Β-catenin Degradation Research Articles

Discovery of a potent β-catenin destabilizer for overcoming the resistance of 5-fluorouracil in colorectal cancer

Wnt/β-catenin signalling is frequently activated in colorectal cancer, in which nuclear β-catenin accumulation contributes to tumour initiation and progression. However, therapeutic agents in clinical use targeting this pathway are lacking. In this report, we describe the synthesis of novel stemona alkaloid analogues and their biological evaluation, among which compound 3 was identified to efficiently inhibit various CRC cells, including 5-fluorouracil-resistant CRC cells. Mechanistically, this study revealed that compound 3 reduced the protein level of β-catenin without affecting its mRNA level, which suggests an alternative mechanism for β-catenin degradation. The expression of downstream proteins, including c-myc, survivin, and cyclin D1, was also significantly inhibited, even in Wnt-activated CRC cells. Briefly, our data highlight the potential of compound 3 as a destabilizer of β-catenin for the treatment of CRC patients.

Elevated Dkk1 Mediates Downregulation of the Canonical Wnt Pathway and Lysosomal Loss in an iPSC Model of Neuronopathic Gaucher Disease.

Gaucher Disease (GD), which is the most common lysosomal storage disorder, is caused by bi-allelic mutations in GBA1—a gene that encodes the lysosomal hydrolase β-glucocerebrosidase (GCase). The neuronopathic forms of GD (nGD) are characterized by severe neurological abnormalities that arise during gestation or early in infancy. Using GD-induced pluripotent stem cell (iPSC)-derived neuronal progenitor cells (NPCs), we have previously reported that neuronal cells have neurodevelopmental defects associated with the downregulation of canonical Wnt signaling. In this study, we report that GD NPCs display elevated levels of Dkk1, which is a secreted Wnt antagonist that prevents receptor activation. Dkk1 upregulation in mutant NPCs resulted in an increased degradation of β-catenin, and there was a concomitant reduction in lysosomal numbers. Consistent with these results, incubation of the mutant NPCs with recombinant Wnt3a (rWnt3a) was able to outcompete the excess Dkk1, increasing β-catenin levels and rescuing lysosomal numbers. Furthermore, the incubation of WT NPCs with recombinant Dkk1 (rDkk1) phenocopied the mutant phenotype, recapitulating the decrease in β-catenin levels and lysosomal depletion seen in nGD NPCs. This study provides evidence that downregulation of the Wnt/β-catenin pathway in nGD neuronal cells involves the upregulation of Dkk1. As Dkk1 is an extracellular Wnt antagonist, our results suggest that the deleterious effects of Wnt/β-catenin downregulation in nGD may be ameliorated by the prevention of Dkk1 binding to the Wnt co-receptor LRP6, pointing to Dkk1 as a potential therapeutic target for GBA1-associated neurodegeneration.

Expression of ubiquitin-specific protease 7 in lung tissue of preterm rats after hyperoxia exposure

Objective To study the expression and significance of ubiquitin-specific protease 7 (USP7) and the key factors of the Wnt signaling pathway in the lung tissue of preterm rats after hyperoxia exposure. Methods A total of 180 preterm neonatal Wistar rats were randomly divided into an air control group, an air intervention group, a hyperoxia control group, and a hyperoxia intervention group, with 45 rats in each group. Lung injury was induced by hyperoxia exposure in the hyperoxia groups. The preterm rats in the intervention groups were given intraperitoneal injection of the USP7 specific inhibitor P5091 (5 mg/kg) every day. The animals were sacrificed on days 3, 5, and 9 of the experiment to collect lung tissue specimens. Hematoxylin-eosin staining was used to observe the pathological changes of lung tissue. RT-PCR and Western blot were used to measure the mRNA and protein expression levels of USP7 and the key factors of the Wnt signaling pathway β-catenin and α-smooth muscle actin (α-SMA) in lung tissue. Results The air groups had normal morphology and structure of lung tissue; on days 3 and 5, the hyperoxia control group showed obvious alveolar compression and disordered structure, with obvious inflammatory cells, erythrocyte diapedesis, and interstitial edema. On day 9, the hyperoxia control group showed alveolar structural disorder and obvious thickening of the alveolar septa. Compared with the hyperoxia control group at the corresponding time points, the hyperoxia intervention group had significantly alleviated disordered structure, inflammatory cell infiltration, and bleeding in lung tissue. At each time point, the hyperoxia groups had a significantly lower radial alveolar count (RAC) than the corresponding air groups (P 0.05). There were no significant differences in the mRNA expression of USP7 and β-catenin and the protein expression of USP7, β-catenin, and α-SMA between the air intervention and air control groups (P > 0.05). Conclusions Hyperoxia exposure can activate the Wnt/β-catenin signaling pathway, and USP7 may participate in hyperoxic lung injury through the Wnt/β-catenin signaling pathway. The USP7 specific inhibitor P5091 may accelerate the degradation of β-catenin by enhancing its ubiquitination, reduce lung epithelial-mesenchymal transition, and thus exert a certain protective effect against hyperoxic lung injury.

