Aberrant EGFR Research Articles

EGFR-dependent signalling reduced and p38 dependent apoptosis required by Gallic acid in Malignant Mesothelioma cells.

The unrestrained EGFR signalling contributes to malignant phenotype in a number of cancers including Malignant Mesotheliomas. Present study was designed to evaluate EGFR-dependent anti-proliferative and apoptotic effects of Gallic acid in transformed Mesothelial (MeT-5A) and Malignant Mesothelioma (SPC212) cells. Gallic acid reduced the viability of Malignant Mesothelioma cells in a concentration and time-dependent manner. However, viability of mesothelial cells reduced only at high concentration and longer time periods. Gallic acid restrained the activation of EGFR, ERK1/2 and AKT proteins and down regulated expression of Cyclin D and Bcl-2 genes, but upregulated the expression of p21 gene in EGF-induced SPC212 cells. GA-induced transitory G1 arrest and triggered mitochondrial and death receptor mediated apoptosis, which requires p38MAPK activation. The data provided here indicate that GA is able to inhibit EGFR dependent proliferation and survival signals and induces p38 pathway dependent apoptosis in Malignant Mesothelioma cells. On the basis of these experimental findings it is worthwhile to investigate further the biological activity of Gallic acid on other Mesothelioma cell lines harbouring aberrant EGFR signals.

ATNT-01ABT-414 MONO- OR COMBINATION THERAPY WITH TEMOZOLOMIDE (TMZ) RECHALLENGE IN PATIENTS WITH RECURRENT GLIOBLASTOMA (GBM) AND AMPLIFIED EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR): A PHASE I STUDY

BACKGROUND: Aberrant EGFR signaling plays a vital role in GBM oncogenesis. ABT-414 is an antibody-drug conjugate with a toxic payload (monomethylauristatin F) linked to ABT-806, an antibody targeted to activated EGFR. This phase I, open-label, 3-arm study (NCT01800695) evaluates ABT-414 mono- or combination therapy with TMZ rechallenge in patients with GBM. Herein, we report outcomes in recurrent GBM. METHODS: Eligible patients (≥18 years) had recurrent supratentorial GBM and Karnofsky performance status ≥70. ABT-414 was given intravenously every 2 weeks with TMZ rechallenge (150-200 mg/m2 days 1-5 q28; Arm B) or as monotherapy (Arm C). ABT-414 dose escalation used a modified continual reassessment method. Primary objectives were to assess ABT-414 safety, maximum tolerated dose (MTD), and recommended phase II dose (RPTD); secondary objectives included antitumor activity evaluation. RESULTS: As of April 2015, 19 Arm B patients were treated in 4 dose groups (0.5-1.5 mg/kg), and 27 Arm C patients were treated at 1.25 mg/kg. In Arms B/C, 10/17 patients had amplified EGFR. Most common grade 3/4 treatment-emergent adverse events in either arm were keratitis (11%/19%), γ-glutamyltransferase increase (16%/0%), convulsions (11%/0%), fatigue (11%/0%), and thrombocytopenia (11%/4%). ABT-414 MTD was 1.5 mg/kg in Arm B and was not reached in Arm C; RPTD is 1.25 mg/kg for both arms. Best objective responses were complete response (CR) in 3 patients and a partial response (PR) in 1 patient in Arm B, and 1 CR and 1 PR in Arm C patients (all confirmed responses were observed in patients with EGFR amplification).Objective response rate among EGFR-amplified subjects was 22.2% (6/27). CONCLUSIONS: RPTD for ABT-414 is 1.25 mg/kg for both combination therapy with TMZ rechallenge and monotherapy. ABT-414 demonstrated a unique safety profile including ocular AEs, and appears to have antitumor activity in recurrent GBM with EGFR amplification.

