Epidermal Growth Factor Receptor Tyrosine Kinase Activity Research Articles

Effects of broth culture filtrate protein of VacA+ Helicobacter pylori on the proliferation and apoptosis of gastric epithelial cells

Background Infection with Helicobacter pylori (H. pylori) may lead to chronic inflammation of the stomach epithelium, mucosal atrophy, imbalance of proliferation and apoptosis of epithelial cells; resulting in chronic gastritis, peptic ulcer, gastric cancer, and many other clinical outcomes. Why and how H. pylorus leads to gastric cancer is not clear yet. Through in vitro experiments, this study evaluated the effects of broth culture filtrate protein (BCF-P) from the supernatant of liquid culture media of H. pylori on proliferation and apoptosis of immortalized human gastric epithelial cell lines (GES-1) and gastric cancer cell lines (AGS). Methods For the study, GES-1 and AGS cell lines mix with BCF-P and epidermal growth factor (EGF). MTT assay and flow cytometry (FCM) determined the levels of proliferation and apoptosis. Detected expression levels of cyclooxygenase-2 (COX-2) and Fas mRNA by reverse transcription (RT)-PCR. Also did analysis of the effects of BCF-P on epidermal growth factor receptor (EGFR) tyrosine kinase activity of GES-1 and AGS cells by non-radioactive enzyme-linked assay. The Student’s t test and one-way analysis of variance (ANOVA) were used for statistical analysis. Results BCF-P inhibited proliferation of GES-1 and AGS cells in a concentration-dependent manner. The inhibition rates are respectively 68.7% in AGS and 61.4% in GES-1. With the same dose and time for inhibiting the proliferation, BCF-P failed to induce apoptosis of GES-1 and AGS cells. Effects of BCF-P reduced the expression of Fas mRNA of GES-1 and AGS cells (P <0.05). This is consistent with the effects of EGF. BCF-P reduced the expression of COX-2 mRNA of AGS cells (P <0.05). This is opposite to the effects of EGF (P <0.05). Effects of BCF-P improved more than three times the EGFR tyrosine kinase activity of GES-1 and AGS cells. Conclusions BCF-P inhibited the proliferation of AGS and GES-1 cells in vitro, unrelated to apoptosis. Effects of BCF-P on gastric epithelial cells in vitro are not equivalent to that of EGF.

Cross-talk between EGFR and T-cadherin: EGFR activation promotes T-cadherin localization to intercellular contacts

Reciprocal cross-talk between receptor tyrosine kinases (RTKs) and classical cadherins (e.g. EGFR/E-cadherin, VEGFR/VE-cadherin) has gained appreciation as a combinatorial molecular mechanism enabling diversification of the signalling environment and according differential cellular responses. Atypical glycosylphosphatidylinositol (GPI)-anchored T-cadherin (T-cad) was recently demonstrated to function as a negative auxiliary regulator of EGFR pathway activation in A431 squamous cell carcinoma (SCC) cells. Here we investigate the reciprocal impact of EGFR activation on T-cad. In resting A431 T-cad was distributed globally over the cell body. Following EGF stimulation T-cad was redistributed to the sites of cell–cell contact where it colocalized with phosphorylated EGFRTyr1068. T-cad redistribution was not affected by endomembrane protein trafficking inhibitor brefeldin A or de novo protein synthesis inhibitor cycloheximide, supporting mobilization of plasma membrane associated T-cad. EGF-induced relocalization of T-cad to cell–cell contacts could be abrogated by specific inhibitors of EGFR tyrosine kinase activity (gefitinib or lapatinib), lipid raft integrity (filipin), actin microfilament polymerization (cytochalasin D or cytochalasin B), p38MAPK (SB203580) or Rac1 (compound4). Erk1/2 inhibitor PD98059 increased phospho-EGFRtyr1068 levels and not only amplified effects of EGF but also per se promoted some relocalization of T-cad to cell–cell contacts. Rac1 activation by EGF was inhibited by gefitinib, lapatinib or SB203580 but amplified by PD98059. Taken together our data suggest that T-cad translocation to cell–cell contacts is sensitive to the activity status of EGFR, requires lipid raft domain integrity and actin filament polymerization, and crucial intracellular signalling mediators include Rac1 and p38MAPK. The study has revealed a novel aspect of reciprocal cross-talk between EGFR and T-cad.

