Β-catenin Gene Research Articles

Investigation of the Apoptosis Inducing and β-catenin Silencing by Tetradentate Schiff Base Zinc(II) Complex on the T-47D Breast Cancer Cells.

Several mechanisms are known for the anticancer effects of cisplatin. However, its most wellknown function involves binding to DNA and activating the DNA damage response. Despite its good effects, the treatment process often leads to chemoresistance and affects the mechanisms that support cell survival, such as pathways that promote cell growth, apoptosis, DNA damage repair, and endocytosis. For this reason, we investigated the effects of a new metal complex (tetradentate Schiff base zinc(II) complex) on breast cancer cells (T-47D). We evaluated its effect on cytotoxicity, apoptosis, and drug resistance in comparison to cisplatin. The results of the MTT test showed that tetradentate Schiff base zinc(II) complex has good cytotoxicity compared to cisplatin. The IC50 values for the [Zn(SB)]Cl2 complex and cisplatin after 72 h of exposure were equal to 42.1 and 276.1 μM, respectively. Real-time PCR assay confirmed that the [Zn(SB)]Cl2 complex activated the mitochondrial pathway of apoptosis and increased the expression of Bak1 and caspase-3 genes significantly compared to cisplatin. More importantly, the [Zn(SB)]Cl2 was able to reduce the expression of the β-catenin gene, which plays a role in drug resistance, by 0.011 compared to the control. Therefore, we can hope for this new complex because, without the help of any β-catenin silencing agent, it was able to inhibit the drug resistance in the T-47D cell line that overexpresses the β-catenin gene.

Vanadium-Doped Mesoporous Bioactive Glass Promotes Osteogenic Differentiation of rBMSCs via the WNT/β-Catenin Signaling Pathway.

Pentavalent vanadium [V(V)] has been studied as bioactive ions to improve the bone defect repair; however, its osteogenic promotion mechanism is still not fully understood so far. In this study, a V-doped mesoporous bioactive glass (V-MBG) was prepared, and its effects on osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) and potential signaling pathways were investigated. The physicochemical characterization revealed that the incorporation of V slightly reduced the specific surface area and increased the mesoporous pore size, and the abundant mesopores of V-MBG were beneficial to the sustained dissolution of V(V) ions as well as calcium, silicon, and phosphorus ions. Cell proliferation results indicated that the high dilution ratio (>16) V-MBG extract markedly promoted the proliferation of rBMSCs compared with the control group and the same dilution ratio MBG extract. Compared with the same dilution ratio MBG extract, diluted V-MBG extracts markedly promoted the secretion of alkaline phosphatase (ALP) and osteocalcin (OCN) protein at day 7 but insignificantly stimulated the runt-related transcription factor 2 (RUNX2) and vascular endothelial growth factor (VEGF) protein synthesis. In depth, the diluted V-MBG extracts remarkably up-regulated the expression of WNT/β-catenin pathway direct target genes, including WNT3a, β-catenin, and AXIN2 genes in contrast to the same dilution ratio MBG extracts, suggesting that the released V(V) ions might promote osteogenic differentiation of rBMSCs via the WNT/β-catenin signaling pathway.

Sirtuin 6 protects against podocyte injury by blocking the renin-angiotensin system by inhibiting the Wnt1/β-catenin pathway.

