Insulin-like growth factor-I (IGF-I) induces epidermal growth factor receptor transactivation and cell proliferation through reactive oxygen species

Insulin-like growth factor-I (IGF-I) plays an important role in proliferation of vascular smooth muscle cells (VSMCs). However, the mechanism that IGF-I induces VSMCs proliferation is not completely understood. In this study, we determined (a) whether and how IGF-I induces transactivation of epidermal growth factor receptor (EGFR) in primary rat aortic VSMCs, (b) the contribution of EGFR to IGF-I-stimulated activation of extracellular signal-regulated kinase (ERK) and cell proliferation, and (c) the role of reactive oxygen species (ROS) in the cellular function. We showed that IGF-I induced phosphorylation of EGFR and ERK1/2 in VSMCs. AG1478, an EGFR inhibitor, inhibited IGF-I-induced phoshorylation of EGFR and ERK1/2. IGF-I stimulated ROS production and Src activation. Antioxidants inhibited IGF-I-induced ROS generation and activation of EGFR, ERK, and Src. Src kinase inhibitor PP1 and Src siRNA blocked IGF-I-induced activation of EGFR and ERK1/2. Inhibition of IGF-I-stimulated EGFR activation inhibited IGF-I-induced VSMC proliferation. These results suggest that (1) IGF-I induces EGFR activation through production of ROS and ROS-mediated Src activation in VSMCs, and (2) EGFR transactivation is required for IGF-I-induced VSMC proliferation.     

Inhibition of proliferation and induction of apoptosis in breast cancer cells by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 ('Iressa') is independent of EGFR expression level

High expression of the epidermal growth factor receptor (EGFR) in breast carcinoma confers a growth advantage to the tumor cells. The EGFR tyrosine kinase inhibitor (EGFR-TKI) ZD1839 ('Iressa') has clinical activity in a wide range of tumor types, although the mechanism(s) by which it exerts its antitumor activity effects remain unclear. We analyzed the ability of ZD1839 to induce apoptosis and/or inhibition of proliferation in breast carcinoma cell lines, as well any association between this ability and the downregulation activity of MAPK and Akt, two recently proposed markers of ZD1839 activity. Proliferation, survival, and activation of Akt and MAPK were evaluated in six human breast cancer cell lines expressing various levels of EGFR and HER2 and exposed to ZD1839. EGFR and HER2 expression levels were determined using specific monoclonal antibodies and FACS analysis. The effects of ZD1839 were independent of EGFR expression levels, but were influenced by high HER2 expression. ZD1839 significantly reduced the rate of [3H]-thymidine incorporation in the four sensitive cell lines, while apoptosis was also induced in two of these cell lines. No correlation was found between the cytostatic or cytotoxic effects of ZD1839 and its ability to downregulate MAPK and Akt activity in the tumor cell lines. Our data suggest that the antitumor activity of ZD1839 is due to a cytostatic effect, and involves apoptosis induction in a subset of sensitive cells only, and that neither MAPK nor Akt is a reliable marker of ZD1839 activity.     

ZD1839 (Gefitinib, 'Iressa'), an epidermal growth factor receptor-tyrosine kinase inhibitor, enhances the anti-cancer effects of TRAIL in human esophageal squamous cell carcinoma

The EGF (epidermal growth factor) receptor-tyrosine kinase inhibitor ZD1839 (Gefitinib, 'Iressa') blocks the cell signaling pathways involved in cell proliferation, survival, and angiogenesis in various cancer cells. TNF-related death apoptosis inducing ligand (TRAIL) acts as an anticancer agent. We investigated the antitumor effects of ZD1839 alone or in combination with TRAIL against human esophageal squamous cell cancer (ESCC) lines. Although all ESCC cells expressed EGF receptor at a protein level, the effect of ZD1839 on cell growth did not correlate with the level of EGFR expression and phosphorylation of EGF receptor protein in ESCC lines. ZD1839 caused a dose-dependent growth arrest at G0-G1 phase associated with increased p27 expression. As TE8 cells are resistant to TRAIL, we tested whether ZD1839 combined with TRAIL induced apoptosis of TE8 cells via the inhibition of EGF receptor signaling by ZD1839. ZD1839 inhibited the phosphorylation of Akt, and enhanced TRAIL-induced apoptosis via activation of caspase-3 and caspase-9, and inactivation of Bcl-xL. Our results indicated that ZD1839 has anti-cancer properties against human esophageal cancer cells. ZD1839 also augmented the anti-cancer activity of TRAIL, even in TRAIL-resistant tumors. These results suggest that treatment with ZD1839 and TRAIL may have potential in the treatment of ESCC patients.     

