/β-catenin signaling is inhibited by Wnt7a, which in limb development signals through a noncanonical pathway involving Lmx1b. Furthermore, we show that LBH is aberrantly overexpressed in mammary tumors of mouse mammary tumor virus (MMTV)-Wnt1-transgenic mice and in aggressive basal subtype human breast cancers that display Wnt/β-catenin hyperactivation. Deregulation of LBH in human basal breast cancer appears to be Wnt/β-catenin dependent, as DKK1 and Wnt7a inhibit LBH expression in breast tumor cells. Overexpression studies indicate that LBH suppresses mammary epithelial cell differentiation, an effect that could contribute to Wnt-induced tumorigenesis. Taken together, our findings link LBH for the first time to the Wnt signaling pathway in both development and cancer and highlight LBH as a potential new marker for therapeutically challenging basal-like breast cancers.
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Catalano, Teresa, Federico Selvaggi, Diana Liberata Esposito, Roberto Cotellese, and Gitana Maria Aceto. "Infectious Agents Induce Wnt/β-Catenin Pathway Deregulation in Primary Liver Cancers." Microorganisms 11, no. 7 (June 22, 2023): 1632. http://dx.doi.org/10.3390/microorganisms11071632.
Abstract:
Interaction between infectious agents and liver tissue, as well as repeated and extreme biological events beyond adaptive capacities, may result in pathological conditions predisposing people to development of primary liver cancers (PLCs). In adults, PLCs mainly comprise hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Various infectious agents in the hepatic microenvironment can destabilize normal liver cell functions by modulating the Wnt/β-catenin pathway components. Among them, hepatotropic viruses B, C, and D are involved in Wnt/β-catenin signaling dysregulation. Other microbial agents, including oncogenic viruses such as Epstein–Barr virus (EBV) and human papilloma virus (HPV), bacteria, e.g., Mycoplasma hyorhinis and Salmonella Typhi, the protozoan parasite Toxoplasma gondii, the fungus Aspergillus flavus, and liver flukes such as Clonorchissinensis or Opisthorchis viverrini, may induce malignant transformation in hepatocytes or in target cells of the biliary tract through aberrant Wnt signaling activation. This review focuses on new insights into infectious agents implicated in the deregulation of Wnt signaling and PLC development. Since the Wnt/β-catenin pathway is a driver of cancer following viral and bacterial infections, molecules inhibiting the complex axis of Wnt signaling could represent novel therapeutic approaches in PLC treatment.
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Chung, Jihyun, Vrajesh Karkhanis, Said Sif, and Robert A. Baiocchi. "Protein Arginine Methyltransferase 5 Supports MYC, Survivin and Cyclin D1 Activity in Aggressive Lymphomas By Regulating the WNT/β-Catenin Pathway." Blood 124, no. 21 (December 6, 2014): 58. http://dx.doi.org/10.1182/blood.v124.21.58.58.
Abstract:
Abstract Introduction: Aggressive histologic subtypes of lymphoma such as mantle cell (MCL) and activated B cell (ABC) are considered incurable and affected patients often have a short median survival despite multimodal therapy. It is well established that altered expression of oncogenes and epigenetic dysregulation of tumor suppressor and regulatory genes promote cellular transformation of normal B cells into malignant lymphoma. Hypermethylation of histone proteins (H3R8 and H4R3) by the protein arginine methyltransferase 5 (PRMT5) enzyme has been documented in multiple cancer types and has been shown to promote tumor cell growth and survival. Importantly, PRMT5 over expression does not occur in normal B cells (resting or activated) and is only detected in malignant lymphoma cells. We have previously shown that PRMT5 regulates the Polycomb-repressive complex 2 (PRC2) complex including EZH2, a core histone-lysine N methyl transferase. EZH2 has tumor suppressor functions and has been shown to regulate WNT antagonist’s gene expression. WNT/β-CATENIN signaling pathway has been associated with increased cell proliferation and survival in various forms of cancers including lymphoma. Until recently, the role of PRMT5 in controlling WNT/β-CATENIN signaling has been unclear. We hypothesized that PRMT5, through its ability to repress EZH2 expression, would control WNT/β-CATENIN signaling and orchestrate downstream pathways that are relevant to lymphomagenesis. Methods: PRMT5 inhibition of patient-derived lymphoma cell lines, primary lymphoma tumor cells and mouse primary Eμ-BRD2 transgenic lymphoma cells by infecting with sh-PRMT5 lentivirus (or sh-GFP control) or a selective small molecule PRMT5 inhibitor (tool compound CMP5). Gene expression was monitored by immunoblotting and reverse transcription (RT) real time PCR. Recruitment of target proteins to promoter regions was examined by ChIP assays. To evaluate PRMT5 and WNT antagonist expression in NHL patient samples, primary tumor samples were collected from 4 patients with MCL. Cellular growth and apoptosis was assessed by proliferation assay and FACS analysis. Results: PRMT5 supports WNT/β-CATENIN activity by direct transcriptional repression of AXIN2 and WIF1 via a PRMT5-EZH2 repressor complex. PRMT5 indirectly supports EZH2 expression via inactivation of the RB-E2F pathway. AXIN2 and WIF1 are two proteins that negatively regulate WNT/bCATENIN. Additionally, PRMT5 inhibition with shRNA or CMP5 leads to repression of the WNT/β-CATENIN signaling pathway by allowing de-repression of AXIN2 and WIF1, leading to decreased nuclear phospho-b-CATENIN and decreased transcription of the target genes CYCLIN D1, c-MYC and SURVIVIN. Reduced nuclear localization of phospho-β-catenin (S675) led to differential enhanced recruitment of co-repressors LSD and HDAC2 (and loss of epigenetic marks H3K4Me3 and H3K9Me3) and loss of activating epigenetic marks H3K9Ac and H3K14Ac on CYCLIN D1, c-MYC and SURVIVIN promoters. We also found that PRMT5 regulates target gene repression in primary blastic variant MCL patient samples and mouse primary lymphoma tumor cells. Significance: Our observations show that PRMT5 is an important epigenetic regulator that governs the expression of its own target genes, the PRC2 program as well as regulating the WNT/β-CATENIN-driven pro-growth and survival genes c-MYC, CYCLIND D1 and SURVIVIN. These results, together with the prevalence of PRMT5 and EZH2 over expression and activation of WNT targets in multiple lymphoma histologic subtypes, suggests that inhibiting PRMT5 is likely to result in removal of repressive histone arginine and lysine marks and promote restoration of normal growth and survival checkpoints in malignant lymphomas. Disclosures No relevant conflicts of interest to declare.
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Wu, Qing-Li, Claudia Zierold, and Erik A. Ranheim. "Dysregulation of Frizzled 6 is a critical component of B-cell leukemogenesis in a mouse model of chronic lymphocytic leukemia." Blood 113, no. 13 (March 26, 2009): 3031–39. http://dx.doi.org/10.1182/blood-2008-06-163303.
Abstract:
Abstract Wnt/Fzd signaling is known to play a key role in development, tissue-specific stem-cell maintenance, and tumorigenesis, particularly through the canonical pathway involving stabilization of β-catenin. We have previously shown that Fzd9−/− mice have a deficiency in pre-B cells at a stage when self-renewing division is occurring in preference to further differentiation, before light chain immunoglobulin recombination. To determine whether pathologic usurpation of this pathway plays a role in B-cell leukemogenesis, we examined the expression of Wnt/Fzd pathway genes in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia. We find that, in the course of leukemogenesis, the expression of Wnt16, Wnt10α, Fzd1, and most dramatically, Fzd6, is progressively up-regulated in the transformed CD5+ B cells of these mice, as are β-catenin protein levels. Elimination of Fzd6 expression by crossing into Fzd6−/− mice significantly delays development of chronic lymphocytic leukemia in this model. Our findings suggest that the self-renewal signals mediated by Wnt/Fzd that are enlisted during B-cell development may be pathologically reactivated in the neoplastic transformation of mature B cells.
