Inhibition of SH2-domain containing inositol phosphatase 2 (SHIP2) in insulin producing INS1E cells improves insulin signal transduction and induces proliferation

Inhibition of the lipid phosphatase SH2-domain containing inositol phosphatase 2 (SHIP2) in L6-C10 muscle cells, in 3T3-L1 adipocytes and in the liver of db/db mice has been shown to ameliorate insulin signal transduction and established SHIP2 as a negative regulator of insulin action. Here we show that SHIP2 inhibition in INS1E insulinoma cells increased Akt, glycogen synthase kinase 3 and extracellular signal-regulated kinases 1 and 2 phosphorylation. SHIP2 inhibition did not prevent palmitate-induced apoptosis, but increased cell proliferation. Our data raise the interesting possibility that SHIP2 inhibition exerts proliferative effects in beta-cells and further support the attractiveness of a specific inhibition of SHIP2 for the treatment of type 2 diabetes.     

The novel mTORC1/2 dual inhibitor INK-128 suppresses survival and proliferation of primary and transformed human pancreatic cancer cells

Pancreatic cancer has one of worst prognosis among all human malignancies around the world, the development of novel and more efficient anti-cancer agents against this disease is urgent. In the current study, we tested the potential effect of INK-128, a novel mammalian target of rapamycin (mTOR) complex 1 and 2 (mTORC1/2) dual inhibitor, against pancreatic cancer cells in vitro. Our results demonstrated that INK-128 concentration- and time-dependently inhibited the survival and growth of pancreatic cancer cells (both primary cells and transformed cells). INK-128 induced pancreatic cancer cell apoptosis and necrosis simultaneously. Further, INK-128 dramatically inhibited phosphorylation of 4E-binding protein 1 (4E-BP1), ribosomal S6 kinase 1 (S6K1) and Akt at Ser 473 in pancreatic cancer cells. Meanwhile, it downregulated cyclin D1 expression and caused cell cycle arrest. Finally, we found that a low concentration of INK-128 significantly increased the sensitivity of pancreatic cancer cells to gemcitabine. Together, our in vitro results suggest that INK-128 might be further investigated as a novel anti-cancer agent or chemo-adjuvant for pancreatic cancer treatment.     

Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration

Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration.     

Sindbis virus replication, is insensitive to rapamycin and torin1, and suppresses Akt/mTOR pathway late during infection in HEK cells

Genetically engineered Sindbis viruses (SIN) are excellent oncolytic agents in preclinical models. Several human cancers have aberrant Akt signaling, and kinase inhibitors including rapamycin are currently tested in combination therapies with oncolytic viruses. Therefore, it was of interest to delineate possible cross-regulation between SIN replication and PI3K/Akt/mTOR signaling. Here, using HEK293T cells as host, we report the following key findings: (a) robust SIN replication occurs in the presence of mTOR specific inhibitors, rapamycin and torin1 or Ly294002 – a PI3K inhibitor, suggesting a lack of requirement for PI3K/Akt/mTOR signaling; (b) suppression of phosphorylation of Akt, mTOR and its effectors S6, and 4E-BP1 occurs late during SIN infection: a viral function that may be beneficial in counteracting cellular drug resistance to kinase inhibitors; (c) Ly294002 and SIN act additively to suppress PI3K/Akt/mTOR pathway with little effect on virus release; and (d) SIN replication induces host translational shut off, phosphorylation of eIF2α and apoptosis. This first report on the potent inhibition of Akt/mTOR signaling by SIN replication, bolsters further studies on the development and evaluation of engineered SIN genotypes in vitro and in vivo for unique cytolytic functions.     

DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells

Pancreatic cancer is one of the most aggressive human malignancies with extremely poor prognosis. The moderate activity of the current standard gemcitabine and gemcitabine-based regimens was due to pre-existing or acquired chemo-resistance of pancreatic cancer cells. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in gemcitabine resistance, and studied the underlying mechanisms. We found that NU-7026 and NU-7441, two DNA-PKcs inhibitors, enhanced gemcitabine-induced cytotoxicity and apoptosis in PANC-1 pancreatic cancer cells. Meanwhile, PANC-1 cells with siRNA-knockdown of DNA-PKcs were more sensitive to gemcitabine than control PANC-1 cells. Through the co-immunoprecipitation (Co-IP) assay, we found that DNA-PKcs formed a complex with SIN1, the latter is an indispensable component of mammalian target of rapamycin (mTOR) complex 2 (mTORC2). DNA-PKcs–SIN1 complexation was required for Akt activation in PANC-1 cells, while inhibition of this complex by siRNA knockdown of DNA-PKcs/SIN1, or by DNA-PKcs inhibitors, prevented Akt phosphorylation in PANC-1 cells. Further, SIN1 siRNA-knockdown also facilitated gemcitabine-induced apoptosis in PANC-1 cells. Finally, DNA-PKcs and p-Akt expression was significantly higher in human pancreatic cancer tissues than surrounding normal tissues. Together, these results show that DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells.     

Differential regulation of Akt/protein kinase B isoforms during cell cycle progression

Phosphatidylinositol 3-kinase pathways play key regulatory roles in cell cycle progression into S phase. In this study, we demonstrated that Akt1/PKBα isoform plays an essential role in G₁/S transition and proliferation. Cells lacking Akt1/PKBα showed an attenuated proliferation as well as G₁/S transition, whereas cells lacking Akt2/PKBβ showed normal proliferation and G₁/S transition. The effect of Akt1/PKBα on cell proliferation and G₁/S transition was completely abolished by swapping pleckstrin homology (PH) domain with that of Akt2/PKBβ. Finally, full activation of Akt/PKB and cyclin D expression was achieved by the Akt1/PKBα or chimeric proteins containing the PH domain of Akt1/PKBα indicating that the PH domain of Akt1/PKBα provides full kinase activity and is necessary for the G₁/S transition.     

Determinants of the tumor suppressor INPP4B protein and lipid phosphatase activities

The tumor suppressor INPP4B is an important regulator of phosphatidyl-inositol signaling in the cell. Reduced INPP4B expression is associated with poor outcomes for breast, prostate, and ovarian cancer patients. INPP4B contains a CX₅R catalytic motif characteristic of dual-specificity phosphatases, such as PTEN. Lipid phosphatase activity of INPP4B has previously been described. In this report we show that INPP4B can dephosphorylate para-nitrophenyl phosphate (pNPP) and 6,8-difluoro-4-methylumbelliferyl (DiFMUP), synthetic phosphotyrosine analogs, suggesting that INPP4B has protein tyrosine phosphatase (PTP) activity. Using mutagenesis, we examined the functional role of specific amino acids within the INPP4B C₈₄₂KSAKDR catalytic site. The K843M mutant displayed increased pNPP hydrolysis, the K846M mutant lost lipid phosphatase activity with no effect on PTP activity, and the D847E substitution ablated PTP activity and significantly reduced lipid phosphatase activity. Further, we show that INPP4B but not PTEN is able to reduce tyrosine phosphorylation of Akt1 and both the lipid and PTP activity of INPP4B likely contribute to the reduction of Akt1 phosphorylation. Taken together our data identified key residues in the INPP4B catalytic domain associated with lipid and protein phosphatase activities and found a robust downstream target regulated by INPP4B but not PTEN.     

Dramatic suppression of colorectal cancer cell growth by the dual mTORC1 and mTORC2 inhibitor AZD-2014

Colorectal cancer is a major contributor of cancer-related mortality. The mammalian target or rapamycin (mTOR) signaling is frequently hyper-activated in colorectal cancers, promoting cancer progression and chemo-resistance. In the current study, we investigated the anti-colorectal cancer effect of a novel mTOR complex 1 (mTORC1) and mTORC2 dual inhibitor: AZD-2014. In cultured colorectal cancer cell lines, AZD-2014 significantly inhibited cancer cell growth without inducing significant cell apoptosis. AZD-2014 blocked activation of both mTORC1 (S6K and S6 phosphorylation) and mTORC2 (Akt Ser 473 phosphorylation), and activated autophagy in colorectal cancer cells. Meanwhile, autophagy inhibition by 3-methyaldenine (3-MA) and hydroxychloroquine, as well as by siRNA knocking down of Beclin-1 or ATG-7, inhibited AZD-2014-induced cytotoxicity, while the apoptosis inhibitor had no rescue effect. In vivo, AZD-2014 oral administration significantly inhibited the growth of HT-29 cell xenograft in SCID mice, and the mice survival was dramatically improved. At the same time, in xenografted tumors administrated with AZD-2014, the activation of mTORC1 and mTORC2 were largely inhibited, and autophagic markers were significantly increased. Thus, AZD-2014 inhibits colorectal cancer cell growth both in vivo and in vitro. Our results suggest that AZD-2014 may be further investigated for colorectal cancer therapy in clinical trials.     

