DLC-1, a Rho GTPase-activating protein with tumor suppressor function, is essential for embryonic development

DLC-1 (deleted in liver cancer 1) is a Rho GTPase-activating protein that is able to inhibit cell growth and suppress tumorigenesis. We have used homologous recombination to inactivate the mouse DLC-1 gene (Arhgap7). Mice heterozygous for the targeted allele were phenotypically normal, but homozygous mutant embryos did not survive beyond 10.5 days post coitum. Histological analysis revealed that DLC-1-/- embryos had defects in the neural tube, brain, heart, and placenta. Cultured fibroblasts from DLC-1-deficient embryos displayed alterations in the organization of actin filaments and focal adhesions.     

Redox regulation of the tumor suppressor PTEN by glutaredoxin 5 and Ycp4

Human PTEN (phosphatase and tensin homolog deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) expressed in Saccharomyces cerevisiae was oxidized in a time- and H₂O₂-concentration-dependent manner. Oxidized hPTEN was reduced by cellular reductants as in human cells. The reduction rate of oxidized hPTEN was monitored in S. cerevisiae mutants in which the genes involved in redox homeostasis had been disrupted. Reduction of hPTEN was delayed in each of S. cerevisiae grx5Δ and ycp4Δ mutants. Expression of Grx5 and Ycp4 in each of the mutants rescued the reduction rate of oxidized hPTEN. Furthermore, an in vitro assay revealed that the human Grx5/GSH system efficiently catalyzed the reduction of oxidized hPTEN. These results suggest that the reduction of oxidized hPTEN is regulated by Grx5 and Ycp4.     

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.     

The RLIP76 N-terminus binds ARNO to regulate PI 3-kinase,Arf6 and Rac signaling,cell spreading and migration

RLIP76 is a multifunctional protein involved in tumor growth and angiogenesis, and a promising therapeutic target in many cancers. RLIP76 harbors docking sites for many proteins, and we have found that it interacts with ARNO, a guanine nucleotide exchange factor for Arf6, and that RLIP76 regulates activation of Rac1 via Arf6, and regulates cell spreading and migration in an ARNO and Arf6-dependent manner. Here we show that ARNO interacts with the RLIP76 N-terminal domain, and this domain was required for RLIP76-dependent cell spreading and migration. We identified two sites in the RLIP76 N-terminus with differential effects on ARNO binding and downstream signaling: Ser29/Ser30 and Ser62. Ser29/30 mutation to Alanine inhibited ARNO interaction and was sufficient to block RLIP76-dependent cell spreading and migration, as well as RLIP76-dependent Arf6 activation. In contrast, RLIP76(S62A) interacted with ARNO and supported Arf6 activation. However, both sets of mutations blocked Rac1 activation. RLIP76-mediated Rac and Arf6 activation required PI3K activity. S29/30A mutations inhibited RLIP76-dependent PI3K activation, but S62A mutation did not. Together these results show that ARNO interaction with the RLIP76 N-terminus regulates cell spreading and motility via PI3K and Arf6, independent of RLIP76 control of Rac.     

Mitochondrial targeting of growth suppressor protein DLC2 through the START domain

Deleted in liver cancer 2 (DLC2) is a candidate tumor suppressor frequently found to be deleted in hepatocellular carcinoma. In this study, we determined the subcellular localization of DLC2. Co-localization and biochemical fractionation studies revealed that DLC2 localized to mitochondria. In addition, the DLC2-containing cytoplasmic speckles were in proximity to lipid droplets. A DLC2 mutant containing the steroidogenic acute regulatory protein-related lipid transfer (START) domain only showed a localization pattern identical to that of DLC2. Taken together, we have provided the first evidence for mitochondrial localization of DLC2 through the START domain. These findings might have implications in liver physiology and carcinogenesis.     

Role of the alpha(1) integrin cytoplasmic tail in the formation of focal complexes, actin organization, and in the control of cell migration

Integrins play a key role in cellular motility; an essential process for embryonic development and tissue morphogenesis, and also for pathological processes such as tumor cell invasion and metastasis. Recently, we showed that the cytoplasmic tail of integrin alpha(1) regulates the formation of focal complexes, F-actin cytoskeleton reorganization, and migration. We now report that the alpha(1) tail directly engages in collagen IV-mediated migration by regulation of the small GTPase Rac1. Deletion variants of the alpha(1) integrin differ in their ability to activate Rac1. Constitutively active Rac1 rescues motility in otherwise immotile cells expressing a truncated alpha(1) integrin without any cytoplasmic tail. In these cells, levels of GTP-Rac1 are constitutively elevated, but kept non-functional in the cytoplasm. The conserved GFFKR motif is sufficient to convey Rac1 activation, but downregulates the amount of GTP-Rac1 in the absence of the alpha(1)-specific sequence PLKKKMEK. This sequence is also required for the recruitment of PI3K to focal adhesions following Rac1 activation. Our results demonstrate that the short alpha(1) cytoplasmic tail is crucial for Rac1 activation and PI3K localization, which in turn results in cytoskeletal rearrangement and subsequent migration.     