Activation of BAD by therapeutic inhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor receptor

Novel cancer chemotherapeutics are required to induce apoptosis by activating pro-apoptotic proteins. Both epidermal growth factor (EGF) and insulin-like growth factor (IGF) provide potent survival stimuli in many epithelia, and activation of their receptors is commonly observed in solid human tumors. Here we demonstrate that blockade of the EGF receptor by a new drug in phase III clinical trails for cancer, ZD1839, potently induces apoptosis in mammary epithelial cell lines and primary cultures, as well as in a primary pleural effusion from a breast cancer patient. We identified the mechanism of apoptosis induction by ZD1839. We showed that it prevents cell survival by activating the pro-apoptotic protein BAD. Moreover, we demonstrate that IGF transactivates the EGF receptor and that ZD1839 blocks IGF-mediated phosphorylation of MAPK and BAD. Many cancer therapies kill tumor cells by inducing apoptosis as a consequence of targeting DNA; however, the threshold at which apoptosis can be triggered through DNA damage is often different from that in normal cells. Our results indicate that by targeting a growth factor-mediated survival signaling pathway, BAD phosphorylation can be manipulated therapeutically to induce apoptosis.     

The activated insulin-like growth factor I receptor induces depolarization in breast epithelial cells characterized by actin filament disassembly and tyrosine dephosphorylation of FAK, Cas, and paxillin.

Insulin-like growth factor I (IGF-I) promotes the motility of different cell types. We investigated the role of IGF-I receptor (IGF-IR) signaling in locomotion of MCF-7 breast cancer epithelial cells overexpressing the wild-type IGF-IR (MCF-7/IGF-IR). Stimulation of MCF-7/IGF-IR cells with 50 ng/ml IGF-I induced disruption of the polarized cell monolayer followed by morphological transition toward a mesenchymal phenotype. Immunofluorescence staining of the cells with rhodamine-phalloidin revealed rapid disassembly of actin fibers and development of a cortical actin meshwork. Activation of phosphatidylinositol (PI)3-kinase downstream of the IGF-IR was necessary for this process, as blocking PI 3-kinase activity with the specific inhibitor LY 294002 at 10 microM prevented disruption of the filamentous actin. In parallel, IGF-IR activation induced rapid and transient tyrosine dephosphorylation of focal adhesion proteins p125 focal adhesion kinase (FAK), p130 Crk-associated substrate (Cas), and paxillin. This process required phosphotyrosine phosphatase (PTP) activity, since pretreatment of the cells with 5 microM phenylarsine oxide (PAO), an inhibitor of PTPs, rescued FAK and its associated proteins Cas and paxillin from IGF-I-induced dephosphorylation. In addition, PAO-pretreated cells were refractory to IGF-I-induced morphological transition. Thus, our findings reveal a new function of the IGF-IR, the ability to depolarize epithelial cells. In MCF-7 cells, mechanisms of IGF-IR-mediated cell depolarization involve PI 3-kinase signaling and putative PTP activities.     

Impaired Shc, Ras, and MAPK activation but normal Akt activation in FL5.12 cells expressing an insulin-like growth factor I receptor mutated at tyrosines 1250 and 1251

The Y1250F/Y1251F mutant of the insulin-like growth factor I receptor (IGF-IR) has tyrosines 1250 and 1251 mutated to phenylalanines and is deficient in IGF-I-mediated suppression of apoptosis in FL5.12 lymphocytic cells. To address the mechanism of loss of function in this mutant we investigated signaling responses in FL5.12 cells overexpressing either a wild-type (WT) or Y1250F/Y1251F (mutant) IGF-IR. Cells expressing the mutant receptor were deficient in IGF-I-induced phosphorylation of the JNK pathway and had decreased ERK and p38 phosphorylation. IGF-I induced phosphorylation of Akt was comparable in WT and mutant expressing cells. The decreased activation of the mitogen-activated protein kinase (MAPK) pathways was accompanied by greatly decreased Ras activation in response to IGF-I. Although phosphorylation of Gab2 was similar in WT and mutant cell lines, phosphorylation of Shc on Tyr(313) in response to IGF-I was decreased in cells expressing the mutant receptor, as was recruitment of Grb2 and Ship to Shc. However, phosphorylation of Shc on Tyr(239), the Src phosphorylation site, was normal. A role for JNK in the survival of FL5.12 cells was supported by the observation that the JNK inhibitor SP600125 suppressed IGF-I-mediated protection from apoptosis. Altogether these data demonstrate that phosphorylation of Shc, and assembly of the Shc complex necessary for activation of Ras and the MAPK pathways are deficient in cells expressing the Y1250F/Y1251F mutant IGF-IR. This would explain the loss of IGF-I-mediated survival in FL5.12 cells expressing this mutant and may also explain why this mutant IGF-IR is deficient in functions associated with cellular transformation and cell migration in fibroblasts and epithelial tumor cells.     

