GSK3β Silencing Research Articles

Glycogen Synthase Kinase-3 Beta (GSK3β) as a Potential Drug Target in Regulating Osteoclastogenesis: An Updated Review on Current Evidence.

Glycogen synthase kinase 3-beta (GSK3β) is a highly conserved protein kinase originally involved in glucose metabolism, insulin activity, and energy homeostasis. Recent scientific evidence demonstrated the significant role of GSK3β in regulating bone remodelling through involvement in multiple signalling networks. Specifically, the inhibition of GSK3β enhances the conversion of osteoclast progenitors into mature osteoclasts. GSK3β is recognised as a pivotal regulator for the receptor activator of nuclear factor-kappa B (RANK)/receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG), phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT), nuclear factor-kappa B (NF-κB), nuclear factor-erythroid 2-related factor 2 (NRF2)/Kelch-like ECH-associated protein 1 (KEAP1), canonical Wnt/beta (β)-catenin, and protein kinase C (PKC) signalling pathways during osteoclastogenesis. Conversely, the inhibition of GSK3β has been shown to prevent bone loss in animal models with complex physiology, suggesting that the role of GSK3β may be more significant in bone formation than bone resorption. Divergent findings have been reported regarding the efficacy of GSK3β inhibitors as bone-protecting agents. Some studies demonstrated that GSK3β inhibitors reduced osteoclast formation, while one study indicated an increase in osteoclast formation in RANKL-stimulated bone marrow macrophages (BMMs). Given the discrepancies observed in the accumulated evidence, further research is warranted, particularly regarding the use of GSK3β silencing or overexpression models. Such efforts will provide valuable insights into the direct impact of GSK3β on osteoclastogenesis and bone resorption.

Abstract 7192: Teriflunomide overcomes gemcitabine resistance by inhibiting the GSK3β/mTOR pathway in pancreatic cancer

Abstract Background: Pancreatic cancer (PC) is a lethal malignancy that is partly attributable to the acquisition of chemoresistance to conventional chemotherapies. Hence, developing strategies to overcome such chemoresistance is essential. Teriflunomide, an active metabolite of leflunomide, has been widely utilized in managing autoimmune disorders, and emerging evidence suggests that it can augment the efficacy of chemotherapy in cancer. We investigated the therapeutic effect of teriflunomide in overcoming resistance to Gemcitabine-based therapy in PC. Methods: We performed docking and genomewide transcriptomic profiling analyses to identify therapeutic targets of Teriflunomide associated with Gemcitabine resistance (Gem-R) in PC. Subsequently, we analyzed the TCGA dataset to determine the prognostic significance of candidate genes in PC patients. A series of experiments were performed in PC cells, and the key findings were validated in patient-derived 3D organoids and a Gem-R Mia-PaCa2 xenograft model. Results: Docking and transcriptomic profiling studies identified GSK3β, MAPK8, and MAPK9 as potential targets of Teriflunomide, which were significantly upregulated in Gem-R cell lines (P < 0.05). GSK3β was independently associated with poor overall and disease-free survival in PC patients. Combined Teriflunomide and Gemcitabine treatment exhibited superior synergistic anti-tumor potential, as evidenced by reducing PC cell proliferation, clonogenicity, invasion, and migration. The combined treatment with Teriflunomide and Gemcitabine primarily inhibited GSK3β phosphorylation, resulting in the silencing of the downstream AKT/mTOR pathway. Lentiviral-based silencing of GSK3β inhibited the AKT/mTOR pathway and reduced the IC50s of Gemcitabine in PC cells. Furthermore, co-administrating both drugs to Gem-R Mia-PaCa-2 xenograft mice significantly reduced tumor growth rates and volumes. These findings were recapitulated in patient-derived 3D-organoids, where the combined treatment resulted in fewer and smaller organoids vs. each drug individually (P < 0.05). Conclusions: We provide novel evidence that Teriflunomide helps overcome resistance to Gemcitabine by inhibiting the GSK3β/AKT/mTOR pathway, which could improve treatment outcomes in PC. Citation Format: Caiming Xu, Yuan Li, Silei Sui, Meiling Chen, Laleh Melstorm, Haiyong Han, Steven T. Rosen, Ajay Goel. Teriflunomide overcomes gemcitabine resistance by inhibiting the GSK3β/mTOR pathway in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7192.

