Aberrant Glycogen Synthase Kinase 3β Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

Background and Purpose

The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer.

Methods

Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined.

Results

Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts.

Conclusion

The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer.     

Integrin-linked kinase regulates chondrocyte shape and proliferation

The interaction of chondrocytes with the extracellular-matrix environment is mediated mainly by integrins. Ligated integrins are recruited to focal adhesions (FAs) together with scaffolding proteins and kinases, such as integrin-linked kinase (Ilk). Ilk binds the cytoplasmic domain of β1-, β2- and β3-integrins and recruits adaptors and kinases, and is thought to stimulate downstream signalling events through phosphorylation of protein kinase B/Akt (Pkb/Akt) and glycogen synthase kinase 3-β (GSK3-β). Here, we show that mice with a chondrocyte-specific disruption of the gene encoding Ilk develop chondrodysplasia, and die at birth due to respiratory distress. The chondrodysplasia was characterized by abnormal chondrocyte shape and decreased chondrocyte proliferation. In addition, Ilk-deficient chondrocytes showed adhesion defects, failed to spread and formed fewer FAs and actin stress fibres. Surprisingly, phosphorylation of Pkb/Akt and GSK3-β is unaffected in Ilk-deficient chondrocytes. These findings suggest that Ilk regulates actin reorganization in chondrocytes and modulates chondrocyte growth independently of phosphorylation of Pkb/Akt and GSK3-β.     

Inactivation of GSK3β and activation of NF-κB pathway via Axl represents an important mediator of tumorigenesis in esophageal squamous cell carcinoma

The receptor tyrosine kinase Axl has been described as an oncogene, and its deregulation has been implicated in the progression of several human cancers. While the role of Axl in esophageal adenocarcinoma has been addressed, there is no information about its role in esophageal squamous cell carcinoma (OSCC). In the current report, we identified, for the first time, deregulation of Axl expression in OSCC. Axl is consistently overexpressed in OSCC cell lines and human tumor samples, mainly in advanced stages of the disease. Blockage of Axl gene expression by small interfering RNA inhibits cell survival, proliferation, migration, and invasion in vitro and esophageal tumor growth in vivo. Additionally, repression of Axl expression results in Akt-dependent inhibition of pivotal genes involved in the nuclear factor-kappaB (NF-κB) pathway and in the induction of glycogen synthase kinase 3β (GSK3β) activity, resulting in loss of mesenchymal markers and induction of epithelial markers. Furthermore, treatment of esophageal cancer cells with the Akt inhibitor wortmannin inhibits NF-κB signaling, induces GSK3β activity, and blocks OSCC cell proliferation in an Axl-dependent manner. Taken together, our results establish a clear role for Axl in OSCC tumorigenesis with potential therapeutic implications.     

FLZ Alleviates the Memory Deficits in Transgenic Mouse Model of Alzheimer’s Disease via Decreasing Beta-Amyloid Production and Tau Hyperphosphorylation

Alzheimer’s disease (AD) is the most common cause of dementia worldwide and mainly characterized by the aggregated β-amyloid (Aβ) and hyperphosphorylated tau. FLZ is a novel synthetic derivative of natural squamosamide and has been proved to improve memory deficits in dementia animal models. In this study, we aimed to investigate the mechanisms of FLZ’s neuroprotective effect in APP/PS1 double transgenic mice and SH-SY5Y (APPwt/swe) cells. The results showed that treatment with FLZ significantly improved the memory deficits of APP/PS1 transgenic mice and decreased apoptosis of SH-SY5Y (APPwt/swe) cells. FLZ markedly attenuated Aβ accumulation and tau phosphorylation both in vivo and in vitro. Mechanistic study showed that FLZ interfered APP processing, i.e., FLZ decreased β-amyloid precursor protein (APP) phosphorylation, APP-carboxy-terminal fragment (APP-CTF) production and β-amyloid precursor protein cleaving enzyme 1 (BACE1) expression. These results indicated that FLZ reduced Aβ production through inhibiting amyloidogenic pathway. The mechanistic study about FLZ’s inhibitory effect on tau phosphorylation revealed t the involvement of Akt/glycogen synthase kinase 3β (GSK3β) pathway. FLZ treatment increased Akt activity and inhibited GSK3β activity both in vivo and in vitro. The inhibitory effect of FLZ on GSK3β activity and tau phosphorylation was suppressed by inhibiting Akt activity, indicating that Akt/GSK3β pathway might be the possible mechanism involved in the inhibitory effect of FLZ on tau hyperphosphorylation. These results suggested FLZ might be a potential anti-AD drug as it not only reduced Aβ production via inhibition amyloidogenic APP processing pathway, but also attenuated tau hyperphosphoylation mediated by Akt/GSK3β.     