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Bezerra Lima, Bruno, Bárbara Faria Fonseca, Nathália da Graça Amado, Débora Moreira Lima, Ronaldo Albuquerque Ribeiro, José Garcia Abreu, and Gerly Anne de Castro Brito. "Clostridium difficile Toxin A Attenuates Wnt/β-Catenin Signaling in Intestinal Epithelial Cells." Infection and Immunity 82, no. 7 (April 7, 2014): 2680–87. http://dx.doi.org/10.1128/iai.00567-13.
Abstract:
ABSTRACTClostridium difficiletoxins A and B (TcdA and TcdB) are homologous glycosyltransferases that inhibit a group of small GTPases within host cells, but several mechanisms underlying their pathogenic activity remain unclear. In this study, we evaluated the effects of TcdA on the Wnt/β-catenin pathway, the major driving force behind the proliferation of epithelial cells in colonic crypts. IEC-6 and RKO cells stimulated with Wnt3a-conditioned medium were incubated with 10, 50, and 100 ng/ml of TcdA for 24 h, resulting in a dose-dependent inhibition of the Wnt signaling, as demonstrated by a T-cell factor (TCF) reporter assay. This was further confirmed by immunofluorescence staining for nuclear localization of β-catenin and Western blotting for β-catenin and c-Myc (encoded by a Wnt target gene). Moreover, our Western blot analysis showed a decrease in the β-catenin protein levels, which was reversed by z-VAD-fmk, a pan-caspase inhibitor. Nonetheless, TcdA was still able to inhibit the Wnt/β-catenin pathway even in the presence of z-VAD-fmk, lithium chloride (a GSK3β inhibitor), or constitutively active β-catenin, as determined by a TCF reporter assay. Furthermore, preincubation of RKO cells with TcdA for 12 h also attenuated Wnt3a-mediated activation of Wnt signaling, suggesting that inactivation of Rho GTPases plays a significant role in that inhibition. Taken together, these findings suggest that attenuation of the Wnt signaling by TcdA is important for TcdA antiproliferative effects.
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Sayitoglu, Muge, Ozden Hatirnaz, Yucel Erbilgin, Fatmahan Atalar, and Ugur Ozbek. "Different Activation of WNT Signaling Pathway in B-Cell and T-Cell Acute Leukemias." Blood 108, no. 11 (November 16, 2006): 4317. http://dx.doi.org/10.1182/blood.v108.11.4317.4317.
Abstract:
Abstract WNT signaling pathway proteins function as hematopoietic growth factors and regulate proliferation in normal T-cell and B-cell development. Recent experimental evidence demonstrated that oncogenic transformation in leukemias of both lymphoid and myeloid lineages is dependent on WNT signaling. Not much is known about activation of WNT signaling pathway, its ligands and receptors in hematopoiesis and leukemia pathogenesis. To define its role in leukemia, we aimed to determine mRNA levels of the critical members of WNT pathway (WNT5A, WNT10B, FZ5, β catenin, APC, TCF-1 and LEF-1) by using quantitative real time PCR in acute lymphoblastic leukemia (ALL) patients (T-cell n=42, B-cell n=46 and pre B-cell n=30) and normal hematopoietic cells (bone marrow n=6, peripheral blood n=10, and CD19+ cells from peripheral blood). These genes expressed varying levels in B-cells, preB-cells and T-cells. In the B-cell leukemia patients, WNT5A was expressed notably (OR=58.05 CI 95% 1.63–1219.55, p>0,001). WNT5A directs Ca++ dependent signaling by PKC and a G protein dependent manner which is an alternative pathway for beta-catenin mediated signaling. Also LEF-1 levels were higher in B-ALL patients and APC expression was down regulated when compared to normal tissue (OR=18.81 CI 95% 0.34–5703, p>0.001 and OR=0.212 CI 95% 0.006–8.816, p=0.001, respectively). It is known that LEF-1 blocks APC mediated β catenin nuclear export and activates transcription of various transforming genes, including cyclin, D1, c-myc, MMP7, and LEF-1 itself. WNT5A or WNT10B proteins were not found to be up regulated in preB-ALL whereas APC and LEF-1 gene expressions were increased compared to normal hematopoietic cells (OR=32.97 CI 95% 0.27–1281, 38 p>0.001 and OR=5.57 CI 95% 0.28–89.51, p=0.01, respectively). We found increased TCF-1 expression (7.4 fold) without any β catenin accumulation in T-ALL patients. It is known that TCF-1 in absence of β catenin functions as a tumor suppressor gene. WNT5A, APC and LEF-1 gene expression levels were also different between T-cell, B-cell and preB cell ALL cases. WNT5A expression had the highest levels in B-ALL compared to T-ALL cases, whereas the highest APC expression levels were observed in preB and T-ALL patients. Also LEF-1 expression levels were significantly different between preB and T-cell ALL patients. Taken together these results indicate that WNT signaling genes have abnormal expression and are active in acute lymphoblastic leukemia. This data suggests different WNT activation mechanisms exist in the leukemic transformation in different hematopoietic cells.
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Stewart, A. Keith, Yuan Xiao Zhu, Maryan Yahyapour, Armen Manoukian, and Sam E. Scanga. "Inhibition of Wnt Pathway Signaling by Thalidomide and Revlimid: Studies in a Drosophila Model System." Blood 104, no. 11 (November 16, 2004): 3356. http://dx.doi.org/10.1182/blood.v104.11.3356.3356.
Abstract:
Abstract High throughput sequencing, gene expression profiling and protein biochemistry in myeloma have all consistently revealed elevated expression of wnt signaling pathways in malignant plasma cells. Indeed, downregulation of the Wnt pathway in myeloma cells has recently been shown to inhibit myeloma cellular proliferation. Preliminary pharmacogenomic studies have also suggested that hyperactivation of the wnt signaling antagonist DKK-1 is associated with response to the immunomodulators thalidomide and revlimid. The mechanism of action for these therapeutically active drugs is however by no means clear as multiple biologic consequences of treatment have been proposed. We report here use of a drosophila model to examine wnt signaling inhibition by these pharmaceuticals. We employed a unique drosophila larval imaginal disc culture system in which wnt pathway activity is monitored through control of LacZ expression by the distalless promoter. In this system 10uM of both thalidomide and revlimid reproducibly inhibit lacZ expression when compared with vehicle controls. Western blots of larva confirmed downregulation of expression of armadillo (the drosophila b-catenin homologue) by both drugs but particularly revlimid. Lithium Chloride is an inhibitor of the drosphila GSK3b homologue shaggy and thus mimics wnt signaling by stabilizing b-catenin. The effect of Lithium could not be overcome by thalidomide or revlimid indicating that the action of these drugs is upstream of shaggy (or GSK3). Next we employed a fly transgenic for wingless which is embryonic lethal. By adding either drug to larval culture medium the lethality of wingless expression was reversed. Indeed drosophila embryos fed thalidomide exhibited developmental plate abnormalities. We next sought evidence that similar effects were evident in revlimid treated human myeloma. As previously reported most myeloma cell lines studied expressed b-catenin and this protein was downregulated by revlimid treatment of human myeloma cell lines co-incident with inhibition of growth as measured by MTT assay. We sought, but failed to find evidence of up-regulation of the wnt signaling pathway antagonist DKK-1 using an ELISA assay on pre and post treatment serum samples in patients responding to thalidomide.The implications of wnt signaling inhibition as a primary or secondary readout of therapeutic efficiency in MM may be of substantial importance in subsequent design of drug therapies or combination therapies.
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Dandekar, Smita C., Eleny Romanos-Sirakis, Faye Pais, Teena Bhatla, Courtney L. Jones, Wallace Bourgeois, Stephen P. Hunger, et al. "Inhibition Of The Wnt Pathway Leads To Improved Chemosensitivity In Pediatric Acute Lymphoblastic Leukemia." Blood 122, no. 21 (November 15, 2013): 1428. http://dx.doi.org/10.1182/blood.v122.21.1428.1428.
