Biphasic Regulation of Caveolin-1 Gene Expression by Fluoxetine in Astrocytes: Opposite Effects of PI3K/AKT and MAPK/ERK Signaling Pathways on c-fos.
ABSTRACT: Previously, we reported that fluoxetine acts on 5-HT2B receptor and induces epidermal growth factor receptor (EGFR) transactivation in astrocytes. Recently, we have found that chronic treatment with fluoxetine regulates Caveolin-1 (Cav-1)/PTEN/PI3K/AKT/glycogen synthase kinase 3? (GSK-3?) signaling pathway and glycogen content in primary cultures of astrocytes with bi-phasic concentration dependence. At low concentrations fluoxetine down-regulates Cav-1 gene expression, decreases membrane content of PTEN, increases PI3K activity and increases phosphorylation of GSK-3? and increases its activity; at high concentrations fluoxetine acts on PTEN/PI3K/AKT/GSK-3? in an inverse fashion. Here, we present the data indicating that acute treatment with fluoxetine at lower concentrations down-regulates c-Fos gene expression via PI3K/AKT signaling pathway; in contrast at higher concentrations fluoxetine up-regulates c-Fos gene expression via MAPK/extracellular-regulated kinase (ERK) signaling pathway. However, acute treatment with fluoxetine has no effect on Cav-1 protein content. Similarly, chronic effects of fluoxetine on Cav-1 gene expression are suppressed by inhibitor of PI3K at lower concentrations, but by inhibitor of MAPK at higher concentrations, indicating that the mechanism underlying bi-phasic regulation of Cav-1 gene expression by fluoxetine is opposing effects of PI3K/AKT and MAPK/ERK signal pathways on c-Fos gene expression. The effects of fluoxetine on Cav-1 gene expression at both lower and higher concentrations are abolished by AG1478, an inhibitor of EGFR, indicating the involvement of 5-HT2B receptor induced EGFR transactivation as we reported previously. However, PP1, an inhibitor of Src only abolished the effect by lower concentrations, suggesting the relevance of Src with PI3K/AKT signal pathway during activation of EGFR.
Project description:The EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway plays prominent roles in malignant transformation, prevention of apoptosis, drug resistance and metastasis. The expression of this pathway is frequently altered in breast cancer due to mutations at or aberrant expression of: HER2, ERalpha, BRCA1, BRCA2, EGFR1, PIK3CA, PTEN, TP53, RB as well as other oncogenes and tumor suppressor genes. In some breast cancer cases, mutations at certain components of this pathway (e.g., PIK3CA) are associated with a better prognosis than breast cancers lacking these mutations. The expression of this pathway and upstream HER2 has been associated with breast cancer initiating cells (CICs) and in some cases resistance to treatment. The anti-diabetes drug metformin can suppress the growth of breast CICs and herceptin-resistant HER2+ cells. This review will discuss the importance of the EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway primarily in breast cancer but will also include relevant examples from other cancer types. The targeting of this pathway will be discussed as well as clinical trials with novel small molecule inhibitors. The targeting of the hormone receptor, HER2 and EGFR1 in breast cancer will be reviewed in association with suppression of the EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway.
Project description:Glycogen synthase kinase (GSK)-3? facilitates interferon (IFN)-? signaling by inhibiting Src homology-2 domain-containing phosphatase (SHP) 2. Mutated phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN) cause AKT activation and GSK-3? inactivation to induce SHP2-activated cellular unresponsiveness to IFN-? in human gastric cancer AGS cells. This study investigated the potential role of galectin-3, which acts upstream of AKT/GSK-3?/SHP2, in gastric cancer cells. Increasing or decreasing galectin-3 altered IFN-? signaling. Following cisplatin-induced galectin-3 upregulation, surviving cells showed cellular unresponsiveness to IFN-?. Galectin-3 induced IFN-? resistance independent of its extracellular ?-galactoside-binding activity. Galectin-3 expression was not regulated by PI3K activation or by a decrease in PTEN. Increased galectin-3 may cause GSK-3? inactivation and SHP2 activation by promoting PDK1-induced AKT phosphorylation at a threonine residue. Overexpression of AKT, inactive GSK-3?R96A, SHP2, or active SHP2D61A caused cellular unresponsiveness to IFN-? in IFN-?-sensitive MKN45 cells. IFN-?-induced growth inhibition and apoptosis in AGS cells were observed until galectin-3 expression was downregulated. These results demonstrate that an increase in galectin-3 facilitates AKT/GSK-3?/SHP2 signaling, causing cellular unresponsiveness to IFN-?.
