Epidermal Growth Factor Receptor Tyrosine Kinase Activity Research Articles

Preclinical Efficacy of Nimotuzumab, an Anti-Egfr Monoclonal Antibody as a Single Agent Therapy in Human GBM u87mg Xenografts

Background: The poor prognosis of patients with high-grade glioma multiform (GBM) has led investigators to the search of new therapeutic strategies. Current treatment includes surgery when possible, radiotherapy and chemotherapy. Molecular-targeted therapies are in the process of clinical testing, and promising agents include monoclonal antibodies. Our study examined the antitumor activity of three different single therapies in nude mice bearing both subcutaneous and orthotopic brain xenografts of the U87MG human GBM cell line. Methods: Cell culture, Histology, Immunohistochemistry, Animal experiments, Statistical analysis. Results: Different groups of treatment included nimotuzumab, a humanized monoclonal antibody that inhibits the EGFR tyrosine kinase activity, or total body irradiation, or the chemotherapeutic agent temozolomide (TMZ). For the control group animals received saline solution instead of the antibody. For the subcutaneous model, only nimotuzumab or TMZ produced a significant delay in tumor growth. In the intracranial model, unlike TMZ, the systemic administration of the antibody did not reduce the tumor growth, despite both therapies inhibited the formation of microsatellites in the brain of mice. The antitumor activity of nimotuzumab was accompanied by a decrease in the microvessel density and the proliferative activity of tumor cells. TMZ only inhibited the tumor cell proliferation but not the formation of new tumor-associated microvessels in xenografts. For radiation therapy, neither antiproliferative nor antiangiogenic activity was found, in accordance with the lack of antitumor activity. Only nimotuzumab reduced the frequency of chemo and radioresistant CD133+ population. Conclusion: Our results illustrate the potential efficacy of nimotuzumab as a single agent against an EGFR-amplified human GBM, a tumor resistant to the therapy with all well-known forms of treatment.

PD08-08: Preclinical Efficacy of the Combination of Met and Src Family Kinase Inhibitors in Triple-Negative Breast Cancer.

Abstract Background: Epidermal growth factor receptor (EGFR), although overexpressed in almost 60% of all triple-negative breast cancers, has yet to be identified as an effective therapeutic target for the treatment of the disease. We have previously shown in cell culture models of triple-negative breast cancer that primary resistance to EGFR tyrosine kinase inhibitors through ligand-independent phosphorylation can be overcome by co-inhibiting EGFR with either Met or Src family kinases (SFKs). We also demonstrated that inhibiting Met kinase activity reduced EGFR and c-Src phosphorylation and inhibiting SFK reduced EGFR phosphorylation. Taken together, these data suggest that EGFR, Met and SFKs regulate the activity of each other through transphosphorylation. This process of transphosphorylation, or crosstalk, allows for the continued activation of key signaling proteins required for proliferation and transformation. Here we demonstrate that inhibiting both Met and SFKs decreases cell viability and eliminates the need for inhibition of EGFR tyrosine kinase activity, as the autophosphorylation sites of EGFR were lost with the combination treatment. Methods: Seven triple-negative (SUM102, SUM149, SUM229, SUM1315, BT20, BT549, MDA-MB-231), two HER2+ (SUM190 and SKBr3), and two ER/PR+ (MCF7 and T47D) breast cancer cell lines were used to test the efficacy of the combination of Met and SFK inhibitors, Arq-197 and dasatinib, respectively. Cell viability after drug treatment was assessed by MTT assays. IC50 values were calculated and drug synergy calculations were performed. Transphosphorylation was measured by immunoblotting using phospho-specific antibodies. Apoptosis was characterized by immunoblotting. Results: All seven triple-negative breast cancer cell lines demonstrated decreased cell viability with the combination of Arq-197 and dasatinib when compared with either drug alone. In contrast, none of the HER2+ or ER/PR+ breast cancer cell lines had any additional loss of viability with the combination treatment. We found that the interactions between Arq-197 and dasatinib were synergistic in several of the breast cancer cell lines. Specifically, in one such cell line (BT20), the combinatorial index values were less than 1.0. Interestingly, in this cell line, the tyrosine phosphorylation levels on residues 1068 and 992 of EGFR were significantly altered with the combination treatment and activation of apoptotic proteins, including PARP, occurred. Conclusions: These data demonstrate that two tyrosine kinases, known to be downstream of EGFR activation, interact to promote resistance to EGFR inhibitors. However, inhibiting both Met and SFKs was sufficient to decrease cell viability and stimulate apoptosis in triple-negative breast cancer cells. Further studies to validate the relevance of these findings are ongoing. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD08-08.