A Phosphoinositide 3-Kinase/Phospholipase Cgamma1 Pathway Regulates Fibroblast Growth Factor-Induced Capillary Tube Formation

Background

The fibroblast growth factors (FGFs) are key regulators of embryonic development, tissue homeostasis and tumour angiogenesis. Binding of FGFs to their receptor(s) results in activation of several intracellular signalling cascades including phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC)γ1. Here we investigated the basic FGF (FGF-2)-mediated activation of these enzymes in human umbilical vein endothelial cells (HUVECs) and defined their role in FGF-2-dependent cellular functions.

Methodology/Principal Findings

We show that FGF-2 activates PLCγ1 in HUVECs measured by analysis of total inositol phosphates production upon metabolic labelling of cells and intracellular calcium increase. We further demonstrate that FGF-2 activates PI3K, assessed by analysing accumulation of its lipid product phosphatidylinositol-3,4,5-P₃ using TLC and confocal microscopy analysis. PI3K activity is required for FGF-2-induced PLCγ1 activation and the PI3K/PLCγ1 pathway is involved in FGF-2-dependent cell migration, determined using Transwell assay, and in FGF-2-induced capillary tube formation (tubulogenesis assays in vitro). Finally we show that PI3K-dependent PLCγ1 activation regulates FGF-2-mediated phosphorylation of Akt at its residue Ser473, determined by Western blotting analysis. This occurs through protein kinase C (PKC)α activation since dowregulation of PKCα expression using specific siRNA or blockade of its activity using chemical inhibition affects the FGF-2-dependent Ser473 Akt phosphorylation. Furthermore inhibition of PKCα blocks FGF-2-dependent cell migration.

Conclusion/Significance

These data elucidate the role of PLCγ1 in FGF-2 signalling in HUVECs demonstrating its key role in FGF-2-dependent tubulogenesis. Furthermore these data unveil a novel role for PLCγ1 as a mediator of PI3K-dependent Akt activation and as a novel key regulator of different Akt-dependent processes.     

Impaired ILK Function Is Associated with Deficits in Hippocampal Based Memory and Synaptic Plasticity in a FASD Rat Model

Fetal Alcohol Spectrum Disorder (FASD) is an umbrella term that encompasses a wide range of anatomical and behavioral problems in children who are exposed to alcohol during the prenatal period. There is no effective treatment for FASD, because of lack of complete characterization of the cellular and molecular mechanisms underlying this condition. Alcohol has been previously characterized to affect integrins and growth factor signaling receptors. Integrin Linked Kinase (ILK) is an effector of integrin and growth-factor signaling which regulates various signaling processes. In FASD, a downstream effector of ILK, Glycogen Synthase Kinase 3β (GSK3β) remains highly active (reduced Ser⁹ phosphorylation). GSK3β has been known to modulate glutamate receptor trafficking and channel properties. Therefore, we hypothesize that the cognitive deficits accompanying FASD are associated with impairments in the ILK signaling pathway. Pregnant Sprague Dawley rats consumed a “moderate” amount of alcohol throughout gestation, or a calorie-equivalent sucrose solution. Contextual fear conditioning was used to evaluate memory performance in 32–33-day-old pups. Synaptic plasticity was assessed in the Schaffer Collateral pathway, and hippocampal protein lysates were used to evaluate ILK signaling. Alcohol exposed pups showed impaired contextual fear conditioning, as compared to control pups. This reduced memory performance was consistent with decrease in LTP as compared to controls. Hippocampal ILK activity and GSK3β Ser^21/9 phosphorylation were significantly lower in alcohol-exposed pups than controls. Increased synaptic expression of GluR2 AMPA receptors was observed with immunoprecipitation of post-synaptic density protein 95 (PSD95). Furthermore, immunoprecipitation of ILK revealed a decreased interaction with GluR2. The ILK pathway appears to play a significant role in memory and synaptic plasticity impairments in FASD rats. These impairments appear to be mediated by reduced GSK3β regulation and increased synaptic stabilization of the calcium-impermeable GluR2 AMPA receptors.     