Abstract:
Abstract Introduction While childhood acute lymphoblastic leukemia (ALL) is highly curable, up to 20% of children will relapse, with dismal prognosis, warranting the need for novel therapies. Previously, using an integrated genomic approach on matched diagnosis-relapse samples, we identified overactivation of the Wnt pathway as a mechanism of disease recurrence at relapse (Hogan et al, Blood 2011). Aberrant Wnt signaling has been linked to cancers of the liver, colon, breast, skin and more recently hematologic malignancies. To validate our findings and determine if Wnt inhibition could restore chemosensitivity in relapsed ALL, we sought to examine directly whether Wnt is activated at relapse in paired samples (examining expression of activated b-catenin and its downstream target Survivin (BIRC5) using multiparameter phosphoflow cytometry) and tested the efficacy of a recently developed small molecule Wnt inhibitor, iCRT14, that specifically interferes with the b-catenin-TCF interaction (Gonzalves et al, PNAS 2011), in ALL cell lines and patient samples. Methods B and T-ALL cell lines were treated with iCRT14 and the expression of target genes were determined by quantitative RT-PCR.10 paired diagnosis-relapse patient samples obtained from the Children’s Oncology Group were washed, fixed and stained simultaneously with caspase 3, CD10, activated b-catenin and survivin and the change in expression of activated b-catenin and survivin from diagnosis to relapse was measured by multiparameter phosphoflow cytometry in each patient by gating on the caspase 3 negative, CD10 positive leukemic blasts. To test the effect of Wnt inhibition on chemosensitivity, B-ALL cell lines were pretreated with iCRT14 for 48 hours prior to incubation with traditional chemotherapy for an additional 24 hours. The response to increasing doses of iCRT14 and chemo, alone and in combination, was assessed by cell viability (Cell Titer-Glo Luminescent Assay (Promega)) and apoptosis (FACS analysis with AnnexinV-PE/7AAD staining (BD Bioscience)). Protein levels of apoptotic markers were assessed. Also, 4 newly diagnosed and 4 relapsed patient samples were treated ex vivo with iCRT14 (20 and 30 uM) and prednisolone, alone and in combination. Drug combination results were analyzed using the Calcusyn program which calculates a Combination Index (CI): CI>1.1=antagonism, 0.9-1.1=additive and <0.9=synergy. Results Previously, we reported that treatment of ALL cell lines with iCRT14, downregulated the mRNA expression of the Wnt target genes BIRC5, axin 2, and c-myc (Romanos et al, ASPHO 2012 # 414). Comparison of Mean Fluorescent Intensity of activated b-catenin and survivin in the 10 pairs showed upregulation of activated b-catenin at relapse in 6 of 10 patients. Survivin expression was increased in 6 patients and in 4 patients the upregulation of activated b-catenin and survivin was concordant. iCRT14 pretreatment of cell lines followed by chemotherapy (prednisolone, etoposide, doxorubicin, cytarabine and 6TG) demonstrated additive to synergistic effects on viability. UOCB1 cells showed synergism with all 5 chemotherapy agents (CI=0.1-0.88). Nalm6 cells were very sensitive to iCRT, hence the combination with chemotherapy showed additive to synergistic effects (CI=0.05-1.I). In Reh cells, all agents besides cytarabine showed robust synergism (CI=0.03-0.55). FACS analysis revealed that iCRT14 alone contributed significantly to apoptosis and combination with chemotherapy further increased cell death with >80% apoptosis by hour 72 with the maximal chemotherapy dose in all cell lines. Change in the protein levels of cleaved PARP and cleaved caspase 3 was seen. The 4 diagnosis patients were very sensitive to prednisolone as expected, precluding synergism with iCRT14. The relapsed patient samples were much less sensitive to prednisolone alone (40% decrease in viability in relapsed patients vs 80% in new diagnoses). Interestingly, all the relapsed patients showed enhanced chemosensitivity with Wnt inhibition. 3 out of 4 relapsed patients showed strong synergism (CI=0.03-0.6) with both doses of iCRT14 and 1 patient showed additive to synergistic effects (CI=0.7 and 1). Conclusion Overactivation of the Wnt pathway may lead to chemoresistance in relapsed ALL. Wnt Inhibition restores chemosensitivity and induces apoptosis in ALL cell lines and primary patient samples making it a potential therapeutic approach. Disclosures: No relevant conflicts of interest to declare.
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Yin, Yuanqin, Fei Li, Songlin Li, Jingjing Cai, Jing Shi, and Youhong Jiang. "TLR4 Influences Hepatitis B Virus Related Hepatocellular Carcinoma by Regulating the Wnt/β-Catenin Pathway." Cellular Physiology and Biochemistry 42, no. 2 (2017): 469–79. http://dx.doi.org/10.1159/000477594.
Abstract:
Background/Aims: We investigated the correlation between toll-like receptor 4 (TLR4) and β-catenin for disclosing the potential pathogenesis of hepatocellular carcinoma (HCC). Methods: Immunohistochemical toolkit was implemented to measure the expression of TLR4 and β-catenin in 98 cases of HCC tissues and adjacent tissues. After setting up the HepG2.2.15 hepatitis B virus (HBV) related HCC cell line, we divided the cells into the control group, TLR4 siRNA group, β-catenin siRNA group, and pcDNA.3.1 TLR4 + β-catenin siRNA group. Western blot, CCK-8 method, Transwell and flow cytometry were used to detect protein expression, cell proliferation, cell migration and invasion as well as cell apoptosis, respectively. Nude mice tumor model was established to observe the effects of TLR4 and β-catenin on the progression of HBV-related HCC in vivo. Results:The positive rates of TLR4 and β-catenin were higher in HCC tissues compared with normal tissues. Both the TLR4 siRNA group and β-catenin siRNA group exhibited a decreased expression of β-catenin. The proliferation, migration and invasion of tumor cells in the above two groups were suppressed, while the cell apoptosis appeared to be stimulated. As suggested by the results from in vivo and in vitro experiments, the up-regulation of TLR4 could antagonize the corresponding effect of β-catenin siRNA. Conclusions: TLR4 can affect the expression of β-catenin and hence influence the progression of HBV-related HCC.
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Gonzalez, Maria, Ahmad Eido, GIUSEPPINA AUGIMERI, and Celina Kleer. "Abstract PO1-24-05: EZH2 histone methyltransferase activity promotes spindle metaplastic breast carcinoma metastasis and is induced by CCN6 knockout/b-catenin/TCF axis." Cancer Research 84, no. 9_Supplement (May 2, 2024): PO1–24–05—PO1–24–05. http://dx.doi.org/10.1158/1538-7445.sabcs23-po1-24-05.