Autophagy facilitates the EGFR-TKI acquired resistance of non-small-cell lung cancer cells

Lung cancer is the leading cause of cancer-related mortality worldwide, with approximately 1.6 million new cases and 1.4 million deaths each year. Non-small-cell lung cancer (NSCLC) accounts for nearly 85% of all cases of lung cancer. Once diagnosed, the 5-year survival rate of NSCLC patients hardly reaches 15% despite many different treatments including surgery, radiotherapy, and chemotherapy being widely used. However, the therapeutic effect of chemotherapy is still not satisfactory in patients with advanced NSCLC and the response rate is only 20e35% with a median survival of 10e12 months. Epidermal growth factor receptor (EGFR) is overexpressed in many solid tumors including NSCLC, and the overexpression of EGFR can affect the pathogenesis of cancer, such as cell proliferation, invasion, and metastasis. Activated EGFR can recruit a number of downstream signaling molecules such as phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) and Ras-Raf-MAPK-ERK kinase (MEK). Thus, EGFR has been the focus of molecular-targeted therapies. Drugs that inhibit the tyrosine kinase activity of EGFR, such as the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, have been developed for the treatment of NSCLC. Although both

Epidermal growth factor receptor expression and signaling are essential in glutamine's cytoprotective mechanism in heat-stressed intestinal epithelial-6 cells

Epidermal growth factor receptor (EGFR) expression and signaling can induce cellular protection after intestinal inflammation. L-Glutamine (GLN) is known to prevent apoptosis after intestinal injury by activating MAPK and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. However, the role of EGFR expression and signaling in GLN-mediated cellular protection in intestinal epithelial-6 (IEC-6) cells after heat stress (HS) is unknown. To address the role of EGFR in GLN-mediated protection, IEC-6 cells were treated with GLN in the presence or absence of EGFR small interfering RNA, the EGFR tyrosine kinase inhibitor AG1478, the ERK1/2 inhibitor PD98059, the p38MAPK inhibitor SB203580, or the PI3-K/Akt inhibitor LY294002 under basal and HS conditions. GLN-mediated cell survival was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Phosphorylated and/or total levels of EGFR, cleaved caspase-3, poly(ADP-ribose) polymerase-1, ERK1/2, p38MAPK, and Akt were assessed by Western blotting. We showed that HS induced a decrease in total, cytoplasmic, and nuclear EGFR levels in IEC-6 cells, which was prevented by GLN supplementation, leading to attenuated apoptosis via EGFR small interfering RNA. Furthermore, the protective effect of GLN was lessened by AG1478, PD98059, and LY294002 but was not affected by SB203580. AG1478 attenuated GLN-mediated increases in ERK1/2 and decreases in p38MAPK phosphorylation. However, AG1478 had no effect on GLN-mediated augmentations in Akt phosphorylation. In summary, EGFR expression was important in the protective mechanism of GLN, as well as GLN-mediated activation of EGFR tyrosine kinase activity. GLN-mediated EGFR signaling activated ERK1/2 and decreased p38MAPK signaling. However, GLN-mediated Akt phosphorylation after HS seems to be independent of EGFR signaling.

