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PubMed2006

Multiple signalling pathways involved in beta2-adrenoceptor-mediated glucose uptake in rat skeletal muscle cells.

1. Beta-adrenoceptor (AR) agonists increase 2-deoxy-[3H]-D-glucose uptake (GU) via beta2-AR in rat L6 cells. The beta-AR agonists, zinterol (beta2-AR) and (-)-isoprenaline, increased cAMP accumulation in a concentration-dependent manner (pEC50=9.1+/-0.02 and 7.8+/-0.02). Cholera toxin (% max increase 141.8+/-2.5) and the cAMP analogues, 8-bromo-cAMP (8Br-cAMP) and dibutyryl cAMP (dbcAMP), also increased GU (196.8+/-13.5 and 196.4+/-17.3%). 2. The adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (50 microM), significantly reduced cAMP accumulation to zinterol (100 nM) (109.7+35.0 to 21.6+4.5 pmol well(-1)), or forskolin (10 microM) (230.1+/-58.0 to 107.2+/-26.3 pmol well(-1)), and partially inhibited zinterol-stimulated GU (217+/-26.3 to 176.1+/-20.4%). The protein kinase A (PKA) inhibitor, 4-cyano-3-methylisoquinoline (100 nM), did not inhibit zinterol-stimulated GU. The PDE4 inhibitor, rolipram (10 microM), increased cAMP accumulation to zinterol or forskolin, and sensitised the GU response to zinterol, indicating a stimulatory role of cAMP in GU. 3. cAMP accumulation studies indicated that the beta2-AR was desensitised by prolonged stimulation with zinterol, but not forskolin, whereas GU responses to zinterol increased with time, suggesting that receptor desensitisation may be involved in GU. Receptor desensitisation was not reversed by inhibition of PKA or Gi. 4. PTX pretreatment (100 ng ml(-1)) inhibited insulin or zinterol-stimulated but not 8Br-cAMP or dbcAMP-stimulated GU. The PI3K inhibitor, LY294002 (1 microM), inhibited insulin- (174.9+/-5.9 to 142.7+/-2.7%) and zinterol- (166.9+/-7.6 to 141.1+/-8.1%) but not 8 Br-cAMP-stimulated GU. In contrast to insulin, zinterol did not cause phosphorylation of Akt. 5. The results suggest that GU in L6 cells involves three mechanisms: (1) an insulin-dependent pathway involving PI3K, (2) a beta2-AR-mediated pathway involving both cAMP and PI3K, and (3) a receptor-independent pathway suggested by cAMP analogues that increase GU independently of PI3K. PKA appears to negatively regulate beta2-AR-mediated GU.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2006

Phosphoinositide 3-kinase controls early and late events in mammalian cell division.

Phosphoinositide 3-kinase (PI3K) plays a crucial role in triggering cell division. To initiate this process, PI3K induces two distinct routes, of which one promotes cell growth and the other regulates cyclin-dependent kinases. Fine-tuned PI3K regulation is also required for later cell cycle phases. Here, we review the multiple points at which PI3K controls cell division and discuss its impact on human cancer.

Animals↗
PubMed2006

PTEN tumor suppressor associates with NHERF proteins to attenuate PDGF receptor signaling.

PTEN, a tumor suppressor frequently inactivated in many human cancers, directly antagonizes the activity of phosphatidylinositol-3-OH kinase (PI3K) by dephosphorylating phosphoinositides. We show here that PTEN interacts directly with the NHERF1 and NHERF2 (Na+/H+ exchanger regulatory factor) homologous adaptor proteins through the PDZ motif of PTEN and the PDZ1 domain of NHERF1 or both PDZ domains of NHERF2. NHERFs were shown to interact directly with platelet-derived growth factor receptor (PDGFR), and we demonstrate the assembly of a ternary complex between PTEN, NHERFs and PDGFR. The activation of the PI3K pathway after PDGFR stimulation was prolonged in NHERF1(-/-) mouse embryonic fibroblasts as compared to wild-type cells, consistent with defective PTEN recruitment to PDGFR in the absence of NHERF1. Depletion of NHERF2 by small interfering RNA similarly increased PI3K signaling. Phenotypically, the loss of NHERF1 enhanced the PDGF-induced cytoskeletal rearrangements and chemotactic migration of the cells. These data indicate that, in normal cells, NHERF proteins recruit PTEN to PDGFR to restrict the activation of the PI3K.