Sirtuins (Sirts) are a family of nicotinamide adenine dinucleotide-dependent protein deacetylases that share diverse cellular functions. Increasing evidence shows that Sirts play a critical role in podocyte injury, which is a major determinant of proteinuria-associated renal disease. Membranous nephropathy (MN) is a typical glomerular disease in which podocyte damage mediates proteinuria development. In this study we investigatedthe molecular mechanisms underlying the regulatory roles of Sirt in podocyte injury in MN patients, rats with cationic bovine serum albumin (CBSA)-induced MN and zymosan activation serum (ZAS)-stimulated podocytes. Compared with healthy controls, MN patients showed significant reduction in intrarenal Sirt1 and Sirt6 protein expression. In CBSA-induced MN rats, significant reduction in intrarenal Sirt1, Sirt3 and Sirt6 protein expression was observed. However, only significant decrease in Sirt6 protein expression was found in ZAS-stimulated podocytes. MN patients showed significantly upregulated protein expression of Wnt1 and β-catenin and renin-angiotensin system (RAS) components in glomeruli. CBSA-induced MN rats exhibited significantly upregulated protein expression of intrarenal Wnt1 and β-catenin and their downstream gene products as well as RAS components. Similar results were observed in ZAS-stimulated podocytes. In ZAS-stimulated podocytes, treatment with a specific Sirt6 activator UBCS039 preserved the protein expression of podocin, nephrin and podocalyxin, accompanied by significant inhibition of the protein expression of β-catenin and its downstream gene products, including Snail1 and Twist; treatment with a β-catenin inhibitor ICG-001 significantly preserved the expression of podocyte-specific proteins and inhibited the upregulation of downstream β-catenin gene products accompanied by significant suppression of the protein expression of RAS components. Thus, we demonstrate that Sirt6 ameliorates podocyte injury by blocking RAS signalling via the Wnt1/β-catenin pathway. Sirt6 is a specific therapeutic target for the treatment of podocyte damage-associated renal disease.

Mesenchymal stem cell exosomes rich in miR-23b-3p affect the Wnt signaling pathway and promote neuronal autophagy to alleviate PD symptoms

This study aims to elucidate the role of miR-23b-3p in mesenchymal stem cell exosomes in regulating the Wnt signaling pathway to promote autophagy of neurons and alleviate Parkinson's disease (PD) symptoms. We generated rat and cellular PD models with 6-OHDA, treated them with mesenchymal stem cell exosomes rich in miR-23b-3p and determined the expression of α-syn and Wnt/β-catenin pathway and autophagy-related genes. In the plasma of PD patients, the levels of miR-23b-3p and the Wnt/β-catenin pathway-related genes β-catenin and DAT were low, while α-syn expression was high. In the PD cell model, miR-23b-3p was downregulated, the Wnt pathway was inhibited, α-syn was upregulated, neuron autophagy was inhibited, and the revitalization of the Wnt/β-catenin pathway could promote the autophagy of neurons. Coculture of miR-23b-3p-enriched exosomes with MN9D cells confirmed that miR-23b-3p-enriched exosomes could promote autophagy in MN9D cells in a PD cell model. Moreover, animal experiments confirmed the results of the cell experiments. Therefore, miR-23b-3p-enriched mesenchymal stem cell exosomes promote neuronal autophagy by regulating the Wnt signaling pathway, thus alleviating PD progression and providing an important basis for the clinical treatment of PD.

Stem Cell Properties of Gastric Cancer Stem-Like Cells under Stress Conditions Are Regulated via the c-Fos/UCH-L3/β-Catenin Axis.

Gastric cancer stem-like cells (GCSCs) possess stem cell properties, such as self-renewal and tumorigenicity, which are known to induce high chemoresistance and metastasis. These characteristics of GCSCs are further enhanced by autophagy, worsening the prognosis of patients. Currently, the mechanisms involved in the induction of stemness in GCSCs during autophagy remain unclear. In this study, we compared the cellular responses of GCSCs with those of gastric cancer intestinal cells (GCICs) whose stemness is not induced by autophagy. In response to glucose starvation, the levels of β-catenin and stemness-related genes were upregulated in GCSCs, while the levels of β-catenin declined in GCICs. The pattern of deubiquitinase ubiquitin C-terminal hydrolase-L3 (UCH-L3) expression in GCSCs and GCICs was similar to that of β-catenin expression depending on glucose deprivation. We also observed that inhibition of UCH-L3 activity reduced β-catenin protein levels. The interaction between UCH-L3 and β-catenin proteins was confirmed, and it reduced the ubiquitination of β-catenin. Our results suggest that UCH-L3 induces the stabilization of β-catenin, which is required to promote stemness during autophagy activation. Also, UCH-L3 expression was regulated by c-Fos, and the levels of c-Fos increased in response to autophagy activation. In summary, our findings suggest that the inhibition of UCH-L3 during nutrient deprivation could suppress stress resistance of GCSCs and increase the survival rates of gastric cancer patients.

Melatonin regulates the periodic growth of secondary hair follicles through the nuclear receptor RORα.