EGFR activity is required for renal tubular cell dedifferentiation and proliferation in a murine model of folic acid-induced acute kidney injury

Proliferation of dedifferentiated intrinsic renal tubular cells has been recognized to be the major cellular event that contributes to renal repair after acute kidney injury (AKI). However, the underlying mechanism that initiates renal tubular dedifferentiation in vivo remains unexplored. Here we investigated whether epidermal growth factor receptor (EGFR) mediates this process in a murine model of folic acid (FA)-induced AKI using waved-2 mice that have reduced tyrosine kinase activity of EGFR and gefitinib, a specific EGFR inhibitor. Administration of FA for 48 h induced EGFR phosphorylation in the kidney of wild-type mice, but this was inhibited in waved-2 mice and wild-type mice given gefitinib. Compared with wild-type mice, waved-2 mice and wild-type mice treated with gefitinib had increased renal dysfunction, histologic damage, and tubular cell apoptosis after FA administration. PAX2, a dedifferentiation marker, and proliferating cell nuclear antigen, a proliferating marker, were highly expressed in renal tubular cells in wild-type mice; however, their expression was largely inhibited in the kidney of waved-2 mice. Inhibition of EGFR with gefitinib also blocked FA-induced expression of these two proteins in wild-type mice. Moreover, FA exposure resulted in phosphorylation of AKT, a downstream signaling molecule of the phosphatidylinositol 3-kinases pathway associated with renal epithelial proliferation in wild-type mice, and its phosphorylation was totally suppressed in waved-2 mice and wild-type mice given gefitinib. Taken together, these results suggest that EGFR activation is essential for initiation of renal tubular cell dedifferentiation and proliferation after AKI.

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Introduction: Epidermal growth factor receptor (EGFR) expression and signaling can induce cellular protection after intestinal inflammation. L-glutamine (GLN) is known to prevent apoptosis after intestinal injury (e.g. heat stress (HS)) by activating mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/ Akt pathways. Hypothesis: We assessed if EGFR expression and signaling are essential in GLN’s cell protective mechanism in intestinal epithelial-6 (IEC-6) cells after HS, and investigated whether PI-3K and MAPK pathways are involved in GLN-mediated EGFR signaling. Methods: GLN-starved IEC-6 cells were treated with GLN with/ without EGFR siRNA (20nM), EGFR tyrosine kinase inhibitor AG1478 (20µM), ERK1/2 inhibitor PD98059 (50µM), p38MAPK inhibitor SB203580 (1µM), or PI3-K/ Akt inhibitor LY294002 (25µM). GLN-mediated cell survival was measured via MTS assay 24h post-HS (44°Cx50min). Total, cytoplasmatic and nuclear levels of EGFR were determined via Western blot under basal and stressed conditions (37°Cx45min; 43°Cx45min). Furthermore, phosphorylated and/or total levels of cleaved caspase-3, PARP1, ERK1/2, p38 MAPK, and Akt were assessed by Western blots under basal conditions (37°Cx45min) and non-lethal HS (43°Cx45min). Statistics were calculated via student’s t-test. Results: Here, we show that HS attenuated total, cytoplasmatic, and nuclear EGFR levels in IEC-6 cells. GLN prevented the decrease of total, cytoplasmatic and nuclear EGFR levels, which attenuated apoptosis, shown via siEGFR. Furthermore, EGFR kinase activity inhibition by AG1478, ERK1/2 kinase inhibition by PD98059 and PI-3 kinase inhibitor LY294002 lessened GLN’s protective effect, but not p38MAPK inhibitor SB203580. AG1478 attenuated GLN-mediated increases in ERK1/2 and decreases in p38MAPK phosphorylation. However, AG1478 had no effect on GLN-mediated augmentations in Akt phosphorylation. Conclusions: In summary, not only EGFR expression was important in GLN’s protective mechanism, but also GLN’s activation of EGFR tyrosine kinase activity. GLN-mediated EGFR signaling activated ERK1/2 and decreased p38MAPK signaling. However, GLN-mediated Akt phosphorylation after HS seems not to be activated via EGFR signaling.

Synthesis of Novel Benzoxazinone Compounds as Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitors

Two benzoxazinone compounds as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors were synthesized and characterized by NMR and high-resolution mass spectrometry (HRMS). An efficient chlorination method was introduced in the synthesis of 4-chloro-2-oxo-2H-benzoxazinone-6-yl acetate. The inhibition activities of the target compounds and the important intermediates for EGFR tyrosine kinase activity in vitro were determined.