Abstract 4258: Genetic profiling of breast cancer confirms a pivotal Role of EGFR pathway in the development of acquired resistance to Tamoxifen in locally recurrent and metastatic breast cancer patients

Abstract Background: Acquisition of resistance to Tamoxifen is a major drawback in the treatment of estrogen receptor (ER)-positive breast cancer (BC) patients. The role of CYP2D6 in predicting response to Tamoxifen is debatable. We assessed the involvement of aberrant EGFR pathway genes expressions and CYP2D6 polymorphism in predicting response to Tamoxifen in a cohort of BC patients from Egypt. Patients and methods: This prospective study included 157 females with hormone receptor positive, locally recurrent inoperable and/or metastatic BC patients treated in the National Cancer Institute, Cairo. Patients were categorized according to their response to Tamoxifen into: responders and refractory groups. RNA and DNA were extracted from tumour and normal tissue samples obtained from all patients. The expression of 92 genes in the EGFR pathway was evaluated using the SABioscience array (Qiagen) with four house-keeping genes and CYP2D6 polymorphism was assessed by PCR. Results: We found that 58.6% of the patients were ER/PR positive, 32.48% were ER positive and 8.91% were PR positive. Response to Tamoxifen correlated significantly with the site of the disease (patients with bone only disease demonstrated better and maintained response compared to those with visceral involvement; p. = 0.005) and CYP2D6 polymorphism (p. = 0.034). CYP2D6 *3, *4 were significantly prevalent in the refractory group (86.6%), whereas variants *10/*10 and *10/*3 were more common in the in the responders (85.5%) (p. = 0.027). RNA profiling of the EGFR pathway showed that 56 genes were differentially over-expressed in the refractory group compared to the responders, of which only JAK1, COL1A1, GAB1, FN1, MKNK1, AKT1, EGFR, NFKB1 and HGDC showed a significant difference between the groups (p<0.05).. Thirty four genes were differentially reduced, of which only STAT5A, RHOA, MAPK9, MAPK10, CCND1 showed significant difference between groups (p<0.05).. Conclusion: The EGFR pathway contributes significantly to patients’ response to Tamoxifen in locally advanced/ metastatic breast cancer. A panel of 13 genes (JAK1, COL1A1, GAB1, FN1, MKNK1, EGFR, NFKB1, HGDC, STAT5A, RHOA, MAPK9, MAPK10, CCND1) in this pathway together with CYP2D6 polymorphisms can predict response to Tamoxifen though this has to be verified in a larger study. Citation Format: Abdel-Rahman N. Zekri, Abeer Bahnassy, Ibrahim Malash, Mohammad Mansour,Sabry Shaarawy, Hoda Abdel-Raouf, Rabab Gaafar. Genetic profiling of breast cancer confirms a pivotal Role of EGFR pathway in the development of acquired resistance to Tamoxifen in locally recurrent and metastatic breast cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4258. doi:10.1158/1538-7445.AM2015-4258

Abstract 1369: Inhibition of Notch- and EGFR signaling reduces cell viability and angiogenesis in glioblastoma multiforme