Suxiao Jiuxin Pill alleviates myocardial ischemia–reperfusion injury through the ALKBH5/GSK3β/mTOR pathway

BackgroundMany studies have shown effective protection from myocardial ischemia–reperfusion injury (MIRI) in animal models, but few, if any, treatments have yielded a substantial reduction in clinical. Several studies showed significant therapeutic effects for the Chinese patent medicine Suxiao Jiuxin Pill (SJP) in MIRI, although the specific molecular mechanisms remain undefined. Recently, increasing evidence indicates an important role for m6A modification in autophagy regulation in MIRI, and SJP has not been investigated in this regard.MethodsIn vivo experiments were performed in a Wistar rat MIRI model. In vitro assays were conducted in hypoxia/reoxygenation (H/R)-treated H9c2 cells. H9c2 cells with ALKBH5 and GSK3β silencing were constructed by lentivirus transfection. TUNEL and Annexin V/PI assays were carried out for apoptosis detection. Then, m6A modification was detected with the EpiQuik m6A RNA methylation quantification kit, and GFP-RFP-LC3B was used to observe dynamic changes in autophagy. The autophagosome structure was assessed by Transmission electron microscopy. qPCR and immunoblot were performed for mRNA and protein analyses, receptively.ResultsSJP significantly mitigated MIRI in rats, reducing infarct size and myocardial apoptosis, and improving left ventricular function. In addition, SJP inhibited autophagy through the GSK3β/mTOR pathway in MIRI rats. In cultured H9c2 cells, SJP significantly inhibited H/R- related apoptosis and autophagic activity through the GSK3β/mTOR pathway. Additionally, SJP enhanced ALKBH5 expression in H/R cardiomyocytes, which is important in impaired m6A modification. Interestingly, ALKBH5 knockdown enhanced autophagy and apoptosis in H/R-induced cells, whereas SJP reversed these effects. Further experiments showed that autophagic activity and apoptosis enhanced by ALKBH5 deficiency are GSK3β/mTOR pathway dependent in H/R-treated H9c2 cells. After SJP administration the above effects were alleviated, suggesting SJP inhibited autophagy through the ALKBH5/GSK3β/mTOR pathway in H/R-induced cardiomyocytes. These effects of SJP were common to its two main constituents, including tetra-methylpyrazine (TMP) and borneol (BOR).ConclusionSJP improves MIRI in rats and alleviates autophagy and apoptosis in H9c2 cells through the ALKBH5/GSK3β/mTOR pathway, thanks to its two major constituents TMP and BOR.

GSK3β is a critical, druggable component of the network regulating the active NOTCH1 protein and cell viability in CLL

NOTCH1 alterations have been associated with chronic lymphocytic leukemia (CLL), but the molecular mechanisms underlying NOTCH1 activation in CLL cells are not completely understood. Here, we show that GSK3β downregulates the constitutive levels of the active NOTCH1 intracellular domain (N1-ICD) in CLL cells. Indeed, GSK3β silencing by small interfering RNA increases N1-ICD levels, whereas expression of an active GSK3β mutant reduces them. Additionally, the GSK3β inhibitor SB216763 enhances N1-ICD stability at a concentration at which it also increases CLL cell viability. We also show that N1-ICD is physically associated with GSK3β in CLL cells. SB216763 reduces GSK3β/N1-ICD interactions and the levels of ubiquitinated N1-ICD, indicating a reduction in N1-ICD proteasomal degradation when GSK3β is less active. We then modulated the activity of two upstream regulators of GSK3β and examined the impact on N1-ICD levels and CLL cell viability. Specifically, we inhibited AKT that is a negative regulator of GSK3β and is constitutively active in CLL cells. Furthermore, we activated the protein phosphatase 2 A (PP2A) that is a positive regulator of GSK3β, and has an impaired activity in CLL. Results show that either AKT inhibition or PP2A activation reduce N1-ICD expression and CLL cell viability in vitro, through mechanisms mediated by GSK3β activity. Notably, for PP2A activation, we used the highly specific activator DT-061, that also reduces leukemic burden in peripheral blood, spleen and bone marrow in the Eµ-TCL1 adoptive transfer model of CLL, with a concomitant decrease in N1-ICD expression. Overall, we identify in GSK3β a key component of the network regulating N1-ICD stability in CLL, and in AKT and PP2A new druggable targets for disrupting NOTCH1 signaling with therapeutic potential.

SiRNA Mediated GSK3β Knockdown Targets Insulin Signaling Pathway and Rescues Alzheimer's Disease Pathology: Evidence from In Vitro and In Vivo Studies.