Differential Effect of Growth Factors on Invasion and Proliferation of Endocrine Resistant Breast Cancer Cells

We have established several breast cancer cell lines that exhibit a permanent ER-depleted phenotype, induced by shRNA transfection of MCF-7 cells, which afford a useful model for studying acquired endocrine resistance. Previously we showed that MDA-231 as well as ER-silenced cells could invade through simulated extracellular matrix components. However, the contribution of individual serum components responsible for cell invasion was not determined. In the present study, an under-agarose gel assay was used to quantitatively assess the invasive movement of two ER-silenced cell lines (pII and YS2.5) in comparison to the parental MCF-7, the ER negative MDA-231, and normal HBL100 cells, as well as a line that was ER-shRNA transfected but failed to exhibit ER down-regulation (YS1.2). We also examined the effect of the growth factors EGF, IGF-1, TGFβ, PDGFC and RANTES on pII cell invasion and proliferation. All breast cancer cell lines which had reduced ER expression exhibited a serum-dependent invasive ability related to the degree of induced ER loss. TGFβ treatment inhibited pII cell proliferation and enhanced their invasive ability but at a relatively high dose. IGF-1 and EGF enhanced pII cell proliferation, with the latter playing the major role in promoting cell invasion. PDGFC did not affect either process although it is highly expressed in pII cells. Differential effects were observed on activation of Akt and ERK1/2 suggesting their involvement as intracellular mediators of EGF induced invasion, in part through the regulation of matrix metalloproteinase activity. Targeting EGF receptor tyrosine kinase activity by erlotinib resulted in significant inhibition of both pII cell proliferation and directional invasion towards EGF suggesting that this drug has potential therapeutic usefulness for preventing spread of particularly endocrine resistant breast cancer.

Deletion of the epidermal growth factor receptor in renal proximal tubule epithelial cells delays recovery from acute kidney injury

To determine the role of epidermal growth factor receptor (EGFR) activation in renal functional and structural recovery from acute kidney injury (AKI), we generated mice with a specific EGFR deletion in the renal proximal tubule (EGFRptKO). Ischemia–reperfusion injury markedly activated EGFR in control littermate mice; however, this was inhibited in either the knockout or wild-type mice given erlotinib, a specific EGFR tyrosine kinase inhibitor. Blood urea nitrogen and serum creatinine increased to a comparable level in EGFRptKO and control mice 24 h after reperfusion, but the subsequent rate of renal function recovery was markedly slowed in the knockout mice. Twenty-four hours after reperfusion, both the knockout and the inhibitor-treated mice had a similar degree of histologic renal injury as control mice, but at day 6 there was minimal evidence of injury in the control mice while both EGFRptKO and erlotinib-treated mice still had persistent proximal tubule dilation, epithelial simplification, and cast formation. Additionally, renal cell proliferation was delayed due to decreased ERK and Akt signaling. Thus, our studies provide both genetic and pharmacologic evidence that proximal tubule EGFR activation plays an important role in the recovery phase after acute kidney injury.

A Novel Role for Epidermal Growth Factor Receptor Tyrosine Kinase and Its Downstream Endoplasmic Reticulum Stress in Cardiac Damage and Microvascular Dysfunction in Type 1 Diabetes Mellitus

Epidermal growth factor receptor tyrosine kinase (EGFRtk) and endoplasmic reticulum (ER) stress are important factors in cardiovascular complications. Understanding whether enhanced EGFRtk activity and ER stress induction are involved in cardiac damage, and microvascular dysfunction in type 1 diabetes mellitus is an important question that has remained unanswered. Cardiac fibrosis and microvascular function were determined in C57BL/6J mice injected with streptozotocin only or in combination with EGFRtk inhibitor (AG1478), ER stress inhibitor (Tudca), or insulin for 2 weeks. In diabetic mice, we observed an increase in EGFRtk phosphorylation and ER stress marker expression (CHOP, ATF4, ATF6, and phosphorylated-eIF2α) in heart and mesenteric resistance arteries, which were reduced with AG1478, Tudca, and insulin. Cardiac fibrosis, enhanced collagen type I, and plasminogen activator inhibitor 1 were decreased with AG1478, Tudca, and insulin treatments. The impaired endothelium-dependent relaxation and -independent relaxation responses were also restored after treatments. The inhibition of NO synthesis reduced endothelium-dependent relaxation in control and treated streptozotocin mice, whereas the inhibition of NADPH oxidase improved endothelium-dependent relaxation only in streptozotocin mice. Moreover, in mesenteric resistance arteries, the mRNA levels of Nox2 and Nox4 and the NADPH oxidase activity were augmented in streptozotocin mice and reduced with treatments. This study unveiled novel roles for enhanced EGFRtk phosphorylation and its downstream ER stress in cardiac fibrosis and microvascular endothelial dysfunction in type 1 diabetes mellitus.