Abstract:
Abstract Background: Metaplastic breast carcinomas (mBrCAs) are a highly aggressive subtype of triple negative breast cancer with histological evidence of deregulated differentiation towards non-glandular components. Previous studies have demonstrated that human mBrCA often exhibit activation of differentiation pathways, including canonical Wnt/b-catenin and EZH2-mediated transcriptional repression. Our lab has identified CCN6 as a tumor suppressor in mBrCA. MMTV-Cre;Ccn6fl/fl (CCN6KO) mice develop spindle mBrCAs and CCN6 is reduced/lost in 68% of human mBrCAs. Recently, we have demonstrated that the tumor suppressor function of CCN6 in spindle mBrCA requires activation of Wnt/b-catenin signaling pathway. Here, we tested the hypothesis that CCN6 KO leads to the upregulation of EZH2 histone methyltransferase promoting mBrCA. Furthermore, we investigated the requirement for the activation of the Wnt/b-catenin pathway in this mechanism. Methods: To test the effect of CCN6/b-catenin on EZH2 gene and protein expression we performed IHC, IF, qRT-PCR, ChIP-Seq, RNA-seq, invasion and adhesion assays, EZH2 reporter assay and immunoblots in mBrCA cell lines and MMTV-Cre;Ccn6fl/fl tumors. To investigate the role of CCN6KO-induced b-catenin activation on EZH2 activity and neoplastic functions we employed three independent approaches: i) Expression of a dominant-negative Tcf4 (dnTcf4) rescued with EZH2-WT, dSET and dNLS domain mutants versus vector in MMTV-Cre;Ccn6fl/fl tumor-derived cells; ii) Expression of a constitutively active mutant (S33Y) b-catenin in concert with treatment with recombinant human CCN6 (rhCCN6; 500 ug/ml) versus control; iii) Syngeneic orthotopic mammary tumor transplants of MMTV-Cre;Ccn6fl/fl were used for in vivo rescue experiments with rhCCN6 or BSA. To assess the therapeutic benefit of inhibiting EZH2 methyltransferase activity, MMTV-Cre;Ccn6fl/fl tumor cells (CCN6KO cells) were implanted orthotopically or intracardially in FVB mice, followed by treatment with EPZ-6438 (a selective EZH2 methyltransferase inhibitor) or vehicle. We tested CCN6, b-catenin, and EZH2 expression by IHC in a cohort of 27 human mBrCA tumor samples. Results: CCN6KO-induced b-catenin/TCF activation mediates EZH2 transcriptional upregulation and the deposition of repressive H3K27me3 in spindle mBrCAs. We found that the invasion program triggered by CCN6KO-induced Wnt/b-catenin requires EZH2 catalytic activity as WT-EZH2 (but not dSET-EZH2) rescued the reduced invasion of CCN6KO-dnTcf4 cells. RNA-seq and 3H3K27 ChIP-seq of CCN6KO-dnTcf4 cells transduced with EZH2-WT or dSET-EZH2 identify specific CCN6KO-b-catenin/Tcf targets that require EZH2 transcriptional repressor function. In vivo, administration of CCN6 protein to MMTV-Cre;Ccn6fl/fl tumor transplants reduces tumor growth and nuclear b-catenin, EZH2 and 3H3K27 in the tumors. Pharmacologic inhibition of EZH2 reduces the growth and metastasis of CCN6KO mBrCA tumors and improves survival. We identify a subset of human spindle mBrCA (54%) that display a CCN6Low/nuclear b-cat/EZH2High phenotype. Conclusion: We found a critical role for EZH2 activation in CCN6-deficient mBrCA tumor phenotypes via b-catenin/TCF Wnt canonical signaling. We demonstrate the effectiveness of pharmacological inhibition of EZH2 methyltransferase activity in reducing primary tumor growth and distant metastasis in mouse models of spindle mBrCA. In clinical samples, low CCN6 is significantly associated with activated b-catenin and high EZH2 in spindle mBrCAs compared to other subtypes. These data reveal a novel tumor suppressor mechanism of CCN6 and provide compelling evidence supporting the potential therapeutic value of CCN6 restoration, b-catenin or EZH2 inhibition as promising approaches for the treatment of spindle mBrCAs. Citation Format: Maria Gonzalez, Ahmad Eido, GIUSEPPINA AUGIMERI, Celina Kleer. EZH2 histone methyltransferase activity promotes spindle metaplastic breast carcinoma metastasis and is induced by CCN6 knockout/b-catenin/TCF axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-05.
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Bouaziz, W., T. Funk-Brentano, H. Lin, E. Hay, and M. Cohen-Solal. "Inhibition of Wnt/B-catenin pathway by sclerostin promotes cartilage maintenance." Osteoarthritis and Cartilage 20 (April 2012): S43—S44. http://dx.doi.org/10.1016/j.joca.2012.02.583.
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Liu, Shan, Zhuo-Hui Luo, Gui-Mei Ji, Wei Guo, Jia-Zhong Cai, Lin-Chun Fu, Juan Zhou, Ying-Jie Hu, and Xiao-Ling Shen. "Cajanolactone A from Cajanus cajan Promoted Osteoblast Differentiation in Human Bone Marrow Mesenchymal Stem Cells via Stimulating Wnt/LRP5/β-Catenin Signaling." Molecules 24, no. 2 (January 12, 2019): 271. http://dx.doi.org/10.3390/molecules24020271.
Abstract:
Cajanolactone A (CLA) is a stilbenoid discovered by us from Cajanus cajan (L.) Millsp. In our study, CLA was found to promote osteoblast differentiation in human bone marrow mesenchymal stem cells (hBMSCs), as judged by increased cellular alkaline phosphatase activity and extracellular calcium deposits, and elevated protein expression of Runx2, collagen-1, bone morphogenetic protein-2, and osteopontin. Mechanistic studies revealed that hBMSCs undergoing osteoblast differentiation expressed upregulated mRNA levels of Wnt3a, Wnt10b, LRP5/6, Frizzled 4, β-catenin, Runx2, and Osterix from the early stage of differentiation, indicating the role of activated Wnt/β-catenin signaling pathway in osteoblast differentiation. Addition of CLA to the differentiation medium further increased the mRNA level of Wnt3a, Wnt10b, Frizzled 4, LRP5, and β-catenin, inferring that CLA worked by stimulating Wnt/LRP5/β-catenin signaling. Wnt inhibitor dickkopf-1 antagonized CLA-promoted osteoblastogenesis, indicating that CLA did not target the downstream of canonical Wnt signaling pathway. Treatment with CLA caused no changes in mRNA expression level, as well as protein secretion of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL), indicating that CLA did not affect the OPG/RANKL axis. Our results showed that CLA, which promoted osteoblast differentiation in hBMSCs, through activating Wnt/LRP5/β-catenin signaling transduction, is a promising anti-osteoporotic drug candidate.
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Lu, Hai-Yan, Tian-Shu Peng, Xiang-Dang Hu, Shuai-Jun Li, Min Luo, Yong-Heng He, and Tian Nie. "Quercetin potentiates the effect of γδ T cells via modulating the expressions of Granzyme B, perforin and IFN-γ and also regulates the Wnt/β-catenin signalling pathway in human colon cancer cells." Bangladesh Journal of Pharmacology 10, no. 2 (April 1, 2015): 251. http://dx.doi.org/10.3329/bjp.v10i2.20387.
Abstract:
<p>Cancer accounts as one of the leading causes of morbidity and mortality. Recent studies focus on the efficiency of phytochemicals in cancer therapy. Influence of quercetin, a flavonoid on the effect of γδ T cells and Wnt/β-catenin signalling pathway in human colon cancer cells (HT55 and HCT116) was investigated. Quercetin at 15-120 µM was observed to markedly reduce the viability of HT55 and HCT116 cells. Quercetin exposure significantly increased γδ T cell proliferation and also raised the expressions of granzyme B (Gra B), perforin (PFP), and interferon- γ (IFN-γ) in γδ T cells. Reduced β-catenin expression with increased expressions of phosphorylated- β-catenin, axin1 and 2 were observed in HT55 and HCT116 cells on exposure to quercetin. However β-actin expression was found to be not much altered. The results suggest that quercetin was able to efficiently potentiate the effect of γδ T cells and modulate Wnt/β-catenin signalling pathway.</p><p> </p>
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Eaton, Charlotte, S. John Liu, Calixto-Hope Lucas, Tim Casey-Clyde, Abrar Choudhury, Vikas Daggubati, Harish Vasudevan, Danielle Swaney, and David Raleigh. "CSIG-37. MERLIN S13 DEPHOSPHORYLATION DRIVES MENINGIOMA WNT SIGNALLING AND CELL PROLIFERATION." Neuro-Oncology 24, Supplement_7 (November 1, 2022): vii47. http://dx.doi.org/10.1093/neuonc/noac209.186.
Abstract:
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.
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Peters, Anthea C., Randy Chung, Charles S. Wong, Leah C. Young, Tony Reiman, and Raymond Lai. "Epigenetic Regulation of the WNT Canonical Pathway in Mantle Cell Lymphoma." Blood 112, no. 11 (November 16, 2008): 3340. http://dx.doi.org/10.1182/blood.v112.11.3340.3340.
