Clostridium difficile toxin B activates the EGF receptor and the ERK/MAP kinase pathway in human colonocytes

BACKGROUND & AIMS: Clostridium difficile toxin B (TxB) mediates acute inflammatory diarrhea characterized by neutrophil infiltration and intestinal mucosal injury. In a xenograft animal model, TxB was shown to induce interleukin (IL)-8 gene expression in human colonic epithelium. However, the precise mechanisms of this TxB response are unknown. The aim of this study was to investigate the TxB-mediated proinflammatory pathway in colonocytes. METHODS: The effect of TxB on epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK) 1/2 signaling pathway and IL-8 gene expression was assessed in nontransformed human colonic epithelial NCM460 cells. TxB regulation of EGFR-ERK1/2 signaling pathways was determined using immunoblot analysis, confocal microscopy, and enzyme-linked immunosorbent assay, whereas IL-8 gene expression was measured by luciferase promoter assay. RESULTS: TxB activates EGFR and ERK1/2 phosphorylation with subsequent release of IL-8 from human colonocytes. Pretreatment with either the EGFR tyrosine kinase inhibitor, AG1478, or an EGFR-neutralizing antibody blocked both TxB-induced EGFR and ERK activation. By using neutralizing antibodies against known ligands of EGFR, we found that the activation of EGFR and ERK1/2 phosphorylation was mediated by transforming growth factor-alpha (TGF-alpha). Inhibition of matrix metalloproteinase (MMP) decreased TGF-alpha secretion and TxB-induced EGFR and ERK activation. Inhibition of MMP, EGFR, and ERK activation significantly decreased TxB-induced IL-8 expression. CONCLUSIONS: TxB signals acute proinflammatory responses in colonocytes by transactivation of the EGFR and activation of the ERK/MAP kinase pathway.     

Helicobacter pylori-Stimulated Interleukin-8 (IL-8) Promotes Cell Proliferation Through Transactivation of Epidermal Growth Factor Receptor (EGFR) by Disintegrin and Metalloproteinase (ADAM) Activation

Helicobacter pylori infection increases the risk of hyperplastic polyps and gastric cancer, but the mechanisms remain to be elucidated. H. pylori was recently shown to transactivate epidermal growth factor receptor (EGFR) through metalloprotease stimulation. The present study was designed to investigate the effect of interleukin-8 (IL-8) induced by H. pylori infection on EGFR transactivation and epithelial cell growth. H. pylori Sydney strain 1 (SS1) having wild-type cag⁺A was used. Phospho-EGFR assay was performed by immunoprecipitation using anti-human EGFR and anti-phosphotyrosine antibodies. DNA synthesis was evaluated by [³H]thymidine uptake using the human gastric cancer cell line, KATO III. H. pylori induced EGFR phosphorylation, and a disintegrin and metalloproteinase (ADAM) inhibitor, KB-R7785, completely suppressed EGFR phosphorylation. IL-8 also induced EGFR phosphorylation, while anti-IL-8 and anti-IL-8 receptor (CXCR1) neutralizing antibodies suppressed EGFR phosphorylation. [³H]Thymidine uptake analysis demonstrated that H. pylori increased DNA synthesis in gastric epithelial cells, and tyrosine kinase inhibitor, MEK inhibitor, and ADAM inhibitor suppressed the DNA synthesis induced by H. pylori. H. pylori-stimulated IL-8 accelerates processing of EGFR ligands through ADAM activation, and cleaved EGFR ligands bind and stimulate EGFR in paracrine and autocrine manners to induce cell proliferation. This may be one of the mechanisms of hyperplastic polyp and gastric cancer development in H. pylori-infected gastric mucosa.     