Induced autocrine signaling through the epidermal growth factor receptor contributes to the response of mammary epithelial cells to tumor necrosis factor alpha

In contrast to the well known cytotoxic effects of tumor necrosis factor (TNF) alpha in many mammary cancer cells, we have found that TNF stimulates the proliferation and motility of human mammary epithelial cells (HMECs). Since the response of HMECs to TNF is similar to effects mediated by epidermal growth factor receptor (EGFR) activation, we explored the potential role of cross-talk through the EGFR signaling pathways in mediating cellular responses to TNF. Using a microarray enzyme-linked immunoassay, we found that exposure to TNF stimulated the dose-dependent shedding of the EGFR ligand transforming growth factor alpha (TGFalpha). Both proliferation and motility of HMECs induced by TNF was prevented either by inhibiting membrane protein shedding with a metalloprotease inhibitor, by blocking epidermal growth factor receptor (EGFR) kinase activity, or by limiting ligand-receptor interactions with an antagonistic anti-EGFR antibody. EGFR activity was also necessary for TNF-induced release of matrix metalloprotease-9, thought to be an essential regulator of mammary cell migration. The cellular response to TNF was associated with a biphasic temporal pattern of extracellular signal-regulated kinase (ERK) phosphorylation, which was EGFR-dependent and modulated by inhibition of metalloprotease-mediated shedding. Significantly, the late phase of ERK phosphorylation, detectable within 4 h after exposure, was blocked by the metalloprotease inhibitor batimastat, indicating that autocrine signaling through ligand shedding was responsible for this secondary wave of ERK activity. Our results indicate a novel and important role for metalloprotease activation and EGFR transmodulation in mediating the cellular response to TNF.     

The Reciprocal Regulation of γ-Synuclein and IGF-I Receptor Expression Creates a Circuit That Modulates IGF-I Signaling

Insulin-like growth factor (IGF) system plays important roles in carcinogenesis and maintenance of the malignant phenotype. Signaling through the IGF-I receptor (IGF-IR) has been shown to stimulate the growth and motility of a wide range of cancer cells. γ-Synuclein (SNCG) is primarily expressed in peripheral neurons but also overexpressed in various cancer cells. Overexpression of SNCG correlates with tumor progression. In the present study we demonstrated a reciprocal regulation of IGF-I signaling and SNCG expression. IGF-I induced SNCG expression in various cancer cells. IGF-IR knockdown or IGF-IR inhibitor repressed SNCG expression. Both phosphatidylinositol 3-kinase and mitogen-activated protein kinase were involved in IGF-I induction of SNCG expression. Interestingly, SNCG knockdown led to proteasomal degradation of IGF-IR, thereby decreasing the steady-state levels of IGF-IR. Silencing of SNCG resulted in a decrease in ligand-induced phosphorylation of IGF-IR and its downstream signaling components, including insulin receptor substrate (IRS), Akt, and ERK1/2. Strikingly, SNCG physically interacted with IGF-IR and IRS-2. Silencing of IRS-2 impaired the interaction between SNCG and IGF-IR. Finally, SNCG knockdown suppressed IGF-I-induced cell proliferation and migration. These data reveal that SNCG and IGF-IR are mutually regulated by each other. SNCG blockade may suppress IGF-I-induced cell proliferation and migration. Conversely, IGF-IR inhibitors may be of utility in suppressing the aberrant expression of SNCG in cancer cells and thereby block its pro-tumor effects.     