Crosstalk of the EphA2 receptor with a serine/threonine phosphatase suppresses the Akt-mTORC1 pathway in cancer cells

Receptor tyrosine kinases of the Eph family play multiple roles in the physiological regulation of tissue homeostasis and in the pathogenesis of various diseases, including cancer. The EphA2 receptor is highly expressed in most cancer cell types, where it has disparate activities that are not well understood. It has been reported that interplay of EphA2 with oncogenic signaling pathways promotes cancer cell malignancy independently of ephrin ligand binding and receptor kinase activity. In contrast, stimulation of EphA2 signaling with ephrin-A ligands can suppress malignancy by inhibiting the Ras-MAP kinase pathway, integrin-mediated adhesion, and epithelial to mesenchymal transition. Here we show that ephrin-A1 ligand-dependent activation of EphA2 decreases the growth of PC3 prostate cancer cells and profoundly inhibits the Akt-mTORC1 pathway, which is hyperactivated due to loss of the PTEN tumor suppressor. Our results do not implicate changes in the activity of Akt upstream regulators (such as Ras family GTPases, PI3 kinase, integrins, or the Ship2 lipid phosphatase) in the observed loss of Akt T308 and S473 phosphorylation downstream of EphA2. Indeed, EphA2 can inhibit Akt phosphorylation induced by oncogenic mutations of not only PTEN but also PI3 kinase. Furthermore, it can decrease the hyperphosphorylation induced by constitutive membrane-targeting of Akt. Our data suggest a novel signaling mechanism whereby EphA2 inactivates the Akt-mTORC1 oncogenic pathway through Akt dephosphorylation mediated by a serine/threonine phosphatase. Ephrin-A1-induced Akt dephosphorylation was observed not only in PC3 prostate cancer cells but also in other cancer cell types. Thus, activation of EphA2 signaling represents a possible new avenue for anti-cancer therapies that exploit the remarkable ability of this receptor to counteract multiple oncogenic signaling pathways.     

START-GAP1/DLC1 is localized in focal adhesions through interaction with the PTB domain of tensin2

START-GAP1, also termed as DLC1, is a negative-regulator for RhoA and Cdc42. START-GAP1 is localized in focal adhesions via the FAT (focal adhesion targeting) domain located in its N-terminal half and interacts with tensin family proteins, that constitutes focal adhesion components. This study has provided evidence that the interaction between START-GAP1 and tensin2 occurs in a PTB domain-dependent manner. It was revealed that FAT3, the third subdomain of the FAT domain divided into five that consists of 39 amino acids, binds directly to the PTB domain of tensin2. This interaction does not require protein phosphorylation, since the interaction was detected with proteins expressed in bacterial expression system. In mammalian genome, there are three genes encoding START domain containing RhoGAPs. START-GAP2/DLC2 and START-GAP3/DLC3, as well as STRT-GAP1/DLC1, bind to the PTB domain of tensin2, presumably due to the presence of highly conserved residues in the center of FAT3. Deletion of this sub-region abrogates the interaction with the tensin PTB domain. Furthermore, D368, H369, G372, F374, P375 and L378 in the highly conserved region of START-GAP1 have been revealed to be essential for the interaction. The tensin2-PTB domain seems to determine the subcellular localization of FAT3. Nevertheless, our study with deletion mutants revealed that FAT3 is essential but not sufficient for the focal adhesion localization of START-GAP1. These results suggest that the interaction between the tensin PTB domain and FAT3 contributes to START-GAP1 localization but only partially. Other factors could affect the START-GAP1 localization.     