Bile acids induce cyclooxygenase-2 expression via the epidermal growth factor receptor in a human cholangiocarcinoma cell line

BACKGROUND & AIMS: Although bile acids have been implicated in colon cancer development, their role in biliary tract carcinogenesis remains unexplored. Because receptor tyrosine kinases and cyclooxygenase (COX)-2 have been implicated in carcinogenesis, we examined the hypothesis that bile acids modulate these enzymes in KMBC cells, a human cholangiocarcinoma cell line. METHODS: The effect of bile acids on epidermal growth factor receptor (EGFR) stimulation, mitogen-activated protein kinase (MAPK) activation, and COX-2 expression was evaluated. RESULTS: Bile acids both induced EGFR phosphorylation and enhanced COX-2 protein expression. Bile acid-induced EGFR phosphorylation was associated with subsequent activation of MAPK p42/44, p38, and c-Jun-N-terminal kinase (JNK). The MAPK inhibitors, PD098059 for MAP or extracellular signal-regulated kinase 1, SB203580 for p38, and BAY 37-9751 for Raf-1, blocked COX-2 induction by bile acids. However, inhibition of JNK activity did not block bile acid-mediated COX-2 induction. CONCLUSIONS: The results show that EGFR is activated by bile acids and functions to induce COX-2 expression by an MAPK cascade. This induction of COX-2 may participate in the genesis and progression of cholangiocarcinomas.     

Signal transduction from membrane to cytoplasm: growth factors and membrane-bound oncogene products increase Raf-1 phosphorylation and associated protein kinase activity.

We have examined the phosphorylation and the serine/threonine-specific kinase activity of the protooncogene product Raf-1 (formerly c-raf) in response to oncogenic transformation or growth-factor treatment of mouse 3T3 cells. Expression of the membrane-bound oncogene products encoded by v-fms, v-src, v-sis, polyoma virus middle-sized tumor antigen, and Ha-ras increased the apparent molecular weight and phosphorylation of the Raf-1 protein, while expression of the nuclear oncogene and protooncogene products encoded by v-fos and c-myc did not. Changes in electrophoretic mobility and phosphorylation occurred rapidly in response to treatment of cells with platelet-derived growth factor, acidic fibroblast growth factor, epidermal growth factor, and the protein kinase C activator phorbol 12-myristate 13-acetate, but not insulin. The phosphorylation of the Raf-1 protein occurred primarily on serine and threonine residues. However, a subpopulation of Raf-1 molecules was phosphorylated on tyrosine residues in cells transformed by v-src or stimulated with platelet-derived growth factor. Transformation by v-src, or treatment with platelet-derived growth factor or phorbol 12-myristate 13-acetate, activated the Raf-1-associated serine/kinase activity as measured in immune-complex kinase assays. These findings suggest that proliferative signals generated at the membrane result in the phosphorylation of the Raf-1 protein and the activation of its serine/threonine kinase activity. Raf-1 activation may thus serve to transduce signals from the membrane to the cytoplasm and perhaps on to the nucleus.     

Aberrant expression of epidermal growth factor receptor and its interaction with protein kinase C δ in inflammation associated neoplastic transformation of human esophageal epithelium in high risk populations

Background and Aim: Esophageal cancer is the second most common cancer among Indian males and is mostly associated with tobacco smoking and alcohol consumption. Epidermal growth factor receptor (EGFR) is a member of Type I tyrosine kinases. Its activation causes the docking of various proteins in its cytosolic tail. In the present study we have analyzed the expression pattern of EGFR, protein kinase C δ (PKCδ), tumor necrosis factor‐α (TNF‐α), nuclear factor κB (NFκB) and the interactions between EGFR and PKCδ in various pathological conditions. Methods: Human esophageal biopsies were obtained from 93 patients with a past history of smoking and alcohol consumption: 20 showed normal mucosa, 40 with dysplasia and 33 squamous cell carcinoma (SCC). These pathological conditions were analyzed immunohistochemically for the presence of EGFR expression and then subsequently analyzed using immunoblot and immunoprecipitation. Results: A statistically significant difference of EGFR overexpression was found between low‐ and high‐grade dysplasia and carcinoma (χ² = 3.3, χ² = 3.42: P = 0.07, 0.33). A statistical significance was observed between dysplasia and SCC and in all histopathological types (χ² = 4, χ² = 4.9; P < 0.05, P = 0.18 and χ² = 26.3, 26.6; P < 0.001). EGFR tyrosine phosphorylation and its association with PKCδ was significantly higher in all histopathological types with χ² = 7.965; P < 0.05 and 4.0830; P = 0.2530. Conclusion: Altogether, our findings reveal that the activation of EGFR and its subsequent interaction with PKCδ under inflammatory conditions might positively be attributed to the transformation of normal esophageal epithelia to SCC, which could explain ongoing inflammation in normal mucosa in a population prone to smoking and alcoholism.     