Anchorage-independent activation of mitogen-activated protein kinase through phosphatidylinositol-3 kinase by insulin-like growth factor I

Insulin-like growth factor I (IGF-I) is a well-established mitogen in human breast cancer cells. We show here that human breast cancer MCF-7 cells, which were prevented from attaching to the substratum and were floating in medium, responded to IGF-I and initiated DNA synthesis. The addition of IGF-I to floating cells induced activation of protein kinase B (PKB)/Akt, as to cells attached to the substratum. In addition, mitogen-activated protein kinase (MAPK)/extracellular response kinase (ERK) and its upstream kinases, ERK kinase (MEK) and Raf-1, were activated by IGF-I in floating cells. While the IGF-I-induced activation of PKB/Akt was inhibited by PI3-K inhibitor LY294002 but not by MEK inhibitor PD98059, the activation of both MEK and ERK by IGF-I was inhibited by both. These findings suggest that the IGF-I signal that leads to stimulation of DNA synthesis of MCF-7 cells is transduced to ERK through PI3-K, only when they are anchorage-deficient.     

The essential role of the death domain kinase receptor-interacting protein in insulin growth factor-I-induced c-Jun N-terminal kinase activation

Insulin-like growth factor I (IGF-I) plays an important role in cell survival, proliferation, and differentiation. Diverse kinases, including AKT/protein kinase B, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), can be activated by IGF-I. Here, we show that the receptor-interacting protein (RIP), a key mediator of tumor necrosis factor-induced NF-kappaB and JNK activation, plays a key role in IGF-I receptor signaling. IGF-I induced a robust JNK activation in wild type but not RIP null (RIP-/-) mouse embryonic fibroblast cells. Reconstitution of RIP expression in the RIP-/- cells restored the induction of JNK by IGF-I, suggesting that RIP is essential in IGF-I-induced JNK activation. Reconstitution experiments with different RIP mutants further revealed that the death domain and the kinase activity of RIP are not required for IGF-I-induced JNK activation. Interestingly, the AKT and ERK activation by IGF-I was normal in RIP-/- cells. The phosphatidylinositol 3-kinase inhibitor, wortmannin, did not affect IGF-I-induced JNK activation. These results agree with previous studies showing that the IGF-I-induced JNK activation pathway is distinct from that of ERK and AKT activation. Additionally, physical interaction of ectopically expressed RIP and IGF-IRbeta was detected by co-immunoprecipitation assays. More importantly, RIP was recruited to the IGF-I receptor complex during IGF-I-induced signaling. Furthermore, we found that IGF-I-induced cell proliferation was impaired in RIP-/- cells. Taken together, our results indicate that RIP, a key factor in tumor necrosis factor signaling, also plays a pivotal role in IGF-I-induced JNK activation and cell proliferation.     

Herstatin, an autoinhibitor of the human epidermal growth factor receptor 2 tyrosine kinase, modulates epidermal growth factor signaling pathways resulting in growth arrest

Herstatin is an autoinhibitor of the ErbB family consisting of subdomains I and II of the human epidermal growth factor receptor 2 (ErbB-2) extracellular domain and a novel C-terminal domain encoded by an intron. Herstatin binds to human epidermal growth factor receptor 2 and to the epidermal growth factor receptor (EGFR), blocking receptor oligomerization and tyrosine phosphorylation. In this study, we characterized several early steps in EGFR activation and investigated downstream signaling events induced by epidermal growth factor (EGF) and by transforming growth factor alpha (TGF-alpha) in NIH3T3 cell lines expressing EGFR with and without herstatin. Herstatin expression decreased EGF-induced EGFR tyrosine phosphorylation and delayed receptor down-regulation despite receptor occupancy by ligand with normal binding affinity. Akt stimulation by EGF and TGF-alpha, but not by fibroblast growth factor 2, was almost completely blocked in the presence of herstatin. Surprisingly, EGF and TGF-alpha induced full activation of MAPK in duration and intensity and stimulated association of the EGFR with Shc and Grb2. Although MAPK was fully stimulated, herstatin expression prevented TGF-alpha-induced DNA synthesis and EGF-induced proliferation. The herstatin-mediated uncoupling of MAPK from Akt activation was also observed in Chinese hamster ovary cells co-transfected with EGFR and herstatin. These findings show that herstatin expression alters EGF and TGF-alpha signaling profiles, culminating in inhibition of proliferation.     