Sporadic Alzheimer's disease (sAD) is a progressive neurodegenerative disorder with dysfunctional insulin signaling and energy metabolism. Emerging evidence suggests impairments in brain insulin responsiveness, glucose utilization, and energy metabolism may be major causes of amyloid precursor protein mishandling. The support for this notion comes from the studies wherein streptozotocin (STZ) induced brain insulin resistance in rodent model resulted in sAD-like neuropathology with cognitive decline. Our previous study showed a compromised insulin signaling pathway, glucose uptake, glucose metabolism, and energy homeostasis in STZ-induced glial-neuronal coculture and in vivo model of sAD. Various components of insulin signaling pathway were examined to understand the metabolic correlation, and GSK3β was selected for gene knockdown strategy to reverse sAD pathology based on the data. In the present study, we have synthesized carboxylated graphene oxide (GO) nanosheets functionalized with PEG and subsequently with polyethylenimine (PEI) to provide attachment sites for GSK3β siRNA. Our results showed that siRNA mediated knockdown of the GSK3β gene reduced expression of amyloid pathway genes (APP and BACE1), which was further confirmed by reduced amyloid beta (Aβ) levels in the in vitro STZ-induced sAD model. GSK3β knockdown also restored insulin signaling, AMPK and Mapk3 pathway by restoring the expression of corresponding candidate genes in these pathways (IR, Glut1/3, Prkaa1/2, Mapk3, BDNF) that reflected improved cellular energy homeostasis, neuronal proliferation, differentiation, maturation, and repair. Behavioral data from Morris water maze (MWM), open field (OF), novel object recognition (NOR), Y maze, and radial arm maze (RAM) tests showed that 0.5 μg nanoformulation (GOc-PP-siRNAGSK3β) intranasally for 7 days improved spatial memory, rescued anxiety like behavior, improved visual and working memory, and rescued exploratory behavior in STZ-induced sAD rats. GSK3β silencing resulted in decreased BACE1 expression and prevented accumulation of Aβ in the cortex and hippocampus. These molecular findings with improved behavioral performances were further correlated with reduced amyloid beta (Aβ) and neurofibrillary tangle (NFTs) formation in the cortex and hippocampus of GOc-PP-siRNAGSK3β administered sAD rats. Therefore, it is conceivable from the present study that nanoparticle-mediated targeting of GSK3β in the sAD appears to be a promising strategy to reverse sAD pathology.

Exendin-4 Reversed the PC12 Cell Damage Induced by circRNA CDR1as/miR-671/GSK3β Signaling Pathway.

The purpose of this paper is to study the effect of circRNA cerebellar degeneration-related protein 1 antisense RNA(CDR1as)/miR-671/GSK3β signaling pathway on PC12 cell injury and the mechanism of Exendin-4 (Ex-4) in PC12 cell injury protection. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to detect the expression levels of circular RNA CDR1as and miR-671 in PC12 cells. By overexpressing or knocking out CDR1as, miR-671, and GSK3β, the role of CDR1as, miR-671, and GSK3β in PC12 cell injury was analyzed. The binding of CDR1as to miR-671 and GSK3β to miR-671 was verified by dual luciferase reporter assay. PC12 cells were treated with 1-methyl-4 phenyl-pyridine ion (MPP+) to construct a PC12 cell damage model. PC12 cell transfection experiments were used to confirm the role of CDR1as/miR-671/GSK3β signal axis in PC12 cell damage, and the role of Ex-4 in the association of circRNA CDR1as/miR-671/GSK3β signaling axis and PC12 cell damage. PC12 cell damage was detected by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cellular lactate dehydrogenase (LDH) release. Ex-4 reversed the phosphorylation levels of PI3K, AKT, and GSK-3β in MPP+-treated PC12 cells, and reduced MPP+-induced PC12 cell damage. CircRNA CDR1as upregulated the expression of GSK3β by sponge miR-671. Ex-4 downregulated CDR1as expression and upregulated miR-671 expression in MPP+-induced PC12 cell. Silencing of CDR1as reduced MPP+-induced PC12 cell damage. CDR1as transfection downregulated the expression of miR-671 in PC12 cells, promoted the expression and phosphorylated of GSK3β, and induced PC12 cell damage. GSK3β silencing reversed CDR1as-induced PC12 cell damage. CDR1as promoted the phosphorylation level of GSK3β in PC12 cells to cause cell damage; Ex-4 reversed the phosphorylation of GSK3β caused by CDR1as in PC12 cells and reduced the PC12 cell damage caused by CDR1as. Ex-4 reverses the damage of PC12 cells induced by CDR1as/miR-671/GSK3β signaling pathway.