Abstract The high-grade glioma, Glioblastoma Multiforme (GBM), is the most prevalent and aggressive brain tumour in adults with a median survival of fifteen months. Thus new therapeutic targets are urgently needed. GBM is characterized by extensive vascularisation why targeting angiogenesis has promising potential in improving the treatment of GBM patients. Aberrant EGFR signaling is another characteristic of GBM, and increased EGFR downstream signaling is believed to correlate with poor prognosis. However, little is known of the involvement of EGFR signaling in GBM angiogenesis. Notch signaling is known for its role in angiogenesis during foetal development and in other cancers, but the importance of Notch signaling in GBM angiogenesis is yet to be elucidated. Recently, it has been shown, that crosstalk between Notch- and EGFR signaling occurs in cancer cells, and aberrant EGFR signaling may therefore play a functional role in GBM angiogenesis through crosstalk with Notch signaling. The aim of this study was to investigate the effect of combined Notch- and EGFR inhibition on GBM cell viability and on ability of GBM cells to induce angiogenesis in endothelial cells. In vitro assays of GBM angiogenesis were established using two patient derived GBM cell cultures, one expressing the mutated EGFR, the EGFRvIII, and one expressing wild type EGFR. The functional relevance of EGFR- and Notch signaling in GBM cells was tested using the tyrosine kinase inhibitor Iressa for EGFR inhibition, and the gamma-secretase inhibitor DAPT for Notch inhibition. We show that combined inhibition of EGFR- and Notch signaling caused additive inhibition of cell viability of both cell cultures in vitro. Abrogated EGFR/Notch signaling in GBM cells further reduced angiogenesis in endothelial cells exposed to conditioned media from treated GBM cells. Moreover, when GBM cells were treated with the inhibitors, this inhibited phosphorylation and activation of the two survival signalling pathways ERK and Akt, and abrogated secretion and expression of the most prominent angiogenic factor - VEGF. The presented results indicate that EGFR- and Notch signaling play a functional role in GBM angiogenesis and that combined treatment with drugs targeting both the EGFR- and Notch pathway could prove beneficial over single drug therapy in targeting both the tumorigenic and angiogenic potential of GBM cells. Citation Format: Mikkel Staberg, Signe Regner Michaelsen, Louise Stobbe Olsen, Mette Kjølhede Nedergaard, Mette Villingshøj, Hans Skovgaard Poulsen. Inhibition of Notch- and EGFR signaling reduces cell viability and angiogenesis in glioblastoma multiforme. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1369. doi:10.1158/1538-7445.AM2015-1369

Development of Resistance to EGFR-Targeted Therapy in Malignant Glioma Can Occur through EGFR-Dependent and -Independent Mechanisms.

Epidermal growth factor receptor (EGFR) is highly amplified, mutated, and overexpressed in human malignant gliomas. Despite its prevalence and growth-promoting functions, therapeutic strategies to inhibit EGFR kinase activity have not been translated into profound beneficial effects in glioma clinical trials. To determine the roles of oncogenic EGFR signaling in gliomagenesis and tumor maintenance, we generated a novel glioma mouse model driven by inducible expression of a mutant EGFR (EGFR*). Using combined genetic and pharmacologic interventions, we revealed that EGFR*-driven gliomas were insensitive to EGFR tyrosine kinase inhibitors, although they could efficiently inhibit EGFR* autophosphorylation in vitro and in vivo. This is in contrast with the genetic suppression of EGFR* induction that led to significant tumor regression and prolonged animal survival. However, despite their initial response to genetic EGFR* extinction, all tumors would relapse and propagate independent of EGFR*. We further showed that EGFR*-independent tumor cells existed prior to treatment and were responsible for relapse following genetic EGFR* suppression. And, the addition of a PI3K/mTOR inhibitor could significantly delay relapse and prolong animal survival. Our findings shed mechanistic insight into EGFR drug resistance in glioma and provide a platform to test therapies targeting aberrant EGFR signaling in this setting.

Genetics of the serrated pathway to colorectal cancer.

Genetics of the serrated pathway to colorectal cancer.

Proteome mapping of epidermal growth factor induced hepatocellular carcinomas identifies novel cell metabolism targets and mitogen activated protein kinase signalling events.

