Role of Phosphoinositide 3-Kinase �� in Glycoprotein VI-mediated Akt Activation in Platelets

Glycoprotein (GP) VI is a critical platelet collagen receptor. Phosphoinositide 3-kinase (PI3K) plays an important role in GPVI-mediated platelet activation, yet the major PI3K isoforms involved in this process have not been identified. In addition, stimulation of GPVI results in the activation of Akt, a downstream effector of PI3K. Thus, we investigated the contribution of PI3K isoforms to GPVI-mediated platelet activation and Akt activation. A protein kinase C inhibitor GF 109203X or a P2Y₁₂ receptor antagonist AR-C69931MX partly reduced GPVI-induced Akt phosphorylation. Platelets from mice dosed with clopidogrel also showed partial Akt phosphorylation, indicating that GPVI-mediated Akt phosphorylation is regulated by both secretion-dependent and -independent pathways. In addition, GPVI-induced Akt phosphorylation in the presence of ADP antagonists was completely inhibited by PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and PI3Kβ inhibitor TGX-221 indicating an essential role of PI3Kβ in Akt activation directly downstream of GPVI. Moreover, GPVI-mediated platelet aggregation, secretion, and intracellular Ca²⁺ mobilization were significantly inhibited by TGX-221, and less strongly inhibited by PI3Kα inhibitor PIK75, but were not affected by PI3Kγ inhibitor AS252424 and PI3Kδ inhibitor IC87114. Consistently, GPVI-induced integrin α_IIbβ₃ activation of PI3Kγ^−/− and PI3Kδ^−/− platelets also showed no significant difference compared with wild-type platelets. These results demonstrate that GPVI-induced Akt activation in platelets is dependent in part on G_i stimulation through P2Y₁₂ receptor activation by secreted ADP. In addition, a significant portion of GPVI-dependent, ADP-independent Akt activation also exists, and PI3Kβ plays an essential role in GPVI-mediated platelet aggregation and Akt activation.     

Cutting edge: differential roles for phosphoinositide 3-kinases, p110gamma and p110delta, in lymphocyte chemotaxis and homing

Despite the established role for PI3Ks in cell migration, the PI3Ks involved in lymphocyte chemotaxis are poorly defined. In this study, we report that p110gamma-deficient T cells, but not B cells, show reduced chemotactic responses to the lymphoid chemokines, CCL19, CCL21, and CXCL12. As B cell and T cell chemotactic responses were both sensitive to the general PI3K inhibitors, wortmannin (WMN) and LY294002, we explored whether B cell responses were affected in mice lacking p110delta, a major PI3K isoform in lymphocytes. B cells deficient in p110delta showed diminished chemotactic responses, especially to CXCL13. Adoptive transfer experiments with WMN-treated wild-type B cells and with p110delta-deficient B cells revealed diminished homing to Peyer's patches and splenic white pulp cords. WMN selectively inhibited CXCR5-dependent B cell homing to Peyer's patches. These observations establish that p110gamma and p110delta function in lymphocyte chemotaxis, and show differential roles for PI3K family members in B and T cell migration.     

Evidence that the lipid phosphatase SHIP-1 regulates T lymphocyte morphology and motility

SHIP-1 negatively regulates the PI3K pathway in hematopoietic cells and has an emerging role in T lymphocyte biology. PI3K and SHIP can regulate cell migration in leukocytes, particularly in neutrophils, although their role in T cell migration has been less clear. Therefore, we sought to explore the role of SHIP-1 in human CD4(+) T lymphocyte cell migration responses to chemoattractants using a lentiviral-mediated expression system and a short hairpin RNA approach. Silencing of SHIP-1 leads to increased basal phosphorylation of protein kinase B/Akt and its substrate GSK3β, as well as an increase in basal levels of polymerized actin, suggesting that SHIP-1 might regulate changes in the cytoskeleton. Accordingly, silencing of SHIP-1 led to loss of microvilli and ezrin/radixin/moesin phosphorylation, which could not be rescued by the PI3K inhibitor Ly294002. There were striking morphological changes, including a loss of microvilli projections, which mirrored changes in wild type cells after stimulation with the chemokine CXCL11. There was no defect in directional T cell migration toward CXCL11 in the SHIP-1-silenced cells but, importantly, there was a defect in the overall basal motility of SHIP-1 knockdown cells. Taken together, these results implicate SHIP-1 as a key regulator of basal PI3K signaling in human CD4(+) T lymphocytes with important phosphatase-independent actions, which together are key for maintaining normal morphology and basal motility.     