GLP-1 promotes osteogenic differentiation of human ADSCs via the Wnt/GSK-3β/β-catenin pathway

Glucagon-like peptide-1 (GLP-1) analogues are promising anti-diabetic drugs which had been shown to have beneficial effects on bone metabolism in clinical practice, but the molecular mechanism remains unclear. In this study, we evaluated whether GLP-1 can affect the “intestine-fat-bone axis” via the Wnt/GSK-3β/β-catenin pathway. We established a diabetic mouse model and then treated mice with GLP-1 analogue liraglutide. The results showed that after liraglutide treatment, glucose tolerance and insulin tolerance were significantly improved in diabetic mice as expected. Moreover, osteogenic markers such as collagenⅠ, Runx2 and OCN were upregulated; and the adipogenic differentiation markers C/EBP-α and PPAR-γ were downregulated, these results indicated that liraglutide could ameliorate the osteogenic metabolism in diabetic mice. In the cell model, human ADSCs (hADSCs) were cultured and induced to undergo osteogenic and adipogenic differentiation under high glucose conditions in vitro and then treated with GLP-1. The results showed that GLP-1 repressed the induction of adipocyte differentiation biomarkers and the secretion of GSK-3β in a dose-dependent manner. In addition, GLP-1 enhanced the expression of osteoblastogenic biomarkers, such as OCN, Runx2 and collagenⅠ, and promoted osteoblastic mineralization. These effects were substantially suppressed by the Wnt signal recombinant human DKK-1 or activated by Wnt pathway agonist LiCl. Silencing of GSK-3β showed that the levels of β-catenin, GSK-3β and Runx2 were significantly increased by 2.46-, 2.05-, 4.44-fold after GLP-1 treatment compared to that observed in the GSK-3β lentiviral group, respectively. We conclude that GLP-1 promotes the osteogenic differentiation of hADSCs via the Wnt/GSK-3β/β-catenin pathway.

Glycogen synthase kinase 3-β inhibition induces lymphangiogenesis through β-catenin-dependent and mTOR-independent pathways.

Lymphatic vessels play an important role in health and in disease. In this study, we evaluated the effects of GSK3-β inhibition on lung lymphatic endothelial cells in vitro. Pharmacological inhibition and silencing of GSK3-β resulted in increased lymphangiogenesis of lung lymphatic endothelial cells. To investigate mechanisms of GSK3-β-mediated lymphangiogenesis, we interrogated the mammalian/mechanistic target of rapamycin pathway and found that inhibition of GSK3-β resulted in PTEN activation and subsequent decreased activation of AKT, leading to decreased p-P70S6kinase levels, indicating inhibition of the mTOR pathway. In addition, consistent with a negative role of GSK3-β in β-catenin stability through protein phosphorylation, we found that GSK3-β inhibition resulted in an increase in β-catenin levels. Simultaneous silencing of β-catenin and inhibition of GSK3-β demonstrated that β-catenin is required for GSK3-β-induced lymphangiogenesis.

Silencing GSK3β instead of DKK1 can inhibit osteogenic differentiation caused by co-exposure to fluoride and arsenic

Chronic exposure to combined fluoride (F) and arsenic (As) continues to be a major public health problem worldwide, attracting the attention of an increasing number of researchers. While bone is the main target organ of syndrome of endemic arsenic poisoning and fluorosis (SEAF), the specific mechanism and targeted intervention remains uncertain. The first question in this study sought to determine the interaction of F and As on the Wnt signaling pathway and its role in osteogenic differentiation in the SEAF population. As can be seen from the data, with the increase in exposure to F, the content of Wnt signaling inhibitor Dickkopf-related protein 1 (DKK1) gradually decreased, but the expression of glycogen synthase kinase-3β (GSK3β), β-catenin and the osteogenic differentiation indicators pro-collagen I alpha 1 (COL1A1) and bone alkaline phosphatase (BALP) were increased. Next, we grouped the SEAF population according to urinary As and found that As can upregulate the GSK3β, β-catenin level and the bone formation bio-marker BALP in serum. But the experiments did not detect any evidence that As can change the content of DKK1 in serum. To better understand the combined effects of F and As on the Wnt signaling pathway, we performed further interaction analysis. These results suggest that the interaction of F and As can inhibit the GSK3β, β-catenin, COL1A1 and BALP. And DKK1 is mainly manifested by the independent effect of F. To further study the role of DKK1 and GSK3β in fluoride-arsenic pollution combined with osteogenic differentiation, we attempted to silence the DKK1 and GSK3β gene in hFOB 1.19 cells. The results show that F, As alone and in combination exposure can up-regulate GSK catenin transcription and protein expression levels and down-regulate DKK1, and COL1A1 and ALP are significantly increased, after silenced the DKK1. The same results did not appear after silenced the GSK3β. F and As alone and in combination exposure did not reverse the inhibition of GSK3β and β-catenin by GSK3β silencing, and COL1A1 and ALP are significantly decreased. The results indicate that silencing GSK3β instead of DKK1 can inhibit osteogenic differentiation caused by co-exposure to fluoride and arsenic. This study can provide a scientific basis for further understanding the causes of bone formation caused by F and As and the improvement of targeted intervention strategies.