Cashmere is the fine bottom hair produced by the secondary hair follicles of the skin. This hair is economically important. Previous studies by our research group have shown that exogenous melatonin (MT) can regulate the periodic growth of secondary hair follicles, induce the secondary development of villi, and alter the expression of some genes related to hair follicle development. Few studies on the regulation of villus growth by MT binding receptors have been published. In this study, MT was implanted subcutaneously behind the ear of Inner Mongolia cashmere goats. RT-qPCR, in situ hybridization, Western blot analysis, immunofluorescence and RNAi techniques were used to investigate the receptors and functions of MT in regulating the development of secondary hair follicles in Inner Mongolia cashmere goats. The results showed that MT binds to the nuclear receptor RORα on dermal papilla stimulates hair follicle development and promotes villus growth. The RORα mRNA expression in the skin of Inner Mongolia cashmere goats was periodic and showed a trend of first increasing and then decreasing. The expression began to increase in February, peaked in April, and reached the lowest level in May. RORα significantly affected the mRNA expression of β-catenin gene, a key gene in hair follicle development, in the presence of MT. It will lay a solid molecular foundation for further research on the regulation mechanism between MT receptor and villus growth and development and to achieve artificial regulation of villus growth time and yield to improve the effect of villus production.

PANORAMA OF GASTRIC AND INTESTINAL CARCINOGENESIS: LITERATURE REVIEW

Purpose. The aim of the review is to analyze the modern data on the molecular mechanisms of stomach and intestinal carcinogenesis, which can be the basis for managing of malignant stomach and intestine neoplasms mortality risks’ reduction.
 Materials and methods. The review includes data from foreign and domestic studies published in the electronic bibliographic databases PubMed, Cyberleninka, Elibrary over the past 10 years.
 Results. It is given the analysis of Wnt and β-catenin gene family role in the pathogenesis of stomach and colon malignant tumors and the carcinogenic potential of Helicobacter pylori in gastric cancer, as well as the driver bacteria contributing to the development of colon cancer.
 Conclusion. Stomach cancer and colon cancer are heterogeneous diseases with diverse molecular and histological subtypes, having both similarities and differences in the patterns of cancer promotion. Progress in transparency genesis of these diseases is associated with an understanding of cytotoxic and genotoxic effects, oxidative stress and chronic inflammation mechanisms.

Ap-2β regulates cranial osteogenic potential via the activation of Wnt/β-catenin signaling pathway

The skull is a fundamental bone that protects the development of brain and consists of several bony elements, such as the frontal and parietal bones. Frontal bone exhibited superior in osteogenic potential and regeneration of cranial defects compared to parietal bone. However, how this regional difference is regulated remains largely unknown. In this study, we identified an Ap-2β transcriptional factor with a higher expression in frontal bone, but its molecular function in osteoblasts needs to be elucidated. We found that Ap-2β knockdown in preosteoblasts leads to reduced proliferation, increased cell death and impaired differentiation. Through RNA-seq analysis, we found that Ap-2β influences multiple signaling pathways including the Wnt pathway, and overexpression of Ap-2β showed increased nuclear β-catenin and its target genes expressions in osteoblasts. Pharmacological activation of Wnt/β-catenin signaling using LiCl treatment cannot rescue the reduced luciferase activities of the β-catenin/TCF/LEF reporter in Ap-2β knockdown preosteoblasts. Besides, transient expression of Ap-2β via the lentivirus system could sufficiently rescue the inferior osteogenic potential in parietal osteoblasts, while Ap-2β knockdown in frontal osteoblasts resulted in reduced osteoblast activity, reduced active β-catenin and target genes expressions. Taken together, our data demonstrated that Ap-2β modulates osteoblast proliferation and differentiation through the regulation of Wnt/β-catenin signaling pathway and plays an important role in regulating regional osteogenic potential in frontal and parietal bone.

Differential expression levels of β-catenin are associated with invasive behavior of both functional and non-functional pituitary neuroendocrine tumor (PitNET).