Co-Depletion of Cathepsin B and uPAR Induces G0/G1 Arrest in Glioma via FOXO3a Mediated p27Kip1 Upregulation

Background

Cathepsin B and urokinase plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied the effect of these molecules on cell cycle progression has not been thoroughly examined.

Methodology/Principal Findings

Cathespin B and uPAR single and bicistronic siRNA plasmids were used to downregulate these molecules in SNB19 and U251 glioma cells. FACS analysis and BrdU incorporation assay demonstrated G0/G1 arrest and decreased proliferation with the treatments, respectively. Immunoblot and immunocyto analysis demonstrated increased expression of p27^Kip1 and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by αVβ3/PI3K/AKT/FOXO pathway as observed by the decreased αVβ3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27^Kip1 and FOXO3a when treated with {"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002 (10 µM) and increased luciferase expression with the siRNA and {"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002 treatments when the FOXO binding promoter region of p27^Kip1 was used. Our treatment also reduced the expression of cyclin D1, cyclin D2, p-Rb and cyclin E while the expression of Cdk2 was unaffected. Of note, the Cdk2-cyclin E complex formation was reduced significantly.

Conclusion/Significance

Our study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27^Kip1 accompanied by the binding of FOXO3a to its promoter. Taken together, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells.     

Class II Phosphoinositide 3-Kinases Contribute to Endothelial Cells Morphogenesis

The question of whether the distinct isoforms of the family of enzymes phosphoinositide 3-kinases (PI3Ks) play redundant roles within a cell or whether they control distinct cellular processes or distinct steps within the same cellular process has gained considerable importance in the recent years due to the development of inhibitors able to selectively target individual isoforms. It is important to understand whether inhibition of one PI3K can result in compensatory effect from other isoform(s) and therefore whether strategies aimed at simultaneously blocking more than one PI3K may be needed. In this study we investigated the relative contribution of distinct PI3K isoforms to endothelial cells (EC) functions specifically regulated by the sphingolipid sphingosine-1-phosphate (S1P) and by high density lipoproteins (HDL), the major carrier of S1P in human plasma. Here we show that a co-ordinated action of different PI3Ks is required to tightly regulate remodelling of EC on Matrigel, a process dependent on cell proliferation, apoptosis and migration. The contribution of each isoform to this process appears to be distinct, with the class II enzyme PI3K-C2β and the class IB isoform p110γ mainly regulating the S1P- and HDL-dependent EC migration and PI3K-C2α primarily controlling EC survival. Data further indicate that PI3K-C2β and p110γ control distinct steps involved in cell migration supporting the hypothesis that different PI3Ks regulate distinct cellular processes.     

Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts: ACTIONS ON CYTOSKELETAL ORGANIZATION,SURVIVAL, AND RESORPTION*

Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (β), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15–20 min to 65–75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kβ and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics.     

Hybrid cells derived from breast epithelial cell/breast cancer cell fusion events show a differential RAF-AKT crosstalk

Background

The biological phenomenon of cell fusion has been linked to several characteristics of tumour progression, including an enhanced metastatogenic capacity and an enhanced drug resistance of hybrid cells. We demonstrated recently that M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics spontaneously fused with MDA-MB-435-Hyg breast cancer cells, thereby giving rise to stable M13MDA435 hybrid cells, which are characterised by a unique gene expression profile and migratory behaviour. Here we investigated the involvement of the PLC-??/??1, PI3K/AKT and RAS-RAF-ERK signal transduction cascades in the EGF and SDF-1?? induced migration of two M13MDA435 hybrid cell clones in comparison to their parental cells.