Simultaneous loss of the DLC1 and PTEN tumor suppressors enhances breast cancer cell migration

The phosphatase and tensin homolog (PTEN) gene is a tumor suppressor frequently deleted or mutated in sporadic tumors of the breast, prostate, endometrium and brain. The protein acts as a dual specificity phosphatase for lipids and proteins. PTEN loss confers a growth advantage to cells, protects from apoptosis and favors cell migration. The deleted in liver cancer 1 (DLC1) gene has emerged as a novel tumor suppressor downregulated in a variety of tumor types including those of the breast. DLC1 contains a Rho GTPase activating domain that is involved in the inhibition of cell proliferation, migration and invasion. To investigate how simultaneous loss of PTEN and DLC1 contributes to cell transformation, we downregulated both proteins by RNA interference in the non-invasive MCF7 breast carcinoma cell line. Joint depletion of PTEN and DLC1 resulted in enhanced cell migration in wounding and chemotactic transwell assays. Interestingly, both proteins were found to colocalize at the plasma membrane and interacted physically in biochemical pulldowns and coimmunoprecipitations. We therefore postulate that the concerted local inactivation of signaling pathways downstream of PTEN and DLC1, respectively, is required for the tight control of cell migration.     

Epigallocatechin gallate (EGCG), a major component of green tea, is a dual phosphoinositide-3-kinase/mTOR inhibitor

The PI3K signaling pathway is activated in a broad spectrum of human cancers, either directly by genetic mutation or indirectly via activation of receptor tyrosine kinases or inactivation of the PTEN tumor suppressor. The key nodes of this pathway have emerged as important therapeutic targets for the treatment of cancer. In this study, we show that (−)-epigallocatechin-3-gallate (EGCG), a major component of green tea, is an ATP-competitive inhibitor of both phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) with K_i values of 380 and 320 nM respectively. The potency of EGCG against PI3K and mTOR is within physiologically relevant concentrations. In addition, EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231 and A549 cells. Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site, agreeing with the finding that EGCG competes for ATP binding. Our results suggest another important molecular mechanism for the anticancer activities of EGCG.     

Tensin2 reduces intracellular phosphatidylinositol 3,4,5-trisphosphate levels at the plasma membrane

Tensins are proposed cytoskeleton-regulating proteins. However, Tensin2 additionally inhibits Akt signalling and cell survival. Structural modelling of the Tensin2 phosphatase (PTPase) domain revealed an active site-like pocket receptive towards phosphoinositides. Tensin2-expressing HEK293 cells displayed negligible levels of plasma membrane phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) under confocal microscopy. However, mock-transfected cells, and Tensin2 cells harbouring a putative phosphatase-inactivating mutation, exhibited significant PtdIns(3,4,5)P₃ levels, which decreased upon phosphatidylinositol 3-kinase inhibition with LY294002. In contrast, wtTensin3, mock and mutant cells were identical in membrane PtdIns(3,4,5)P₃ and Akt phosphorylation. In vitro lipid PTPase activity was however undetectable in isolated recombinant PTPase domains of both Tensins, indicating a possible loss of structural stability when expressed in isolation. In summary, we provide evidence that Tensin2, in addition to regulating cytoskeletal dynamics, influences phosphoinositide-Akt signalling through its PTPase domain.     

Targeting of mTORC1/2 by dihydroevocarpine induces cytotoxicity in acute myeloid leukemia

Interactions between the tumor cells and bone marrow (BM) microenvironment promote survival, growth, and chemoresistance of acute myeloid leukemia (AML). The mTOR pathway plays a key role in mediating the AML-BM microenvironment interactions. Here, we report the anti-AML activity of a natural monomer extracted from the Chinese medicinal herb Evodia rutaecarpa, dihydroevocarpine. Our results showed that dihydroevocarpine-induced cytotoxicity, apoptosis, and G0/G1 arrest in AML cells, and inhibited the tumor growth in an AML xenograft model. Importantly, our study revealed that the dihydroevocarpine treatment inhibited the mTOR pathway via suppressing the mTORC1/2 activity, and thus overcame the protective effect of the BM microenvironment on AML cells. Taken together, our findings suggest that dihydroevocarpine could be used as a potential anti-AML agent alone or a therapeutic adjunct in AML therapy, particularly in the presence of the BM microenvironment.     