Project description:The activity of the phosphatase and tensin homologue (PTEN) is known to be suppressed via post-translational modification. However, the mechanism and physiological significance by which post-translational modifications lead to PTEN suppression remain unclear. Here we demonstrate that PTEN destabilization is induced by EGFR- or oncogenic PI3K mutation-mediated AKT activation in cervical cancer. EGFR/PI3K/AKT-mediated ubiquitination and degradation of PTEN are dependent on the MKRN1 E3 ligase. These processes require the stabilization of MKRN1 via AKT-mediated phosphorylation. In cervical cancer patients with high levels of pAKT and MKRN1 expression, PTEN protein levels are low and correlate with a low 5-year survival rate. Taken together, our results demonstrate that PI3K/AKT signals enforce positive-feedback regulation by suppressing PTEN function.
Project description:Idiopathic Pulmonary Fibrosis is a lethal fibrotic disease characterized by the unrelenting proliferation and persistence of fibroblasts in a type I collagen-rich matrix that result in an expanding reticular network of fibrotic tissue. However, the underlying mechanism responsible for the persistence of myofibroblasts in IPF remains unclear. During normal tissue repair, unwanted fibroblasts are eliminated during collagen-matrix contraction by a mechanism whereby high PTEN activity suppresses Akt. We have previously found that FoxO3a, a transcriptional activator of apoptosis-inducing proteins, is inactivated in IPF fibroblasts resulting from aberrantly high PI3K/Akt activity due to inappropriately low PTEN activity. Here we demonstrate that this low FoxO3a activity confers IPF fibroblasts with resistance to collagen-mediated apoptosis. We show that the mechanism by which low FoxO3a activity confers IPF fibroblasts with an apoptotic resistant phenotype involves suppression of Fas expression as a result of down regulation of cav-1 expression via a PTEN/Akt-dependent pathway. We demonstrate that PTEN over-expression or Akt inhibition increases FoxO3a expression in IPF fibroblasts, resulting in up-regulation of caveolin-1. We show that FoxO3a binds to the cav-1 promoter region and ectopic expression of FoxO3a transcriptionally increases cav-1 mRNA and protein expression. In turn, we show that overexpression of caveolin-1 increases Fas levels and caspase-3/7 activity and promotes IPF fibroblast apoptosis on polymerized type I collagen. We have found that the expression of caveolin-1, Fas and cleaved caspase-3 proteins in fibroblasts within the fibroblastic foci of IPF patient specimens is low. Our data indicate that the pathologically altered PTEN/Akt axis inactivates FoxO3a down-regulating cav-1 and Fas expression. This confers IPF fibroblasts with an apoptosis-resistant phenotype and may be responsible for IPF progression.