Results

Analysis of the migratory behaviour by using the three-dimensional collagen matrix migration assay showed that M13SV1-EGFP-Neo cells as well as M13MDA435 hybrid cells, but not the breast cancer cell line, responded to EGF stimulation with an increased locomotory activity. By contrast, SDF-1?? solely stimulated the migration of M13SV1-EGFP-Neo cells, whereas the migratory activity of the other cell lines was blocked. Analysis of signal transduction cascades revealed a putative differential RAF-AKT crosstalk in M13MDA435-1 and -3 hybrid cell clones. The PI3K inhibitor Ly294002 effectively blocked the EGF induced migration of M13MDA435-3 hybrid cells, whereas the EGF induced locomotion of M13MDA435-1 hybrid cells was markedly increased. Analysis of RAF-1 S259 phosphorylation, being a major mediator of the negative regulation of RAF-1 by AKT, showed decreased pRAF-1 S259 levels in LY294002 treated M13MDA435-1 hybrid cells. By contrast, pRAF-1 S259 levels remained unaltered in the other cell lines. Inhibition of PI3K/AKT signalling by Ly294002 relieves the AKT mediated phosphorylation of RAF-1, thereby restoring MAPK signalling.

Conclusions

Here we show that hybrid cells could evolve exhibiting a differential active RAF-AKT crosstalk. Because PI3K/AKT signalling has been chosen as a target for anti-cancer therapies our data might point to a possible severe side effect of AKT targeted cancer therapies. Inhibition of PI3K/AKT signalling in RAF-AKT crosstalk positive cancer (hybrid) cells could result in a progression of these cells. Thus, not only the receptor (activation) status, but also the activation of signal transduction molecules should be analysed thoroughly prior to therapy.     

TGF-β signalling-related markers in cancer patients with bone metastasis

We measured transforming growth factor (TGF)-β-dependent biomarkers in plasma and in peripheral blood mononuclear cells (PBMCs) to identify suitable pharmacodynamic markers for future clinical trials with TGF-β inhibitors. Forty-nine patients with bone metastasis were enrolled in the study, including patients with breast (n=23) and prostate cancer (n=15). Plasma TGF-β1 levels were elevated in more than half of the cancer patients (geometric mean 2.63 ng ml^-1) and positively correlated with increased platelet factor 4 (PF4) levels, parathyroid-related protein (PTHrP), von Willebrand Factor (vWF) and interleukin (IL)-10. PBMC were stimulated ex vivo to determine the individual biological variability of an ex vivo assay measuring pSMAD expression. This assay performed sufficiently well to allow its future use in a clinical trial of a TGF-β inhibitor.     

Role of phosphoinositide 3OH-kinase in autocrine transformation by PDGF-BB

Phosphoinositide 3OH-kinases (PI3K) are a family of lipid kinases that activates signalling pathways important for migration, cytoskeletal rearrangements, and cell survival. These processes are important hallmarks in transformation. We have evaluated the functional role of PI3K for development of a transformed morphology and migratory responses of murine fibroblasts (NIH/sis and COL1A1/NIH3T3 cell lines) stimulated in an autocrine fashion by constitutive expression of platelet-derived growth factor-BB (PDGF-BB). We show that prolonged treatment with the specific PI3K inhibitor LY294002, induced a reversion of the transformed morphology, and prevented density-independent growth and focus formation. Functional PI3K was also required for development of the transformed morphology of NIH/sis and COL1A1/NIH3T3. Furthermore, treatment with LY294002 completely perturbed random migration of the cells. In addition our data show that, in the signalling pathways downstream of PI3K, activation of the small GTPase Rac was a prerequisite for the transformation signal. Our data also indicate the presence of a suramin-insensitive PI3K activity. Most likely this was due to the presence of a suramin-insensitive intracellular PDGFR pool that allowed activation of PI3K located in intracellular compartments. In conclusion these data show that intact PI3K activity was required for the morphological alterations and the enhanced migratory response that are hallmarks for PDGF induced autocrine transformation.     

Non-redundant Roles of Phosphoinositide 3-Kinase Isoforms �� and �� in Glycoprotein VI-induced Platelet Signaling and Thrombus Formation

Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cγ2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3Kα or -β strongly and selectively suppressed GPVI-induced Ca²⁺ mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cγ2 by PI3Kα/β. That PI3Kα and -β have a non-redundant function in GPVI-induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca²⁺ increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3Kα/β was blocked or p85α was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kδ or -γ. Furthermore, PI3Kα/β, but not PI3Kγ, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kα and -β isoforms are required for full GPVI-dependent platelet Ca²⁺ signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kα an interesting new target for anti-platelet therapy.     