AMP-activated protein kinase regulates β-catenin protein synthesis by phosphorylating serine/arginine-rich splicing factor 9

β-catenin is a multi-functional protein with a central role in regulating embryonic development and tissue homeostasis. The abnormal accumulation of β-catenin, due to disrupted β-catenin degradation or unregulated β-catenin synthesis, causes the development of cancer. A recent study showed that the overexpression of proto-oncogene serine/arginine-rich splicing factor 9 (SRSF9) promotes β-catenin accumulation via binding β-catenin mRNA and enhancing its translation in a manner that is dependent on the mechanistic target of rapamycin (mTOR). However, the regulation of the interaction between SRSF9 and mRNA of β-catenin remains unclear. Here, we show that AMP-activated protein kinase (AMPK) phosphorylates SRSF9 at the RNA-interacting SWQDLKD motif that plays a major role in determining substrate specificity. The phosphorylation by AMPK inhibits the binding of SRSF9 to β-catenin mRNA and suppresses β-catenin protein synthesis caused by SRSF9 overexpression without changing the β-catenin mRNA levels. Our findings suggest that AMPK activators are potential therapeutic targets for SRSF9-derived overproduction of β-catenin in cancer cells.

EPB41 suppresses the Wnt/β-catenin signaling in non-small cell lung cancer by sponging ALDOC.

Despite advancements in therapeutic options, the overall prognosis for non-small-cell lung cancer (NSCLC) remains poor. Further exploration of the etiology and targets for novel treatments is crucial for managing NSCLC. In this study, we revealed the significant potential of EPB41 for inhibiting NSCLC proliferation, invasion and metastasis in vitro and in vivo. Consistent with its tumor suppressor role in NSCLC, the expression of EPB41 in NSCLC specimens evidently decreased compared to that in normal tissues, and low EPB41 expression was associated with poor prognoses for NSCLC patients. We further demonstrated the importance of EPB41 protein as a novel inhibitor of the Wnt signaling, which regulates β-Catenin stability, and elucidated the crucial role of the EPB41/ALDOC/GSK3β/β-Catenin axis in NSCLC. Suppression of EPB41 expression in cancer cells elevated the levels of free ALDOC protein released from the EPB41-ALDOC complex, leading to disassembly of the β-catenin destruction complex, reduced proteolytic degradation of β-catenin, elevated cytoplasmic accumulation and nuclear translocation of β-catenin, thereby activating the expression of multiple oncogenes and, thus, NSCLC pathogenesis. Our study highlights the potential of EPB41 as a future therapeutic target for lung cancer.

Multifaceted WNT Signaling at the Crossroads Between Epithelial-Mesenchymal Transition and Autophagy in Glioblastoma.