Autocrine effect of EGFR ligands on the pro-inflammatory response induced by PM2.5 exposure in human bronchial epithelial cells

Human exposure to PM(2.5) (particulate matter with an aerodynamic diameter below 2.5 μm) is known to be responsible for airway inflammation and may also induce airway remodelling. In respiratory epithelial cells exposed to PM(2.5), releases of pro-inflammatory cytokines such as granulocyte macrophage-colony stimulating factor (GM-CSF) and growth factor ligands of the epidermal growth factor receptor (EGFR) are increased. The present study aimed at determining the involvement of EGFR ligands by autocrine effects in PM(2.5)-induced GM-CSF release. PM(2.5) exposure triggers GM-CSF release by human bronchial epithelial (HBE) cells. This release is dependent on EGFR activation by ligand binding as it is inhibited by AG1478, an inhibitor of EGFR tyrosine kinase activity as well as by a neutralizing anti-EGFR antibody. The use of conditioned medium from cells previously exposed to PM(2.5) demonstrates that PM(2.5)-exposed cells release soluble EGFR ligands able to induce GM-CSF release by an autocrine manner. It was further demonstrated by inhibiting tumour-necrosis factor-alpha converting enzyme (TACE) that is involved in some EGFR ligand shedding. TAPI-2 and GM-6001, two TACE inhibitors, prevented the PM(2.5)-induced GM-CSF release as well as the silencing of TACE by siRNA. We provide evidence that the pro-inflammatory response induced by PM(2.5) exposure on HBE cells, results from an autocrine effect of EGFR ligands released by TACE activity. This autocrine loop by eliciting and sustaining inflammation could contribute to exacerbation of airway remodelling in respiratory-compromised individuals.

Thermostable direct hemolysin diminishes tyrosine phosphorylation of epidermal growth factor receptor through protein kinase C dependent mechanism

BackgroundAdequate evidence mounts to the fact that several bacteria and their toxins have protective or curative roles in colorectal cancers. Thermostable direct hemolysin (TDH), produced by Vibrio parahaemolyticus, down regulates cell proliferation in colon carcinoma cell lines. TDH induces Ca2+ influx from an extracellular environment accompanied by protein kinase C phosphorylation. Activated protein kinase C inhibits the tyrosine kinase activity of epidermal growth factor receptor (EGFR), the rational target of anti-colorectal cancer therapy. MethodsImmunoblotting analyses were performed to ascertain protein kinase C activation, EGFR status, EGFR phosphorylation and mitogen activated protein kinase (MAPK) activity. Flow cytometry analysis and ELISA reconfirmed tyrosine phosphorylation of EGFR and ERK activations, respectively. PKC-α siRNA knockdown was done to corroborate the involvement of PKC-α in the undertaken study. ResultsOur study showed the translocation of PKC-α from cytosol to the membrane fraction in colon carcinoma cell lines on incubation with TDH. The EGFR tyrosine kinase activity exhibited a down regulation on TDH treatment which involved PKC-α, as confirmed by siRNA knockdown. Also ERK phosphorylation occurred on PKC-α activation. Conclusion: TDH activated PKC‐α down regulates EGFR tyrosine kinase activity by MEK dependent mechanism involving MAPK. General significanceIn this study we have seen that TDH has an implication in EGFR based therapeutic approach in colorectal cancer via PKC mediated mechanism.