Animals↗
PubMed2006

Suppression of Egr-1 transcription through targeting of the serum response factor by oncogenic H-Ras.

The transcription factor Egr-1 functions as a key regulator in cellular growth, differentiation, and apoptosis. The loss of Egr-1 expression is closely associated with tumor development, although the molecular mechanism behind the suppression of Egr-1 is largely unknown. In this report, we show that growth factor-induced transcriptional activation of Egr-1 gene is downregulated by chronic expression of oncogenic H-Ras in NIH3T3 fibroblasts. Our results demonstrate that phosphoinositide 3-kinase (PI3K) signaling is necessary for oncogenic H-Ras-mediated reduction of Egr-1 gene expression. Aberrant activation of PI3K signaling by oncogenic Ras decreased the level of serum response factor (SRF) protein through the acceleration of proteolysis, which resulted in decreased SRF binding to the serum response element (SRE) sites within the Egr-1 promoter, leading to the suppression of Egr-1 transcription. Inhibition of PI3K signaling restored the downregulation of SRF and Egr-1 expression caused by oncogenic Ras. Our findings suggest a novel signaling mechanism by which prolonged activation of oncogenic H-Ras can trigger the loss of tumor suppressor Egr-1 through the PI3K pathway in NIH3T3 fibroblast model cell lines.

3T3 Cells↗
PubMed2004

beta1-Integrins induce phosphorylation of Akt on serine 473 independently of focal adhesion kinase and Src family kinases.

Adhesion by means of beta1-integrins induces the phosphorylation of Akt, an event strictly dependent on the activity of the phosphatidylinositol 3-kinase (PI3K). Binding of the p85 regulatory subunit of PI3K to phosphorylated tyrosine 397 in focal adhesion kinase (FAK) is considered to be the mechanism of cell adhesion-induced activation of class Ia PI3K. Here we show that PI3K-dependent phosphorylation of Akt in response to ligation of beta1-integrins occurs efficiently in the absence of FAK tyrosine phosphorylation. Akt S473 phosphorylation was strongly promoted both in cells expressing the integrin subunit splice variant beta1B, which is unable to activate FAK, and in FAK knockout cells. In addition, we found this phosphorylation to be independent of the Src family kinases Src, Fyn and Yes. These results indicate that a major pathway for adhesion-dependent activation of PI3K/Akt is triggered by the membrane proximal part of the beta1 subunit in a FAK and Src-independent manner.

Animals↗
PubMed2005

Phosphoinositide 3-kinase is required for human adipocyte differentiation in culture.

OBJECTIVE: Phosphoinositide 3-kinase (PI3K) is required for murine adipocyte differentiation. However, a recent report concluded that PI3K was not involved in the differentiation of human preadipocytes into adipocytes. We have re-examined the role of PI3K in human preadipocyte differentiation, enrolling more patients and using more adipogenic indices. METHODS: Human preadipocytes, isolated from nine patients, were induced to differentiate in the presence or absence of 100 nM wortmannin. After 12-15 days, triacylglycerol accumulation and the expression of adipogenic markers (fatty acid synthase and adiponectin) were measured. RESULTS: A significant inhibition in triacylglycerol accumulation and in the induction of fatty acid synthase protein expression was observed, but there was no effect on adiponectin protein expression. CONCLUSION: Inhibition of PI3K reduces the differentiation of human preadipocytes into adipocytes, suggesting a role for this enzyme in the human adipogenic process.