Abstract:
Abstract Dysregulation of the WNT signalling pathway is an important event in the pathogenesis of several types of cancer, including colon cancer and acute leukemia. When a WNT protein binds to its receptor, glycogen synthase kinase (GSK)-3β is inactivated by phosphorylation (i.e. pGSK-3β) allowing β-catenin to accumulate in the nucleus and promote transcription of proliferative genes. Conversely, a protein complex that includes GSK-3β initiates destruction of β-catenin if no WNT protein binds its receptor or if an antagonist interferes with WNT ligand-receptor interaction. We previously demonstrated that the WNT canonical pathway is often constitutively active in mantle cell lymphoma (MCL), a subtype of B-cell non-Hodgkin lymphoma. We showed that GSK-3β is phosphorylated, thus inactive, in two-thirds of MCL cases, and that a high level of pGSK-3β correlates with both β-catenin expression and poor patient survival. In order to elucidate the mechanisms of constitutive WNT signalling in MCL, we investigated the RNA expression of six WNT pathway antagonists including WIF-1 using fresh tumour samples and three types of cultured MCL cells. In vivo WIF-1 expression was abundant only in tumours shown to lack pGSK-3β expression. We also examined the regulation of these WNT antagonists by promoter methylation by treating cultured cells with demethylating agent 5-aza-2’-deoxycytidine. The WNT pathway inhibitors were often negatively regulated in MCL-derived cell lines by epigenetic DNA methylation; inhibitors that were not expressed at baseline showed expression after demethylation treatment in at least one MCL cell line. Methylation-specific PCR confirmed that the promoter region of WIF-1 was methylated in almost half of MCL tumour samples. Our data demonstrates that in MCL several antagonists of the canonical WNT pathway are negatively regulated by promoter methylation, thereby allowing aberrant WNT pathway activation. This data confirms that WNT signalling is important in MCL pathogenesis, and identifies potential therapeutic targets for MCL.
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Dao, Kim-Hien T., Michael D. Rotelli, Curtis L. Petersen, Brie R. Brown, Whitney D. Nelson, Jane E. Yates, Amy Hanlon Newell, Susan B. Olson, Brian J. Druker, and Grover C. Bagby. "FANCL Ubiquitinates Beta-Catenin and Enhances Its Nuclear Function." Blood 118, no. 21 (November 18, 2011): 1335. http://dx.doi.org/10.1182/blood.v118.21.1335.1335.
Abstract:
Abstract Abstract 1335 Fanconi anemia (FA) is associated with a hereditary predisposition to bone marrow failure. The proteins encoded by the FANC genes are primarily involved in DNA repair responses through the formation of a large, multisubunit complex that has E3 ubiquitin ligase activity (Annual Review of Genetics 2009;43:223). FA hematopoietic stem cells display defective stem cell properties and limited replicative potential. However, the molecular basis for how a FA genetic background contributes to those defects remains poorly understood. Here we provide evidence that FANCL, which has E3 ubiquitin ligase activity, enhances beta-catenin activity (Figure A) and protein expression. Beta-catenin is a nuclear effector of canonical Wnt signaling. The Wnt/beta-catenin pathway is active in normal hematopoietic stem cells in the native bone marrow environment and disruption of this signaling pathway results in defective hematopoietic stem cells (Nature 2003;423:409). To test whether FANCL positively regulates beta-catenin through its ubiquitination activity, we performed cell-based ubiquitination assays. We show that FANCL functionally ubiquitinates beta-catenin (Figure B) and that ubiquitin chain extension can occur via non-lysine-48 ubiquitin linkages. Accumulating evidence reveal diverse, non-proteolytic biological roles for proteins modified by atypical ubiquitin chains (EMBO Reports 2008;9:536). Our data suggests that FANCL may enhance the protein function of beta-catenin via ubiquitination with atypical ubiquitin chains. Importantly, we demonstrate that suppression of FANCL expression in human CD34+ cord blood stem cells reduces beta-catenin expression (Figure C) and multilineage progenitor expansion. These results demonstrate a role for the FA pathway in regulating Wnt/beta-catenin signaling. Therefore, diminished Wnt/beta-catenin signaling may be an important underlying molecular defect in FA hematopoietic stem cells leading to their accelerated loss. A, LEF-TCF-luciferase reporter assay showing increasing beta-catenin activity in 293FT cells with increasing FANCL expression compared with vector-control (VC) (n=4). B, Immunoprecipitation of beta-catenin in cells transfected with vector-control or FANCL and probed for hemagglutinin (HA)-tagged ubiquitin shows increased ubiquitinated forms of beta-catenin with FANCL expression (n=4). C, shRNA suppression of FANCL expression in CD34+ cord blood stem cells results in decreased beta-catenin expression compared with a scramble control (Scr) by immunofluorescence analysis (three different shRNA constructs, n=3 for each construct). Disclosures: No relevant conflicts of interest to declare.
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Filipovich, Alexandra, Rajesh Kumar Gandhirajan, Iris Gehrke, Julian Paesler, Simon Jonas Poll-Wolbeck, Sabrina Uhrmacher, Magdalena Hertweck, et al. "Lack of WNT Pathway Co-Receptors as a Possible Reason for Chronic Lymphoctic Leukemia (CLL) Cells Resistance to Dickkopf-1 (DKK1)." Blood 114, no. 22 (November 20, 2009): 1598. http://dx.doi.org/10.1182/blood.v114.22.1598.1598.
Abstract:
Abstract Abstract 1598 Poster Board I-624 WNT signaling is known to regulate cell survival. Components of the WNT signaling cascade were previously shown to be upregulated in CLL cells in comparison to healthy B cells. Accordingly, this pathway is thought to be responsible for the enhanced survival of CLL cells. The canonical WNT pathway is initiated by proteins of the WNT ligand family, which bind to a receptor complex composed of the Frizzled proteins (Fz), and low-density lipoprotein (LDL) receptor-related proteins (LRP5/6). Dickkopf (DKK1) is known to antagonize WNT/beta-catenin signaling by direct high-affinity binding to the WNT coreceptor LRP5/6, thereby inhibiting interaction of LRP5/6 with the WNT-Frizzled complex. DKK1 coreceptor Kremen2 (KRM2) is speculated to play an important role in modulating DKK1 dependent antagonisation of WNT/beta-catenin signaling. Inactivation of WNT pathway by DKK1 could lead to an increased apoptosis in CLL cells. The purpose of this study was to investigate the effect of DKK1 in CLL cells in vitro. B-cells from CLL patients and JVM-3 cells were incubated in presence or absence of DKK1 (0.3 μg/ml) for 24 and 48 hours. Survival was measured by flow cytometry and caspase-3, -7 activation. To prove dose dependency, CLL cells were incubated with different doses of DKK1. Furthermore, the expression status of intracellular WNT signaling components was analysed by immunoblotting. The expression of receptors LRP6 and KRM2 was determined using real time PCR. Furthermore, we measured the expression of DKK1 in healthy and CLL B-cells. The flow cytometry analysis showed that incubating primary B-cells with DKK1 increased overall mean survival after 24 hours (vehicle control 81.3 ± 11.7 % versus DKK11 91.1 ± 5.3%) and after 48 hours (vehicle control 65.8 ± 13.5% versus DKK1 76.2 ± 8.2%). Accordingly, the activity of caspases-3 and -7 in these cultures was significantly reduced after DKK1 stimulation. The level of phosphorylated LRP6 decreased after incubating cells with DKK1 for 24 h in a dose-dependent fashion. β-catenin and c-myc expression was increased compared to untreated samples. Expression of LRP6 and KRM2, acquired by means of real-time PCR was lower in CLL cells, than in healthy B-cells. Similar, the basal amount of DKK1 was higher in CLL cells. Summing up, unlike other WNT active cancers such as multiple myeloma, addition of DKK1 to culture of CLL cells does not lead to inactivation of the WNT pathway. In contrast, in CLL the addition of DKK1 even increases CLL cell survival in vitro. This could be explained by a lack of WNT pathway-coreceptors LRP6 and KRM2 on the membrane of CLL cells. The unexpected increase of WNT pathway proteins might be caused by unspecific binding to other receptors of the WNT pathway than to LRP6. High basal expression of DKK1 in primary CLL cells may be referred to the fact, that DKK1 is itself a target gene of Lef-1, which is known to be highly upregulated in CLL cells. Further investigations on this controversy in WNT-signalling in CLL cells are needed. Disclosures Hallek: BayerScheringAG: Honoraria, Research Funding.