Tu1825 Protease-Activated Receptor 2 Exerts a Tonic Anti-Apoptotic Effect in HT-29 Colonic Epithelial Cells

Backround: The myofibroblast (MFB) has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer but the mechanism(s) involved remains incompletely understood. Recent evidence suggests that TNF-α is a central regulator of multiple inflammatory signaling cascades. One important target of TNF-α may be the signaling pathway downstream of the epidermal growth factor receptor (EGFR), a member of the ErbB family of receptors, which has been strongly associated with intestinal repair and the development of several human cancers. Methods: The human colonic myofibroblast cell line 18Co was grown to confluence on 35x10mm cell culture dishes and was used from passages 10-14. 18Co cells were exposed to TNF-α (10 ng/ml) and EGF (5 ng/ ml) under varying conditions. EGFR protein expression, EGFR tyrosine phosphorylation at Y845 and Y1068, p42/44 MAPK phosphorylation, and COX-2 expression were assessed by Western blot analysis. Live cell imaging was performed using Quantum dot technology. Experiments were also performed using primary myofibroblasts harvested from human colon tissue. Results: Treatment of 18Co cells with TNF-α led to a time-dependent increase in cell surface EGFR expression, an effect that was completely prevented by inhibition of protein synthesis with cycloheximide. Stimulation of up-regulated EGFR with EGF or HBEGF was associated with enhanced EGFR tyrosine kinase activity, prolonged ERK activation, and a synergistic increase in COX-2 expression compared to 18Co cells treated with EGF andHB-EGF alone. Importantly, TNF-α also increased EGFR expression in primarymyofibroblasts isolated from human colon tissue. Conclusions: Our results imply that TNF-α induced a significant upregulation of EGFR in human colonic myofibroblasts that is associated with enhanced signaling and increased functional activity (ERK, COX-2). Induction of EGFR expression by TNF-α provides a plausible mechanism to explain the exaggerated cellular responsiveness that characterizes IBD, and which may contribute to a microenvironment that predisposes to colitis-associated cancer.

Abstract LB-99: EGF receptor tyrosine kinase defines critical prognostic genes of stage IA lung adenocarcinoma

Abstract Non-small cell lung cancer (NSCLC) is the commonest and the most fatal histological subtype of lung cancer. About 10-30% of patients diagnosed as stage IA (the earliest stage of a lung tumor, smaller than three centimeters in diameter with no evidence of regional lymph node and/or regional metastasis) and submitted to surgery die due to recurrence. Therefore, the identification of prognostic biomarkers for stage IA lung adenocarcinoma with poor prognosis is of great importance to select patients who will be benefited by adjuvant therapy. To the best of our knowledge, there is not a set of genes that identifies these patients. It is known that epidermal growth factor (EGF) signaling pathway is closely related to aggressive phenotypes of lung and other cancers. EGFR tyrosine kinase-specific inhibitor, namely gefitinib is expected to alter the gene expression patterns caused by EGFR tyrosine kinase activity. We performed microarray assays in order to obtain the entire gene expression time course profile of human primary lung epithelial cells that were stimulated with EGF in both the presence and absence of gefitinib. The time courses are composed of 19 time points along 48 hours. The data were subjected to a mathematical analysis, namely the State Space Model (SSM) in order to select genes with gene expressions altered by gefitinib. One hundred thirty nine genes were identified. These genes were used as expression signatures to train a risk scoring model that classifies patients in high- or low-risk (risk of dying in five years). This model was trained by using a data set composed of 253 North American patients with lung adenocarcinomas. Then, the predictive ability of the risk scoring model was examined in two independent cohorts composed of North American and Japanese patients. The model enabled the statistically significant identification of high-risk stage IA lung adenocarcinoma in both cohorts, with hazard ratios (HRs) for death of 7.16 (P=0.029) for North American and 10.98 (P=0.008) for Japanese. The set of 139 genes altered by gefitinib includes several ones that are involved in biological aspects of cancer phenotypes but are yet unknown to be involved in EGF signaling. This result strongly re-emphasizes that EGF signaling status underlies aggressive phenotype of cancer cells, and also suggests the first set of genes that are useful for the identification of stage IA lung adenocarcinoma patients with poor prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-99. doi:1538-7445.AM2012-LB-99