Adipocytes↗
PubMed2003

A new selective AKT pharmacological inhibitor reduces resistance to chemotherapeutic drugs, TRAIL, all-trans-retinoic acid, and ionizing radiation of human leukemia cells.

It is now well established that the reduced capacity of tumor cells of undergoing cell death through apoptosis plays a key role both in the pathogenesis of cancer and in therapeutic treatment failure. Indeed, tumor cells frequently display multiple alterations in signal transduction pathways leading to either cell survival or apoptosis. In mammals, the pathway based on phosphoinositide 3-kinase (PI3K)/Akt conveys survival signals of extreme importance and its downregulation, by means of pharmacological inhibitors of PI3K, considerably lowers resistance to various types of therapy in solid tumors. We recently described an HL60 leukemia cell clone (HL60AR cells) with a constitutively active PI3K/Akt pathway. These cells were resistant to multiple chemotherapeutic drugs, all-trans-retinoic acid (ATRA), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Treatment with two pharmacological inhibitors of PI3K, wortmannin and Ly294002, restored sensitivity of HL60AR cells to the aforementioned treatments. However, these inhibitors have some drawbacks that may severely limit or impede their clinical use. Here, we have tested whether or not a new selective Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (Akt inhibitor), was as effective as Ly294002 in lowering the sensitivity threshold of HL60 cells to chemotherapeutic drugs, TRAIL, ATRA, and ionizing radiation. Our findings demonstrate that, at a concentration which does not affect PI3K activity, the Akt inhibitor markedly reduced resistance of HL60AR cells to etoposide, cytarabine, TRAIL, ATRA, and ionizing radiation. This effect was likely achieved through downregulation of expression of antiapoptotic proteins such as c-IAP1, c-IAP2, cFLIP(L), and of Bad phosphorylation on Ser 136. The Akt inhibitor did not influence PTEN activity. At variance with Ly294002, the Akt inhibitor did not negatively affect phosphorylation of protein kinase C-zeta and it was less effective in downregulating p70S6 kinase (p70S6K) activity. The Akt inhibitor increased sensitivity to apoptotic inducers of K562 and U937, but not of MOLT-4, leukemia cells. Overall, our results indicate that selective Akt pharmacological inhibitors might be used in the future for enhancing the sensitivity of leukemia cells to therapeutic treatments that induce apoptosis or for overcoming resistance to these treatments.

Antineoplastic Agents↗
PubMed2001

Mechanisms of regulation of cell adhesion and motility by insulin receptor substrate-1 in prostate cancer cells.

LNCaP cells are human prostatic cancer cells that have a frame-shift mutation of the tumor suppressor gene PTEN and do not express the insulin receptor substrate-1 (IRS-1), a major substrate of the type 1 insulin-like growth factor receptor (IGF-IR). Ectopic expression of IRS-1 in LNCaP cells increases cell adhesion and decreases cell motility by an IGF-I-independent mechanism. We show now that these effects of IRS-1 are accompanied by serine phosphorylation of IRS-1 and are inhibited by inhibitors of phosphatidylinositol 3-kinase (PI3K). We have confirmed the requirement for PI3K activity and serine phosphorylation by the use of IRS-1 mutants, expressed in LNCaP cells. Serine phosphorylation inhibits IGF-I-induced tyrosyl phosphorylation of IRS-1, which is restored by the expression of wild-type PTEN or by inhibition of PI3K activity. Finally, IRS-1 in LNCaP cells co-immunoprecipitates with integrin alpha 5 beta 1, and the association is again IGF-I-independent. We conclude that in LNCaP cells, IRS-1 is serine phosphorylated by PI3K, generating effects that are different, and even opposite, from those generated by IGF-I.

Adenocarcinoma↗
PubMed2001

Phosphoinositide-3 kinase-PKB/Akt pathway activation is involved in fibroblast Rat-1 transformation by human T-cell leukemia virus type I tax.