Tumor cells can employ epithelial-mesenchymal transition (EMT) or autophagy in reaction to microenvironmental stress. Importantly, EMT and autophagy negatively regulate each other, are able to interconvert, and both have been shown to contribute to drug-resistance in glioblastoma (GBM). EMT has been considered one of the mechanisms that confer invasive properties to GBM cells. Autophagy, on the other hand, may show dual roles as either a GBM-promoter or GBM-suppressor, depending on microenvironmental cues. The Wingless (WNT) signaling pathway regulates a plethora of developmental and biological processes such as cellular proliferation, adhesion and motility. As such, GBM demonstrates deregulation of WNT signaling in favor of tumor initiation, proliferation and invasion. In EMT, WNT signaling promotes induction and stabilization of different EMT activators. WNT activity also represses autophagy, while nutrient deprivation induces β-catenin degradation via autophagic machinery. Due to the importance of the WNT pathway to GBM, and the role of WNT signaling in EMT and autophagy, in this review we highlight the effects of the WNT signaling in the regulation of both processes in GBM, and discuss how the crosstalk between EMT and autophagy may ultimately affect tumor biology.

β-Catenin Regulates Wound Healing and IL-6 Expression in Activated Human Astrocytes.

Reactive astrogliosis is prominent in most neurodegenerative disorders and is often associated with neuroinflammation. The molecular mechanisms regulating astrocyte-linked neuropathogenesis during injury, aging and human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) are not fully understood. In this study, we investigated the implications of the wingless type (Wnt)/β-catenin signaling pathway in regulating astrocyte function during gliosis. First, we identified that HIV-associated inflammatory cytokines, interleukin (IL)-1β and tumor necrosis factor (TNF)-α induced mediators of the Wnt/β-catenin pathway including β-catenin and lymphoid enhancer-binding factor (LEF)-1 expression in astrocytes. Next, we investigated the regulatory role of β-catenin on primary aspects of reactive astrogliosis, including proliferation, migration and proinflammatory responses, such as IL-6. Knockdown of β-catenin impaired astrocyte proliferation and migration as shown by reduced cyclin-D1 levels, bromodeoxyuridine incorporation and wound healing. HIV-associated cytokines, IL-1β alone and in combination with TNF-α, strongly induced the expression of proinflammatory cytokines including C-C motif chemokine ligand (CCL)2, C-X-C motif chemokine ligand (CXCL)8 and IL-6; however, only IL-6 levels were regulated by β-catenin as demonstrated by knockdown and pharmacological stabilization. In this context, IL-6 levels were negatively regulated by β-catenin. To better understand this relationship, we examined the crossroads between β-catenin and nuclear factor (NF)-κB pathways. While NF-κB expression was significantly increased by IL-1β and TNF-α, NF-κB levels were not affected by β-catenin knockdown. IL-1β treatment significantly increased glycogen synthase kinase (GSK)-3β phosphorylation, which inhibits β-catenin degradation. Further, pharmacological inhibition of GSK-3β increased nuclear translocation of both β-catenin and NF-κB p65 into the nucleus in the absence of any other inflammatory stimuli. HIV+ human astrocytes show increased IL-6, β-catenin and NF-κB expression levels and are interconnected by regulatory associations during HAND. In summary, our study demonstrates that HIV-associated inflammation increases β-catenin pathway mediators to augment activated astrocyte responses including migration and proliferation, while mitigating IL-6 expression. These findings suggest that β-catenin plays an anti-inflammatory role in activated human astrocytes during neuroinflammatory pathologies, such as HAND.

SRY is a Key Mediator of Sexual Dimorphism in Hepatic Ischemia/Reperfusion Injury.