Neonatal Lethality in Knockout Mice Expressing the Kinase-Dead Form of the Gefitinib Target GAK Is Caused by Pulmonary Dysfunction

Gefitinib (Iressa) is an inhibitor of the epidermal growth factor receptor (EGFR) that has shown promising activity in the treatment of patients with non-small cell lung cancer (NSCLC). However, adverse side effects of gefitinib treatment, such as respiratory dysfunction, have limited the therapeutic benefit of this targeting strategy. The present results show that this adverse effect can be attributed to the inhibition of the novel gefitinib target GAK (Cyclin G-associated kinase), which is as potently inhibited by the drug as the tyrosine kinase activity of EGFR. Knockout mice expressing the kinase-dead form of GAK (GAK-kd) died within 30 min after birth primarily due to respiratory dysfunction. Immunohistochemical analysis revealed that surfactant protein A (SP-A) was abundant within alveolar spaces in GAK-kd+/+ mice but not in GAK-kd-/- pups. E-cadherin and phosphorylated EGFR signals were also abnormal, suggesting the presence of flat alveolar cells with thin junctions. These results suggest that inhibition of GAK by gefitinib may cause pulmonary alveolar dysfunction, and the present study may help prevent side effects associated with gefitinib therapy in NSCLC patients.

Inhibition of the epidermal growth factor receptor by erlotinib prevents immortalization of human cervical cells by Human Papillomavirus type 16

The Human Papillomavirus type-16 (HPV-16) E6 and E7 oncogenes are selectively retained and expressed in cervical carcinomas, and expression of E6 and E7 is sufficient to immortalize human cervical epithelial cells. Expression of the epidermal growth factor receptor (EGFR) is often increased in cervical dysplasia and carcinoma, and HPV oncoproteins stimulate cell growth via the EGFR pathway. We found that erlotinib, a specific inhibitor of EGFR tyrosine kinase activity, prevented immortalization of cultured human cervical epithelial cells by the complete HPV-16 genome or the E6/E7 oncogenes. Erlotinib stimulated apoptosis in cells that expressed HPV-16 E6/E7 proteins and induced senescence in a subpopulation of cells that did not undergo apoptosis. Since immortalization by HPV E6/E7 is an important early event in cervical carcinogenesis, the EGFR is a potential target for chemoprevention or therapy in women who have a high risk for cervical cancer.

Akt1 interacts with epidermal growth factor receptors and hedgehog signaling to increase stem/transit amplifying cells in the embryonic mouse cortex

A subset of precursors in the embryonic mouse cortex and in neurospheres expresses a higher level of the serine/threonine kinase Akt1 than neighboring precursors. We reported previously that the functional significance of high Akt1 expression was enhanced Akt1 activity, resulting in an increase in survival, proliferation, and self-renewal of multipotent stem/transit amplifying cells. Akt1 can interact with a number of signaling pathways, but the extrinsic factors that are required for specific effects of elevated Akt1 expression have not been identified. In this study we addressed the contributions of signaling via epidermal growth factor (EGF) and hedgehog (Hh) receptors. In EGF receptor-null precursors or following transient inhibition of EGF receptor tyrosine kinase activity, elevating Akt1 by retroviral transduction could still increase survival and proliferation but could not increase self-renewal. We also found that elevated Akt1 expression induced the expression of EGF receptors (EGFRs) in wild-type precursors. Several extrinsic factors, including Shh, can induce EGFR expression by cortical precursors, and we found that elevating Akt1 allowed them to respond to a subthreshold concentration of Shh to induce EGFRs. In precursors that lack the Hh receptor smoothened, however, elevating Akt1 did not increase EGFR expression or self-renewal, though it could still stimulate proliferation. These findings suggest that a subset of precursors in the embryonic cortex that express an elevated level of Akt1 can respond to lower concentrations of Shh than neighboring precursors, resulting in an increase in their expression of EGFRs. Signaling via EGFRs is required for their self-renewal.