Phosphoinositide 3-Kinase C2�� Regulates RhoA and the Actin Cytoskeleton through an Interaction with Dbl

The regulation of cell morphology is a dynamic process under the control of multiple protein complexes acting in a coordinated manner. Phosphoinositide 3-kinases (PI3K) and their lipid products are widely involved in cytoskeletal regulation by interacting with proteins regulating RhoGTPases. Class II PI3K isoforms have been implicated in the regulation of the actin cytoskeleton, although their exact role and mechanism of action remain to be established. In this report, we have identified Dbl, a Rho family guanine nucleotide exchange factor (RhoGEF) as an interaction partner of PI3KC2β. Dbl was co-immunoprecipitated with PI3KC2β in NIH3T3 cells and cancer cell lines. Over-expression of Class II phosphoinositide 3-kinase PI3KC2β in NIH3T3 fibroblasts led to increased stress fibres formation and cell spreading. Accordingly, we found high basal RhoA activity and increased serum response factor (SRF) activation downstream of RhoA upon serum stimulation. In contrast, the dominant-negative form of PI3KC2β strongly reduced cell spreading and stress fibres formation, as well as SRF response. Platelet-derived growth factor (PDGF) stimulation of wild-type PI3KC2β over-expressing NIH3T3 cells strongly increased Rac and c-Jun N-terminal kinase (JNK) activation, but failed to show similar effect in the cells with the dominant-negative enzyme. Interestingly, epidermal growth factor (EGF) and PDGF stimulation led to increased extracellular signal-regulated kinase (Erk) and Akt pathway activation in cells with elevated wild-type PI3KC2β expression. Furthermore, increased expression of PI3KC2β protected NIH3T3 from detachment-dependent death (anoikis) in a RhoA-dependent manner. Taken together, these findings suggest that PI3KC2β modulates the cell morphology and survival through a specific interaction with Dbl and the activation of RhoA.     

Isolation and characterization of a putative liver progenitor population after treatment of fetal rat hepatocytes with TGF-beta

Bone morphogenetic protein-2 (BMP-2), a member of transforming growth factor-beta superfamily, plays a crucial role in migration and metastasis of human cancer cells. Integrins are the major adhesive molecules in mammalian cells. Here we found that BMP-2 directed the migration and increased cell surface and mRNA expression of beta1 integrin in human chondrosarcoma cancer cells (JJ012). Pretreated of JJ012 cells with phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002) or Akt inhibitor inhibited the BMP-2-mediated migration and integrin expression. BMP-2 increased the phosphorylation of p85 subunit of PI3K and serine 473 of Akt. In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited BMP-2-mediated cells migration and integrin upregulation. Stimulation of JJ012 cells with BMP-2 induced IkappaB kinase (IKKalpha/beta) phosphorylation, IkappaB phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. Furthermore, the BMP-2-mediated increasing of IKKalpha/beta phosphorylation, IkappaB phosphorylation, and p65 Ser(536) phosphorylation were inhibited by Ly294002 and Akt inhibitor. Co-transfection with p85 and Akt mutants also reduced the BMP-2-induced kappaB-luciferase activity. Taken together, these results suggest that the BMP-2 acts through PI3K/Akt, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of beta1 integrin and contributing the migration of human chondrosarcoma cells.     

Inhibition of chemokine (CXC motif) ligand 12/chemokine (CXC motif) receptor 4 axis (CXCL12/CXCR4)-mediated cell migration by targeting mammalian target of rapamycin (mTOR) pathway in human gastric carcinoma cells