BackgroundHepatocellular carcinoma (HCC) is on the rise and the sixth most common cancer worldwide. To combat HCC effectively research is directed towards its early detection and the development of targeted therapies. Given the fact that epidermal growth factor (EGF) is an important mitogen for hepatocytes we searched for disease regulated proteins to improve an understanding of the molecular pathogenesis of EGF induced HCC. Disease regulated proteins were studied by 2DE MALDI-TOF/TOF and a transcriptomic approach, by immunohistochemistry and advanced bioinformatics.ResultsMapping of EGF induced liver cancer in a transgenic mouse model identified n = 96 (p < 0.05) significantly regulated proteins of which n = 54 were tumour-specific. To unravel molecular circuits linked to aberrant EGFR signalling diverse computational approaches were employed and this defined n = 7 key nodes using n = 82 disease regulated proteins for network construction. STRING analysis revealed protein-protein interactions of > 70% disease regulated proteins with individual proteins being validated by immunohistochemistry. The disease regulated network proteins were mapped to distinct pathways and bioinformatics provided novel insight into molecular circuits associated with significant changes in either glycolysis and gluconeogenesis, argine and proline metabolism, protein processing in endoplasmic reticulum, Hif- and MAPK signalling, lipoprotein metabolism, platelet activation and hemostatic control as a result of aberrant EGF signalling. The biological significance of the findings was corroborated with gene expression data derived from tumour tissues to evntually define a rationale by which tumours embark on intriguing changes in metabolism that is of utility for an understanding of tumour growth. Moreover, among the EGF tumour specific proteins n = 11 were likewise uniquely expressed in human HCC and for n = 49 proteins regulation in human HCC was confirmed using the publically available Human Protein Atlas depository, therefore demonstrating clinical significance.ConclusionNovel insight into the molecular pathogenesis of EGF induced liver cancer was obtained and among the 37 newly identified proteins several are likely candidates for the development of molecularly targeted therapies and include the nucleoside diphosphate kinase A, bifunctional ATP-dependent dihydroyacetone kinase and phosphatidylethanolamine-binding protein1, the latter being an inhibitor of the Raf-1 kinase.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1312-z) contains supplementary material, which is available to authorized users.

Exoming into Rare Skin Disease: EGFR Deficiency

Dermatologists are frequently asked to see patients with pustular eruptions caused by EGFR mAbs or tyrosine kinase inhibitors. In this issue, Campbell et al. describe an infant with severely inflammed skin and bowel and lung disease caused by a homozygous mutation in the EGFR gene. This commentary discusses the power of exome sequencing in disease gene discovery within the rare genodermatoses and the role of aberrant EGFR signaling in a subset of monogenic skin and epithelial syndromes.

Abstract DDT01-03: Discovery and preclinical pharmacology of JNJ-61186372: A novel bispecific antibody targeting EGFR and cMET

Abstract NSCLC with activating mutations in the EGFR gene are associated with high response rates to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, but invariably acquired resistance emerges over time. A primary cause of resistance is the emergence of secondary mutations in EGFR which neutralize the effectiveness of TKIs. In addition, the cMet pathway is often activated, either through MET gene amplification, overexpression of cMet protein, or an increase in the ligand HGF, to provide a compensatory survival pathway conferring resistance to EGFR TKIs. We have designed a bispecific EGFR-cMet antibody (JNJ-61186372) with a unique set of mechanisms of action resulting in anti-tumor activity in the EGFR mutant setting, with or without cMet pathway activation. We have demonstrated three mechanisms of action that contribute to the activity of JNJ-61186372: 1) inhibition of ligand-induced phosphorylation of both EGFR and cMet, 2) receptor degradation in vivo, and 3) enhanced ADCC activity. JNJ-61186372 inhibited EGF-induced phosphorylation of EGFR in cell lines with either wild-type (WT) EGFR or activating mutations in EGFR. In the same cell lines, JNJ-61186372 inhibited HGF-induced phosphorylation of cMet. JNJ-61186372 also blocked pERK and pAkt with similar IC50 values in EGFR-WT and EGFR mutant cell lines, indicating that downstream signaling pathways were inhibited. Total protein levels of both EGFR and cMet were decreased in xenograft tumor models following treatment with JNJ-61186372 compared to tumors from mice treated with PBS control suggesting that one mechanism by which JNJ-61186372 suppresses EGFR and cMet activity in vivo is through degradation of both receptors. The third mechanism of action is directing immune cells to kill tumor cells. JNJ-61186372 is produced with low levels of fucosylation, which translates to an enhanced antibody-dependent cellular cytotoxicity (ADCC). These three mechanisms of action of JNJ-61186372 provide a distinct preclinical profile for targeting both EGFR and cMET in a single bispecific antibody. JNJ-61186372 demonstrated efficacy in multiple in vivo tumor models with EGFR mutations, including both cell line and patient-derived xenografts. Importantly, JNJ-61186372 effectively inhibited tumor growth in models with mutant EGFR and cMet activation, whereas single agent EGFR inhibitors were less effective. The preclinical data support the clinical development of JNJ-61186372 in patients with lung cancer and other malignancies associated with aberrant EGFR and cMET signaling. Citation Format: Sheri L. Moores, Mark Chiu, Barbara Bushey, Kristen Chevalier, Peter Haytko, Joost Neijssen, Paul Parren, Janine Schuurman, Mark Anderson, Ricardo Attar, Robert Kramer, Matthew V. Lorenzi. Discovery and preclinical pharmacology of JNJ-61186372: A novel bispecific antibody targeting EGFR and cMET. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr DDT01-03. doi:10.1158/1538-7445.AM2014-DDT01-03