Molecular imaging of epidermal growth factor receptor kinase activity

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is commonly altered in different tumor types, leading to abnormally regulated kinase activity and excessive activation of downstream signaling cascades, including cell proliferation, differentiation, and migration. To investigate the EGFR signaling events in real time and in living cells and animals, here we describe a multidomain chimeric reporter whose bioluminescence can be used as a surrogate for EGFR kinase activity. This luciferase-based reporter was developed in squamous cell carcinoma cells (UMSCC1) to generate a cancer therapy model for imaging EGFR. The reporter is designed to act as a phosphorylated substrate of EGFR and reconstitutes luciferase activity when it is not phosphorylated, thereby providing a robust indication of EGFR inhibition. We validated the reporter in vitro and demonstrated that its activity could be differentially modulated by EGFR tyrosine kinase inhibition with erlotonib or receptor activation with epidermal growth factor. Further experiments in vivo demonstrated quantitative and dynamic monitoring of EGFR tyrosine kinase activity in xenograft. Results obtained from these studies provide unique insight into pharmacokinetics and pharmacodynamics of agents that modulate EGFR activity, revealing the usefulness of this reporter in evaluating drug availability and cell targeting in both living cells and mouse models.

Metabolic profiles as predictive biomarkers of erlotinib-induced adverse effects in patients with non-small cell lung cancer.

2593 Background: Erlotinib is an orally available, selective inhibitor of EGFR tyrosine kinase activity used in the treatment of patients with non-small cell lung cancer (NSCLC), and skin rash is a...

Lactobacillus Casei Prevents Osmotic Stress-Induced Disruption of Tight Junctions (TJ) and Adherens Junctions (AJ) in CACO-2 Cell Monolayers by a PKC-Dependent Mechanism, but Independent of EGF Receptor Tyrosine Kinase Activity

right ears of mice (N=5) were injected with SbS and the left ears were injected with vehicle as control. The first treatment was administered at 1 hour after 5x10^6 pfu Ad-VEGF-A164 was injected into both ears. The second SbS/vehicle injections were administered 24 hours later. An inhibitory effect of SbS was clearly evident by day 7 with reduced new vessel formation in the right ear compared to the left. The effect of SbS remained evident until the end of the experiment on day 21. 4) DSS (4% for 5 days) was administered to mice to induce colitis. Daily gavage of Sb significantly attenuated weight-loss (N=5,p<0.01). DSS treated mice had significantly increased blood vessel density and volume compared to healthy mice. Sb treatment significantly reduced the neo-vascularization associated with inflammation in the colon. CONCLUSIONS: Sb inhibits VEGFR signaling and reduces neo-vascularization In Vitro and in two separate In Vivo models. Our findings indicate that the probiotic yeast S boulardii can modulate angiogenesis in intestinal injury and repair, which provides a novel mechanism for its beneficial effects.

Abstract 1086: RHA plays an important role in EGFR-mediated gene transcription in cancer cells

Abstract EGF treatment induces the translocation of its receptor (i.e. EGFR) from the cell surface into the nucleus. Nuclear expression of EGFR is negatively correlated with overall survival of cancer patients and EGFR nuclear translocation is important for cancer cell's resistance to the treatment with Cetuximab, cisplatin and radiation. Moreover, EGFR in the nucleus can associates with other proteins including STAT3, STAT5A, DNA-PK and PCNA to regulate cell transformation, proliferation, and DNA repair and replication. It has been demonstrated that nuclear EGFR regulates gene transcription through its binding to an AT-rich sequence (ATRS) of target gene promoter. However, EGFR does not have a DNA-binding domain and there is no evidence to support the direct binding of EGFR to the specific DNA sequence. Thus, identification of a DNA-binding partner of nuclear EGFR is crucial for us to understand how EGFR regulates gene transcription in the nucleus. Using a non-biased approach, we identified RNA helicase A (i.e. RHA), which is a highly conserved, multiple functional transcriptional activator, could interact with nuclear EGFR upon EGF stimulation in several cancer cells. The EGFR/RHA complex then associated with gene promoter through binding of RHA to the AT-rich sequence of the promoter to activate its transcription. Knockdown of RHA expression was found to abrogate the binding of EGFR to its target gene promoter, thereby reducing EGF/EGFR-induced gene expression. Moreover, interruption of EGFR-RHA interaction decreased EGFR-induced promoter activity. Interestingly, we found that the EGFR/RHA-regulated gene promoter activity is dependent on the EGFR tyrosine kinase activity but independent of RHA helicase activity. Finally, we observed a positive correlation among nuclear expression of EGFR, RHA and cyclin D1 in human breast cancer tissue samples. These results, taken together, indicate that RHA is an important mediator for EGFR-induced gene transactivation in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1086. doi:10.1158/1538-7445.AM2011-1086

Abstract 5459: Association of CYP1A1 and CYP3A5 polymorphisms with pharmacokinetics of erlotinib in patients with non-small cell lung cancer