Rhein protects the myocardiac cells against hypoxia/reoxygention-induced injury by suppressing GSK3β activity

BackgroundRhein, an anthraquinone compound isolated from rhubarb, has been shown to protect the pancreatic β cells from hyperglycemia induced apoptosis in our previous studies. PurposeIn the present study, we examined whether rhein can protect myocardial cells against ischemia reperfusion (I/R)-induced apoptosis and investigated the underlying mechanism. MethodsWe used an in vitro model of myocardial hypoxia/reoxygenation (H/R) injury. H9c2 cells were incubated with rhein for 1 h and then subjected to hypoxia for 6 h, followed by reoxygenation for 2 h. Cells viability, apoptosis and ROS were assayed for the treated cells. AKT, p-AKT, GSK3β, p- GSK3β, P38 and p-P38 proteins were analyzed using Western blotting. PI3K/AKT inhibitor, LY294002, and GSK3β siRNA were also used to determine the signaling pathways involved in the protection by rhein. ResultsRhein increased viability, decreased apoptosis and ROS production, of the cells that were exposed to H/R. Rhein also increased the phosphorylation of AKT and GSK3β, an effect that was eliminated by LY294002. GSK3β silencing by siRNA showed similar effect as LY294002. The p-P38 level was upregulated by H/R and downregulated in the presence of rhein; however, the p-P38 downregulation was completely abolished by GSK3β silencing. ConclusionRhein protects myocardial H9c2 cells against hypoxia/reoxygenation induced injury via AKT/ GSK3β/p38 pathway.

Environmental Enrichment and Social Isolation Mediate Neuroplasticity of Medium Spiny Neurons through the GSK3 Pathway

SUMMARYResilience and vulnerability to neuropsychiatric disorders are linked to molecular changes underlying excitability that are still poorly understood. Here, we identify glycogen-synthase kinase 3β (GSK3β) and voltage-gated Na+ channel Nav1.6 as regulators of neuroplasticity induced by environmentally enriched (EC) or isolated (IC) conditions—models for resilience and vulnerability. Transcriptomic studies in the nucleus accumbens from EC and IC rats predicted low levels of GSK3β and SCN8A mRNA as a protective phenotype associated with reduced excitability in medium spiny neurons (MSNs). In vivo genetic manipulations demonstrate that GSK3β and Nav1.6 are molecular determinants of MSN excitability and that silencing of GSK3β prevents maladaptive plasticity of IC MSNs. In vitro studies reveal direct interaction of GSK3β with Nav1.6 and phosphorylation at Nav1.6T1936 by GSK3β. A GSK3β-Nav1.6T1936 competing peptide reduces MSNs excitability in IC, but not EC rats. These results identify GSK3β regulation of Nav1.6 as a biosignature of MSNs maladaptive plasticity.

Abstract B83: The role of inflammatory pathways on cell survival and epithelial-mesenchymal transition in head and neck cancer