Abstract 743: Potent anti-tumor activity of the MEK1/2 inhibitor MEK162 in human non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (SCCHN) cell lines

Abstract BACKGROUND: MAPK pathway dysregulation is frequently seen in cancer. For NSCLC, this may be due to driver mutations or gene amplification in EGFR, KRAS, or BRAF, though tumors without such mutations frequently display pathway hyperactivity. Aberrant EGFR signaling in SCCHN may lead to hyperactivity of the MAPK or PI3K pathways. MEK162 (ARRY-438162) is a potent, tight binding, uncompetitive MEK inhibitor currently in clinical development for treatment of solid tumors. MEK162 has an IC50 of 10nM against purified MEK1/2. We evaluated whether MEK162 would inhibit NSCLC and SCCHN cell lines and whether specific molecular alterations predicted anti-tumor activity. We also evaluated the combination of MEK162 in combination with BYL719, a potent PI3K alpha inhibitor. METHODS: The 50% inhibitory concentration (IC50) of MEK162, alone or in combination with BYL719, was determined in 40 human NSCLC and 30 SCCHN cell lines in vitro. Effects on MAPK and PI3K/AKT pathway signaling were studied by western blot analysis. Effects of MEK162 alone or in combination with BYL719 on cell cycle progression were also determined by flow cytometry. RESULTS: Using previously published cut-offs for sensitivity in cell lines, among NSCLC cell lines, 20/40 (50%) were sensitive to MEK162 (IC50 ≤ 500nM), 9/40 were intermediately sensitive (IC50 500nM-1µM), and 11/40 were resistant (IC50 ≥ 1µm). Using the same cut-offs, 80% (24/30) of SCCHN cell lines were sensitive, 2/30 were intermediately sensitive, and 4/30 were resistant to MEK162. MEK162 exposure resulted in increased cells in G0/G1 and decreased cells in S-phase. MEK162 decreased phosphorylated ERK (p-ERK) in all cell lines treated and resulted in an increase in phosphorylated AKT (p-AKT), presumably as a compensatory result from MAPK pathway inhibition. Combined treatment with MEK162 and BYL719 resulted in synergistic growth inhibition for all cell lines and decreased levels of both p-ERK and p-AKT levels at 24 hours. CONCLUSION: MEK162 potently inhibited growth of human NSCLC and SCCHN cell lines in most cell lines tested and led to synergistic inhibition when combined with BYL719. The compensatory increase in pAKT seen with MEK162 was abrogated with dual treatment with MEK162 and BYL719, demonstrating that synergy may be mediated by simultaneous blockade of MAPK and PI3K/AKT pathways. These data provide a preclinical rationale for evaluating MEK162, alone or in combination with BYL719, in metastatic NSCLC and SCCHN. Supported by 1K23CA149079, P50 CA090440, V Foundation for Cancer Research, Jonsson Comprehensive Cancer Center, Wolfen Family Lung Cancer Research Program, Stiles Program in Oncology, National Lung Cancer Partnership and One Ball Matt Memorial Golf Tournament. Citation Format: Deborah JL Wong, Edward B. Garon, Danielle D. Silveira, Naeimeh Kamranpour, Sharon Pitts, Meenal Chalukya, Habib Hamidi, Steven Dubinett, Ronald Linnartz, Richard S. Finn, Dennis J. Slamon. Potent anti-tumor activity of the MEK1/2 inhibitor MEK162 in human non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (SCCHN) cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 743. doi:10.1158/1538-7445.AM2014-743