Abstract Background: Erlotinib is a selective inhibitor of EGFR tyrosine kinase activity used for the treatment of patients with NSCLC. It has been reported that ABCB1 polymorphisms affect pharmacokinetics and adverse events of erlotinib in Japanese patients (Hamada A, 2009 AACR meeting). Erlotinib is metabolized by CYP3A4, CYP3A5, CYP1A1, and CYP1A2. The purpose of this study was to investigate the association of CYP1A1 and CYP1A5 single nucleotide polymorphisms (SNPs) with inter-individual variability of erlotinib pharmacokinetics. Methods: Patients with NSCLC were treated with single-agent oral erlotinib 150 mg/day. Plasma levels of erlotinib were measured by high-performance liquid chromatography on days 1 and at steady state (&amp;gt;day 8). DNA was obtained from whole blood, and genotyping was carried out using an Applied Biosystems TaqMan SNP Genotyping Assay on an ABI PRISM® 7900HT system. Results: Fifty patients (mean age, 67 years) were enrolled in the study. Histological classifications were: adenocarcinoma (n=41), squamous cell carcinoma (n=7), and unknown (n=2). Smoking history was indicated as: never smoker (n=23), former smoker (n=24), and current smoker (n=3). For the CYP3A5 6986A&amp;gt;G polymorphism, the frequencies of wild-type (AA), heterozygote (GA), and homozygote (GG) were 6%, 34%, and 60%, respectively. For the CYP1A1 2455A&amp;gt;G polymorphism, the frequencies of wild-type (AA), heterozygote (GA), and homozygote (GG) were 64%, 24%, and 12%, respectively. The mean (±SD) maximum concentrations (Cmax), trough concentrations (Ctrough) on day 1, and steady-state trough concentrations (Css) on &amp;gt;day 8 were 1.66±0.73 μg/mL, 0.77±0.5 μg/mL, and 1.5±0.8 μg/mL, respectively. Lower exposure levels of erlotinib were observed in patients carrying the CYP3A5 6986AA allele than in patients carrying one or two G alleles (GA, GG). The Cmax on day 1 in patients carrying the CYP3A5 AA and GA/GG alleles were 0.76±0.27 μg/mL and 1.78±0.69 μg/mL, respectively. (p=0.0198) On the other hand, patients carrying the CYP1A1 2455GG allele had higher Css than in patients carrying the CYP1A1 2455AA or the CYP1A1 2455GA alleles (2.3±1.16 μg/mL vs. 1.4±0.69 μg/mL, p=0.0161) Conclusion: These results suggested that the CYP3A5 6986A&amp;gt;G and CYP1A1 2455A&amp;gt;G polymorphisms affect the pharmacokinetics of erlotinib in Japanese patients. Further studies involving a larger sample size will be required to evaluate whether measurement of the CYP3A5 and the CYP1A1 polymorphisms may help to optimize erlotinib treatment in individual patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5459. doi:10.1158/1538-7445.AM2011-5459

EPIDERMAL GROWTH FACTOR RECEPTOR TARGETED THERAPIES FOR SOLID TUMOURS

The majority of human epithelial cancers is frequently characterized by a functional activation of the epidermal growth factor receptor (EGFR)-driven- pathways. Today, two classes of EGFR inhibitors are routinely used in the clinic: anti-EGFR monoclonal antibodies such as cetuximab and panitumumab and small-molecule inhibitors of the EGFR tyrosine kinase activity such as erlotinib and gefitinib. Anti-EGFR therapies have been approved in several countries for the treatment of metastatic non–small-cell lung cancer, colorectal cancer, squamous-cell carcinoma of the head and neck, and pancreatic cancer. This article summarizes the clinical evidence of the anticancer activity of anti-EGFR treatment, and considers the current, and controversial, clinical issues with respect to their optimal use in the treatment of patients with cancer. Mechanisms of resistance to anti-EGFR treatment are also briefly discussed.