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Chung, Jihyun, Vrajesh Karkhanis, Sif Said, and Robert A. Baiocchi. "Protein Arginine Methyltransferase 5 Regulates WNT/β-Catenin Target Gene Expression in at Multiple Levels." Blood 128, no. 22 (December 2, 2016): 4106. http://dx.doi.org/10.1182/blood.v128.22.4106.4106.
Abstract:
Abstract Introduction: It is well established that altered expression of oncogenes and epigenetic dysregulation of tumor suppressor and regulatory genes promotes lymphomagenesis. Over-expression of the type II protein arginine methyltransferase (PRMT5) enzyme has been associated with increased cell proliferation and survival in both solid and blood cancers. We have reported that PRMT5 is essential for EBV-driven B cell transformation and that it regulates the EZH2, EED and SUZ12, components of the Polycomb-repressive complex 2 (PRC2) complex via transcriptional silencing of RBL2and hyper-phosphorylation of RB1 in aggressive lymphomas. Here we report a mechanistic assessment of how PRMT5 over-expression supports the WNT/β-CATENIN pathway at multiple levels and drives oncogenic β-CATENIN target genes in lymphoma. Methods: PRMT5 inhibition of patient-derived lymphoma cell lines, primary lymphoma tumor cells and mouse primary Eμ-BRD2 transgenic lymphoma cells was achieved by infecting with sh-PRMT5 lentivirus (or sh-GFP control), a selective small molecule PRMT5 inhibitor (Alinari et al, Blood 2015, CMP5) or CRISPR/CAS9 PRMT5 deletion. Gene expression was monitored by immunoblotting and reverse transcription (RT) real time PCR. Recruitment of target proteins to promoter regions was examined by ChIP-PCR assays. To evaluate PRMT5 and WNT antagonist expression in NHL patient samples, primary tumor samples were collected from 4 patients with MCL. Cellular growth and apoptosis was assessed by MTS proliferation assay and FACS analysis. WIF1 protein detection in cell culture media was performed by ELISA. Results: PRMT5 regulated WNT/β-CATENIN signaling by direct transcriptional repression of AXIN2 and WIF1, two proteins that negatively regulate this pathway. PRMT5 inhibition with shRNA, CRISPR/CAS9 deletion, or CMP5 led to restored expression of AXIN2 and WIF1 transcript and protein that was associated with transcriptional repression of WNT/β-CATENIN target genes CYCLIN D1, MYC, and SURVIVIN. With PRMT5 inhibition, we observed differential enhanced recruitment of co-repressors LSD and HDAC2 and loss of associated epigenetic marks H3K4Me3 and H3K9Me (associated with the LSD demethylase) and H3K9Ac and H3K14Ac (associated with HDAC2) at each b-CATENIN target promoter. PRMT5 inhibition was found to reduce recruitment of co-activators CBP and MLL1 and respective epigenetic mark H3K4me3. The de-repression of AXIN2 and WIF1 was associated with loss of phospho-AKT (S450, S473) and down-stream survival pathways. Reduction in phospho-AKT was attributed to physical association between AXIN2 and the down-stream activity of secreted WIF1 in media of lymphoma cells treated with PRMT5 inhibition or CRISPR/CAS9 PRMT5 deletion. Furthermore, reduction of phospho-AKT prevented dimerization of MLL1 leading to dissociation of the BCL9-Pygopus TCF1/b-CATENIN transcriptional activation complex at MYC, CYCLIND1, and SURVIVIN promoters. Our observations show that PRMT5 is an important epigenetic regulator that governs the expression WNT/β-CATENIN-driven oncogenes c-MYC, CYCLIND D1 and SURVIVIN. PRMT5 inhibition restores regulation at several levels that converge on AKT signaling; (i) AXIN2-AKT interaction and (ii) WIF1 inhibition of WNT signaling. The restored regulation occurs via modulation of the downstream transcriptional machinery that is supported by constitutive AKT activity. This data identifies a novel pathway to interfere with WNT/β-CATENIN signaling and validates the driver role orchestrated by PRMT5 in lymphoma. Disclosures Baiocchi: Essanex: Research Funding.
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Chiarini, Francesca, Francesca Paganelli, Alberto M. Martelli, and Camilla Evangelisti. "The Role Played by Wnt/β-Catenin Signaling Pathway in Acute Lymphoblastic Leukemia." International Journal of Molecular Sciences 21, no. 3 (February 7, 2020): 1098. http://dx.doi.org/10.3390/ijms21031098.
Abstract:
Acute lymphoblastic leukemia (ALL) is an aggressive hematologic neoplastic disorder that arises from the clonal expansion of transformed T-cell or B-cell precursors. Thanks to progress in chemotherapy protocols, ALL outcome has significantly improved. However, drug-resistance remains an unresolved issue in the treatment of ALL and toxic effects limit dose escalation of current chemotherapeutics. Therefore, the identification of novel targeted therapies to support conventional chemotherapy is required. The Wnt/β-catenin pathway is a conserved signaling axis involved in several physiological processes such as development, differentiation, and adult tissue homeostasis. As a result, deregulation of this cascade is closely related to initiation and progression of various types of cancers, including hematological malignancies. In particular, deregulation of this signaling network is involved in the transformation of healthy HSCs in leukemic stem cells (LSCs), as well as cancer cell multi-drug-resistance. This review highlights the recent findings on the role of Wnt/β-catenin in hematopoietic malignancies and provides information on the current status of Wnt/β-catenin inhibitors with respect to their therapeutic potential in the treatment of ALL.
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Koch, Ute, Anne Wilson, Monica Cobas, Rolf Kemler, H. Robson MacDonald, and Freddy Radtke. "Simultaneous loss of β- and γ-catenin does not perturb hematopoiesis or lymphopoiesis." Blood 111, no. 1 (January 1, 2008): 160–64. http://dx.doi.org/10.1182/blood-2007-07-099754.
Abstract:
Hematopietic stem cells (HSCs) maintain life-long hematopoiesis in the bone marrow via their ability to self-renew and to differentiate into all blood lineages. Although a central role for the canonical wnt signaling pathway has been suggested in HSC self-renewal as well as in the development of B and T cells, conditional deletion of β-catenin (which is considered to be essential for Wnt signaling) has no effect on hematopoiesis or lymphopoiesis. Here, we address whether this discrepancy can be explained by a redundant and compensatory function of γ-catenin, a close homolog of β-catenin. Unexpectedly, we find that combined deficiency of β- and γ-catenin in hematopoietic progenitors does not impair their ability to self-renew and to reconstitute all myeloid, erythroid, and lymphoid lineages, even in competitive mixed chimeras and serial transplantations. These results exclude an essential role for canonical Wnt signaling (as mediated by β- and/or γ-catenin) during hematopoiesis and lymphopoiesis.
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Kamran, Hamza, Ariz Akhter, Hassan Rizwan, Meer-Taher Shahbani-Rad, Ghaleb Elyamany, Douglas A. Stewart, and Adnan Mansoor. "Plasmablastic Lymphoma: Synergism betweenWnt/B-Cateninand theRAS Signalling Molecules May Identify Potential Targets for Therapy." Blood 136, Supplement 1 (November 5, 2020): 36. http://dx.doi.org/10.1182/blood-2020-140651.