Although research continues to elucidate the molecular mechanism underlying pituitary tumor pathogenesis, limited information is available on the potential role and expression profile of β-catenin in functional and non-functional pituitary neuroendocrine tumors (PitNETs). In the current study, 104 pituitary samples (tumors and cadaveric healthy pituitary tissues) were included and the gene and protein expression levels of β-catenin were assessed by Real-Time PCR and immunohistochemistry, respectively. The correlation between expression level of β-catenin and tumor invasive feature and size as well as patient age, gender, and hormonal level was measured. The data showed that PitNET samples expressed higher levels of the β-catenin gene and protein compared to healthy pituitary tissues. Although there was no difference in β-catenin expression level between non-functioning (NF-PitNETs) and growth hormone-producing tumors (GH-PitNETs), both tumor types showed significantly elevated β-catenin levels compared to healthy pituitary tissues. The high level of β-catenin in the invasive functional and non-functional tumors is indicative of the association of β-catenin with PitNETs invasion. The expression pattern of the β-catenin gene and protein was consistently and significantly associated with these tumor types. The correlation between β-catenin and insulin-like growth factor 1 (IGF-1) in GH-PitNETs indicates the potential relevance of β-catenin and IGF-1 for GH-PitNETs. The simultaneous increase in the expression of β-catenin gene and protein level in PitNET tissues and their relationship to the tumor severity indicates the possible contributing role of β-catenin and its underlying signaling mediators in PitNET pathogenesis.

Effect of grape seed extract on β-catenin gene expression and hyperglycemia in rats induced by streptozotocin

Amaç: Tip 2 Diabetes Mellitus, insülin direnci ve/veya insülin eksikliği veya yetersizliği durumunda yüksek kan şekeri ile karakterize metabolik bir hastalıktır. Wnt/β-katenin sinyal yolu, tüm fizyolojik süreçlerde rol oynar. Yoldaki herhangi bir kusur diyabetin gelişmesine neden olur. Bu çalışmanın amacı, deneysel Tip 2 diyabetik sıçanlarda üzüm çekirdeği ekstresinin (GSE) vücut ağırlığı, kan şekeri ve β-katenin geni ekspresyonu üzerindeki etkisini belirlemektir. Gereç ve Yöntem: Sıçanlar Kontrol, Diyabetik Kontrol ve Tedavi grupları (100, 200, 400 mg/kg GSE) olmak üzere beş gruba ayrıldı. Diyabet ve tedavi gruplarındaki ratlara yüksek yağlı diyet ve düşük doz streptozotosin (35 mg/ kg) verildi. Deney süresince sıçanların vücut ağırlıkları haftalık olarak kaydedildi. Suda çözünen ekstraktlar dört hafta boyunca gavaj yoluyla uygulandı ve çalışmada tedavi sonunda hayvanlardan alınan karaciğer, pankreas ve kan örnekleri kullanıldı. Bulgular: GSE tedavisinin diyabete bağlı genel kilo kaybı üzerine etkisinin minimal olduğu, 100 ve 400 mg/kg GSE dozlarının kan şekerini düşürdüğü gözlendi (p<0,05). β-katenin gen ekspresyonu sonuçlarına göre karaciğer dokusu (p>0,05) ve pankreas dokusu (p<0,05) açısından gruplar arasında istatistiksel olarak anlamlı fark yoktu. Öneri: Elde edilen bulgulara göre üzüm çekirdeği diyabetin tedavisinde umut verici olabilir.

Berberine nanostructures attenuate ß-catenin, a key component of epithelial mesenchymal transition in lung adenocarcinoma

Lung cancer (LC) is the leading cause of cancer-related deaths globally. It accounts for more than 1.9 million cases each year due to its complex and poorly understood molecular mechanisms that result in unregulated cell proliferation and metastasis. β-Catenin is a developmentally active protein that controls cell proliferation, metastasis, polarity and cell fate during homeostasis and aids in cancer progression via epithelial–mesenchymal transition. Therefore, inhibition of the β-catenin pathway could attenuate the progression of LC. Berberine, an isoquinoline alkaloid which is known for its anti-cancer and anti-inflammatory properties, demonstrates poor solubility and bioavailability. In our study, we have encapsulated berberine into liquid crystalline nanoparticles to improve its physiochemical functions and studied if these nanoparticles target the β-catenin pathway to inhibit the human lung adenocarcinoma cell line (A549) at both gene and protein levels. We observed for the first time that berberine liquid crystalline nanoparticles at 5 µM significantly attenuate the expression of the β-catenin gene and protein. The interaction between berberine and β-catenin was further validated by molecular simulation studies. Targeting β-catenin with berberine nanoparticles represents a promising strategy for the management of lung cancer progression.