Could changes in the regulation of the PI3K/PKB/Akt signaling pathway and cell cycle be involved in astrocytic tumor pathogenesis and progression?

The most frequent alterations found in astrocytomas are two major groups of signaling proteins: the cell cycle and the growth factor-regulated signaling pathways. The aim of our study was to detect changes in expression of the following proteins: the tumor suppressors PTEN, p53, and p21Waf1/Cip1, glial fibrillary acidic protein (GFAP, as a marker of astroglial differentiation), the phosphorylated form of protein kinase B/Akt (PKB/Akt), which is downstream to the epidermal growth factor receptor (EGFR), and MDM2, which degrades p53. Paraffin-embedded astrocytoma tissue samples from 89 patients were divided into low grade (grade I-II; 42 samples) and high grade astrocytomas (grade III-IV; 47 samples). Mouse monoclonal antibodies against GFAP, PTEN, PKB/Akt phosphorylated on serine 473, EGFR, p53, p21Waf1/Cip1 and MDM2 were used, followed by standard indirect immunohistochemical method. EGFR protein was detected in 29 % of low grade and in 60 % of high grade astrocytomas. The expression of phosphorylated PKB/Akt was found in roughly the same proportions: in 86% of low grade and in 79% of high grade astrocytomas. PTEN was not found in most of astrocytomas, 64% of low grade and 74% of high grade tumors showed no PTEN staining. Overexpression of the mutated form of p53 or loss of p53 expression, however, was found in about 63% in both groups of astrocytomas with no differences between them. GFAP expression was decreased in tumor astrocytes compared to normal astrocytes and this decreased with grading. GFAP positive tumor cells were detected in only 50% of low grade, and 32% of high grade astrocytomas. The level of MDM2 expression was similar in both grades. Loss of p21Waf1/Cip1 expression was shown in 20% of low and in 45% of high grade tumors. In the subgroup of high grade tumors with wild type p53, 86% showed p21Waf1/Cip1 expression, whereas in the subgroup of high grade tumors with altered p53, only 35% displayed p21Waf1/Cip1. We conclude that EGFR expression increases with astrocytoma grading. EGFR activation may subsequently lead to stimulation of the PKB/Akt survival pathway. PTEN defects may also participate in aggressive tumor behaviour through activation of the PKB/Akt pathway. The alteration of p53 supports the finding that the cell cycle regulation is also disrupted during development of astrocytomas. The changes in PTEN and p53 expression, and activation of PKB/Akt are events in the early stages of astrocytomagenesis. EGFR is one of the factors, which drives the progression of astrocytomas from low to high grade stage.     

Expression of platelet-derived endothelial cell growth factor in inflammatory bowel disease