Epidermal growth factor upregulates serotonin transporter in human intestinal epithelial cells via transcriptional mechanisms

Serotonin transporter (SERT) regulates extracellular availability of serotonin and is a potential pharmacological target for gastrointestinal disorders. A decrease in SERT has been implicated in intestinal inflammatory and diarrheal disorders. However, little is known regarding regulation of SERT in the intestine. Epidermal growth factor (EGF) is known to influence intestinal electrolyte and nutrient transport processes and has protective effects on intestinal mucosa. Whether EGF regulates SERT in the human intestine is not known. The present studies examined the regulation of SERT by EGF, utilizing Caco-2 cells grown on Transwell inserts as an in vitro model. Treatment with EGF from the basolateral side (10 ng/ml, 24 h) significantly stimulated SERT activity (∼2-fold, P < 0.01) and mRNA levels compared with control. EGF increased the activities of the two alternate promoter constructs for human SERT gene: SERT promoter 1 (hSERTp1, upstream of exon 1a) and SERT promoter 2 (hSERTp2, upstream of exon 2). Inhibition of EGF receptor (EGFR) tyrosine kinase activity by PD168393 (1 nM) blocked the stimulatory effects of EGF on SERT promoters. Progressive deletions of the SERT promoter indicated that the putative EGF-responsive elements are present in the -672/-472 region of the hSERTp1 and regions spanning -1195/-738 and -152/+123 of hSERTp2. EGF markedly increased the binding of Caco-2 nuclear proteins to the potential AP-1 cis-elements present in EGF-responsive regions of hSERTp1 and p2. Overexpression of c-jun but not c-fos specifically transactivated hSERTp2, with no effects on hSERTp1. Our findings define novel mechanisms of transcriptional regulation of SERT by EGF via EGFR at the promoter level that may contribute to the beneficial effects of EGF in gut disorders.

Epidermal Growth Factor Receptor Mediates the Vascular Dysfunction But Not the Remodeling Induced by Aldosterone/Salt

Pathophysiological aldosterone (aldo)/mineralocorticoid receptor signaling has a major impact on the cardiovascular system, resulting in hypertension and vascular remodeling. Mineralocorticoids induce endothelial dysfunction, decreasing vasorelaxation in response to acetylcholine and increasing the response to vasoconstrictors. Activation of the epidermal growth factor receptor (EGFR) is thought to mediate the vascular effects of aldo, but this has yet to be demonstrated in vivo. In this study, we analyzed the molecular and functional vascular consequences of aldo-salt challenge in the waved 2 mouse, a genetic model with a partial loss of EGFR tyrosine kinase activity. Deficient EGFR activity is associated with global oxidative stress and endothelial dysfunction. A decrease in EGFR activity did not affect the arterial wall remodeling process induced by aldo-salt. By contrast, normal EGFR activity was required for the aldo-induced enhancement of phenylephrine- and angiotensin II-mediated vasoconstriction. In conclusion, this in vivo study demonstrates that EGFR plays a key role in aldosterone-mediated vascular reactivity.

Activation of kinin B 1 receptor increases the release of metalloproteases-2 and -9 from both estrogen-sensitive and -insensitive breast cancer cells

The kinin B 1 receptor (B 1R) agonist Lys-des[Arg 9]-bradykinin (LDBK) increases proliferation of estrogen-sensitive breast cancer cells by a process involving activation of the epidermal growth factor receptor (EGFR) and downstream signaling via the ERK1/2 mitogen-activated protein kinase pathway. Here, we investigated whether B 1R stimulation induced release of the extracellular matrix metalloproteases MMP-2 and MMP-9 via ERK-dependent pathway in both estrogen-sensitive MCF-7 and -insensitive MDA-MB-231 breast cancer cells. Cells were stimulated with 1–100 nM of the B 1R agonist for variable time-points. Western blotting and gelatin zymography were used to evaluate the presence of MMP-2 and MMP-9 in the extracellular medium. Stimulation of B 1R with as little as 1 nM LDBK induced the accumulation of these metalloproteases in the medium within 5–30 min of stimulation. In parallel, immunocytochemistry revealed that metalloprotease levels in the breast cancer cells declined after stimulation. This effect was blocked either by pre-treating the cells with a B 1R antagonist or by transfecting with B 1R-specific siRNA. Activation of the ERK1/2 pathway and EGFR transactivation was required for release of metalloproteases because both the MEK1 inhibitor, PD98059, and AG1478, an inhibitor of the EGFR-tyrosine kinase activity, blocked this event. The importance of EGFR-dependent signaling was additionally confirmed since transfection of cells with the dominant negative EGFR mutant HERCD533 blocked the release of metalloproteases. Thus, activation of B 1R is likely to enhance breast cancer cells invasiveness by releasing enzymes that degrade the extracellular matrix and thereby favor metastasis.