Regulation of lamellipodia formation and cell invasion by CLIP-170 in invasive human breast cancer cells

Lamellipodia formation necessary for cell invasion is regulated by Rac1. We report here that lamellipodia formation and three-dimensional invasion were significantly promoted by HGF and serum, respectively, in invasive human breast cancer cells. Rac1 formed a complex with CLIP-170, IQGAP1, and kinesin in serum-starved cells, and stimulation of the cells with HGF and serum caused the partial release of IQGAP1 and kinesin from Rac1-CLIP-170 complex. The HGF-induced release of the proteins and promotion of lamellipodia formation were inhibited by an inhibitor of PI3K. Moreover, downregulation of CLIP-170 by siRNA released IQGAP1 and kinesin from Rac1 and promoted lamellipodia formation and invasion, independent of HGF and serum. The results suggest that promotion of lamellipodia formation and invasion by HGF or serum requires PI3K-dependent release of IQGAP1 and kinesin from Rac1-CLIP-170 complex and that CLIP-170 prevents cells from the extracellular stimulus-independent lamellipodia formation and invasion by tethering IQGAP1 and kinesin to Rac1.     

Rac signaling in breast cancer: a tale of GEFs and GAPs

Rac GTPases, small G-proteins widely implicated in tumorigenesis and metastasis, transduce signals from tyrosine-kinase, G-protein-coupled receptors (GPCRs), and integrins, and control a number of essential cellular functions including motility, adhesion, and proliferation. Deregulation of Rac signaling in cancer is generally a consequence of enhanced upstream inputs from tyrosine-kinase receptors, PI3K or Guanine nucleotide Exchange Factors (GEFs), or reduced Rac inactivation by GTPase Activating Proteins (GAPs). In breast cancer cells Rac1 is a downstream effector of ErbB receptors and mediates migratory responses by ErbB1/EGFR ligands such as EGF or TGFα and ErbB3 ligands such as heregulins. Recent advances in the field led to the identification of the Rac-GEF P-Rex1 as an essential mediator of Rac1 responses in breast cancer cells. P-Rex1 is activated by the PI3K product PIP3 and Gβγ subunits, and integrates signals from ErbB receptors and GPCRs. Most notably, P-Rex1 is highly overexpressed in human luminal breast tumors, particularly those expressing ErbB2 and estrogen receptor (ER). The P-Rex1/Rac signaling pathway may represent an attractive target for breast cancer therapy.     

S1P(2) receptors mediate inhibition of glioma cell migration through Rho signaling pathways independent of PTEN

Sphingosine 1-phosphate (S1P) induced the inhibition of glioma cell migration. Here, we characterized the signaling mechanisms involved in the inhibitory action by S1P. In human GNS-3314 glioblastoma cells, the S1P-induced inhibition of cell migration was associated with activation of RhoA and suppression of Rac1. The inhibitory action of S1P was recovered by a small interference RNA specific to S1P₂ receptor, a carboxyl-terminal region of Gα12 or Gα13, an RGS domain of p115RhoGEF, and a dominant-negative mutant of RhoA. The inhibitory action of S1P through S1P₂ receptors was also observed in both U87MG glioblastoma and 1321N1 astrocytoma cells, which have no protein expression of a phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These results suggest that S1P₂ receptors/G_12/13-proteins/Rho signaling pathways mediate S1P-induced inhibition of glioma cell migration. However, PTEN, recently postulated as an indispensable molecule for the inhibition of cell migration, may not be critical for the S1P₂ receptor-mediated action in glioma cells.     

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.     

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.     

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.     

Altered Ca signaling in cancer cells: Proto-oncogenes and tumor suppressors targeting IP3 receptors

Proto-oncogenes and tumor suppressors critically control cell-fate decisions like cell survival, adaptation and death. These processes are regulated by Ca^2 + signals arising from the endoplasmic reticulum, which at distinct sites is in close proximity to the mitochondria. These organelles are linked by different mechanisms, including Ca^2 +-transport mechanisms involving the inositol 1,4,5-trisphosphate receptor (IP₃R) and the voltage-dependent anion channel (VDAC). The amount of Ca^2 + transfer from the endoplasmic reticulum to mitochondria determines the susceptibility of cells to apoptotic stimuli. Suppressing the transfer of Ca^2 + from the endoplasmic reticulum to the mitochondria increases the apoptotic resistance of cells and may decrease the cellular responsiveness to apoptotic signaling in response to cellular damage or alterations. This can result in the survival, growth and proliferation of cells with oncogenic features. Clearly, proper maintenance of endoplasmic reticulum Ca^2 + homeostasis and dynamics including its links with the mitochondrial network is essential to detect and eliminate altered cells with oncogenic features through the apoptotic pathway. Proto-oncogenes and tumor suppressors exploit the central role of Ca^2 + signaling by targeting the IP₃R. There are an increasing number of reports showing that activation of proto-oncogenes or inactivation of tumor suppressors directly affects IP₃R function and endoplasmic reticulum Ca^2 + homeostasis, thereby decreasing mitochondrial Ca^2 + uptake and mitochondrial outer membrane permeabilization. In this review, we provide an overview of the current knowledge on the proto-oncogenes and tumor suppressors identified as IP₃R-regulatory proteins and how they affect endoplasmic reticulum Ca^2 + homeostasis and dynamics.     