Project description:The contribution of zinc-mediated neuronal death in the process of both acute and chronic neurodegeneration has been increasingly appreciated. Phosphatase and tensin homologue, deleted on chromosome 10 (PTEN), the major tumor suppressor and key regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, plays a critical role in neuronal death in response to various insults. NEDD4-1-mediated PTEN ubiquitination and subsequent degradation via the ubiquitin proteosomal system have recently been demonstrated to be the important regulatory mechanism for PTEN in several cancer types. We now demonstrate that PTEN is also the key mediator of the PI3K/Akt pathway in the neuronal response to zinc insult. We used primary cortical neurons and neuroblastoma N2a cells to show that zinc treatment results in a reduction of the PTEN protein level in parallel with increased NEDD4-1 gene/protein expression. The reduced PTEN level is associated with an activated PI3K pathway as determined by elevated phosphorylation of both Akt and GSK-3 as well as by the attenuating effect of a specific PI3K inhibitor (wortmannin). The reduction of PTEN can be attributed to increased protein degradation via the ubiquitin proteosomal system, as we show NEDD4-1 to be the major E3 ligase responsible for PTEN ubiquitination in neurons. Moreover, PTEN and NEDD4-1 appear to be able to counter-regulate each other to mediate the neuronal response to zinc. This reciprocal regulation requires the PI3K signaling pathway, suggesting a feedback loop mechanism. This study demonstrates that NEDD4-1-mediated PTEN ubiquitination is crucial in the regulation of PI3K/Akt signaling by PTEN during the neuronal response to zinc, which may represent a common mechanism in neurodegeneration.
Project description:MiR-19a, a member of mir-17-92 microRNA clusters, has been demonstrated to promote cell proliferation and angiogenesis via regulating the PI3K/AKT pathway, the major insulin signaling pathway. However, whether miR-19a plays an important role in glycogen synthesis in hepatocytes remains unknown. Here, we define the impact of miR-19a on glycogen synthesis and IL-6-induced reduced glycogenesis in hepatocytes and its underlying mechanisms. Our studies indicate that miR-19a was down-regulated in the livers of db/db mice and mice injected with IL-6, as well as mouse NCTC 1469 hepatocytes and HEP 1-6 hepatocytes treated by IL-6. We found that over-expression of miR-19a in NCTC 1469 cells and HEP 1-6 cells led to increased activation of the AKT/GSK pathway and synthesis of glycogen, whereas down-regulation of miR-19a impaired AKT/GSK phosphorylation and glycogenesis. Over-expression of miR-19a ameliorated IL-6-induced reduced glycogen synthesis in hepatocytes. Moreover, we identified PTEN as the target of miR-19a by a luciferase assay. Down-regulation of PTEN rescued the effects of miR-19a suppression on the activation of the AKT/GSK pathway and improved glycogenesis in NTC 1469 cells. These findings show for the first time that miR-19a might activate the AKT/GSK pathway and glycogenesis via down-regulation of PTEN expression.
Project description:Amplification of the gene encoding the epidermal growth factor (EGF) receptor (EGFR) occurs commonly in glioblastoma, leading to activation of downstream kinases including phosphatidylinositol 3'-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). Here, we show that phosphorylation of mTOR and its downstream substrate rpS6 (ribosomal protein S6) are robust biomarkers for the antiproliferative effect of EGFR inhibitors. Inhibition of EGFR signaling correlated with decreased abundance of phosphorylated mTOR (p-mTOR) and rpS6 (p-rpS6) in cells wild type for the gene encoding PTEN (phosphatase and tensin homolog on chromosome 10), a negative regulator of PI3K. In contrast, inhibition of EGFR signaling failed to affect p-mTOR or p-rpS6 in cells mutant for PTEN, which are resistant to EGFR inhibitors. Although the abundance of phosphorylated Akt (p-Akt) decreased in response to inhibition of EGFR signaling, Akt was dispensable for signaling between EGFR and mTOR. We identified an Akt-independent pathway linking EGFR to mTOR that was critically dependent on protein kinase C (PKC). Consistent with these observations, the abundance of EGFR generally correlated with phosphorylation of rpS6 and PKC in primary human glioblastoma tumors, and correlated poorly with phosphorylation of Akt. Inhibition of PKC led to decreased viability of glioma cells regardless of PTEN or EGFR status, suggesting that PKC inhibitors should be tested in glioma. These findings underline the importance of signaling between EGFR and mTOR in glioma, identify PKCalpha as essential to this network, and question the necessity of Akt as a critical intermediate coupling EGFR and mTOR in glioma.