Increased expression of epidermal growth factor receptor induces sequestration of extracellular signal-related kinases and selective attenuation of specific epidermal growth factor-mediated signal transduction pathways

Increased expression of the epidermal growth factor receptor (EGFR) is common in cancer and correlates with neoplastic progression. Although the biology of this receptor has been the subject of intense investigation, surprisingly little is known about how increased expression of the wild-type EGFR affects downstream signal transduction in cells. We show that increasing the expression of the receptor results in dramatic shifts in signaling with attenuation of EGF-induced Ras, extracellular signal-related kinases (ERKs), and Akt activation, as well as amplification of STAT1 and STAT3 signaling. In this study, we focus on the mechanism of attenuated ERK signaling and present evidence suggesting that the mechanism of attenuated ERK signaling in EGFR-overexpressing cells is a sequestration of ERKs at the cell membrane in EGFR-containing complexes. Increased expression of the EGFR results in an aberrant localization of ERKs to the cell membrane. Furthermore, ERKs become associated with the EGFR in a physical complex in EGFR-overexpressing cells but not in control cells. The EGFR-ERK association is detected in unstimulated cells or on exposure to a low concentration of EGF; under these conditions, ERK activation is minimal. Exposure of these cells to saturating concentrations of EGF results in a decreased membrane localization of ERKs, a concomitant dissociation of ERKs from the EGFR, and restores ERK activation. A similar association can be detected between the EGFR and MEK1 in receptor-overexpressing cells, suggesting that multiple components of the ERK signaling pathway may become trapped in complexes with the EGFR. These findings can be demonstrated in cells transfected to express high levels of the EGFR as well as in cancer cells which naturally overexpress the EGFR and, thus, may be representative of altered EGFR signaling in human cancer.     

The effect of ZD1839 (Iressa), an epidermal growth factor receptor tyrosine kinase inhibitor, in combination with cisplatin, on apoptosis in SCC-15 cells

The aim of this study was to determine the effect of ZD1839 on growth and apoptosis in SCC-15 (a human head and neck cancer cell line) lone, or in combination with cisplatin. High expression of the epidermal growth factor receptor has been implicated in the development of squamous cell carcinomas of head and neck. ZD1839 ('Iressa') is an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor that blocks signal transduction pathways implicated in proliferation and survival of cancer cells, and other host-dependent processes promoting cancer growth. Here, growth arrest was observed with 3.64 microm ZD1839. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (sMTT) viability assay revealed a significant decrease (P < 0.001) in the percentage of surviving cells upon treatment with ZD1839 and cisplatin compared with cisplatin or ZD1839 on their own. Combined therapy of 3.64 microm ZD1839 for 24 h, prior to administration of 100 microm cisplatin, significantly (P < 0.001) and additively increased the cytotoxicity effect of cisplatin. p53-independent apoptosis was seen with cisplatin treatment, a novel finding. These data support the use of ZD1839 in anti-cancer therapy, and particularly in combination therapy. Cisplatin may induce p53-independent apoptosis. Over-expression of Bcl-2 in head and neck squamous cell carcinoma tumour cell lines is unlikely to be a general mechanism to protect these cells from apoptosis.     

Epidermal growth factor receptor signaling modulates apoptosis via p38alpha MAPK-dependent activation of Bax in intestinal epithelial cells

Previous studies have demonstrated that the proapoptotic protein Bax plays an important role in the elevated enterocyte apoptosis that occurs during the intestinal adaptation response to massive small bowel resection (SBR). Additionally, epidermal growth factor receptor (EGFR) activation prevents SBR-induced enterocyte apoptosis. The present study aims to delineate the relationship between EGFR activity and intestinal epithelial cell apoptosis. Treatment of model intestinal epithelial cells (RIEC-18) with both a selective EGFR inhibitor (ZD1839) and EGFR small interfering RNA knockdown resulted in a dramatic increase in apoptosis, accompanied by rapid phosphorylation of p38alpha. Concurrently, Bax underwent conformational changes consistent with activation and translocated to mitochondria. In contrast, EGF stimulation enhanced cell survival by attenuating p38alpha phosphorylation, Bax conformational change, mitochondrial trafficking, and apoptosis. These results demonstrate that that diminished EGFR activity initiates the intrinsic pathway of apoptosis through p38alpha-dependent Bax activation in intestinal epithelial cells. These finding provide mechanistic insight into the role that EGFR signaling plays in the regulation of enterocyte apoptosis following massive intestinal loss.     