Activated phosphoinositide 3-kinase (PI3K) and its downstream target Akt are essential for the fibroblast transformation induced by many viral products. Tax, encoded by human T-cell leukemia virus type I (HTLV-I), has been demonstrated to induce the transformation of rat fibroblast Rat-1 cell through NF-kappaB activation. By stable transfection of Rat-1 cells with expressing constructs of Tax and its mutant M47, which is defective in HTLV-I LTR transactivation, we selected their transformed clones, which have characteristics of NF-kappaB activation and colony formation beyond the cell monolayer (a malignant phenotype). However, these two characteristics in the transformed clones of Tax and M47 disappear after these cells have been treated with wortmannin, a specific inhibitor of PI3K. Further, increased activity of the PI3K/Akt is observed in the transformed clones of Tax and M47 as compared to the clones of empty vector Neo and the M148, which is defective in NF-kappaB activation and cell transformation. Increased activity of PI5K is present in the transformed clones of both Tax and M47 and in the M148 clone as compared to that in the Neo cell. It is known that the efficiency of Tax-induced cell transformation is not high; a minority of Tax-expressing clones show transformation, although the majority of Tax-expressing clones show activated NF-kappaB. A Tax-expressing, nontransformed clone after transfection with an active form of the catalytic subunit of PI3K, p110alpha, becomes transformed. Consistent with these results, a Tax highly-expressing human T-cell line MT2 exhibits both higher polyphosphoinositide turnover and higher activities of PI3K and PI5K than those of Jurkat or MT1 and HTLV-I-negative and a Tax-unexpressing cell line, respectively. These results demonstrate that the activation of the PI3K/Akt signaling pathway, excepting for the NF-kappaB, is also required for the cell transformation induced by Tax.

Androstadienes↗
PubMed2001

The tyrosine phosphatase SHP-2 is required for mediating phosphatidylinositol 3-kinase/Akt activation by growth factors.

SHP-2 is a ubiquitously expressed non-transmembrane tyrosine phosphatase with two SH2 domains. Multiple reverse-genetic studies have indicated that SHP-2 is a required component for organ and animal development. SHP-2 wild-type and homozygous mutant mouse fibroblast cells in which the N-terminal SH2 domain was target-deleted were used to examine the function of SHP-2 in regulating Phosphatidylinositol 3-Kinase (PI3K) activation by growth factors. In addition, SHP-2 and various mutants were introduced into human glioblastoma cells as well as SHP-2(-/-) mouse fibroblasts. We found that EGF stimulation and EGFR oncoprotein (DeltaEGFR) expression independently induced the co-immunoprecipitation of the p85 subunit of PI3K with SHP-2. Targeted deletion of the N-terminal SH2 domain of SHP-2 severely impaired PDGF- and IGF-induced Akt phosphorylation. Ectopic expression of SHP-2 in U87MG gliobastoma cells elevated EGF-induced Akt phosphorylation, and the effect was abolished by mutation of its N-terminal SH2 domain. Likewise, the reconstitution of SHP-2 expression in the SHP-2(-/-) cells enhanced Akt phosphorylation induced by EGF while rescuing that induced by PDGF and IGF. Further lipid kinase activity assays confirmed that SHP-2 modulation of Akt phosphorylation correlated with its regulation of PI3K activation. Based on these results, we conclude that SHP-2 is required for mediating PI3K/Akt activation, and the N-terminal SH2 domain is critically important for a "positive" role of SHP-2 in regulating PI3K pathway activation.

Animals↗
PubMed2002

Induction of human MDR1 gene expression by 2-acetylaminofluorene is mediated by effectors of the phosphoinositide 3-kinase pathway that activate NF-kappaB signaling.