Antitumor Activity of Luteolin Against Ehrlich Solid Carcinoma in Rats via Blocking Wnt/β-Catenin/SMAD4 Pathway.

Background Ehrlich solid carcinoma (ESC) is characterized by rapid proliferation and a short survival time. Because Ehrlich ascites carcinoma (EAC) resembles human cancer cells, Ehrlich solid and ascetic forms are commonly used to determine the anticancer effects of various compounds. Luteolin is a flavonoid compound found in many dietary sources, including carrots, peppers, celery, olive oil, peppermint, and oregano. Luteolin has potent anti-inflammatory, antidiabetic, antitumor, and antiapoptotic activities. Aims This study aims to investigate the antitumor activity of luteolin against ESC in rats by affecting the Wnt/β-catenin/SMAD4 pathway. Methods We introduced 0.15 ml of Ehrlich cells (2 × 106) ESC into the left hind thighs of rats. After eight days of inoculation, the rats orally received 25 mg/kg of luteolin daily. We stained sections of tumor tissues with Masson's trichrome. We used another part of the tumor tissue to assess gene and protein expression of Wnt, β-catenin, E-cadherin, and SMAD4. Results Treatment of carcinoma rats with luteolin increased the mean survival time and reduced tumor volume and weight. In addition, examination of tumor tissue stained with Masson's trichrome showed loosely to densely packed collagen fibers in between neoplastic cells and scattered papillary expansion of a loose blue band of collagen expression along the covering adipose connective tissue and extending in a fine strand in between muscle fibers, which was ameliorated by treating rats with luteolin. Finally, treating ESC in rats with luteolin overexpressed E-cadherin and downregulated Wnt, β-catenin, and SMAD4. Conclusions We found luteolin has antineoplastic activity against ESC by reducing tumor size and weight while improving the structure of muscle cells. It works by suppressing Wnt, β-catenin, and SMAD4, resulting in decreased tumor cell proliferation and differentiation. Additionally, luteolin overexpresses E-cadherin, leading to reduced tumor cell invasion and metastasis.

Cigarette smoking induces lung cancer tumorigenesis via upregulation of the WNT/β-catenin signaling pathway

Lung cancer has the highest mortality rate compared to any other cancer worldwide, and cigarette smoking is one of the major etiological factors. How cigarette smoke (CS) induces tumorigenesis in healthy cells is still not completely understood. In this study, we treated healthy human bronchial epithelial cells (16HBE14o) with 1 % cigarette smoke extract (CSE) for one week. The CSE exposed cells showed upregulation of WNT/β-catenin pathway genes like WNT3, DLV3, AXIN and β-catenin, 30 oncology proteins were found to be upregulated after CSE treatment. Further, we explored whether the role of extracellular vesicles (EVs) obtained from CSE exposed cells can induce tumorigenesis. We observed that CSE EVs induced migration of healthy 16HBE14o cells by upregulation of various oncology proteins in recipient cells like AXL, EGFR, DKK1, ENG, FGF2, ICAM1, HMOX1, HIF1a, SERPINE1, SNAIL, HGFR, PLAU which are related to WNT signaling, epithelial mesenchymal transition (EMT) and Inflammation, whereas inflammatory marker, GAL-3 and EMT marker, VIM were downregulated. Moreover, β-catenin RNA was found in CSE EVs, upon treatment of these EVs to healthy cells, the β-catenin gene level was decreased in recipient cells compared to healthy 16HBE14o cells, indicating the utilisation of β-catenin RNA in healthy cells. Overall, our study suggests that CS treatment can induce tumorigenesis of healthy cells by upregulating WNT/β-catenin signaling in vitro and human lung cancer patients. Therefore targeting the WNT/β-catenin signaling pathway is involved in tumorigenesis inhibition of this pathway could be a potential therapeutic approach for cigarette smoke induced lung cancer.

Transcription of WNT Genes in Hematopoietic Niche's Mesenchymal Stem Cells in Multiple Myeloma Patients with Different Responses to Treatment.