Evaluation of radioiodinated quinazoline derivative as a new ligand for EGF receptor tyrosine kinase activity using SPECT

A radioiodinated analog of PD153035 (m-IPQ) was evaluated as a potential epidermal growth factor receptor tyrosine kinase (EGFR-TK) activity imaging ligand for SPECT. The 50% inhibition concentration (IC₅₀) value of m-IPQ for EGFR-TK phosphorylation inhibition was evaluated and compared to various EGFR-TK inhibitors. [(125)I]m-IPQ was synthesized by iododestannylation reaction. Biodistribution study of [(125)I]m-IPQ was conducted in normal mice and tumor-bearing mice. The selectivity and binding characteristics (B(max) and K(d)) were analyzed. The quinazoline derivative m-IPQ was found to have high inhibitory potency (IC₅₀: 50.5 ± 3.5 nM) and selectivity toward EGFR-TK. In vivo biodistribution studies of [(125)I]m-IPQ demonstrated its rapid clearance and low retention in normal tissue. On the other hand, high tumor uptake was observed. However, the increase in [(125)I]m-IPQ uptake in the stomach as a deiodination parameter was found. Thus, [(125)I]m-IPQ showed low in vivo stability. The selectivity toward EGFR-TK of m-IPQ was confirmed by the pretreatment experiment with EGFR-TK specific inhibitors, PD153035, Genistein. [(125)I]m-IPQ bound to single population of binding sites with high affinity and kinetic parameter. In addition, [(125)I]m-IPQ was bound to EGFR-TK according to the amount of EGFR-TK expression in the tumor. [(125)I]m-IPQ showed a relatively high tumor accumulation with selective EGFR-TK binding. Moreover, the tumor uptake of [(125)I]m-IPQ might be reflected in the amount of EGFR-TK expression in the tumor. These good characteristics of [(125)I]m-IPQ suggested that a ¹²³I-labeled counterpart, [¹²³I]m-IPQ, would have great potential for EGFR-TK imaging with SPECT. However, the in vivo stability of this compound needs to improve.

Cancer stem cell, niche and EGFR decide tumor development and treatment response: A bio-computational simulation study

Recent research in cancer biology has suggested the hypothesis that tumors are initiated and driven by a small group of cancer stem cells (CSCs). Furthermore, cancer stem cell niches have been found to be essential in determining fates of CSCs, and several signaling pathways have been proven to play a crucial role in cellular behavior, which could be two important factors in cancer development. To better understand the progression, heterogeneity and treatment response of breast cancer, especially in the context of CSCs, we propose a mathematical model based on the cell compartment method. In this model, three compartments of cellular subpopulations are constructed: CSCs, progenitor cells (PCs), and terminal differentiated cells (TCs). Moreover, (1) the cancer stem cell niche is, considered by modeling its effect on division patterns (symmetric or asymmetric) of CSCs, and (2) the EGFR signaling pathway is integrated by modeling its role in cell proliferation, apoptosis. Our simulation results indicate that (1) a higher probability for symmetric division of CSC may result in a faster expansion of tumor population, and for a larger number of niches, the tumor grows at a slower rate, but the final tumor volume is larger; (2) higher EGFR expression correlates to tumors with larger volumes while a saturation function is observed, and (3) treatments that inhibit tyrosine kinase activity of EGFR may not only repress the tumor volume, but also decrease the CSCs percentages by shifting CSCs from symmetric divisions to asymmetric divisions. These findings suggest that therapies should be designed to effectively control or eliminate the symmetric division of CSCs and to reduce or destroy the CSC niches.

Effects of vandetanib on adenoma formation in a dextran sodium sulphate enhanced ApcMIN/+ mouse model

The Apc(MIN/+) mouse is a well-characterised model of intestinal tumourigenesis in which animals develop macroscopically detectable adenomas. However, most of the adenomas are formed in the small intestine and resolution of events in the colon, the most relevant site for human disease, is limited. Inducing colitis with dextran sodium sulphate (DSS) can selectively enhance the development of lesions in the colon. We demonstrated that a DSS pre-treatment is well tolerated and effective at inducing colon adenomas in an Apc(MIN/+) mouse model. We then investigated the effect of inhibiting vascular endothelial growth factor (VEGFR)- and epidermal growth factor receptor (EGFR)-dependent signalling pathways on the development of adenomas induced in DSS-pretreated (DSS/Apc(MIN/+)) or non-DSS-pretreated (Apc(MIN/+)) mice using vandetanib (ZD6474), a potent and selective inhibitor of VEGFR and EGFR tyrosine kinase activity. Eight-week old Apc(MIN/+) mice were given either drinking water or 1.8% DSS and then vandetanib (ZD6474) (50 mg/kg/day) or vehicle by oral gavage for 28 days and sacrificed 24 h after the last dose and assessed for adenoma formation in the intestines. DSS pre-treatment was well tolerated and significantly enhanced formation of adenomas in the colon of control Apc(MIN/+) mice. Vandetanib treatment significantly reduced adenoma formation in the small intestine by 68% (P=0.001) and the colon by 77% (from 13.8 to 3.1, P=0.01) of DSS-pretreated Apc(MIN/+) mice. In the Apc(MIN/+) group, vandetanib also reduced the mean number of adenomas in the small intestine by 76% (P<0.001) and in the colon by 60% (from 3.9 to 1.5, P=0.1). DSS-pre-treatment increased the resolution of the model, allowing us to confirm statistically significant effects of vandetanib on the development and growth of colon adenomas in the Apc(MIN/+) mouse. Moreover these preclinical data provide a rationale for studying the effects of vandetanib in early stages of intestinal cancer in the clinic.