The expression of P-glycoprotein encoded by the multidrug resistance (MDR1) gene is associated with the emergence of the MDR phenotype in cancer cells. Human MDR1 and its rodent homolog mdr1a and mdr1b are frequently overexpressed in liver cancers. However, the underlying mechanisms are largely unknown. The hepatocarcinogen 2-acetylaminofluorene (2-AAF) efficiently activates rat mdr1b expression in cultured cells and in Fisher 344 rats. We recently reported that activation of rat mdr1b in cultured cells by 2-AAF involves a cis-activating element containing a NF-kappaB binding site located -167 to -158 of the rat mdr1b promoter. 2-AAF activates IkappaB kinase (IKK), resulting in degradation of IkappaBbeta and activation of NF-kappaB. In this study, we report that 2-AAF could also activate the human MDR1 gene in human hepatoma and embryonic fibroblast 293 cells. Induction of MDR1 by AAF was mediated by DNA sequence located at -6092 which contains a NF-kappaB binding site. Treating hepatoma cells with 2-AAF activated phosphoinositide 3-kinase (PI3K) and its downstream effectors Rac1, and NAD(P)H oxidase. Transient transfection assays demonstrated that constitutively activated PI3K and Rac1 enhanced the activation of the MDR1 promoter by 2-AAF. Treatment of hepatoma cells with 2-AAF also activated another PI3K downstream effector Akt. Transfection of recombinant encoding a dominant activated Akt also enhanced the activation of MDR1 promoter activation by 2-AAF. These results demonstrated that 2-AAF up-regulates MDR1 expression is mediated by the multiple effectors of the PI3K signaling pathway.

2-Acetylaminofluorene↗
PubMed2002

ETV6-NTRK3 transformation requires insulin-like growth factor 1 receptor signaling and is associated with constitutive IRS-1 tyrosine phosphorylation.

Congenital fibrosarcoma (CFS) and cellular mesoblastic nephroma (CMN) are pediatric spindle cell malignancies that share two specific cytogenetic abnormalities: trisomy of chromosome 11 and a t(12;15)(p13;q25) translocation. The t(12;15) rearrangement creates a transcriptionally active fusion gene that encodes a chimeric oncoprotein, ETV6-NTRK3 (EN). EN transforms NIH3T3 fibroblasts through constitutive activation of both the Ras-mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol-3'kinase (PI3K)-Akt pathway. However, the role of trisomy 11 in CFS and CMN remains unknown. In this study we demonstrate elevated expression of the chromosome 11p15.5 insulin-like growth factor 2 gene (IGF2) in CFS and CMN tumors. Moreover, we present evidence that an intact IGF signaling axis is essential for in vitro EN-mediated transformation. EN only very weakly transformed so-called R-murine fibroblasts derived from mice with a targeted disruption of the IGF1 receptor gene (IGFRI), but transformation activity was fully restored in R- cells engineered to re-express IGFRI (R+ cells). We also observed that the major IGFRI substrate, insulin-receptor substrate-1 (IRS-1), was constitutively tyrosine phosphorylated and could be co-immunoprecipitated with EN in either R- or R+ cells expressing the EN oncoprotein. IRS-1 association with Grb2 and PI3K p85, which link IGFRI to the Ras-MAPK and PI3K-Akt pathways, respectively, was enhanced in both cell types in the presence of EN. However, activation of the Ras-MAPK and PI3K-Akt pathways was markedly attenuated in EN-expressing R- cells compared to EN-transformed R+ cells. This suggests that IRS-1 may be functioning as an adaptor in EN signal transduction, but that a link to EN transformation pathways requires the presence of IGFRI. Our findings indicate that an intact IGF signaling axis is essential for EN transformation, and are consistent with a role for trisomy 11 in augmenting this pathway in EN expressing tumors.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Selective PDZ protein-dependent stimulation of phosphatidylinositol 3-kinase by the adenovirus E4-ORF1 oncoprotein.