To identify the role and mechanism of a male specific gene, SRY, in I/R-induced hepatic injury. Males are more vulnerable to I/R injury than females. However, the mechanism of these sex-based differences remains poorly defined. Clinicopathologic data of patients who underwent hepatic resection were identified from an international multi-institutional database. Liver specific SRY TG mice were generated, and subjected to I/R insult with their littermate WT controls in vivo. In vitro experiments were performed by treating primary hepatocytes from TG and WT mice with hypoxia/reoxygen-ation stimulation. Clinical data showed that postoperative aminotransferase level, incidence of overall morbidity and liver failure were markedly higher among 1267 male versus 508 female patients who underwent hepatic resection. SRY was dramatically upregulated during hepatic I/R injury. Overexpression of SRY in male TG mice and ectopic expression of SRY in female TG mice exacerbated liver I/R injury compared with WTs as manifested by increased inflammatory reaction, oxidative stress and cell death in vivo and in vitro. Mechanistically, SRY interacts with Glycogen synthase kinase-3β (GSK-3β) and β-catenin, and promotes phosphorylation and degradation of β-catenin, leading to suppression of the downstream FOXOs, and activation of NF-κBand TLR4 signaling. Furthermore, activation of β-catenin almost completely reversed the SRYoverexpression-mediated exacerbation of hepatic I/R damage. SRY is a novel hepatic I/R mediator that promotes hepatic inflammatory reaction, oxidative stress and cell necrosis via inhibiting Wnt/β-catenin signaling, which accounts for the sex-based disparity in hepatic I/R injuries.

PHF20 Promotes Glioblastoma Cell Malignancies Through a WISP1/BGN-Dependent Pathway.

Glioblastoma (GBM) stem cells are resistant to cancer therapy, and therefore responsible for tumor progression and recurrence after conventional therapy. However, the molecular mechanisms driving the maintenance of stemness and dedifferentiation are poorly understood. In this study, we identified plant homeodomain finger-containing protein 20 (PHF20) as a crucial epigenetic regulator for sustaining the stem cell-like phenotype of GBM. It is highly expressed in GBM and tightly associated with high levels of aggressiveness of tumors and potential poor prognosis in GBM patients. Knockout of PHF20 inhibits GBM cell proliferation, as well as its invasiveness and stem cell-like traits. Mechanistically, PHF20 interacts with WDR5 and binds to the promoter regions of WISP1 for its expression. Subsequently, WISP1 and BGN act in concert to regulate the degradation of β-Catenin. Our findings have identified PHF20 as a key driver of GBM malignant behaviors, and provided a potential target for developing prognosis and therapy.

Small Molecule Destabilizer of β-Catenin and Ras Proteins Antagonizes Growth of K-Ras Mutation-Driven Colorectal Cancers Resistant to EGFR Inhibitors.

Oncogenic K-Ras mutations in colorectal cancer (CRC) combined with APC mutations worsen CRC prognosis and lower drug effectiveness. Thus, inhibition of both Wnt/β-catenin and Ras-MAPK signaling may be a rational strategy to improve the treatment of this cancer. To identify a novel compound inhibiting both Wnt/β-catenin and Ras-MAPK signaling in CRC. We developed a two-part screening system consisting of analysis of TOP flash reporter cells and then potential toxicity effects on primary neural stem cells (NSCs). We then screened 2000 chemical compounds and tested efficacy of candidates against isogenic colon cancer cells harboring wild-type or mutant K-Ras. We employed immunohistochemistry and immunocytochemistry to determine marker signatures associated with development of disease phenotypes. We identified CPD0857, a compound that inactivates Wnt/β-catenin signaling and promotes ubiquitin-dependent proteasomal degradation of β-catenin and Ras proteins. CPD0857 effectively decreased proliferation and increased apoptosis of CRC cell lines, and overcame resistance of CRC harboring APC and K-Ras mutations to treatment with an EGFR monoclonal antibody (mAb). Moreover, CPD0857 attenuated invasiveness of highly migratory CRC cells in vitro. Accordingly, xenograft mice treated with CPD0857 showed slower tumor growth and significant decreases in both β-catenin and Ras protein expression. CPD0857 may be a potential drug for treating aggressive CRC carrying mutations that aberrantly activate Wnt/β-catenin and Ras-ERK pathways.