Background: Platelet-derived endothelial cell growth factor (PD-ECGF) is reported to be highly expressed in tumors and inflammatory tissues, but its expression and role in inflammatory bowel disease (IBD) are still unclear. In this study we examined the location and tissue density of cells immunoreactive for PD-ECGF in the colonic mucosa of IBD. Methods: Paraffin-embedded sections of colonic tissue from patients with ulcerative colitis (UC) or Crohn's disease (CD) were immunostained for PD-ECGF. As controls, noninflamed mucosa of IBD, as well as normal colonic mucosa from patients with colorectal cancer, were used. Also, cancer tissues were evaluated. In addition, changes in the expression of PD-ECGF in human umbilical vein endothelial cells (HUVEC) after treatment with inflammatory cytokines and angiogenic factors, as well as after coculture with colon cancer cell lines, were evaluated by flow cytometry. Results: In normal colonic mucosa and noninflamed mucosa of IBD, PD-ECGF expression was negligible. In inflamed colonic mucosa, strong expression was observed, predominantly in macrophages and fibroblasts. Vascular endothelial cells of the inflamed colonic mucosa, but not of normal colonic mucosa or of neoplastic tissues, stained for PD-ECGF, and the microvessel density was significantly increased in the severely inflamed mucosa. Flow cytometry demonstrated that PD-ECGF was constitutively expressed in HUVEC. Inflammatory cytokines and vascular endothelial growth factor (VEGF) increased its expression, whereas basic fibroblast growth factor (bFGF) decreased it. Coculture with colon cancer cell lines in direct contact, but not in those without contact, also resulted in an important decrease in the expression of PD-ECGF in HUVEC. Conclusions: Autocrine production of PD-ECGF by endothelial cells may be a mechanism of inflammatory angiogenesis, but not tumor angiogenesis, and may be particularly important for the maintenance of damaged vasculature in IBD. Received: September 17, 2001 / Accepted: September 6, 2002 Acknowledgments. This study was supported partly by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and partly by a grant from the Ministry of Health, Labour, and Welfare of Japan. Reprint requests to: S. Saito, Present address: Department of Surgery, Chigasaki Municipal Hospital, 5-15-1 Honson, Chigasaki 253-0042, Japan     

Differential activation of epidermal growth factor (EGF) receptor downstream signaling pathways by betacellulin and EGF

To determine the downstream signaling pathways regulated by betacellulin (BTC) in comparison with epidermal growth factor (EGF), we used Chinese hamster ovary cells overexpressing the human EGF receptor (ErbB1/EGFR). The overall time-dependent activation of EGFR autophosphorylation was identical in cells treated with 1 nm BTC or 1.5 nm EGF. Analysis of site-specific EGFR phosphorylation demonstrated that the BTC and EGF tyrosine phosphorylation of Y1086 was not significantly different. In contrast, the autophosphorylation of Y1173 was markedly reduced in BTC-stimulated cells, compared with EGF stimulation that directly correlated with a reduced BTC stimulation of Shc tyrosine phosphorylation, Ras, and Raf-1 activation. On the other hand, Y1068 phosphorylation was significantly increased after BTC stimulation, compared with EGF in parallel with a greater extent of Erk phosphorylation. Expression of a dominant interfering MEK kinase 1 (MEKK1) and Y1068F EGFR more efficiently blocked the enhanced Erk activation by BTC, compared with EGF. Interestingly BTC had a greater inhibitory effect on apoptosis, compared with EGF, and expression of Y1068F EGFR abolished this enhanced inhibitory effect. Together, these data indicated that although BTC and EGF share overlapping signaling properties, the ability of BTC to enhance Erk activation occurs independent of Ras. The increased BTC activation results from a greater extent of Y1068 EGFR tyrosine phosphorylation and subsequent increased recruitment of the Grb2-MEKK1 complex to the plasma membrane, compared with EGF stimulation. The increased Erk activation by BTC associated with antiapoptotic function.     

Activation of the Raf-1/MEK/ERK cascade by bile acids occurs via the epidermal growth factor receptor in primary rat hepatocytes

Bile acids have been reported to activate several different cell signaling cascades in rat hepatocytes. However, the mechanism(s) of activation of these pathways have not been determined. This study aims to determine which bile acids activate the Raf-1/MEK/ERK cascade and the mechanism of activation of this pathway. Taurodeoxycholic acid (TDCA) stimulated (+235%) the phosphorylation of p(74) Raf-1 in a time (5 to 20 minutes) and concentration-dependent (10 to 100 micromol/L) manner. Raf-1 and ERK activities were both significantly increased by most bile acids tested. Deoxycholic acid (DCA) was the best activator of ERK (3.6-fold). A dominant negative Ras (N17) construct expressed in primary hepatocytes prevented the activation of ERK by DCA. The epidermal growth factor receptor (EGFR)-specific inhibitor (AG1478) significantly inhibited (approximately 81%) the activation of ERK by DCA. DCA rapidly (30 to 60 seconds) increased phosphorylation of the EGFR (approximately 2-fold) and Shc (approximately 4-fold). A dominant negative mutant of the EGFR (CD533) blocked the ability of DCA to activate ERK. In conclusion, these results show that DCA activates the Raf-1/MEK/ERK signaling cascade in primary hepatocytes primarily via an EGFR/Ras-dependent mechanism.     