Met Receptor Acts Uniquely for Survival and Morphogenesis of EGFR-Dependent Normal Mammary Epithelial and Cancer Cells

Mammary gland development and breast cancer growth require multiple factors both of endocrine and paracrine origin. We analyzed the roles of Epidermal Growth Factor Receptor (EGFR) and Hepatocyte Growth Factor Receptor (Met) in mammary epithelial cells and mammary tumor cells derived from a mutated-ErbB2 transgenic mice. By using highly specific tyrosine kinase inhibitors we found that MCF-10A and NMuMG mammary epithelial cell lines are totally dependent on EGFR activation for their growth and survival. Proliferation and 3D-morphogenesis assays showed that HGF had no role in maintaining mammary cell viability, but was the only cytokine able to rescue EGFR-inhibited mammary cells. Insulin-Like Growth Factor-I (IGF-I), basic-Fibroblast Growth Factor (b-FGF) and Neuregulin, which are well known mammary morphogenic factors, did not rescue proliferation or morphogenesis in these cell lines, following EGFR inhibition. Similarly, ErbB2-driven tumor cells are EGFR-dependent and also display HGF-mediated rescue. Western-blot analysis of the signaling pathways involved in rescue after EGFR inhibition indicated that concomitant ERK1/2 and AKT activation was exclusively driven by Met, but not by IGF-I or b-FGF. These results describe a unique role for EGFR and Met in mammary epithelial cells by showing that similar pathways can be used by tumorigenic cells to sustain growth and resist to EGFR-directed anti-tumorigenic drugs.     

Carbachol-stimulated transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T(84) cells is mediated by intracellular ca(2+), PYK-2, and p60(src)

Ca(2+)-dependent agonists, such as carbachol (CCh), stimulate epidermal growth factor receptor (EGFR) transactivation and mitogen-activated protein kinase activation in T(84) intestinal epithelial cells. This pathway constitutes an antisecretory mechanism by which CCh-stimulated chloride secretion is limited. Here, we investigated mechanisms underlying CCh-stimulated epidermal growth factor receptor (EGFR) transactivation. Thapsigargin (TG, 2 microM) stimulated EGFR and extracellular signal-regulated kinase (ERK) phosphorylation in T(84) cells. Inhibition of either EGFR or ERK activation, with tyrphostin AG1478 (1 microM) and PD 98059 (20 microM), respectively, potentiated chloride secretory responses to TG, as measured by changes in short-circuit current (I(sc)) across T(84) cells. CCh (100 microM) stimulated tyrosine phosphorylation and association of the Ca(2+)-dependent tyrosine kinase, PYK-2, with the EGFR, which was inhibited by the Ca(2+) chelator, BAPTA (20 microM). The calmodulin inhibitor, fluphenazine (50 microM) inhibited CCh-stimulated PYK-2 association with the EGFR and phosphorylation of EGFR and ERK. CCh also induced tyrosine phosphorylation of p60(src) and association of p60(src) with both PYK-2 and the EGFR. The Src family kinase inhibitor, PP2 (20 nM-20 microM) attenuated CCh-stimulated EGFR and ERK phosphorylation and potentiated chloride secretory responses to CCh. We conclude that CCh-stimulated transactivation of the EGFR is mediated by a pathway involving elevations in intracellular Ca(2+), calmodulin, PYK-2, and p60(src). This pathway represents a mechanism that limits CCh-stimulated chloride secretion across intestinal epithelia.     

Nicotine-induced phosphorylation of Akt through epidermal growth factor receptor and Src in PC12h cells

Nicotine treatment triggers calcium influx into neuronal cells, which promotes cell survival in a number of neuronal cells. Phosphoinositide (PI) 3-kinase and downstream PI3-kinase target Akt have been reported to be important in the calcium-mediated promotion of survival in a wide variety of cells. We investigated the mechanisms of nicotine-induced phosphorylation of Akt in PC12h cells, in comparison with nicotine-induced ERK phosphorylation. Nicotine induced Akt phosphorylation in a dose-dependent manner. A nicotinic acetylcholine receptor (nAChR) alpha7 subunit-selective inhibitor had no significant effect on nicotine-induced Akt phosphorylation, while a non-selective nAChR antagonist inhibited the phosphorylation. L-type voltage-sensitive calcium channel (VSCC) antagonists, calmodulin antagonist, and Ca2+/calmudulin-dependent protein kinase (CaM kinase) inhibitor prevented the nicotine-induced Akt phosphorylation. Three epidermal growth factor receptor (EGFR) inhibitors prevented the nicotine-induced phosphorylation of both extracellular signal-regulated protein kinase (p42/44 MAP kinase, ERK) and Akt. In contrast, an inhibitor of the Src family tyrosine kinase prevented the nicotine-induced Akt phosphorylation but not ERK phosphorylation. These results suggested that nicotine induces the activation of both PI3-kinase/Akt and ERK pathways via common pathways including non-alpha7-nAChRs, L-type VSCC, CaM kinase II and EGFR in PC12h cells, but Src family tyrosine kinases only participate in the pathway to activate Akt.     