Abstract 770: Overcoming Trastuzumab resistance with the novel pan-erbb inhibitor AZD8931

Abstract Approximately 15-20% of all invasive breast carcinomas overexpress the HER-2 receptor, a membrane surface-bound receptor tyrosine kinase upstream of critical proliferation and cell survival pathways. Trastuzumab, a humanized monoclonal antibody to the extracellular domain of HER-2, has proven to be a beneficial treatment for patients diagnosed as HER-2+, but does have its limitations. A majority of advanced HER-2 positive breast cancer patients will develop resistance to the therapy within the first year while many others do not respond to the drug when used alone. The purpose of this study was to determine whether AZD8931, a reversible pan-ERBB inhibitor that equipotently inhibits the tyrosine kinase-activity of EGFR, HER-2, and HER-3, could reverse this resistance in established trastuzumab-resistant BT474 and SKBR3 cell lines. To account for tumor heterogeneity in HER-2+ tumors, response to AZD8931 was examined in both ER-positive (BT474) and ER-negative (SKBR3) cell lines. Proliferation assays were used to assess the efficacy of AZD8931 alone and in combination with trastuzumab. Results showed significantly diminished cell growth in all tested cell lines, including those resistant to trastuzumab, at clinically relevant concentrations of AZD8931 (<2µM). Furthermore, when AZD8931 was tested in conjunction with trastuzumab, the resistance to trastuzumab was reversed using clinically relevant doses of trastuzumab. Using combination indices, it was found that AZD8931 synergizes well with trastuzumab and could be used as a combination therapy for both trastuzumab-sensitive and trastuzumab-resistant breast cancer patients. Western blot analyses confirmed the results of cell proliferation assays as p-EGFR, p-HER-2, p-HER-3, p-Akt, and p-MAPK, were all reduced after treatment with AZD8931. Thus, AZD8931 alone or in combination with trastuzumab may be an effective therapy in HER2+ breast cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 770. doi:1538-7445.AM2012-770

Genetic or Pharmacologic Blockade of EGFR Inhibits Renal Fibrosis

Although enhanced activation of the EGF receptor (EGFR) associates with the development and progression of renal fibrosis, the mechanisms linking these observations are not completely understood. Here, after unilateral ureteral obstruction (UUO), wild-type mice exhibited sustained EGFR phosphorylation in the kidney and developed renal fibrosis that was more severe than the renal fibrosis observed in waved-2 mice, which have reduced EGFR tyrosine kinase activity. Waved-2 mice also showed fewer renal tubular cells arrested at G2/M, reduced expression of α-smooth muscle actin (α-SMA), downregulation of multiple genes encoding profibrogenic cytokines, including TGF-β1, and dephosphorylation of Smad3, STAT3, and ERK1/2. Administration of the specific EGFR inhibitor gefitinib recapitulated this phenotype in wild-type mice after UUO. Furthermore, inactivation of either EGFR or STAT3 reduced UUO-induced expression of lipocalin-2, a molecule associated with the pathogenesis of CKD. In cultured renal interstitial fibroblasts, inhibition of EGFR also abrogated TGF-β1- or serum-induced phosphorylation of EGFR, STAT3, ERK1/2, and Smad3 as well as expression of α-SMA and extracelluar matrix proteins. Taken together, these data suggest that EGFR may mediate renal fibrogenesis by promoting transition of renal epithelial cells to a profibrotic phenotype, increased production of inflammatory factors, and activation of renal interstitial fibroblasts. Inhibition of EGFR may have therapeutic potential for fibrotic kidney disease.