Epidermal growth factor partially restores colonic ion transport responses in mouse models of chronic colitis

BACKGROUND & AIMS: Epidermal growth factor receptor (EGFR) activation, which plays an important role in regulating intestinal ion transport, can alleviate clinical symptoms such as diarrhea in patients with ulcerative colitis and promote mucosal restitution in animal models of colitis. Here, we investigate whether EGFR can regulate colonic ion transport in the setting of colitis. METHODS: Distal colon from control mice and mice with colitis was retained for immunohistochemistry or mounted in Ussing chambers. Ion transport responses across the tissues to the calcium agonist carbachol and the adenosine 3',5'-cyclic monophosphate agonist forskolin were measured with or without epidermal growth factor (EGF) pretreatment. RESULTS: EGF pretreatment of normal colonic mucosa inhibited ion transport responses to carbachol and forskolin but potentiated the reduced ion transport responses seen in dextran sulfate sodium (DSS)-treated and mdr1a knockout mouse colon. Ion substitution studies and the sodium transport inhibitor amiloride showed that sodium movement primarily accounted for the potentiating effect of EGF in DSS-treated tissues, despite decreased sodium channel expression. EGF potentiation of transport responses in DSS-treated colon was completely blocked by the cytoskeletal disruptor cytochalasin D and the phosphatidylinositol 3-kinase inhibitor wortmannin, whereas the novel and conventional protein kinase C isoform inhibitor G?6850 and the extracellular signal-regulated kinase inhibitor PD98059 partially reduced EGF effects. EGFR epithelial distribution and transforming growth factor alpha expression were also altered in DSS-treated tissues. CONCLUSIONS: Chronic inflammation uncovers a potentiating effect of EGFR activation on epithelial electrogenic sodium absorption that would be expected to ameliorate diarrheal symptoms associated with colitis.     

Enhanced insulin signaling via Shc in human breast cancer

Insulin is a mild mitogen and has been shown to potentiate mitogenic influence of other growth factors. Because hyperinsulinemia and/or overexpression of insulin receptors have been linked to development, progression, and outcome of breast cancer, we attempted to evaluate the mechanism of these associations. We have compared the expression of insulin receptors and the magnitude of insulin signaling in breast tumors and adjacent normal mammary tissue samples obtained from 20 patients. We observed that insulin binding more than doubled in the tumors as compared with the normal tissue (P <.01 by paired t test). Insulin signaling to Shc, judged by the magnitude of its phosphorylation, was also significantly enhanced in the tumors. In contrast, the phosphorylation of the insulin-receptor substrate-1 (IRS-1), Akt, and mitogen-activated protein (MAP) kinase were identical in the tumorous and normal mammary tissues. Finally, tumors displayed significantly increased amounts of farnesylated p21 Ras and geranylgeranylated Rho-A (P <.01), consistent with Shc-dependent activation of farnesyl (FTase) and geranylgeranyl transferases (GGTase) in the tumor tissue. We conclude that the mechanism of the mitogenic influence of insulin in breast cancer may include increased expression of insulin receptors, preferential hyperphosphorylation of Shc, and increased amounts of prenylated p21 Ras and Rho-A in tumor tissue as compared with adjacent normal mammary tissue.     