While PDZ domain-containing proteins represent cellular targets for several different viral oncoproteins, including human papillomavirus E6, human T-cell leukemia virus type 1 Tax, and human adenovirus E4-ORF1, the functional consequences for such interactions have not been elucidated. Here we report that, at the plasma membrane of cells, the adenovirus E4-ORF1 oncoprotein selectively and potently stimulates phosphatidylinositol 3-kinase (PI3K), triggering a downstream cascade of events that includes activation of both protein kinase B and p70S6-kinase. This activity of E4-ORF1 could be abrogated by overexpression of its PDZ-protein targets or by disruption of its PDZ domain-binding motif, which was shown to mediate complex formation between E4-ORF1 and PDZ proteins at the plasma membrane of cells. Furthermore, E4-ORF1 mutants unable to activate the PI3K pathway failed to transform cells in culture or to promote tumors in animals, and drugs that block either PI3K or p70S6-kinase inhibited E4-ORF1-induced transformation of cells. From these results, we propose that the transforming and tumorigenic potentials of the adenovirus E4-ORF1 oncoprotein depend on its capacity to activate PI3K through a novel PDZ protein-dependent mechanism of action.

Adenovirus E4 Proteins↗
PubMed2003

The RAS signal transduction pathway and its role in radiation sensitivity.

RAS has been shown to increase radiation resistance. Upstream and downstream pathways from RAS could thus be targets for manipulation of radiosensitivity. EGFR expression and AKT phosphorylation are also associated with the response to radiation. A retrospective study evaluating EGFR and AKT in patients treated with multimodality therapy found a significant association between P-AKT and treatment failure. Moreover, these data are strengthened by in vitro studies showing that inhibition of EGFR, RAS, PI3K, and AKT radiosensitized cancer cell lines. We have previously shown that PI3K is a mediator of RAS-induced radiation resistance. We now suggest that EGFR, which is upstream of PI3K, may also mediate resistance through a common pathway. In addition to EGFR and RAS, PTEN can also regulate the PI3K pathway. Identifying a common signal for EGFR, RAS, or PTEN that results in radiation resistance may uncover targets for developing molecular-based radiosensitization protocols for tumors resistant to radiation and thus improve local control.

Animals↗
PubMed2004

Effects of a conditionally active v-ErbB and an EGF-R inhibitor on transformation of NIH-3T3 cells and abrogation of cytokine dependency of hematopoietic cells.

Epidermal growth factor (EGF) and its cognate receptor (EGF-R) are often dysregulated in human neoplasia. Moreover, EGF-R-transformed cell lines have constitutive EGF-R activity, which makes elucidation of its effects difficult to determine. In the following studies, the effects of a novel conditionally activated form of EGF-R, v-ErbB:ER, on the morphological transformation of NIH-3T3 cells and the abrogation of hematopoietic cell cytokine dependence were investigated. The v-ErbB ES-4 oncogene was fused to the hormone binding domain of the estrogen receptor (ER). This construct, v-ErbB:ER, requires beta-estradiol or 4-OH tamoxifen for activation. v-ErbB:ER conditionally transformed NIH-3T3 cells and abrogated cytokine dependence of hematopoietic cells. Stimulation of v-ErbB:ER activity resulted in the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and Raf/MEK/ERK kinase cascades. To determine the importance of these signal transduction pathways, the conditionally transformed hematopoietic cells were treated with EGF-R, PI3K and MEK inhibitors. The EGF-R inhibitor AG1478 effectively inhibited MEK, ERK and Akt activation, and induced apoptosis when the cells were grown in response to v-ErbB:ER. Apoptosis was observed at 100- to 1000-fold lower concentrations of AG1478 when the cells were grown in response to v-ErbB:ER as opposed to IL-3. Furthermore, the parental, BCR-ABL- and Raf-transformed cells were only susceptible to the apoptosis-inducing effects of AG1478 at the highest concentrations demonstrating the specificity of these inhibitors. MEK or PI3K inhibitors suppressed ERK or Akt activation, respectively, and induced apoptosis in the v-ErbB:ER-responsive cells. However, MEK and PI3K inhibitors only induced apoptosis at 1000-fold higher concentrations than the EGFR inhibitor. This novel v-ErbB:ER construct and these conditionally transformed cell lines will be useful to further elucidate ErbB-mediated signal transduction and to determine the effectiveness of various inhibitors in targeting different aspects of EGF-R-mediated signal transduction and malignant transformation.