The innate immune effector ISG12a promotes cancer immunity by suppressing the canonical Wnt/\u03b2-catenin signaling pathway

The ability to harness innate immunity is a promising solution for improving cancer immunotherapy. Interferon (IFN) induces expression of IFN-stimulated genes (ISGs) by activating the JAK-STAT signaling pathway to promote innate immunity and inhibit malignant tumor growth, but the functions and mechanisms of most ISGs in cancer regulation are unknown. As an innate immune effector, ISG12a promotes the innate immune response to viral infection. In this study, ISG12a was found to be expressed at low levels in gastrointestinal cancer, represented by hepatocellular cancer (HCC) and gastric cancer (GC), and it identified as a tumor suppressor that affects clinical prognosis. ISG12a silencing accelerated the malignant transformation and epithelial–mesenchymal transition of cancer cells. Mechanistically, ISG12a promoted β-catenin proteasomal degradation by inhibiting the degradation of ubiquitinated Axin, thereby suppressing the canonical Wnt/β-catenin signaling pathway. Notably, β-catenin was identified as a transcription factor for PD-L1. Inhibition of Wnt/β-catenin signaling by ISG12a suppressed expression of the immune checkpoint PD-L1, rendering cancer cells sensitive to NK cell-mediated killing. This study reveals a mechanism underlying the anticancer effects of IFN. Some ISGs, as represented by ISG12a, may be useful in cancer therapy and prevention. The identified interrelations among innate immunity, Wnt/β-catenin signaling, and cancer immunity may provide new insight into strategies that will improve the efficiency of immunotherapy.

Thymoquinone inhibits the proliferation and invasion of esophageal cancer cells by disrupting the AKT/GSK‐3β/Wnt signaling pathway via PTEN upregulation

Although thymoquinone (TQ) has been reported to exert antitumor activity against various types of human cancers without evident toxicity, limited studies have reported the effects of TQ on esophageal cancer. Here, we showed that TQ induced cell cycle arrest in the G2/M phase and significantly inhibited cell proliferation and invasion. Further investigation of the potential mechanism revealed that TQ increased the levels of p53 and p21 but significantly reduced the expression of Cyclin B1, Cyclin A, and Cyclin E. Moreover, TQ led to a decrease in Bcl-2 and an increase in cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, and Bax, indicating that TQ induced apoptosis by activating the intrinsic mitochondrial apoptosis pathway. Western blotting showed that TQ disrupted the PI3K/AKT pathway by upregulating PTEN, thus modulating GSK-3β activity, increasing β-catenin degradation, and decreasing decreased MMP-2 and MMP-9 levels in Eca109 cells. However, these changes were attenuated by disrupting PTEN function (using a potent inhibitor) or downregulating PTEN expression. In addition, in vivo results showed that the efficacy of TQ as an antitumor agent in a mouse xenograft tumor model. In conclusion, TQ suppressed human esophageal cancer cells proliferation and invasion both in vitro and in vivo and could provide a novel therapeutic approach for esophageal cancer.

Abstract 1444: c-FLIP antagonizes the survival of extracellular matrix detached cancer cells

Abstract The metastasis of cancer cells to distant sites accounts for over 90% of cancer related deaths. In order to metastasize, cancer cells must be able to survive during extracellular matrix (ECM) detachment, namely by blocking anoikis – caspase dependent cell death – and restoring metabolic deficiencies. Such barriers during tumorigenesis are abrogated through activation of oncogenes, and/or inhibition of tumor suppressors. Elucidating the signaling pathways involved in cancer cell metastasis is a crucial step in designing effective therapeutic interventions. Surprisingly, our investigation has revealed that cellular FLICE-like Inhibitory Protein (c-FLIP), a protein known for its anti-apoptotic function, antagonizes the survival of ECM-detached cancer cells. Additionally, we demonstrated that oncogenic signaling results in the downregulation of c-FLIP expression further suggesting that cancer cells can benefit from diminished FLIP levels. We observed that this repression of c-FLIP expression is a consequence of MEK-ERK signaling. Our study also suggests that the mechanism underlying this paradoxical c-FLIP pathway involves the induction of β-catenin degradation through inhibition of GSK-3β phosphorylation by p38 MAPK. This results in the ubiquitination and subsequent degradation of β-catenin which could contribute to reduced proliferation and migration. Citation Format: Matyas Abel Tsegaye, Kyle McGeehan, Mati Nemera, Zachary T. Schafer. c-FLIP antagonizes the survival of extracellular matrix detached cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1444.