Abstract:
Background:Plasmablastic lymphoma (PBL), is a rare aggressive B-cell lymphoma that shares many overlapping characteristics with activated B-cell type diffuse large B-cell lymphoma (ABC-DLBCL) and multiple myeloma (MM). High expression ofWnt/β-cateninpathway molecules has been linked with several aspects of tumour biology in ABC-DLBCL and MM. In MMWnt/β-cateninplay critical role in chemoresistance, while high FOXP1 in ABC-DLBCL exert poor prognosis through up-regulation of theWnt/β-cateninsignalling pathway. There is strong evidence that enhanced crosstalk between the Wnt/β-catenin andRASpathways impact tumorigenesis and metastasis of cancerous stem cells in various cancers. In breast cancer; targeting theWnt/β-cateninand RAS pathways with small molecular inhibitors have shown effective results. The role of the Wnt/β-catenin signalling pathway and its corresponding linkage toRASsignalling molecules remained unknown in PBL. This pilot study provides preliminary data in relation to expressionof Wnt/β-cateninandRASpathways molecules in PBL in contrast to ABC-DLBCL. Method:FFPE RNA from diagnostic tissue samples in PBL patients (n=31) were compared with ABC-DLBCLs (n=18) patients for keyWnt/β-cateninandRASpathway molecules expression, utilizing nCounter (NanoString Technologies) platform. Qlucore Omics Explorer software was employed with defined criteria (fold change >2.0; p<0.01 and q <0.05) for statistical analysis. Gene Set Enrichment Analysis (GSEA) from 5 publicly available gene data sets was used to analyze the expression of other accompanying pathways. Result:We identified significant differential expression of mRNA related toWnt/β-cateninsignalling between ABC-DLBCL and PBL (Figure 1). Expression ofWnt/β-cateninsignalling inhibitors (CXXC4, SFRP2, and DKK1) were significantly higher among PBL compared to ABC-DLBCL (8.12-3.22 log fold difference). In divergence, molecules linked withWnt/β-cateninsignalling activation were elevated in PBL when compared to ABC-DLBCL (FZD3andWNT10B). The GESA analysis proved that the RAS pathway was significantly up-regulated in PBL patients compared to ABC-DLBCL. In particular, the expression of crucial RAS pathway genes such asNRAS, RAF1, SHC1, andSOS1was significantly up-regulated in PBL patients when compared to ABC-DLBCL patients (Figure 2). Conclusion:Our data suggest that the expression ofWnt/B-catenintarget genes and ligands are enhanced in PBL patients along with the up-regulation of theRASsignalling pathway molecules as compared to ABC-DLBCL. The heightened expression of crucialWnt/B-catenininhibitors does not down-regulate the Wnt/β-catenin signalling. We anticipate that the combined down-regulation of theWnt/ β-cateninandRASpathways by targeting its key members (RAF1, NRAS, FZD3) may serve to contain tumor progression in PBL, hence impacting prognosis. Disclosures Stewart: Gilead:Honoraria;Sandoz:Honoraria;Teva:Honoraria;Amgen:Honoraria;Celgene:Honoraria;Abbvie:Honoraria;Roche:Honoraria;Janssen:Honoraria;Novartis:Honoraria;AstraZeneca:Honoraria.
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Shooshtarizadeh, Peiman, Ryan Chen, and Tarik Moroy. "The Zinc Finger Transcription Factor Growth Factor Independence 1b (Gfi1b) Regulates The Wnt/Beta-Catenin Signaling Pathway In Hematopoietic Stem Cells Through Interaction With Inhibitory Proteins." Blood 122, no. 21 (November 15, 2013): 2417. http://dx.doi.org/10.1182/blood.v122.21.2417.2417.
Abstract:
Abstract Hematopoietic stem cells (HSCs) reside in the bone marrow in specific niches at the border between bone cells and the bone marrow (endosteal niche) or around blood vessels (perivascular niche). In the endosteal niche, HSCs are maintained at low oxygen levels in a quiescent (dormant) state by adhesion to niche cells. We have previously shown that Gfi1b restricts the expansion and proliferation of HSCs as well as their mobilization or re-localization into peripheral blood. We have proposed that Gfi1b exerts this function by regulating the expression of surface molecules such as integrins on HSCs that are required to maintain them in their bone marrow niche at a quiescent state. The objective of this study was to gain more insight into the precise molecular mechanisms by which Gfi1b regulates HSCs dormancy and mobilization and to obtain insights that may be exploited in the future to improve stem cell therapies or the expansion of human hematopoietic stem cells for clinical use. Immune precipitation and mass spectrometry identified a series of Gfi1b interacting proteins, most notably a group of regulators of the canonical Wnt/beta-catenin pathway. Independent protein IP validation of these findings suggested that Gfi1b can interact with several inhibitors of the canonical Wnt/beta catenin pathway namely with APC (Adenomatous polyposis coli) a tumor suppressor protein and important factor in the beta-catenin destruction complex, with the DNA helicase and chromatin remodeling factor CHD8, which silences beta catenin mediated transcription, with CtBP which antagonizes beta-catenin activity and is part of the LSD1/CoRest histone demethylase complex and with the direct beta-catenin inhibitors TLE1 and TLE3 (also called Groucho). Of particular interest was that the interactions between the Groucho proteins and Gfi1b were dependent on a previously unidentified Groucho binding domain (GBD) in Gfi1b. This is a well-conserved six-amino acid stretch that is found in the middle part of the Gfi1b protein. In addition, the binding of CtBP was dependent on the presence of the 20 amino acid N-terminal SNAG domain in Gfi1b that also mediates LSD1 binding. Using luciferase reporter gene assays (TOP/FOP reporter assay), we found that Gfi1b was able to significantly up-regulates TCF/beta-catenin-dependent transcription upon activation by LiCl or Wnt3A in HEK293 cells. This activity of Gfi1b was dependent on both the presence of the SNAG domain and the newly identified Groucho binding domain. Also, Gfi1b was able to reverse partially the inhibitory effect of CtBP and TLE3 on beta-catenin activity in the TOP/FOP reporter assays. To obtain further evidence that Gfi1b is indeed implicated in regulating the Wnt/beta catenin signaling pathway in hematopoietic stem cells, we FACS sorted Lin-Kit1+Sca+ hematopoietic progenitors (LSK cells) from wt and Gfi1b deficient mice and tested them for expression of Wnt effector genes using a Wnt signaling specific PCR array. We observed that the majority of Wnt target genes were significantly down regulated in Gfi1 deficient LSKs compared to wt LSKs. Among the genes affected the most were typical Wnt targets such as Axin2, Frz7, Tcf4, Klf5, Vegfa and Ccnd1. To show that Gfi1b is able to regulate Wnt pathway effectors in vivo in HSCs, we crossed Gfi1b flox/flox, Mx-Cre mice with animals that carry a NLS-lacZ reporter gene under the control of the endogenous Axin2 promoter/enhancer region. Treatment with pIpC, which deletes Gfi1b correlated with a significant decrease of Axin2 expression in HSCs and MPP1, which are high Gfi1b expressing cells. The Axin2 reporter was not affected by Gfi1b deletion in MPP2 or GMPs, which express low levels or no Gfi1b. The canonical Wnt/b-catenin signaling pathway is recognized as one of the elements that are critically important in the regulation of HSC function. Here we have identified Gfi1b as a potential new player in the Wnt-beta catenin signaling pathway. Our data suggest that Gfi1b acts on at least two inhibitory complexes of this pathway, on the TLE family of Groucho proteins and the CtBP/LSD1 complex and regulates effectors of the Wnt/beta-catenin signaling cascade. We propose therefore that Gfi1b may titer the level of activation of the Wnt/beta-catenin signaling pathway in HSCs, which offers an explanation of the hematopoietic stem cell phenotype seen in mice lacking Gfi1b. Disclosures: No relevant conflicts of interest to declare.
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Huang, Jian, and Peter S. Klein. "Maintenance of Hematopoietic Stem Cells Through Regulation of Wnt and mTOR Pathways." Blood 120, no. 21 (November 16, 2012): 2309. http://dx.doi.org/10.1182/blood.v120.21.2309.2309.