Project description:Anthrax lethal toxin (LeTx) is a cytotoxic virulence factor that causes cell cycle arrest and cell death in various cell types. However, susceptibility to the cytotoxic effects varies depending on cell types. In proliferating monocytes, LeTx has only transient cytotoxic effects due to activation of the phosphoinositide 3-kinase (PI3K)-AKT-mediated adaptive responses. To date, the mechanism of LeTx in activating PI3K-AKT signaling axis is unknown. This study shows that the histone deacetylase 8 (HDAC8) is involved in activating PI3K-AKT signaling axis through down-regulating the phosphatase and tensin homolog 1 (PTEN) in human monocytic THP-1 cells. The HDAC8-specific activator TM-2-51 and inhibitor PCI-34051 enhanced and prevented, respectively, AKT activation and cell cycle progression in LeTx-treated cells. Furthermore, HDAC8 induced tri-methylation of histone H3 lysine 27 (H3K27me3), which is known to suppress PTEN expression, through at least in part down-regulating the H3K27me3 eraser Jumonji Domain Containing (JMJD) 3. Importantly, the JMJD3-specific inhibitor GSK-J4 induced AKT activation and protected cell cycle arrest in LeTx-treated cells, regardless the presence of HDAC8 activity. Collectively, this study for the first time demonstrated that HDAC8 activity determines susceptibility to cell cycle arrest induced by LeTx, through regulating the PI3K-PTEN-AKT signaling axis.
Project description:Squamous cell lung carcinoma (SCC) accounts for 30% of patients with NSCLC and to date, no molecular targeted agents are approved for this type of tumor. However, recent studies have revealed several oncogenic mutations in SCC patients, including an alteration of the PI3K/AKT pathway, i.e. PI3K point mutations and amplification, AKT mutations and loss or reduced PTEN expression. Prompted by our observation of a correlation between PTEN loss and FAK phosphorylation in a cohort of patients with stage IV SCC, we evaluated the relevance of PTEN loss in cancer progression as well as the efficacy of a new combined treatment with the pan PI3K inhibitor buparlisip and the FAK inhibitor defactinib. An increase in AKT and FAK phosphorylation, associated with increased proliferation and invasiveness, paralleled by the acquisition of mesenchymal markers, and overexpression of the oncomir miR-21 were observed in SKMES-1-derived cell clones with a stable reduction of PTEN. Notably, the combined treatment induced a synergistic inhibition of cell proliferation, and a significant reduction in cell migration and invasion only in cells with reduced PTEN. The molecular mechanisms underlying these findings were unraveled using a specific RTK array that showed a reduction in phosphorylation of key kinases such as JNK, GSK-3 ?/?, and AMPK-?2, due to the concomitant decrease in AKT and FAK activation. In conclusion, the combination of buparlisib and defactinib was effective against cells with reduced PTEN and warrants further studies as a novel therapeutic strategy for stage IV SCC patients with loss of PTEN expression.
Project description:Glycogen synthase kinase 3 (GSK-3) is a serine/threonine (S/T) protein kinase. Although GSK-3 originally was identified to have functions in regulation of glycogen synthase, it was subsequently determined to have roles in multiple normal biochemical processes as well as various disease conditions. GSK-3 is sometimes referred to as a moonlighting protein due to the multiple substrates and processes which it controls. Frequently, when GSK-3 phosphorylates proteins, they are targeted for degradation. GSK-3 is often considered a component of the PI3K/PTEN/AKT/GSK-3/mTORC1 pathway as GSK-3 is frequently phosphorylated by AKT which regulates its inactivation. AKT is often active in human cancer and hence, GSK-3 is often inactivated. Moreover, GSK-3 also interacts with WNT/?-catenin signaling and ?-catenin and other proteins in this pathway are targets of GSK-3. GSK-3 can modify NF-?B activity which is often expressed at high levels in cancer cells. Multiple pharmaceutical companies developed small molecule inhibitors to suppress GSK-3 activity. In addition, various natural products will modify GSK-3 activity. This review will focus on the effects of small molecule inhibitors and natural products on GSK-3 activity and provide examples where these compounds were effective in suppressing cancer growth.