Ctnnb1 transcriptional upregulation compensates for Mdm2/p53-mediated β-catenin degradation in neutrophils following cardioembolic stroke

BackgroundA better understanding of the mechanism(s) underlying cardioembolic stroke can promote recovery and reduce the risk of recurrent embolisms. MethodsPeripheral blood mononuclear cell (PBMC) gene expression datasets from cardioembolic patients and healthy controls were obtained from the Gene Expression Omnibus (GEO) database (GSE58294). The Limma software package was utilized to identify differentially-expressed genes (DEGs). Protein-protein interaction (PPI) analysis of the DEGs was performed using STRING. A weighted gene co-expression network analysis (WGCNA) was used to build a gene co-expression network. In vitro experiments assessed the effects on neutrophils exposed to oxygen and glucose-deprived (OGD) cortical neurons. An in vivo murine model of thromboembolic stroke was constructed through thrombin injection to examine effects on circulating neutrophils. Mechanistic in vitro studies were conducted using the proteasome inhibitor MG132, the p53-Mdm2 binding inhibitor Nutlin-3a, Mdm2 small-interfering RNA (siRNA), and Ctnnb1 siRNA. ResultsDEG analysis identified 44 upregulated and 66 downregulated genes in cardioembolic stroke PBMCs. PPI analysis of these DEGs yielded one eight-node protein module with β-catenin (CTNNB1) as the central hub protein. Integration of the DEGs with WGCNA-derived hub genes revealed the key hub DEGs CTNNB1 and mouse double minute 2 (MDM2). Follow-up experiments revealed Mdm2, p53, and phospho-β-catenin upregulation in neutrophils exposed to OGD neurons in vitro and following thromboembolic stroke in vivo. Mechanistic studies revealed that neutrophils transcriptionally upregulate Ctnnb1 expression to compensate for Mdm2/p53-mediated β-catenin degradation induced by exposure to OGD neurons, thereby promoting neutrophil survival. ConclusionCompensatory Ctnnb1 transcriptional upregulation in neutrophils induced by ischemic neuron exposure may be involved in promoting neutrophil survival following cardioembolic stroke.

Anti-melanogenic effect of the aqueous ethanol extract of Ginkgo biloba leaf in B16F10 cells

Ginkgo biloba leaf extract (GLE) shows diverse biological effects, and it is used as an ingredient in some skincare products. GLE and a few purified compounds are known to inhibit melanogenesis. However, there are very few reports on the molecular mechanisms underlying the regulation of melanogenesis by GLE. We investigated the anti-melanogenic effect of a GLE, an aqueous ethanol extract of Ginkgo biloba leaves, and the molecular mechanisms involved. B16F10 murine melanoma cells were treated with various concentrations of GLE (0–100 μg/mL), together with 0.1 nM α-melanocyte stimulating hormone (α-MSH), which was used to induce melanogenesis. Cell proliferation, total melanin content, cellular reactive oxygen species (ROS) content, and cellular tyrosinase activity were measured. The cellular levels of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein 1 (TYRP1), and the phosphorylation levels of Akt, glycogen synthase kinase 3β (GSK3β), and β-catenin were analyzed. GLE inhibited α-MSH-induced melanogenesis and ROS generation, without causing cytotoxicity. GLE decreased cellular tyrosinase activity and the levels of MITF, tyrosinase, and TYRP1. GLE decreased the phosphorylation of Akt (Ser473) and GSK3β (Ser9) and increased the phosphorylation of β-catenin (Ser33/37/Thr41). Our results suggest that GLE can inhibit α-MSH-induced melanogenesis in B16F10 cells by downregulating the expression of melanogenic proteins, i.e., MITF, tyrosinase, and TYRP1. The downregulation of melanogenic protein expression appears to result from stimulating β-catenin degradation, which is induced by phosphorylation by GSK3β that is activated via inhibition of Akt. GLE’s antioxidant activity can also play a role in its anti-melanogenic effect.

MiR-23a-3p increases endometrial receptivity via CUL3 during embryo implantation.