Identification of a novel Ser/Thr protein phosphatase Ppq1 as a negative regulator of mating MAP kinase pathway in Saccharomyces cerevisiae

The specificity and efficiency of cell signaling is largely governed by the complex formation of signaling proteins. The precise spatio-temporal control of the complex assembly is crucial for proper signaling and cell survival. Protein phosphorylation is a key mechanism of signal processing in most of cell signaling networks. Phosphatases, along with kinases, control the phosphorylation state of many proteins and thus play a critical role in the precise regulation of signaling at each stage such as activation, propagation, and adaptation. Identification and functional analysis of pathway-specific phosphatase is, therefore, crucial for the understanding of cell signaling mechanisms. Here, we have developed a novel screening strategy to identify pathway-specific phosphatases, in which the entire repertoire of cell’s phosphatases was tethered to a signaling complex and the changes in signaling response were monitored. As a model target, we have chosen the mating MAP kinase pathway in the budding yeast, which is composed of three kinases and Ste5 scaffold protein. Using this strategy, a putative Ser/Thr phosphatase, Ppq1, was identified to be mating-specific. Results show that Ppq1 down-regulates mating signaling by targeting at or upstream of the terminal MAP kinase Fus3 in the cascade. The catalytic activity of Ppq1 as a phosphatase was confirmed in vitro and is necessary for its function in the regulation of mating signaling. Overall, the data suggest that Ppq1 functions as a negative regulator of mating MAPK pathway by dephosphorylating target pathway protein(s) and plays a key role in the control of the background signaling noise.     

Phosphoinositide-dependent pathways in mouse sperm are regulated by egg ZP3 and drive the acrosome reaction

Sperm of many animals must complete an exocytotic event, the acrosome reaction, in order to fuse with eggs. In mammals, acrosome reactions are triggered during sperm contact with the egg extracellular matrix, or zona pellucida, by the matrix glycoprotein ZP3. Here, we show that ZP3 stimulates production of phosphatidylinositol-(3,4,5)-triphosphate in sperm membranes. Phosphatidylinositol-3-kinase antagonists that prevent acrosome reactions and fertilization in vitro, while generation of this phosphoinositide in the absence of ZP3 triggered acrosome reactions. Downstream effectors of phosphatidylinositol-(3,4,5)-triphosphate in sperm include the protein kinases, Akt and PKCzeta. These studies outline a signal transduction pathway that plays an essential role in the early events of mammalian fertilization.     

Comparison of phosphatidylinositol-3-kinase signalling within a panel of human colorectal cancer cell lines with mutant or wild-type PIK3CA

Recent studies have identified conserved missense mutations in PIK3CA, the gene encoding the catalytic phosphatidylinositol-3-kinase subunit p110alpha, in a variety of human cancers. Further investigation demonstrated that PIK3CA mutations lead to increased basal phosphatidylinositol-3-kinase activity, promoting cell growth and invasion [Samuels, Y., Diaz, L.A., Jr., Schmidt-Kittler, O., Cummins, J.M., Delong, L., Cheong, I., Rago, C., Huso, D.L., Lengauer, C., Kinzler, K.W., Vogelstein, B. and Velculescu, V.E. (2005) Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 7, 561-573]. A panel of commonly used colorectal cancer cell lines was screened for these PIK3CA mutations. Constitutive and IGF-1-stimulated phosphatidylinositol-3-kinase activity, signal response and duration were assessed. In the assays used no differences distinguished cells carrying PIK3CA mutations indicating that these mutations did not significantly alter growth factor stimulated or steady state phosphatidylinositol-3-kinase activity in normal cell culture conditions.     