Abstract Like many solid cancers, in head and neck cancers (HNC) the presence of metastases is a well-known prognostic factor associated with worst survival. Studies to date indicate that chronic stimulation with tumor necrosis factor alpha (TNF-α) has the potential to enhance several tumor cell characteristics, such as cell survival, proliferation, and migration, promoting metastasis. It is believed that TNF-α stimulation leads to the activation of different inflammatory pathways, such as the nuclear factor-kappa B (NF-kB) and AKT pathway, inhibiting GSK-3β-mediated phosphorylation and degradation of Snail and β-catenin, stabilizing them and contributing to the process of epithelial-mesenchymal transition (EMT), through which metastasis occurs. By targeting distinct components mediating these pathways, microRNAs can post-transcriptionally regulate these biologic processes, playing an important role in cancer progression. By using quantitative fluorescence microscopy (High-Content Analysis--HCA), we sought to investigate the roles of the aforementioned signaling pathways and of selected microRNAs, in EMT of HNC. To do so, FADU cells derived from an oropharynx HNC were transfected with microRNAs or siRNAs against different components of inflammatory pathways (P65/RelA, AKT1, GSK3β, and beta-catenin), followed by stimulation with TNF-α (20 ng/mL) for a period of 72 h. Cells were immunostained using nucleic acid and cytoplasmic dyes, as well as antibodies against markers of cell cycle (Cyclin B1), apoptosis (cleaved Caspase-7), and EMT (N-Cadherin, Vimentin and SNAIL1/2). TNF-α stimulation altered several phenotypic features associated with EMT, including loss of cell-cell contact, acquisition of fibroblast morphology, increased expression of all EMT markers evaluated, as well as reduced expression of E-cadherin. In line, TNF-α stimulation enhanced the migratory potential of nonproliferating mitomycin-treated FADU cells (HCA cell migration assay, Oris Pro). The siRNA-knockdown of RelA and AKT1 led to a reduction in the nuclear levels of SNAIL, while silencing of GSK3β increased its levels. Additionally, in the absence of TNF-α stimulation, silencing of inflammatory pathway components led to a reduction in the number of cells. Importantly, silencing of RelA and also AKT1 had a major impact in cell survival, drastically increasing the percentage of apoptotic cells and reducing cell numbers to a level similar to a control cytotoxic siRNA against ubiquitin. Functional evaluation of the 30 microRNAs revealed that some of them targeted elements of the evaluated pathways, promoting or repressing the phenotypic changes associated with TNF-induced EMT. Our results provide evidence of the molecular mechanisms mediating TNF-α-induced epithelial-mesenchymal transition in a well-established head and neck cancer cell model. Moreover, we highlight potential microRNA targets, whose post-transcriptional regulation is at least partially responsible for the modulation of the EMT process. Altogether, our work provides further understanding of the mechanisms contributing to cancer metastasis, and may be further explored in order to develop new therapeutic agents for head and neck cancer. Citation Format: Bruno Sangiorgi, Ildercílio Mota de Souza Lima, Josiane Lilian Schiavinato, Wilson Araújo Silva, Jr., Dimas Tadeu Covas, Marco Antônio Zago, Rodrigo Alexandre Panepucci. The role of inflammatory pathways on cell survival and epithelial-mesenchymal transition in head and neck cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B83.

The regulation of Hh/Gli1 signaling cascade involves Gsk3\u03b2- mediated mechanism in estrogen-derived endometrial hyperplasia

The present study was undertaken to explore the functional involvement of Hh signaling and its regulatory mechanism in endometrial hyperplasia. Differential expression of Hh signaling molecules i.e., Ihh, Shh, Gli1 or Gsk3β was observed in endometrial hyperplasial (EH) cells as compared to normal endometrial cells. Estradiol induced the expression of Hh signaling molecules and attenuated the expression of Gsk3β whereas anti-estrogen (K1) or progestin (MPA) suppressed these effects in EH cells. Cyclopamine treatment or Gli1 siRNA knockdown suppressed the growth of EH cells and reduced the expression of proliferative markers. Estradiol also induced the nuclear translocation of Gli1 which was suppressed by both MPA and K1 in EH cells. While exploring non-canonical mechanism, LY-294002 (Gsk3β activator) caused a decrease in Gli1 expression indicating the involvement of Gsk3β in Gli1 regulation. Further, Gsk3β silencing promoted the expression and nuclear translocation of Gli1 demonstrating that Gsk3β serves as a negative kinase regulator of Gli1 in EH cells. Similar attenuation of Hh signaling molecules was observed in rats with uterine hyperplasia undergoing anti-estrogen treatment. The study suggested that Hh/Gli1 cascade (canonical pathway) as well as Gsk3β-Gli1 crosstalk (non-canonical pathway) play crucial role in estrogen-dependent cell proliferation in endometrial hyperplasia.

MiR-26a enhances invasive capacity by suppressing GSK3β in human lung cancer cells

Lung cancer is the common cause of death from cancer, and most lung cancer patients die of metastasis. MicroRNAs (miRNAs) function as either oncogenes or tumor suppressors, playing crucial role not only in tumorigenesis, but also in tumor invasion and metastasis. There are several studies showed that miR-26a is involved in carcinogenesis, however, its role in tumor metastasis need to be elucidated. In this study, we showed that ectopic expression of miR-26a enhanced migration and invasion of lung cancer cells. Glycogen synthase kinase-3β (GSK3β) was identified as a direct target of miR-26a. GSK3β expression negatively correlated with miR-26a expression in lung cancer tissues. Silencing of GSK3β achieved similar effect as miR-26a over-expression; over-expression of GSK3β reversed the enhanced effect of miR-26a on lung cancer cell migration and invasion. Further study indicated that miR-26a increased β-catenin expression and nuclear translocation. C-myc and cyclin D1, the downstream genes of β-catenin, were also up-regulated by miR-26a. Furthermore, xenograft study showed that miR-26a promoted lung cancer cell growth in vivo, and suppressed GSK3β expression. Collectively, our results demonstrated that miR-26a enhanced metastatic potential of lung cancer cells via activation of β-catenin pathway by targeting GSK3β, suggesting the potential applicability of miR-26a as a target for cancer treatment.