A receptive endometrium is required in a successful embryo implantation. The ubiquitination-induced β-catenin degradation is related to the implantation failure.This study aimed to elucidate whether miR-23a-3p regulates endometrial receptivity via the modulation of β-catenin ubiquitination.The expressions of miR-23a-3p and CUL3 were detected in endometrial epithelial cells (EECs) isolated from pregnant mice and in hormone-induced EEC-like Ishikawa cells. The ubiquitination experiment was performed to explore the effect of CUL3 and miR-23a-3p on β-catenin ubiquitination level. The trophoblast attachment was detected by co-culturing JAR (choriocarcinoma cell line) spheroids with Ishikawa cell monolayers. miR-23a-3p was upregulated while CUL3 was downregulated in EECs at day 4 after pregnancy compared with day 1, as well as in hormone-induced Ishikawa cells. miR-23a-3p positively regulated the protein level of β-catenin without affecting the mRNA level. The ubiquitination and degradation of β-catenin was suppressed by miR-23a-3p, while it was promoted by CUL3. Immunoprecipitation confirmed the binding between CUL3 and β-catenin. Luciferase reporter assay confirmed the target relationship between miR-23a-3p and CUL3. The ubiquitination of β-catenin was modulated by the miR-23a-3p/CUL3 pathway. The overexpression of miR-23a-3p promoted JAR spheroid attachments in Ishikawa cells. miR-23a-3p is beneficial for the endometrial receptivity and embryo implantation, whose mechanism is partly through the modulation of CUL3/β-catenin.

Magnesium deficiency with high calcium-to-magnesium ratio promotes a metastatic phenotype in the CT26 colon cancer cell line.

Magnesium (Mg) plays important roles in maintaining genomic stability and cellular redox. Mg also serves as nature's physiological calcium (Ca) channel antagonist, controlling intracellular Ca entry. Because Ca is the most important second messenger, its intracellular concentration is tightly regulated. Excess intracellular Ca can activate aberrant signaling pathways leading to the acquisition of pathological characteristics and cell injury. Several epidemiological studies have linked Mg deficiency (MgD) and increased Ca:Mg ratios with higher incidences of colon cancer and increased mortality. While it is estimated that less than 50% of the US population consumes the recommended daily allowance for Mg, Ca supplementation is widespread. Therefore, we studied the effect of MgD, with variable Ca:Mg ratios on cellular oxidative stress, cell migration, calpain activity, and associated signaling pathways using the CT26 colon cancer cell line. MgD (with Ca:Mg ratios >1) elevated intracellular Ca levels, calpain activity and TRPM7 expression, as well as oxidative stress and cell migration, consistent with observed degradation of full-length E-cadherin, β-catenin, and N-terminal FAK. MgD was accompanied by enhanced degradation of IκBα and the transactivation domain containing the C-terminus of NF-κB p65 (RelA). MgD-exposed CT26 cells exhibited increased p53 degradation and aneuploidy, markers of genomic instability. By contrast, these pathological changes were not observed when CT26 were cultured under MgD conditions where the Ca:Mg ratio was kept at 1. Together, these data support that exposure of colon cancer cells to MgD with physiological Ca concentrations (or increasing Ca:Mg ratios) leads to the acquisition of a more aggressive, metastatic phenotype.

Ubiquitin-specific protease 49 attenuates IL-1β-induced rat primary chondrocyte apoptosis by facilitating Axin deubiquitination and subsequent Wnt/β-catenin signaling cascade inhibition.

Osteoarthritis (OA) is an age-related chronic joint degenerative disease. Interleukin 1 beta (IL-1β) is considered a marker for the progression of OA. In this study, we found that Ubiquitin-Specific Peptidase 49 (USP49) was significantly less expressed in OA patients compared with healthy individuals. Treating primary rat chondrocytes with different concentrations of IL-1β resulted in decreased Usp49 expression, while Usp49 overexpression could attenuate IL-1β-induced chondrocyte apoptosis by promoting Axin deubiquitination. The deubiquitination of Axin led to the accumulation of the protein, which in turn resulted in β-catenin degradation and Wnt/β-catenin signaling cascade inhibition. Interestingly, we also found that [6]-gingerol, an anti-OA drug, could upregulate the protein level of Usp49 and suppress the Wnt/β-catenin signaling cascade in primary rat chondrocytes. Taken together, our study not only demonstrates that Usp49 can negatively regulate the progression of OA by inhibiting the Wnt/β-catenin signaling cascade, but also elucidates the underlying molecular mechanisms.