Knockdown of Akt isoforms by RNA silencing suppresses the growth of human prostate cancer cells in vitro and in vivo

The serine/threonine kinase Akt has three highly homologous isoforms in mammals: Akt1, Akt2, and Akt3. Recent studies indicate that Akt is often constitutively active in many types of human malignancy. Here we investigated the expression and function of Akt isoforms in human prostatic carcinoma cells. Initially, we used Western blotting to examine Akt expression in four human prostate cancer cell lines. Next, small-interfering RNAs (siRNAs) specific for Akt isoforms were used to elucidate their role on the in vitro and in vivo growth of prostate cancer cells. Expression of Akt1 and Akt2 was detected in all cells tested, but Akt3 was expressed only in cancer cells that did not express androgen receptors. All synthetic siRNAs against Akt isoforms suppressed their expression and inhibited the growth of cancer cells in vitro. Furthermore, atelocollagen-mediated systemic administration of siRNAs significantly reduced the growth of tumors that had been subcutaneously xenografted. These results suggest that targeting Akt isoforms could be an effective treatment for prostate cancers.     

Polarization of endosomal SNX27 in migrating and tumor-engaged natural killer cells

Polarization is a critical mechanism for the proper functioning of many cell types. For lymphocytes, it is essential in a variety of processes, including migration from the blood to other tissue sites and vice versa. In NK cells and CTLs, the cytotoxic granule delivery mechanism requires polarization for granule movement to the immunological synapse (IS), in killing tumor and virus-infected cells. Recently, it has become apparent that endosomes are also involved in the cytotoxic mechanism. Using an in vitro conjugation approach, we show that in NK-92 cells, endosomal Sorting Nexin 27 (SNX27) polarizes to the IS during tumor cell engagement in a distinct compartment adjacent to the cytotoxic granules. We also show that SNX27 polarizes to the apical membrane, opposite the uropod, during NK cell migration. These previously unreported results indicate that SNX27 is a participant in NK cell polarization, as a mediator or target of the mechanism.     

Associations of the PTEN − 9C>G polymorphism with insulin sensitivity and central obesity in Chinese

Background

Phosphatase and tensin homolog on chromosome 10 gene (PTEN) is known as a tumor-suppressor gene. Previous studies demonstrated that PTEN dysfunction affects the function of insulin. However, investigations of PTEN single nucleotide polymorphisms (SNPs) and IR-related disease associations are limited. The aim of the present study was to investigate whether its polymorphism could be involved in the risk of metabolic syndrome (MetS).

Methods

The genotype frequency of PTEN − 9C>G polymorphism was determined by using a Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) method in 530 subjects with MetS and 202 healthy control subjects of the Han Ethnic Chinese population in a case–control analysis.

Results

The PTEN − 9C>G polymorphism was not associated with MetS or its hyperglycemia, hypertension and hypertriglyceridemia components. In the control individuals aged < 60 years or ≥ 60 years, the CG genotype individuals had lower insulin sensitivity than CC individuals (P < 0.05). In the < 60-year-old MetS group and normal glucose tolerance (NGT) subgroup, the CG individuals had lower insulin sensitivity and higher waist circumference (WC) and waist-height-ratio (WHtR) than CC individuals (P < 0.05). Multiple linear regression analysis showed that the PTEN polymorphism (P = 0.001) contributed independently to 4.2% (adjusted R²) of insulin sensitivity variance (estimated by Matsuda ISI), while age (P = 0.004), gender (P = 0.000) and the PTEN polymorphism (P = 0.032) contributed independently to 5.6% (adjusted R²) of WHtR variance.

Conclusions

The CG genotype of PTEN − 9C>G polymorphism was not associated with MetS and some of its components as well. However, it may not only decrease insulin sensitivity in the healthy control and MetS in pre-elderly or NGT subjects, but may also increase the risk of central obesity among these MetS individuals.