Insulin Induces Swelling-dependent Activation of the Epidermal Growth Factor Receptor in Rat Liver

The aim of the study was to analyze whether the proliferative effects of insulin in rat liver involve cross-signaling toward the epidermal growth factor receptor (EGFR) and whether this is mediated by insulin-induced hepatocyte swelling. Studies were performed in the perfused rat liver and in primary rat hepatocytes. Insulin (35 nmol/liter) induced phosphorylation of the EGFR at position Tyr(845) and Tyr(1173), but not at Tyr(1045), suggesting that EGF is not involved in insulin-induced EGFR activation. Insulin-induced EGFR phosphorylation and subsequent ERK1/2 phosphorylation were sensitive to bumetanide, indicating an involvement of insulin-induced hepatocyte swelling. In line with this, hypoosmotic (225 mosmol/liter) hepatocyte swelling also induced EGFR and ERK1/2 activation. Insulin- and hypoosmolarity-induced EGFR activation were sensitive to inhibition by an integrin-antagonistic RGD peptide, an integrin beta1 subtype-blocking antibody, and the c-Src inhibitor PP-2, indicating the involvement of the recently described integrin-dependent osmosensing/signaling pathway (Schliess, F., Reissmann, R., Reinehr, R., vom Dahl, S., and Häussinger, D. (2004) J. Biol. Chem. 279, 21294-21301). As shown by immunoprecipitation studies, insulin and hypoosmolarity induced a rapid, RGD peptide-, integrin beta1-blocking antibody and PP-2-sensitive association of c-Src with the EGFR. As for control, insulin-induced insulin receptor substrate-1 phosphorylation remained unaffected by the RGD peptide, PP-2, or inhibition of the EGFR tyrosine kinase activity by AG1478. Both insulin and hypoosmolarity induced a significant increase in BrdU uptake in primary rat hepatocytes, which was sensitive to RGD peptide-, integrin beta1-blocking antibody, PP-2, AG1478, and PD098059. It is concluded that insulin- or hypoosmolarity-induced hepatocyte swelling triggers an integrin- and c-Src kinase-dependent EGFR activation, which may explain the proliferative effects of insulin.

Abstract 1207: Transactivation of EGFR/ErbB2 receptor by TNF abrogates TNF-induced apoptosis in bronchial epithelial cells and non-small cell lung cancer cells