CXCL12/CXCR4 plays an important role in metastasis of gastric carcinoma. Rapamycin has been reported to inhibit migration of gastric cancer cells. However, the role of mTOR pathway in CXCL12/CXCR4-mediated cell migration and the potential of drugs targeting PI3K/mTOR pathway remains unelucidated. We found that CXCL12 activated PI3K/Akt/mTOR pathway in MKN-45 cells. Stimulating CHO-K1 cells expressing pEGFP-C1-Grp1-PH fusion protein with CXCL12 resulted in generation of phosphatidylinositol (3,4,5)-triphosphate, which provided direct evidence of activating PI3K by CXCL12. Down-regulation of p110β by siRNA but not p110α blocked phosphorylation of Akt and S6K1 induced by CXCL12. Consistently, p110β-specific inhibitor blocked the CXCL12-activated PI3K/Akt/mTOR pathway. Moreover, CXCR4 immunoprecipitated by anti-p110β antibody increased after CXCL12 stimulation and G(i) protein inhibitor pertussis toxin abrogated CXCL12-induced activation of PI3K. Further studies demonstrated that inhibitors targeting the PI3K/mTOR pathway significantly blocked the chemotactic responses of MKN-45 cells triggered by CXCL12, which might be attributed primarily to inhibition of mTORC1 and related to prevention of F-actin reorganization as well as down-regulation of active RhoA, Rac1, and Cdc42. Furthermore, rapamycin inhibited the secretion of CXCL12 and the expression of CXCR4, which might form a positive feedback loop to further abolish upstream signaling leading to cell migration. Finally, we found cells expressing high levels of cxcl12 were sensitive to rapamycin in its activity inhibiting migration as well as proliferation. In summary, we found that the mTOR pathway played an important role in CXCL12/CXCR4-mediated cell migration and proposed that drugs targeting the mTOR pathway may be used for the therapy of metastatic gastric cancer expressing high levels of cxcl12.     

Chloroquine or Chloroquine-PI3K/Akt Pathway Inhibitor Combinations Strongly Promote γ-Irradiation-Induced Cell Death in Primary Stem-Like Glioma Cells

We asked whether inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is highly active in cancer stem cells (CSCs) and upregulated in response to genotoxic treatments, promote γ-irradiationγIR)-induced cell death in highly radioresistant, patient-derived stem-like glioma cells (SLGCs). Surprisingly, in most cases the inhibitors did not promote γIR-induced cell death. In contrast, the strongly cytostatic {"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002 and PI-103 even tended to reduce it. Since autophagy was induced we examined whether addition of the clinically applicable autophagy inhibitor chloroquine (CQ) would trigger cell death in SLGCs. Triple therapy with CQ at doses as low as 5 to 10 µM indeed caused strong apoptosis. At slightly higher doses, CQ alone strongly promoted γIR-induced apoptosis in all SLGC lines examined. The strong apoptosis in combinations with CQ was invariably associated with strong accumulation of the autophagosomal marker LC3-II, indicating inhibition of late autophagy. Thus, autophagy-promoting effects of PI3K/Akt pathway inhibitors apparently hinder cell death induction in γ-irradiated SLGCs. However, as we show here for the first time, the late autophagy inhibitor CQ strongly promotes γIR-induced cell death in highly radioresistant CSCs, and triple combinations of CQ, γIR and a PI3K/Akt pathway inhibitor permit reduction of the CQ dose required to trigger cell death.     

The TLR3, PI3K,survin, FasL,and Fas genes as major risk factors of occurrence and development of cervical cancer disease

To investigate the role of TLR3/PI3K signals in the occurrence and development of cervical cancer disease, TLR3–siRNA was used to block key signaling pathways involved in cervical cancer metastasis that are pivotal to metastatic tumor cells but not to normal cells under ordinary physiologic conditions. Results show that tumor U14 cell growth, migration and invasion in TLR3–siRNA treatment group were significantly decreased. Through LY294002 suppressing targeted gene, the LY294002 treatment specifically and significantly knocked down the expressions of tumor TLR3 and PI3K proteins in cervical cancer mice. Furthermore, expressions of tumor Survivin and FasL proteins were markedly suppressed, whereas expressions of Fas protein were upregulated in LY294002 treatment group mice. LY294002 treatment suppressed tumor growth and increased the thymus and spleen indeces and survival days of cervical cancer mice. This study demonstrates that TLR3–siRNA and LY294002 treatments can markedly suppress cervical cancer cell invasion and tumor growth and increase survival life by silencing targeted genes.     

Hepatocyte Growth Factor Increases Vascular Endothelial Growth Factor-A Production in Human Synovial Fibroblasts through c-Met Receptor Pathway

Background

Angiogenesis is essential for the progression of osteoarthritis (OA). Hepatocyte growth factor (HGF) is an angiogenic mediator, and it shows elevated levels in regions of OA. However, the relationship between HGF and vascular endothelial growth factor (VEGF-A) in OA synovial fibroblasts (OASFs) is mostly unknown.