3T3 Cells↗
PubMed2005

Alternative phospholipase D/mTOR survival signal in human breast cancer cells.

Cancer cells generate survival signals to suppress default apoptotic programs that protect from cancer. Phosphatidylinositol-3-kinase (PI3K) generates a survival signal that is frequently dysregulated in human cancers. Phospholipase D (PLD) has also been implicated in signals that promote survival. One of the targets of PLD signaling is mTOR (mammalian target of rapamycin), a critical regulator of cell cycle progression and cell growth. We report here that elevated PLD activity in the MDA-MB-231 human breast cancer cell line generates an mTOR-dependent survival signal that is independent of PI3K. In contrast, MDA-MB-435S breast cancer cells, which have very low levels of PLD activity, are dependent on PI3K for survival signals. The data presented here identify an alternative survival signal that is dependent on PLD and mTOR and is active in a breast cancer cell line where the PI3K survival pathway is not active.

Breast Neoplasms↗
PubMed2006

beta1-integrin-mediated signaling essentially contributes to cell survival after radiation-induced genotoxic injury.

Integrin-mediated adhesion to extracellular matrix proteins confers resistance to radiation- or drug-induced genotoxic injury. To analyse the underlying mechanisms specific for beta1-integrins, wild-type beta1A-integrin-expressing GD25beta1A cells were compared to GD25beta1B cells, which express signaling-incompetent beta1B variants. Cells grown on fibronectin, collagen-III, beta1-integrin-IgG or poly-l-lysine were exposed to 0-6 Gy X-rays in presence or depletion of growth factors and phosphatidylinositol-3 kinase (PI3K) inhibitors (LY294002, wortmannin). In order to test the relevance of these findings in tumor cells, human A-172 glioma cells were examined under the same conditions after siRNA-mediated silencing of beta1-integrins. We found that beta1A-integrin-mediated adhesion to fibronectin, collagen-III or beta1-IgG was essential for cell survival after radiation-induced genotoxic injury. Mediated by PI3K, pro-survival beta1A-integrin/Akt signaling was critically involved in this process. Additionally, the beta1-integrin downstream targets p130Cas and paxillin-impaired survival-regulating PI3K-dependent JNK. In A-172 glioma cells, beta1-integrin knockdown and PI3K inhibition confirmed the central role of beta1-integrins in Akt- and p130Cas/paxillin-mediated prosurvival signaling. These findings suggest beta1-integrins as critical regulators of cell survival after radiation-induced genotoxic injury. Elucidation of the molecular circuitry of prosurvival beta1-integrin-mediated signaling in tumor cells may promote the development of innovative molecular-targeted therapeutic antitumor strategies.

Animals↗
PubMed2006

eEF1A2 activates Akt and stimulates Akt-dependent actin remodeling, invasion and migration.

eEF1A2 (eukaryotic protein elongation factor 1 alpha 2) is a protein translation factor that is likely a human oncogene by virtue of its capacity to transform mammalian cells and its high expression in tumors of the ovary, breast and lung. Here, we show that expression of eEF1A2 is sufficient to stimulate the formation of filopodia in BT549 human breast cancer cells and non-transformed Rat2 cells. Filopodia formation in eEF1A2-expressing cells is dependent on the activity of phosphatidylinositol-3 kinase (PI3K), and the ROCK and Akt kinases. Furthermore, eEF1A2 expression is sufficient to activate Akt in a PI3K-dependent fashion and inactivation of eEF1A2 by short interfering RNA reduces Akt activity. Using breast cancer cell line BT 549, we show that eEF1A2 expression stimulates cell migration and invasion in a largely PI3K- and Akt-dependent manner. These results suggest that eEF1A2 regulates oncogenesis through Akt and PI3K-dependent cytoskeletal remodeling.

Actins↗