Mesenchymal stromal cells (MSCs) are involved in bone tissue remodeling due to their ability to differentiate into osteoblasts and to influence osteoclasts' activity. Multiple myeloma (MM) is associated with bone resorption. During disease progression, MSCs acquire a tumor-associated phenotype, losing their osteogenic potential. The process is associated with impaired osteoblasts/osteoclasts balance. The WNT signaling pathway plays a major role in maintaining the balance. In MM, it functions in an aberrant way. It is not known yet whether the WNT pathway is restored in patients' bone narrow after treatment. The aim of the study was to compare the level of WNT family gene transcription in the bone marrow MSCs of healthy donors and MM patients before and after therapy. The study included healthy donors (n = 3), primary patients (n = 3) and patients with different response status to therapy (bortezomib-containing induction regimens) (n = 12). The transcription of the WNT and CTNNB1 (encoding β-catenin) genes was accessed using qPCR. The mRNA quantity of ten WNT genes, as well as CTNNB1 mRNA encoding β-catenin, a key mediator in canonical signaling, was evaluated. The observed differences between the groups of patients indicated that aberrant functioning of the WNT pathway was retained after treatment. The differences that we detected for WNT2B, WNT9B and CTNNB1 suggested their possible application as prognostic molecular markers.

Antitumor activity of galaxamide involved in cell apoptosis and stemness by inhibiting Wnt/β-catenin pathway in cervical cancer.

Our previous work reported that galaxamide, a cyclopeptide extracted from the seaweed Galaxaura filamentosa, showed antiproliferative activity against HeLa cells by MTT assay. In this study, the growth-inhibitory effects of galaxamide in HeLa cells and xenograft mouse models were investigated. It was found galaxamide significantly inhibited cell growth, colony formation, migration, and invasion and induced cell apoptosis by inhibiting the Wnt signaling pathway in HeLa cells. RNA sequencing revealed that galaxamide regulated stemness by Wnt6 signaling pathway in HeLa cells. By analyzing The Cancer Genome Atlas database, Wnt6 was found to be negatively/positively correlated with stemness- and apoptosis-related genes in human cervical cancer. Cancer stem-like cells (CSCs) isolated and enriched from HeLa cells demonstrated elevated Wnt6 and β-catenin genes compared with nonstem HeLa cells. After galaxamide treatment, CSCs showed abrogation of sphere-forming ability, along with inhibition of stemness-related and Wnt pathway genes. Galaxamide treatment was also accompanied by the induction of apoptosis in HeLa cells, which was consistent with the results in BALB/c nude mice. Our results provide evidence that suppression of stemness by downregulating the Wnt signaling pathway is the molecular mechanism by which galaxamide effectively inhibits cell growth and induces apoptosis in cervical cancer cells.

β-Catenin signaling in alveolar macrophages enhances lung metastasis through a TNF-dependent mechanism.

The main cause of malignancy-related mortality is metastasis. Although metastatic progression is driven by diverse tumor-intrinsic mechanisms, there is a growing appreciation for the contribution of tumor-extrinsic elements of the tumor microenvironment, especially macrophages, which correlate with poor clinical outcomes. Macrophages consist of bone marrow-derived and tissue-resident populations. In contrast to bone marrow-derived macrophages, the transcriptional pathways that govern the pro-metastatic activities of tissue-resident macrophages (TRMs) remain less clear. Alveolar macrophages (AMs) are a TRM population with critical roles in tissue homeostasis and metastasis. Wnt/β-catenin signaling is a hallmark of cancer and has been identified as a pathologic regulator of AMs in infection. We tested the hypothesis that β-catenin expression in AMs enhances metastasis in solid tumor models. Using a genetic β-catenin gain-of-function approach, we demonstrated that (a) enhanced β-catenin in AMs heightened lung metastasis; (b) β-catenin activity in AMs drove a dysregulated inflammatory program strongly associated with Tnf expression; and (c) localized TNF-α blockade abrogated this metastatic outcome. Last, β-catenin gene CTNNB1 and TNF expression levels were positively correlated in AMs of patients with lung cancer. Overall, our findings revealed a Wnt/β-catenin/TNF-α pro-metastatic axis in AMs with potential therapeutic implications against tumors refractory to the antineoplastic actions of TNF-α.