RNA Interference Silencing of Glycogen Synthase Kinase 3β Inhibites Tau Phosphorylation in Mice with Alzheimer Disease.

To explore the effect of glycogen synthase kinase 3β (GSK-3β) silencing on Tau-5 phosphorylation in mice suffering Alzheimer disease (AD). GSK-3β was firstly silenced in human neuroblastoma SH-SY5Y cells using special lentivirus (LV) and the content of Tau (A-12), p-Tau (Ser396) and p-Tau (PHF-6) proteins. GSK-3β was also silenced in APP/PS1 mouse model of AD mice, which were divided into three groups (n = 10): AD, vehicle, and LV group. Ten C57 mice were used as control. The memory ability of mice was tested by square water maze, and the morphological changes of hippocampus and neuron death were analyzed by haematoxylin-eosin staining. Moreover, the levels of Tau and phosphorylated Tau (p-Tau) were detected by western blotting and immunohistochemistry, respectively. The lentivirus-mediated GSK-3β silencing system was successfully developed and silencing GSK-3β at the cellular level reduced Tau phosphorylation obviously. Moreover, GSK-3β silence significantly improved the memory ability of AD mice in LV group compared with AD group (P < 0.05) according to the latency periods and error numbers. As for the hippocampus morphology and neuron death, no significant change was observed between LV group and normal control. Immunohistochemical detection and western blotting revealed that the levels of Tau and p-Tau were significantly down-regulated after GSK-3β silence. Silencing GSK-3β may have a positive effect on inhibiting the pathologic progression of AD through down-regulating the level of p-Tau.

GSK3β inhibition protects the immature brain from hypoxic-ischaemic insult via reduced STAT3 signalling

Hypoxic-ischaemic (HI) injury is an important cause of neurological morbidity in neonates. HI leads to pathophysiological responses, including inflammation and oxidative stress that culminate in cell death. Activation of glycogen synthase kinase 3β (GSK3β) and the signal transducer and activator of transcription (STAT3) promotes brain inflammation.The purpose of this study was to test whether inhibition of GSK3β signalling protects against neonatal HI brain injury. Mice were subjected to HI at postnatal day (PND) 9 and treated with a selective GSK3β inhibitor, SB216763. Brain injury and caspase-3 activation, anti-oxidant and inflammatory mRNA responses and activation of STAT3 were analysed. Our results show that HI reduced phosphorylation of GSK3β, thus promoting its kinase activity. The GSK3β inhibitor reduced caspase-3 activation and neuronal cell death elicited by HI and reverted the effects of HI on gene expression of the anti-oxidant enzyme sod2 and mitochondrial factor pgc1α. The HI insult activated STAT3 in glial cells and GSK3β inhibition attenuated STAT3 phosphorylation and its nuclear translocation following HI. Further, GSK3β inhibition reduced HI-induced gene expression of pro-inflammatory cytokines tnfα and Il-6, while promoted the anti-inflammatory factor Il-10.In summary, data show that GSK3β inhibition is neuroprotective in neonatal HI brain injury likely via reduced pro-inflammatory responses by blocking STAT3 signalling. Our study suggests that pharmacological interventions built upon GSK3β silencing strategies could represent a novel therapy in neonatal brain injury.

MiRNA-99b-3p functions as a potential tumor suppressor by targeting glycogen synthase kinase-3β in oral squamous cell carcinoma Tca-8113 cells.

Dysregulation of microRNAs (miRNAs) has been associated with carcinogenesis in oral squamous cell carcinoma (OSCC). In the present study, we investigated the expression and function of miR-99b-3p in human OSCC. We found that the expression levels of miR-99b-3p decreased in 21 clinical OSCC samples (84%). Furthermore, ectopic expression of miR-99b-3p inhibited OSCC cell proliferation by downregulating glycogen synthase kinase-3β (GSK3β), an miR-99b-3p' target gene, at the mRNA and protein levels, both in vitro and in vivo. Moreover, the silencing of GSK3β recapitulated the cellular and molecular effects in a similar manner to the overexpression of miR-99b-3p, which included inhibition of OSCC cell proliferation and suppression of p65 (RelA) and G1 regulators (cyclin D1, CDK4 and CDK6) in vitro. Our data suggest that miR-99b-3p functions as a tumor suppressor in OSCC via GSK3β downregulation.

Lentiviral silencing of GSK-3β in adult dentate gyrus impairs contextual fear memory and synaptic plasticity.