Abstract [Background] Epidermal Growth Factor Receptor (EGFR) and other ErbB receptors; ErbB2-4, through cytokine and growth factor stimulation, are upregulated in pneumonitis and lung cancer, suggesting that the activation of ErbB receptors is response to pneumonitis and lung cancer in humans. TNF is a cytokine with many biological properties including both anti- and pro-apoptotic signaling pathways. However, the molecular switch, which determines TNF regulation of these two different functions is not well characterized. We previously presented that TNF transactivated both EGFR and ErbB2 via Src kinase activity that promote the survival response to TNF. In the present study, we tested the hypothesis that EGFR/ErbBs activity regulates TNF-mediated bronchial epithelial and non-small cell lung cancer cell survival. [Method] Bronchial epithelial, Beas-2B cells and non-small cell lung cancer, NCI-H292 cells were used for these studies. Cells were treated with TNF (100 ng/ml) in the presence of either PI 3-kinase inhibitor (wortmannin or LY294002), MAP kinase inhibitor (U0126), MMP inhibitor (GM6001 or TAPI-1 or −2) or EGFR tyrosine kinase inhibitor (Erlotinib). Apoptosis and signal transduction pathway activation were determined by TUNEL staining and Western blot analysis, respectively. [Results] In Beas-2B and NCI-H292 cells, TNF-induced apoptosis was significantly enhanced in the presence of EGFR tyrosine kinase inhibitor. Similarly, treatment of Beas-2B and NCI-H292 cells with TNF failed to increase apoptosis except in the presence of PI 3-kinase inhibitor, which increased the apoptotic response 10-fold. Treatment with TNF in the presence of MAP kinase inhibitor, did not increase apoptosis. In Beas 2B and NCI-H292 cells, TNF stimulated EGFR/ErbB2 phosphorylation within 5 min. EGFR/ErbB2 transactivation by TNF was blocked with GM6001 and TAPI-1, which inhibited MMP activity, especially TACE. Furthermore, in the presence of neutralizing antibodies for HB-EGF, TGF-α and EGF, EGFR/ErbB2 transactivation by TNF was inhibited, suggesting TNF-stimulated EGFR/ErbB2 transactivation via TACE cleavage of HB-EGF, TGF- α and EGF abrogates TNF-induced apoptosis in human bronchial epithelial cell and lung cancer cell. TNF stimulated AKT activation via EGFR tyrosine kinase activation. Akt was shown to be a downstream target of TNF-activated EGFR/ErbB2. [Conclusion] We demonstrate that TNF stimulates EGFR/ErbB2 transactivation via ADAM/MMP in a manner that requires HB-EGF, TGF- α and EGF with AKT as a critical downstream regulator of TNF-induced anti-apoptosis. This novel observation has significant implications for understanding the role of EGFR/ErbB2 in maintaining human bronchial epithelial cell homeostasis in an environment of inflammation, injury/repair, such as inflammation-associated carcinogenesis and tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1207.

Abstract 5542: Activation of epidermal growth factor receptor potentiates effects of radiation in A549 lung cancer cells

Abstract Introduction : For patients with advanced non-small cell lung cancer (NSCLC), chemotherapy alone or in combination with radiation (IR) is considered the standard treatment. However, after the documentation of the over-expression of epidermal growth factor receptor (EGFR) in 50% to 80% of NSCLCs and transforming growth factor-alpha in 81% to 93% of NSCLCs, EGFR has emerged as an important target molecule in NSCLC therapy. The cellular effects of EGFR activation include increased proliferation, promotion of cell motility, adhesion, invasion, angiogenesis and enhanced cell survival. However, activation of EGFR in some tumor cell lines excessively expressing EGFR resulted in cellular apoptosis and growth inhibition. This study attempts to define the role of EGFR kinase activity in sensitizing the responsiveness of EGFR-overexpressed NSCLC cells to IR. Methods: Cell survival, proliferation, cell cycle distribution, reporter activity of EGR-1 and protein expression was studied by colony forming assays, cell counting, flow cytometry, 14Cchloramphenicol acetyl transferase (CAT) reporter assay and Western analysis respectively in A549 and H460 cells. Results: Interestingly, we observed that EGFR activation mediated sensitization of IR effects in A549 cells over-expressing EGFR. Tyrosine kinase inhibitor (TKI), AG1478 blocked EGF effect on sensitization of IR in A549 cells but not in H460 cells. To understand the molecular mechanism underlying the response of A549 cells to IR and EGFR, we investigated the effects of EGFR signaling and IR on expression of early growth response (EGR-1), a transcription factor that sensitizes IR therapy, in both IR-sensitive and insensitive lung cancer cell lines. However, we observed that EGR-1 is not involved in EGFR signaling-mediated IR sensitization since inhibition of Erk (EGFR induces expression of Egr-1 through activation of Erk1/2), did not block EGFR-mediated sensitization of IR effect. Further, we observed that JNK kinase activity mediated EGF-dependent IR effects. Interestingly, acute induction of serum EGF expression level was observed in the patients who responded to IR therapy. Conclusions: Results from this study show that EGFR activation and IR synergistically activate JNK signaling that leads to cellular apoptosis that has important implications for anti-EGFR therapy or IR therapy. Although, preclinical studies have shown that EGFR inhibition can render an additive or synergistic effect with radiotherapy in NSCLCs, clinical benefits are modest. This study suggests that tyrosine kinase activity of EGFR might be required for IR sensitivity. Thus, the inhibition of EGFR by TKIs may lead to reduced responsiveness of NSCLCs to IR thereby compromising the treatment efficacy. Understanding the role of EGFR activity in sensitization of NSCLC cells to IR is therefore extremely important for both radiotherapy and the EGFR TKI-based chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5542.