TNF-α induces upregulation of EGFR expression and signaling in human colonic myofibroblasts

The myofibroblast has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer, but the mechanism(s) involved remains incompletely understood. Recent evidence suggests that TNF-α is a central regulator of multiple inflammatory signaling cascades. One important target of TNF-α may be the signaling pathway downstream of the epidermal growth factor receptor (EGFR), which has been associated with many human cancers. Here, we show that long-term exposure of 18Co cells, a model of human colonic myofibroblasts, with TNF-α led to a striking increase in cell surface EGFR expression, an effect that was completely inhibited by cycloheximide. Subsequent EGFR binding by EGF and heparin binding (HB)-EGF was associated with enhanced EGFR tyrosine kinase activity, prolonged ERK activation, and a significant increase in cyclooxygenase-2 (COX-2) expression compared with 18Co cells treated with EGF and HB-EGF alone. TNF-α also increased EGFR expression and signaling in primary myofibroblasts isolated from human colon tissue. TNF-α-induced upregulation of EGFR may be a plausible mechanism to explain the exaggerated cellular responsiveness that characterizes inflammatory bowel disease and that may contribute to a microenvironment that predisposes to colitis-associated cancer through enhanced COX-2 expression.

Studies of cell signaling in a reconstructed human epidermis exposed to sensitizers: IL-8 synthesis and release depend on EGFR activation

Models of reconstructed human epidermis (RHE) holding proliferating and fully differentiated cultured keratinocytes allow in vitro investigation of early molecular and cellular epidermal events during the complex response of keratinocytes at the onset of allergic contact dermatitis (ACD) or sensitization. In this study, data collected on RHE exposed to well-characterized sensitizing chemicals, such as dinitrofluorobenzene, oxazolone, cinnamaldehyde and isoeugenol, revealed a transient expression of IL-8 mRNA in association with abundant IL-8 cell release. Investigations of keratinocyte signaling illustrate transient activation by tissue exposure to sensitizing chemicals of the epidermal growth factor receptor (EGFR). This activation of EGFR tyrosine kinase is involved in the expression and release of IL-8. The IL-8 release appears also to be partially dependent on p38 and ERK 1/2 MAPK activation. Moreover, data suggest that heparin-binding EGF-like growth factor (HB-EGF) expression and release induced after exposure of RHE to sensitizing chemicals are also under the control of EGFR tyrosine kinase activity, independently of the IL-8 expression and release. Mechanistic approach of keratinocyte responses in the context of RHE underlying regulation of expression and release of epidermal cytokines and growth factors after topical application of sensitizing chemicals is proposed to identify biomarkers which could then be analysed for in vitro toxicological screening of new or undefined compounds.

Design, synthesis and biological evaluation of N-phenylsulfonylnicotinamide derivatives as novel antitumor inhibitors

A series of novel N-phenylsulfonylnicotinamide derivatives (1–24) have been synthesized and evaluated as potential EGFR tyrosine kinase (TK) inhibitors. Among all the compounds, compound 10 (5-bromo-N-(4-chlorophenylsulfonyl)nicotinamide) showed the most potent growth inhibitory activity against EGFR TK and antiproliferative activity of MCF-7 cancer cell line in vitro, with IC50 value of 0.09 and 0.07μM. Docking simulation was performed to insert compound 10 into the EGFR TK active site to determine the probable binding model. Based on the preliminary results, compound 10 with potent inhibitory activity to tumor growth may be a potential anticancer agent.

Successful Treatment of Non-small Cell Lung Cancer with Gefitinib after Severe Erlotinib-related Hepatotoxicity

Gefitinib and erlotinib are first-generation small molecular inhibitors of EGFR tyrosine kinase activity. To the best of our knowledge, to date, two reports have stated that patients with NSCLC who develop severe hepatotoxicity secondary to gefitinib treatment can be safely switched to erlotinib. However, the reverse situation has not been reported. Here, we present the first case with non-small cell lung cancer harboring EGFR mutation who developed grade 3/4 hepatotoxicity after initiation of erlotinib, which resolved when therapy was changed to gefitinib. As far as we know, this is the first report showing the efficacy of gefitinib for a non-small cell lung cancer patient who developed severe hepatotoxicity while under erlotinib therapy.