Abstract:
Abstract Abstract 2309 Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to differentiate into all lineages of the blood. The signaling pathways regulating hematopoietic stem cell (HSCs) self-renewal and differentiation are not well understood. We are very interested in understanding the roles of glycogen synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in HSCs. In our previous study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a b-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disruption of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated by b-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal such that inhibition of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. In the current study, we found that suppression of the mammalian target of rapamycin (mTOR) pathway, an established nutrient sensor, combined with activation of canonical Wnt/ß-catenin signaling, allows the ex vivo maintenance of human and mouse long-term HSCs under cytokine-free conditions. We also show that combining two clinically approved medications that activate Wnt/ß-catenin signaling and inhibit mTOR increases the number of long-term HSCs in vivo. Disclosures: No relevant conflicts of interest to declare.
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Abed, Shurooq Hussamaldeen, and Yossra Saleh Khudhur. "Decreased β-catenin Expression in First Trimester Villi and Decidua of Patients with Recurrent Spontaneous Miscarriage." Medical Journal of Tikrit University 29, no. 2 (December 31, 2023): 53–60. http://dx.doi.org/10.25130/mjotu.29.1.8.
Abstract:
Accumulating evidence suggests that Wnt/β-catenin pathways play an important role in the physiological processes of endometrial development and differentiation, preimplantation embryo development, and blastocyst implantation. The objective of the current study was to signify the association between Wnt/β-catenin signalling pathway and recurrent spontaneous miscarriage and examine the expression of β-catenin first-trimester villi and decidua in women with spontaneous miscarriage. A comparative cross-sectional study was conducted in Saladin General Hospital from the 1st of January to the 1st of October 2021. A convenient sample of 80 pregnant women was enrolled in the current study and consisted of group A (40 pregnant women who were presented with first or second miscarriage) and group B (40 pregnant women who were presented with recurrent spontaneous miscarriage). There was a significant association between decreased β-catenin and recurrent miscarriage as 77.5% of the women who presented with recurrent spontaneous miscarriage had decreased levels of β-catenin, while only 32.5% of those with first or second miscarriage had decreased levels of β-catenin. The results of the current study implied that β-catenin is a valuable biomarker for unexplained recurrent spontaneous miscarriage. The women who presented with recurrent spontaneous miscarriage had a decreased expression of β-catenin .
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Coelho, Barbara Paranhos, Mariana B. Prado, Frederico M. Ferreira, Tiago G. dos Santos, Helder I. Nakaya, and Marilene H. Lopes. "Abstract 325: The cellular prion protein is involved in the modulation of Wnt signaling in glioblastoma." Cancer Research 83, no. 7_Supplement (April 4, 2023): 325. http://dx.doi.org/10.1158/1538-7445.am2023-325.
Abstract:
Abstract Glioblastoma (GBM) is an aggressive, invasive and therapy resistant brain tumor. Our research group has been unraveling the role of the cellular prion protein (PrPC) on GBM biology. PrPC is a scaffold protein that interacts with a plethora of partners, and it is important for proliferation and stemness maintenance of glioblastoma stem-like cells (GSCs), that are responsible for the aggressiveness and resistance to therapy of GBM. GSCs have aberrant activation of several signaling pathways that control self-renewal and differentiation, such as the Notch, Hedgehog (Hh) and Wnt pathways. Additionally, PrPC was proposed as a modulator of the Wnt signaling pathway on proliferating intestinal epithelial cells due to its binding to beta-catenin and upregulation of the transcriptional activity of the beta-catenin/TCF7L2 complex. Therefore, we aim to further explore this connection between Wnt signaling and PrPC in GBM. We generated U87 and U251 GBM PrPC knockout cells (PrPC-KO) by CRISPR-Cas9. RNA sequencing analysis comparing PrPC-KO GBM cells with wild-type controls resulted in 28 differentially expressed genes (DEGs) for U87 cells and 10 DEGs for U251 cells that are components of pathways related to Wnt signaling. We chose to analyze the expression of the upregulated DEGs CAMK2A, NFATC4, WISP1, PPP2R2B, SFRP2; and the downregulated DEGs RAC3 and SHH by qPCR. Our results indicate that the Wnt-Ca2+ pathway may be activated in U87 PrPC -KO cells, due to increased expression of NFATC4 and CAMK2A. Regarding the canonical Wnt signaling, it appears to be a balance in the expression of genes that activate or inhibit these pathways in U87 PrPC-KO cells, since WISP1 and PPP2R2B were upregulated and SFRP2 was downregulated. When analyzing the expression of the same genes in U251 PrPC-KO cells, however, we observed a different expression profile than what was found for U87 cells. Apart from RAC3, the expression of all genes analyzed were downregulated. Importantly, expression of SHH was downregulated in both U87 and U251 PrPC-KO cells. When we analyzed protein levels of Wnt signaling components, we found a decrease of beta-catenin and Wnt5a/b in U87 PrPC-KO, demonstrating a downregulation of the canonical signaling. Moreover, we found by immunofluorescence that beta-catenin distribution on the cellular membrane was altered in U87 PrPC-KO cells. Our results demonstrate, therefore, a potential involvement of PrPC in modulating Wnt signaling in GBM. As a scaffolding protein, PrPC may be contributing to GBM biology by increasing the effectiveness of Wnt signaling, by localizing beta-catenin on important regions of the tumor cell membrane, or by insulating beta-catenin from degradation. Altogether, PrPC may be a promising target for the development of novel therapy strategies against GBM. Supported by FAPESP (2019/14952-0) Citation Format: Barbara Paranhos Coelho, Mariana B. Prado, Frederico M. Ferreira, Tiago G. dos Santos, Helder I. Nakaya, Marilene H. Lopes. The cellular prion protein is involved in the modulation of Wnt signaling in glioblastoma [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 325.
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Isaacs, James, Andrew B. Nixon, Emily Bolch, Katie Quinn, Kimberly Banks, Brent Allen Hanks, and John H. Strickler. "Blood-based genomic profiling of cell-free DNA (cfDNA) to identify microsatellite instability (MSI-H), tumor mutational burden (TMB) and Wnt/B-Catenin pathway alterations in patients with gastrointestinal (GI) tract cancers." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): 3552. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.3552.
Abstract:
3552 Background: MSI-H cancers are responsive to immune checkpoint blockade (ICB), but nearly half of all patients experience primary or early treatment resistance. Activation of the WNT/B-Catenin pathway can lead to immune exclusion and may drive resistance to ICB. Methods: 12 patients had stage III (N = 1) or IV (N = 11) MSI-H GI tract (small bowl, colon, or rectal) cancers. Blood samples were obtained after (N = 5) or during (N = 5) ICB. 2 patients did not receive ICB. Blood samples from 8 patients with microsatellite stable (MSS) metastatic colorectal cancer were included as controls. The Guardant Health (Redwood City, CA) Omni 2.0 mb panel was used to analyze cfDNA. We analyzed MSI-H status, TMB, and mutations within the WNT/B-Catenin pathway, including APC, RNF43 and CTNNB1. Results: Of 12 patients with MSI-H GI cancers, 1 sample failed enrichment due to hemolysis. MSI-H was not detected in 2 patients with a history of MSI-H in tissue; however these patients had a complete response to ICB at the time of blood collection. The Omni panel identified MSI-H in the remaining 9 patients with MSI-H disease in tissue. Among 8 control patients with MSS disease in tissue, MSI-H was not detected. Median TMB (mutations/Mb) was greater for MSI-H specimens (109; range 30-807) than for MSS specimens (13; range 6-24). All 8 patients with MSS GI cancers were identified to have APC mutations, and none were found to have CTNNB1 or RNF43 mutations. Of 9 evaluable MSI-H GI cancers, 2 had APC mutations alone. The remaining 7 carried RNF43 mutations (G659fs). All patients with RNF43 mutations were found to have disease progression while on ICB. Among these 7 patients with RNF43 mutations, 6 had additional mutations in APC or CTNNB1. Conclusions: Blood based genomic profiling can identify MSI-H cancers. Patients with MSI-H cancers resistant to ICB in this cohort have mutations in RNF43 as well as additional mutations in APC or CTNNB1, suggesting that co-activation of the WNT/B-Catenin pathway may be biologically important. Further study of the role of WNT/B-Catenin pathway activation in ICB resistance will be pursued using tumor tissue from this cohort.