Abstract LB016: Immunotherapeutic potential of ST316, a peptide antagonist of β-catenin

Abstract The transcription factor β-catenin is a key player in many cellular processes, including stem cell renewal, cellular homeostasis and inflammation. Deregulation of β-catenin occurs frequently in several cancer types by mutation or overexpression of the β-catenin gene or mutation of negative regulators such as Adenomatous Polyposis Coli (APC). ST316 is a novel peptide antagonist that specifically interferes with the association of β-catenin with its co-activator BCL9, disrupting β-catenin nuclear localization and attenuating target gene expression. In an orthotopic triple-negative breast cancer (TNBC) model, ST316 demonstrates potent anti-tumor activity, resulting in 84% tumor growth inhibition (TGI) (p<0.001 vs. vehicle control). In addition to its cell-autonomous mechanisms, deregulated Wnt/β-catenin signaling can promote tumorigenesis by impacting the tumor microenvironment. Reprogramming of immunosuppressive M2-like Tumor Associated Macrophages (TAMs) toward an immune-promoting program (M1-like) is an attractive cancer immunotherapeutic strategy. Here we explored ST316 potential for macrophage repolarization toward the M1-like phenotype, activation of cytotoxic T-cells in macrophage/T-cell co-culture assays and cooperation of ST316 with anti-PD-1 to enhance anti-tumor activity in vivo. Initial studies demonstrate that human macrophages derived from Peripheral Blood Mononuclear Cells (hPBMCs) and subsequently committed to the M2-like identity are reprogrammed toward an M1-like phenotype upon ST316 exposure. ST316 treatment dose-dependently suppressed expression of the M2 marker CD163 by flow cytometry and quantitative PCR, resulting in 100-fold increase in the M1/M2 ratio without substantial impact on cell viability. Importantly, T-cell viability and activation markers associated with M1-state (CD80, CD86) were not affected by ST316 at the concentrations used in these studies. Further, in co-cultures of M2 macrophage with T cells, ST316 exposure resulted in a three-fold increase in T-cell activation compared to control M2/T cell co-cultures, as measured by intracellular IFN-γ staining. Finally, in an orthotopic TNBC model in vivo, subtherapeutic ST316 enhanced the anti-tumor activity of anti-PD-1 [85% TGI with combination, compared to 51% TGI with anti-PD1 alone (p<0.01) and -9% TGI with subtherapeutic ST316 alone (p<0.001)]. Anti-tumor activity was accompanied by an increase the M1/M2 ratio. Overall, these results support the immuno-therapeutic potential of ST316 and extend the application range of ST316 to include Wnt-driven cancers with poor clinical response to immune checkpoint blockade and other immunotherapeutic agents. Citation Format: Claudio Scuoppo, Lila Ghamsari, Erin Gallagher, Siok Leong, Mark Koester, Rick Ramirez, Jerel Gonzales, Gene Merutka, Barry Kappel, Abi Vainstein-Haras, Jim Rotolo. Immunotherapeutic potential of ST316, a peptide antagonist of β-catenin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB016.

Protein Interaction Network Analysis of β-catenin to Map its Crucial Interacting Genes using Systems Biology Approach

β-catenin (CTNNB1), a scaffold protein, plays a vital role in embryonic development, adult tissue homeostasis, neurodegenerative diseases, bone diseases, chronic obstructive pulmonary diseases, wound healing, and pigment disorders. β-catenin is the crucial component in the Wnt-signaling pathway. β-catenin levels in the cytosol balance the Wnt-signaling pathway. The activated Wnt-signaling pathway allows the accumulation of β-catenin in the nucleus and further act as a promoter to initiate transcription of several oncogenes responsible for carcinogenesis. In the present study, we have used a systems biology approach to map β-catenin interactors. Primary protein-protein interaction databases such as PubChem, CTD, and STITCH databases were used to collect all the experimentally valid β-catenin interactors and their subcellular location was traced using UniProtKB database. The interactome was constructed and analyzed by utilizing STRING and Cytoscape tools. MCODE, Cytoscape tool, was utilized to construct and analyze the sub-networks. Correlation between modular seed proteins and β-catenin was studied using UALCAN database. Functional enrichment analysis was done using DAVID database. Cytohubba, a Cytoscape tool was utilized to identify the top gene interactors of β-catenin. Further, the expression and gene ontology of each β-catenin gene interactor were analyzed using UALCAN and CleuGO tools. The analysis reveals β-catenin interactors, TP53, EP300, RPS27A, UBC, HDAC1, SRC, AKT1, EGFR, HSP90AA1, and CREBBP as the first top gene interactors. This study uses a systems biology-driven approach and successfully identifies and understands the biological roles of the top β-catenin interactors.