CKIP-1 regulates macrophage proliferation by inhibiting TRAF6-mediated Akt activation

Macrophages play pivotal roles in development, homeostasis, tissue repair and immunity. Macrophage proliferation is promoted by macrophage colony-stimulating factor (M-CSF)-induced Akt signaling; yet, how this process is terminated remains unclear. Here, we identify casein kinase 2-interacting protein-1 (CKIP-1) as a novel inhibitor of macrophage proliferation. In resting macrophages, CKIP-1 was phosphorylated at Serine 342 by constitutively active GSK3β, the downstream target of Akt. This phosphorylation triggers the polyubiquitination and proteasomal degradation of CKIP-1. Upon M-CSF stimulation, Akt is activated by CSF-1R-PI3K and then inactivates GSK3β, leading to the stabilization of CKIP-1 and β-catenin proteins. β-catenin promotes the expression of proliferation genes including cyclin D and c-Myc. CKIP-1 interacts with TRAF6, a ubiquitin ligase required for K63-linked ubiquitination and plasma membrane recruitment of Akt, and terminates TRAF6-mediated Akt activation. By this means, CKIP-1 inhibits macrophage proliferation specifically at the late stage after M-CSF stimulation. Furthermore, CKIP-1 deficiency results in increased proliferation and decreased apoptosis of macrophages in vitro and CKIP-1^−/− mice spontaneously develop a macrophage-dominated splenomegaly and myeloproliferation. Together, these data demonstrate that CKIP-1 plays a critical role in the regulation of macrophage homeostasis by inhibiting TRAF6-mediated Akt activation.     

Glycogen synthase kinase 3β promotes osteosarcoma invasion and migration via regulating PTEN and phosphorylation of focal adhesion kinase

Aim: Typical features of human osteosarcoma are highly invasive and migratory capacities. Our study aimed to investigate the roles of glycogen synthase kinase 3β (GSK3β) in human osteosarcoma metastasis.Methods: GSK3β expressions in clinical osteosarcoma tissues with or without metastasis were examined by immunohistochemical staining. The expressions of GSK3β, p-GSK3β^Ser9, and p-GSK3β^Tyr216 in human osteoblast cells (hFOB1.19) and human osteosarcoma cells (MG63, SaOS-2, and U2-OS) were detected by Western blotting. The GSK3β activity was measured by non-radio isotopic in vitro kinase assay. Migration and invasion abilities of MG-63 cells treated with small-molecular GSK3β inhibitors were respectively examined by monolayer-based wound-healing assay and transwell assay. The mRNA expressions of GSK3β, matrix metalloproteinase-2 (MMP-2), MMP-9, phosphatase with tensin homology (PTEN), and focal adhesion kinase (FAK) were detected after siRNA transfection for 72 h. Meanwhile, protein expressions of GSK3β, FAK, p-FAK^Y397, PTEN, MMP-2, and MMP-9 were measured by Western blotting.Results: Clinical osteosarcoma tissues with metastasis showed higher GSK3β expressions. MG63 and U2-OS cells that were easy to occur metastasis showed significantly higher expressions and activities of GSK3β than SaOS-2 cells. Inhibition of GSK3β with small-molecular GSK3β inhibitors in MG63 cells significantly attenuated cell migration and invasion. These effects were associated with reduced expressions of MMP-2 and MMP-9. Moreover, increased PTEN and decreased p-FAK^Y397 expressions were observed following GSK3β knockdown by siRNA transfection. Conclusion: GSK3β might promote osteosarcoma invasion and migration via pathways associated with PTEN and phosphorylation of FAK.     

MicroRNA-34A inhibits the growth,invasion and metastasis of gastric cancer by targeting PDGFR and MET expression

Within the family of RTKs (receptor tyrosine kinases), PDGFR (platelet-derived growth factor receptor) has been implicated in carcinogenesis and tumour development. miRNAs (microRNAs), which can target the mRNAs (messenger RNAs) of cancer-associated genes, are abnormally expressed in various cancers. In this study, our aim was to identify the miRNAs that target PDGFR-α/β and to study the functions of these miRNAs. miR-34a was predicted to target PDGFR, and luciferase reporter assays showed that miR-34a could directly target PDGFR. Meanwhile, we found that miR-34a was down-regulated in gastric cancer tissues and was associated with metastasis. Our findings showed that miR-34a could inhibit gastric cancer cell migration, invasion and proliferation, but these tumourigenic properties were only partially restored when PDGFR-α/β was overexpressed. In subsequent experiments, we found that the overexpression of both PDGFR and MET could completely restore the gastric cancer tumourigenic properties. Moreover, the cancer-associated cell signalling pathway was studied, and we found that miR-34a could inhibit Akt [PKB (protein kinase B)] phosphorylation, which was restored by the overexpression of both PDGFR and MET. In conclusion, miR-34a may act as a potential tumour suppressor in gastric cancer and is associated with the mechanisms of gastric cancer metastasis; miR-34a can inhibit gastric cancer tumourigenesis by targeting PDGFR and MET through the PI3K (phosphoinositide 3-kinase)/Akt pathway.     