Histone H4R3 symmetric di-methylation by Prmt5 protects against cardiac hypertrophy via regulation of Filip1L/β-catenin

Background and purposeAlthough histone lysine methylation has been extensively studied for their participation in pathological cardiac hypertrophy, the potential regulatory role of histone arginine methylation remains to be elucidated. The present study focused on H4R3 symmetric di-methylation (H4R3me2s) induced by protein arginine methyltransferase 5 (Prmt5), and explored its epigenetic regulation and underlying mechanisms in cardiomyocyte hypertrophy. Methods and results1. The expressions of Prmt5 and H4R3me2s were suppressed in cardiac hypertrophy models in vivo and in vitro; 2. Prmt5 silencing or its inhibitor EPZ, or knockdown of cooperator of Prmt5 (Copr5) to disrupt H4R3me2s, facilitated cardiomyocyte hypertrophy, whereas overexpression of wild type Prmt5 rather than the inactive mutant protected cardiomyocytes against hypertrophy; 3. ChIP-sequence analysis identified Filip1L as a target gene of Prmt5-induced H4R3me2s; 4. Knockdown or inhibition of Prmt5 impaired Filip1L transcription and subsequently prevented β-catenin degradation, thus augmenting cardiomyocyte hypertrophy. ConclusionsThe present study reveals that Prmt5-induced H4R3me2s ameliorates cardiomyocyte hypertrophy by transcriptional upregulation of Filip1L and subsequent enhancement of β-catenin degradation. Deficiency of Prmt5 and the resulting suppression of H4R3me2s might facilitate the development of pathological cardiac hypertrophy. Prmt5 might serve as a key epigenetic regulator in pathological cardiac hypertrophy.

New molecular insights into dual inhibitors of tankyrase as Wnt signaling antagonists: 3D-QSAR studies on 4H-1,2,4-triazole derivatives for the design of novel anticancer agents

Genetic mutations in APC or CNTBB1 gene with aberrant canonical Wnt/β-catenin pathway are responsible for more than 90% of colorectal carcinogenesis. Tankyrases (TNKS) are known to downregulate Wnt signaling by stabilizing AXIN protein through poly(ADP ribose)polymerization or PARSylation process and subsequently, promoting degradation of intracellular β-catenin. Tankyrase enzymes are modulated by a range of known inhibitors that bind individually to any of the nicotinamide or induced adenosine pockets or as dual binding antagonists. Hence, for designing dual tankyrase inhibitors as Wnt signaling antagonist; we carried out 3D-QSAR studies using a data set of 51 molecules of reported 3,4,5-trisubstituted 4H-1,2,4-triazole derivatives. These reported 51 molecules were divided into a training set (39 molecules) and test set (12 molecules), aligned and subjected to generate CoMFA, CoMSIA, and HQSAR models. CoMFA analysis showed q2 value of 0.694, r2ncv value of 0.991 and r2pred value of 0.641. Optimized CoMSIA analysis (SEHA) showed q2 value of 0.624, r2ncv value of 0.909 and r2pred value of 0.850. Both internal and external validations were performed for generated models of CoMFA and CoMSIA (SEHA) and satisfactory results were obtained. HQSAR analysis showed q2, r2, and r2pred values of 0.781, 0.901, and 0.811, respectively. Applicability domain was also found to be satisfactory with all compounds falling within the range and no outlier was observed. Contour maps from all studies provided significant results with identification of desired spatial arrangement of different atoms or functional groups in a molecule. Triazole ring system-based molecules were reported as potent tankyrase inhibitors. These noteworthy results were employed for the design of different triazole derivatives as potent tankyrase inhibitors, wherein a series of 20 different molecules were designed for evaluation of their potentials as novel tankyrase inhibitors.