β-Catenin interacts with the TAZ1 and TAZ2 domains of CBP/p300 to activate gene transcription

The transcriptional co-regulator β-catenin is a critical member of the canonical Wnt signaling pathway, which plays an important role in regulating cell fate. Deregulation of the Wnt/β-catenin pathway is characteristic in the development of major types of cancer, where accumulation of β-catenin promotes cancer cell proliferation and renewal. β-catenin gene expression is facilitated through recruitment of co-activators such as histone acetyltransferases CBP/p300; however, the mechanism of their interaction is not fully understood. Here we investigate the interaction between the C-terminal transactivation domain of β-catenin and CBP/p300. Using a combination of pulldown assays, isothermal titration calorimetry, and nuclear resonance spectroscopy we determine the disordered C-terminal region of β-catenin binds promiscuously to the TAZ1 and TAZ2 domains of CBP/p300. We then map the interaction site of the C-terminal β-catenin transactivation domain onto TAZ1 and TAZ2 using chemical-shift perturbation studies. Luciferase-based gene reporter assays indicate Asp750-Leu781 is critical to β-catenin gene activation, and mutagenesis revealed that acidic and hydrophobic residues within this region are necessary to maintain TAZ1 binding. These results outline a mechanism of Wnt/β-catenin gene regulation that underlies cell development and provides a framework to develop methods to block β-catenin dependent signaling.

Moshen granule ameliorates membranous nephropathy by blocking intrarenal renin-angiotensin system signalling via the Wnt1/β-catenin pathway

BackgroundMembranous nephropathy (MN) is one of the cardinal causes of nephrotic syndrome in adults, but an adequate treatment regimen is lacking. PurposeWe assessed the effect of Moshen granule (MSG) on patients with MN and cationic bovine serum albumin (CBSA)-induced rats. We further identified the bioactive components of MSG and revealed the underlying molecular mechanism of its renoprotective effects. MethodsWe determined the effect of MSG on patients with MN and CBSA-induced rats and its components on podocyte injury in zymosan-activated serum (ZAS)-elicited podocytes and revealed their regulatory mechanism on the Wnt/β-catenin/renin-angiotensin system (RAS) signalling axis. ResultsMSG treatment improved renal function and reduced proteinuria in MN patients and significantly reduced proteinuria and preserved the protein expression of podocin, nephrin, podocalyxin and synaptopodin in CBSA-induced MN rats. Mechanistically, MSG treatment significantly inhibited the protein expression of angiotensinogen, angiotensin converting enzyme and angiotensin II type 1 receptor, which was accompanied by inhibition of the protein expression of Wnt1 and β-catenin and its downstream gene products, including Snail1, Twist, matrix metalloproteinase-7, plasminogen activator inhibitor-1 and fibroblast-specific protein 1, in CBSA-induced MN rats. We further identified 81 compounds, including astragaloside IV (AGS), calycosin, barleriside A and geniposidic acid, that preserve the podocyte-specific protein expression in ZAS-induced podocytes. Among these four compounds, AGS exhibited the strongest inhibitory effects on podocyte protein expression. AGS treatment significantly inhibited the protein expression of RAS components and Wnt1 and β-catenin and its downstream gene products in ZAS-induced podocytes. In contrast, the inhibitory effect of AGS on podocyte-specific proteins, β-catenin downstream gene products and RAS components was partially abolished in ZAS-induced podocytes treated with ICG-001 and β-catenin siRNA. ConclusionThis study first demonstrates that AGS mitigates podocyte injury by inhibiting the activation of RAS signalling via the Wnt1/β-catenin pathway by both pharmacological and genetic methods. Therefore, AGS might be considered a new β-catenin inhibitor that inhibits the Wnt1/β-catenin pathway to retard MN in patients.