Methodology/Principal Findings

Here we found that stimulation of OASFs with HGF induced concentration- and time-dependent increases in VEGF-A expression. Pretreatment with PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002), Akt inhibitor, or mTORC1 inhibitor (rapamycin) blocked the HGF-induced VEGF-A production. Treatment of cells with HGF also increased PI3K, Akt, and mTORC1 phosphorylation. Furthermore, HGF increased the stability and activity of HIF-1 protein. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that c-Met, PI3K, Akt, and mTORC1 signaling pathways were potentially required for HGF-induced HIF-1α activation.

Conclusions/Significance

Taken together, our results provide evidence that HGF enhances VEGF-A expression in OASFs by an HIF-1α-dependent mechanism involving the activation of c-Met/PI3K/Akt and mTORC1 pathways.     

ErbB4 expression in neural progenitor cells (ST14A) is necessary to mediate neuregulin-1beta1-induced migration

Activation of the receptor tyrosine kinase ErbB4 leads to various cellular responses such as proliferation, survival, differentiation, and chemotaxis. Two pairs of naturally occurring ErbB4 isoforms differing in their juxtamembrane (JMa/JMb) and C termini (cyt1/cyt2) have been described. To examine the role of ErbB4 in neuron migration, we cloned and stably transfected each of the four ErbB4 isoforms in ST14A cells (a neural progenitor cell line derived from the striatum of embryonic day 14 rats) endogenously expressing the other members of the ErbB family: ErbB1, ErbB2, and ErbB3. Using immunoprecipitation assays, we showed that the neuregulin-1beta1 (NRG1beta1) stimulus induced ErbB4 tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3K) recruitment and activation (as demonstrated by Akt phosphorylation) either directly (ErbB4 cyt1 isoform) or indirectly (ErbB4 cyt2 isoform). We examined the ability of the four ErbB4 isoforms to induce chemotaxis and cell proliferation in response to NRG1beta1 stimulation. Using migration assays, we observed that only ErbB4-expressing cells stimulated with NRG1beta1 showed a significant increase in migration, whereas the growth rate remained unchanged. Additional assays showed that inhibition of PI3K (but not of phospholipase Cgamma) dramatically reduced migratory activity. Our data show that ErbB4 signaling via PI3K activation plays a fundamental role in controlling NRG1beta1-induced migration.     

Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway

Ovarian cancer is the leading cause of death from gynecological malignancy for women. The amplification of the PI3K catalytic subunit (p110) and the lost function of PTEN are frequently detected in ovarian cancer cells. PI3K plays an important role in tumorigenesis. To specifically inhibit PI3K activity in ovarian cancer cells, we constructed small interfering RNA (siRNA) against p110. The expression of p110 siRNA significantly decreased cell migration, invasion, and proliferation compared to the siSCR control cells. The expression of p110 siRNA induced CDK inhibitor p27^KIP1 levels, and decreased levels of cyclin D1, CDK4, and phosphorylated retinoblastoma protein. PI3K transmits the mytogenic signal through AKT. AKT has three isoforms in the cells: AKT1, AKT2 and AKT3. We found that inhibition of AKT1 is sufficient to affect cell migration, invasion, and proliferation. Expression of AKT1 siRNA had a similar effect as p110 siRNA in the cells. We showed the roles of specific PI3K and AKT isoforms in the cells, which are important to understanding the mechanism of PI3K/AKT signaling in ovarian cancer cells. Both p110 and AKT1 siRNA-expressing cells decreased the activation of p70S6K1. Inhibition of p70S6K1 activity by its siRNA also decreased cell migration, invasion, and proliferation associated with the induction of p27^KIP1 levels, and with the inhibition of cell cycle-associated proteins including cyclin D1, CDK2, and phosphorylated retinoblastoma protein. This study demonstrates the important role of the PI3K/AKT/mTOR/p70S6K1 pathway in cell proliferation, migration, and invasion in ovarian cancer cells by using siRNA-mediated gene silencing as a reverse genetic method.