Incomplete surgical resection of ductal carcinomas in situ results in activation of ERBB2 in residual breast cancer cells

ERBB2 amplification and consecutive overexpression is a predictor for poor prognosis in breast cancer patients. In addition, incomplete resection of ERBB2-overexpressing tumors leads to increased proliferation of residual breast cancer cells. While the local release of cytokines is thought to be responsible for the malignant behavior of remaining tumor tissue, the exact mechanism is still unknown. We have analyzed epidermal growth factor receptor (EGFR), activated (p)EGFR, and activated (p)ERBB2 protein expression in ERBB2-overexpressing and in non-ERBB2-overexpressing tumors from patients who underwent breast surgery and consecutive re-excision for involved margins, and compared expression levels by immunohistochemistry. While overall ERBB2 protein expression in the initial and the re-excised sample were comparable, we observed an increase in pERBB2 in ductal carcinomas in situ in both, ERBB2-overexpressing (16/21 vs 24/24; P=0.018, chi(2) test) and non-ERBB2-overexpressing tumors (3/28 vs 5/12; P=0.025, chi(2) test). pERBB2 was not increased in invasive tumors, regardless on whether the samples had been taken from a ERBB2-overexpressing (9/25 vs 6/17; P=0.261, chi(2) test) or a non-ERBB2-overexpressing tumor (1/27 vs 0/8; P=0.581, chi(2) test). EGFR expression was only detected in 1/47 ERBB2-overexpressing primary tumors and 2/48 non-ERBB2-overexpressing tumors, and was undetectable in re-excised specimen. Taken together, we have demonstrated an increase in ERBB2 receptor activation in incompletely resected preinvasive breast cancer. We hypothesize that receptor phosphorylation is caused by growth factor stimulation in response to intraoperative tissue damage, and perioperative inhibition of specific cytokines could become a promising therapeutic strategy.     

Constitutive activation of Stat3 signaling abrogates apoptosis in squamous cell carcinogenesis in vivo

Field cancerization predisposes the upper aerodigestive tract mucosa to the formation of multiple primary tumors, when exposed to environmental carcinogens. Up-regulation of epidermal growth factor receptor occurs early in squamous cell carcinogenesis and is critical for the loss of growth control in a variety of human cancers, including head and neck squamous cell carcinomas. In these tumor cells in culture, epidermal growth factor receptor stimulation initiates signaling via persistent activation of selective STAT proteins. To determine the timing of Stat3 activation in head and neck carcinogenesis, we studied the expression and constitutive activation of Stat3 in tumors and normal mucosa from patients with head and neck cancer compared with mucosa from controls without cancer. Stat3 was up-regulated and constitutively activated in both primary human head and neck tumors as well as in normal mucosa from these cancer patients compared with control normal mucosa from patients without cancer. In vivo liposome-mediated gene therapy with a Stat3 antisense plasmid efficiently inhibited Stat3 activation, increased tumor cell apoptosis, and decreased Bcl-x(L) expression in a head and neck xenograft model. These findings provide evidence that constitutively activated Stat3 is an early event in head and neck carcinogenesis that contributes to the loss of growth control by an anti-apoptotic mechanism.     

Raf-1, a potential therapeutic target, mediates early steps in endometriosis lesion development by endometrial epithelial and stromal cells

Endometriosis is a hormone-sensitive gynecological disorder characterized by the benign growth of endometrial-like tissue in the pelvic cavity. Endometriotic lesions composed of endometrial stromal cells (ESC) and glandular epithelial cells (EEC) are thought to arise from menstrual endometrial tissue reaching the pelvic cavity via retrograde menstruation. The cause of endometriotic lesion formation is still not clear. Recent evidence suggest that cytokines may play a role in the early development of endometriosis lesions. Because cytokines and growth factors signal via the v-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) kinase pathway, we have examined the role of Raf-1 in early steps of endometriosis lesion formation, specifically attachment of endometrial cells to peritoneal mesothelial cells (PMC) and invasion of endometrial cells through PMC (trans-mesothelial invasion). Raf-1 antagonist GW5074 decreased attachment to PMC and trans-mesothelial invasion by primary EEC and ESC. Raf-1 also mediated TGFβ-induced trans-mesothelial invasion by the established, low-invasive EEC line EM42. TGFβ treatment of EEC resulted in Raf-1 phosphorylation at S338 and phosphorylation of ERK, suggesting that TGFβ activates Raf-1 signaling in these cells. GW5074 had little effect on ESC proliferation but inhibited EEC growth significantly under reduced serum conditions. Antagonizing Raf-1 activity and expression via GW5074 and specific Raf-1 small interfering RNA, respectively, did not alter EEC resistance to growth inhibition by TGFβ. Raf-1 inhibition blocked induction of EEC growth by epidermal growth factor. Our data suggest that Raf-1 may mediate pathologic steps involved in early endometriosis lesion formation and may be a mediator of TGFβ and epidermal growth factor actions in endometriosis.     