LRIG1 dictates the chemo-sensitivity of temozolomide (TMZ) in U251 glioblastoma cells via down-regulation of EGFR/topoisomerase-2/Bcl-2

In the current study, we aimed to understand the potential role of leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) in TMZ-resistance of U251 glioma cells. We established TMZ-resistant U251 clones (U251/TMZ cells), which expressed low level of LRIG1, but high levels of epidermal growth factor receptor (EGFR), topoisomerase-2 (Topo-2) and Bcl-2. Depletion of LRIG1 by the targeted RNA interference (RNAi) upregulated EGFR/Topo-2/Bcl-2 in U251 cells, and the cells were resistant to TMZ. Reversely, over-expression of LRIG1 in U251 cells downregulated EGFR/Topo-2/Bcl-2 expressions, and cells were hyper-sensitive to TMZ. Our data suggested EGFR-dependent mammalian target of rapamycin (mTOR) activation was important for Topo-2 and Bcl-2 expressions in U251/TMZ cells. The EGFR inhibitor and the mTOR inhibitor downregulated Topo-2/Bcl-2 expressions, both inhibitors also restored TMZ sensitivity in U251/TMZ cells. Finally, inhibition of Topo-2 or Bcl-2 by targeted RNAi(s) knockdown or by the corresponding inhibitor re-sensitized U251/TMZ cells to TMZ, indicating that both Topo-2 and Bcl-2 were important for TMZ resistance in the resistant U251 cells. Based on these results, we concluded that LRIG1 inhibits EGFR expression and the downstream signaling activation, interferes with Bcl-2/Topo-2 expressions and eventually sensitizes glioma cells to TMZ.     

Role of the PDK1-PKB-GSK3 pathway in regulating glycogen synthase and glucose uptake in the heart

In order to investigate the importance of the PDK1-PKB-GSK3 signalling network in regulating glycogen synthase (GS) in the heart, we have employed tissue specific conditional knockout mice lacking PDK1 in muscle (mPDK1-/-), as well as knockin mice in which the protein kinase B (PKB) phosphorylation site on glycogen synthase kinase-3alpha (GSK3alpha) (Ser21) and GSK3beta (Ser9) is changed to Ala. We demonstrate that in hearts from mPDK1-/- or double GSK3alpha/GSK3beta knockin mice, insulin failed to stimulate the activity of GS or induce its dephosphorylation at residues that are phosphorylated by GSK3. We also establish that in the heart, both GSK3 isoforms participate in the regulation of GS, with GSK3beta playing a more prominent role. This contrasts with skeletal muscle where GSK3beta is the major regulator of insulin-induced GS activity. Despite the inability of insulin to stimulate glycogen synthesis in hearts from the mPDK1-/- or double GSK3alpha/GSK3beta knockin mice, these animals possessed normal levels of cardiac glycogen, demonstrating that total glycogen levels are regulated independently of insulin's ability to stimulate GS in the heart and that mechanisms such as allosteric activation of GS by glucose-6-phosphate and/or activation of GS by muscle contraction, could operate to maintain normal glycogen levels in these mice. We also demonstrate that in cardiomyocytes derived from the mPDK1-/- hearts, although the levels of glucose transporter type 4 (GLUT4) are increased 2-fold, insulin failed to stimulate glucose uptake, providing genetic evidence that PDK1 plays a crucial role in enabling insulin to promote glucose uptake in cardiac muscle.     

PTEN, a negative regulator of PI3 kinase signalling, alters tau phosphorylation in cells by mechanisms independent of GSK-3

Deregulation of PTEN/Akt signalling has been recently implicated in the pathogenesis of Alzheimer's disease (AD), but the effects on the molecular processes underlying AD pathology have not yet been fully described. Here we report that overexpression of PTEN reduces tau phosphorylation in CHO cells. This effect was abrogated by mutant PTEN constructs with either a catalytically inactive point mutation (C124S) or with only inactive lipid phosphatase activity (G129E), suggesting an indirect, lipid phosphatase-dependent process. The predominant effects of PTEN on tau appeared to be mediated by reducing ERK1/2 activity, but were independent of Akt, GSK-3, JNK and the tau phosphatases PP1 and PP2A. Our studies provide evidence for an effect of PTEN on the phosphorylation of tau in AD pathogenesis, and provide some insight into the mechanisms through which deregulation of PTEN may contribute towards the progression of tauopathy.