Epidermal growth factor receptor-dependent PI3K-activation promotes restitution of wounded human gastric epithelial monolayers

Restitution is a crucial event during the healing of superficial injury of the gastric mucosa involving epithelial cell sheet movement into the damaged area. We demonstrated that growth factors promote the restitution of human gastric epithelial cells. However, the intracellular signaling pathways that transmit extracellular cues as well as regulate basal and growth factor-stimulated gastric epithelial cell migration are still unclear. Herein, confluent human gastric epithelial cell monolayers (HGE-17) or primary cultures of gastric epithelial cells were wounded with a razor blade and the migration response was analyzed in presence or absence of TGFalpha or of pharmacological inhibitors of signaling proteins. Kinase activation profile analysis and phase-contrast microscopy were also performed in parallel. We report that ERK1/2 and Akt activities are rapidly stimulated following wounding of HGE-17 cells. Treatment of confluent HGE-17 cells or primary cultures of gastric epithelial cells with the phosphatidylinositol 3-kinase inhibitor LY294002, but not the MEK1 inhibitor, PD98059, significantly inhibits basal and TGFalpha-induced migration following wounding. Conversely, treatment of wounded HGE-17 cells with phosphatidylinositol(3,4,5)-triphosphate is sufficient to stimulate basal cell migration by 235%. In addition, pp60c-src kinase activity and tyrosine phosphorylation of epidermal growth factor receptors (EGFR) are also rapidly enhanced after wounding and pharmacological inhibition of both these activities strongly attenuates basal and TGFalpha-induced migration as well as Akt phosphorylation levels. In conclusion, the present results indicate that EGFR-dependent PI3K activation promotes restitution of wounded human gastric epithelial monolayers.     

Targeted inhibition of the epidermal growth factor receptor-tyrosine kinase by ZD1839 ('Iressa') induces cell-cycle arrest and inhibits proliferation in prostate cancer cells

The epidermal growth factor (EGF) plays a role in the development of prostate cancer, which becomes essential after androgen resistance has emerged. The EGF receptor (EGFR) is therefore a potential target for anticancer therapy. We evaluated the effects of ZD1839 ('Iressa'), an orally active EGFR-tyrosine kinase inhibitor, on prostate cancer cell lines. The effects of ZD1839 were evaluated on the anchorage dependent and independent growth of androgen-responsive (LNCaP) and androgen-independent (DU145 and PC3) cells by a cell proliferation assay, cell counting, and soft agar analysis. Flow cytometric analysis and Western blotting were used to assess the effects on the cell-cycle and on protein expression levels, respectively. ZD1839 caused a dose- and time-dependent growth inhibition in all three cell lines. A dose-dependent supra-additive increase in growth inhibition was observed when ZD1839 was combined with the antiandrogen flutamide or ionizing radiation (IR). The antiproliferative effect of ZD1839 was mainly cytostatic and associated with a block in the G(0)/G(1) phase of the cell-cycle, evident after about 12 h of treatment. In the DU145 cells this block was associated with an increase in expression of the CDK inhibitor p27(Kip1), both in the cytoplasmic and nuclear fractions. The increase in p27(Kip1) was not evident in the LNCaP and PC3 cells. No changes were observed in the expression of cyclin D1 protein. These results demonstrate the antiproliferative effects of ZD1839 on the growth of prostate cancer cells and suggest that inhibition of EGFR-associated signal transduction pathway might represent a promising novel therapeutic strategy for the treatment of prostate cancer.     

Multiple mechanisms are responsible for transactivation of the epidermal growth factor receptor in mammary epithelial cells

The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.