Attempts have been made to use glycogen synthase kinase-3 beta (GSK3β) inhibitors for prophylactic treatment of neurocognitive conditions. However the use of lithium, a non-specific inhibitor of GSK3β results in mild cognitive impairment in humans. The effects of global GSK3β inhibition or knockout on learning and memory in healthy adult mice are also inconclusive. Our study aims to better understand the role of GSK3β in learning and memory through a more regionally, targeted approach, specifically performing lentiviral-mediated knockdown of GSK3β within the dentate gyrus (DG). DG-GSK3β-silenced mice showed impaired contextual fear memory retrieval. However, cue fear memory, spatial memory, locomotor activity and anxiety levels were similar to control. These GSK3β-silenced mice also showed increased induction and maintenance of DG long-term potentiation (DG-LTP) compared to control animals. Thus, this region-specific, targeted knockdown of GSK3β in the DG provides better understanding on the role of GSK3β in learning and memory.

Ethanol-induced transcriptional activation of programmed cell death 4 (Pdcd4) is mediated by GSK-3β signaling in rat cortical neuroblasts.

Ingestion of ethanol (ETOH) during pregnancy induces grave abnormalities in developing fetal brain. We have previously reported that ETOH induces programmed cell death 4 (PDCD4), a critical regulator of cell growth, in cultured fetal cerebral cortical neurons (PCNs) and in the cerebral cortex in vivo and affect protein synthesis as observed in Fetal Alcohol Spectrum Disorder (FASD). However, the mechanism which activates PDCD4 in neuronal systems is unclear and understanding this regulation may provide a counteractive strategy to correct the protein synthesis associated developmental changes seen in FASD. The present study investigates the molecular mechanism by which ethanol regulates PDCD4 in cortical neuroblasts, the immediate precursor of neurons. ETOH treatment significantly increased PDCD4 protein and transcript expression in spontaneously immortalized rat brain neuroblasts. Since PDCD4 is regulated at both the post-translational and post-transcriptional level, we assessed ETOH’s effect on PDCD4 protein and mRNA stability. Chase experiments demonstrated that ETOH does not significantly impact either PDCD4 protein or mRNA stabilization. PDCD4 promoter-reporter assays confirmed that PDCD4 is transcriptionally regulated by ETOH in neuroblasts. Given a critical role of glycogen synthase kinase 3β (GSK-3β) signaling in regulating protein synthesis and neurotoxic mechanisms, we investigated the involvement of GSK-3β and showed that multifunctional GSK-3β was significantly activated in response to ETOH in neuroblasts. In addition, we found that ETOH-induced activation of PDCD4 was inhibited by pharmacologic blockade of GSK-3β using inhibitors, lithium chloride (LiCl) and SB-216763 or siRNA mediated silencing of GSK-3β. These results suggest that ethanol transcriptionally upregulates PDCD4 by enhancing GSK-3β signaling in cortical neuroblasts. Further, we demonstrate that canonical Wnt-3a/GSK-3β signaling is involved in regulating PDCD4 protein expression. Altogether, we provide evidence that GSK-3β/PDCD4 network may represent a critical modulatory point to manage the protein synthetic anomalies and growth aberrations of neural cells seen in FASD.

Regulation of MDA-MB-231 cell proliferation by GSK-3β involves epigenetic modifications under high glucose conditions

Hyperglycemia is a critical risk factor for development and progression of breast cancer. We have recently reported that high glucose induces phosphorylation of histone H3 at Ser 10 as well as de-phosphorylation of GSK-3β at Ser 9 in MDA-MB-231 cells. Here, we elucidate the mechanism underlying hyperglycemia-induced proliferation in MDA-MB-231 breast cancer cells. We provide evidence that hyperglycemia led to increased DNA methylation and DNMT1 expression in MDA-MB-231 cells. High glucose condition led to significant increase in the expression of PCNA, cyclin D1 and decrease in the expression of PTPN 12, p21 and PTEN. It also induced hypermethylation of DNA at the promoter region of PTPN 12, whereas hypomethylation at Vimentin and Snail. Silencing of GSK-3β by siRNA prevented histone H3 phosphorylation and reduced DNMT1 expression. We show that chromatin obtained after immunoprecipitation with phospho-histone H3 was hypermethylated under high glucose condition, which indicates a cross-talk between DNA methylation and histone H3 phosphorylation. ChIP-qPCR analysis revealed up-regulation of DNMT1 and metastatic genes viz. Vimentin, Snail and MMP-7 by phospho-histone H3, which were down-regulated upon GSK-3β silencing. To the best of our knowledge, this is the first report which shows that interplay between GSK-3β activation, histone H3 phosphorylation and DNA methylation directs proliferation of breast cancer cells.