Altered Regulation of Akt Signaling with Murine Cerebral Malaria,Effects on Long-Term Neuro-Cognitive Function,Restoration with Lithium Treatment

Neurological and cognitive impairment persist in more than 20% of cerebral malaria (CM) patients long after successful anti-parasitic treatment. We recently reported that long term memory and motor coordination deficits are also present in our experimental cerebral malaria model (ECM). We also documented, in a murine model, a lack of obvious pathology or inflammation after parasite elimination, suggesting that the long-term negative neurological outcomes result from potentially reversible biochemical and physiological changes in brains of ECM mice, subsequent to acute ischemic and inflammatory processes. Here, we demonstrate for the first time that acute ECM results in significantly reduced activation of protein kinase B (PKB or Akt) leading to decreased Akt phosphorylation and inhibition of the glycogen kinase synthase (GSK3β) in the brains of mice infected with Plasmodium berghei ANKA (PbA) compared to uninfected controls and to mice infected with the non-neurotrophic P. berghei NK65 (PbN). Though Akt activation improved to control levels after chloroquine treatment in PbA-infected mice, the addition of lithium chloride, a compound which inhibits GSK3β activity and stimulates Akt activation, induced a modest, but significant activation of Akt in the brains of infected mice when compared to uninfected controls treated with chloroquine with and without lithium. In addition, lithium significantly reversed the long-term spatial and visual memory impairment as well as the motor coordination deficits which persisted after successful anti-parasitic treatment. GSK3β inhibition was significantly increased after chloroquine treatment, both in lithium and non-lithium treated PbA-infected mice. These data indicate that acute ECM is associated with abnormalities in cell survival pathways that result in neuronal damage. Regulation of Akt/GSK3β with lithium reduces neuronal degeneration and may have neuroprotective effects in ECM. Aberrant regulation of Akt/GSK3β signaling likely underlies long-term neurological sequelae observed in ECM and may yield adjunctive therapeutic targets for the management of CM.     

AIF inhibits tumor metastasis by protecting PTEN from oxidation

Apoptosis-inducing factor (AIF) exerts dual roles on cell death and survival, but its substrates as a putative oxidoreductase and roles in tumorigenesis remain elusive. Here, we report that AIF physically interacts with and inhibits the oxidation of phosphatase and tensin homolog on chromosome ten (PTEN), a tumor suppressor susceptible for oxidation-mediated inactivation. More intriguingly, we also identify PTEN as a mitochondrial protein and the ectopic expression of mitochondrial targeting sequence-carrying PTEN almost completely inhibits Akt phosphorylation in PTEN-deficient cells. AIF knockdown causes oxidation-mediated inactivation of the lipid phosphatase activity of PTEN, with ensuing activation of Akt kinase, phosphorylation of the Akt substrate GSK-3β, and activation of β-catenin signaling in cancer cells. Through its effect on β-catenin signaling, AIF inhibits epithelial–mesenchymal transition (EMT) and metastasis of cancer cells in vitro and in orthotopically implanted xenografts. Accordingly, the expression of AIF is correlated with the survival of human patients with cancers of multiple origins. These results identify PTEN as the substrate of AIF oxidoreductase and reveal a novel function for AIF in controlling tumor metastasis.     

Endoglin mediates fibronectin/α5β1 integrin and TGF-β pathway crosstalk in endothelial cells

Both the transforming growth factor β (TGF-β) and integrin signalling pathways have well-established roles in angiogenesis. However, how these pathways integrate to regulate angiogenesis is unknown. Here, we show that the extracellular matrix component, fibronectin, and its cellular receptor, α5β1 integrin, specifically increase TGF-β1- and BMP-9-induced Smad1/5/8 phosphorylation via the TGF-β superfamily receptors endoglin and activin-like kinase-1 (ALK1). Fibronectin and α5β1 integrin increase Smad1/5/8 signalling by promoting endoglin/ALK1 cell surface complex formation. In a reciprocal manner, TGF-β1 activates α5β1 integrin and downstream signalling to focal adhesion kinase (FAK) in an endoglin-dependent manner. α5β1 integrin and endoglin form a complex on the cell surface and co-internalize, with their internalization regulating α5β1 integrin activation and signalling. Functionally, endoglin-mediated fibronectin/α5β1 integrin and TGF-β pathway crosstalk alter the responses of endothelial cells to TGF-β1, switching TGF-β1 from a promoter to a suppressor of migration, inhibiting TGF-β1-mediated apoptosis to promote capillary stability, and partially mediating developmental angiogenesis in vivo. These studies provide a novel mechanism for the regulation of TGF-β superfamily signalling and endothelial function through crosstalk with integrin signalling pathways.