Targeting EGFR activity in blood vessels is sufficient to inhibit tumor growth and is accompanied by an increase in VEGFR-2 dependence in tumor endothelial cells

Epidermal growth factor receptor (EGFR) targeting agents such as kinase inhibitors reduce tumor growth and progression. We have previously reported that EGFR is not only expressed by the tumor cells but by the tumor endothelial cells (EC) as well (Amin, D. N., Hida, K., Bielenberg, D. R., Klagsbrun, M., 2006. Tumor endothelial cells express epidermal growth factor receptor (EGFR) but not ErbB3 and are responsive to EGF and to EGFR kinase inhibitors. Cancer Res. 66, 2173–80). Thus, targeting tumor blood vessel EGFR may be a viable strategy for tumor growth inhibition. We describe here a melanoma xenograft model where the tumor cells express very little or no EGFR but the tumor blood vessels express activated EGFR. The EGFR kinase inhibitor, gefitinib (Iressa), retarded tumor growth with a size decrease of 38% compared to control mice, ostensibly due to targeting of the blood vessels. EC were isolated from tumors of gefitinib-treated mice. These EC were unable to proliferate in response to EGF and displayed relatively weaker activation of MAPK and AKT signaling in response to EGF compared to tumor EC isolated from vehicle-treated mice. In contrast, the tumor EC from gefitinib-treated mice expressed higher levels of VEGFR-2 both at the mRNA and protein level. In addition, these cells were less sensitive to EGFR kinase inhibitors in vitro but more sensitive to a VEGFR-2 kinase inhibitor. These results suggest that in tumor EC from gefitinib-treated mice there is a switch from dependence on EGFR activity to signaling via VEGFR-2. Our data provide a molecular rationale for combination therapies targeting both EGF and VEGF signaling on the tumor vasculature.     

Fibronectin-induced proliferation in thyroid cells is mediated by alphavbeta3 integrin through Ras/Raf-1/MEK/ERK and calcium/CaMKII signals

We recently demonstrated in an immortalized thyroid cell line that integrin stimulation by fibronectin (FN) simultaneously activates two signaling pathways: Ras/Raf/MAPK kinase (Mek)/Erk and calcium Ca2+/calcium calmodulin-dependent kinase II (CaMKII). Both signals are necessary to stimulate Erk phosphorylation because CaMKII modulates Ras-induced Raf-1 activity. In this study we present evidence that extends these findings to normal human thyroid cells in primary culture, demonstrating its biological significance in a more physiological cell model. In normal thyroid cells, immobilized FN-induced activation of p21Ras and Erk phosphorylation. This pathway was responsible for FN-induced cell proliferation. Concurrent increase of intracellular Ca2+ concentration and CaMKII activation was observed. Both induction of p21Ras activity and increase of intracellular Ca2+ concentration were mediated by FN binding to alphavbeta3 integrin. Inhibition of the Ca2+/CaMKII signal pathway by calmodulin or CaMKII inhibitors completely abolished the FN-induced Erk phosphorylation. Binding to FN induced Raf-1 and CaMKII to form a protein complex, indicating that intersection between Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways occurred at Raf-1 level. Interruption of the Ca2+/CaMKII signal pathway arrested cell proliferation induced by FN. We also analyzed thyroid tumor cell lines that displayed concomitant aberrant integrin expression and signal transduction. These data confirm that integrin activation by FN in normal thyroid cells generates Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways and that both are necessary to stimulate cell proliferation, whereas in thyroid tumors integrin signaling is altered.