The role of NF-κB in the pathogenesis of glioma

Activation of NF-κB affects multiple aspects of cancer biology including cell survival and resistance to treatment. Glioblastoma (GBM) is the most common primary malignant tumor of the brain in adults and is resistant to treatment. Recent studies have reported that NF-κB activation in GBM is widespread and have elucidated the underlying regulatory mechanisms. EGFR gene amplification and mutation are among the key genetic alterations in GBM, and aberrant EGFR signaling is a key activator of NF-κB in GBM. In this review we discuss the evidence for activation of NF-κB in GBM and the key signaling pathways involved. Substantial evidence suggests a role for NF-κB in the pathogenesis of GBM and its resistance to treatment, indicating that NF-κB pathways may be useful targets for treatment.

Effect of trametinib in combination with panitumumab and trastuzumab on tumor growth in an orthotopic xenograft model of human pancreatic cancer.

190 Background: Aberrant MAPK and EGFR family signaling are key drivers of pancreatic ductal adenocarcinoma(PDAC). We hypothesized that combination trametinib(MEK1/2 inhibitor), panitumumab(EGFR inhibitor) and trastuzumab(Her2 inhibitor) would more effectively suppress tumor growth than any of these monotherapies. Methods: Patient-derived PDAC cell line MAD09-366 was exposed to trametinib, panitumumab, trastuzumab, and combination therapies in vitro. Western blot analysis was performed on treated cell lysates. Athymic, nude mice were orthotopically implanted with patient-derived PDAC xenografts(MAD09-366, 08-608, and 08-738). Established murine tumors were treated with control, trametinib (0.3mg/kg, qDay), panitumumab (500ug, BIW), trastuzumab (200ug, BIW) or in combination. MRI was used to assess tumor response. Results: Two of 3 PDACs were Kras mutant, 2 of 3 demonstrated increased Her2 activity, and all 3 showed increased EGFR activity. In vitro studies showed increased growth inhibition of triple-therapy-treated cells relative to control or each inhibitor alone. Western blot analysis revealed that EGF stimulation increased Ras pathway signaling in this Kras-mutant cell line. With EGF stimulation, the greatest Ras pathway signaling inhibition was seen in triple-therapy-treated cells. In vivo studies in all PDAC xenografts revealed that triple therapy significantly decreased tumor growth rate relative to control, trametinib alone, panitumumab alone, or panitumumab plus trastuzumab. In 2 of 3 PDACs assessed, triple therapy was superior to trametinib plus panitumumab. Average tumor size in MAD08-738 triple-therapy-treated mice decreased by 9.3%. Conclusions: Triple therapy with trametinib, panitumumab, and trastuzumab demonstrated the greatest in vitro Ras signaling blockade. In vivo, this combination produced significant tumor growth inhibition or regression in all PDAC tumors studied. This regimen should be considered for a future clinical trial in pancreatic cancer patients.

New negative feedback regulators of Egfr signaling in Drosophila.

The highly conserved epidermal growth factor receptor (Egfr) pathway is required in all animals for normal development and homeostasis; consequently, aberrant Egfr signaling is implicated in a number of diseases. Genetic analysis of Drosophila melanogaster Egfr has contributed significantly to understanding this conserved pathway and led to the discovery of new components and targets. Here we used microarray analysis of third instar wing discs, in which Egfr signaling was perturbed, to identify new Egfr-responsive genes. Upregulated transcripts included five known targets, suggesting the approach was valid. We investigated the function of 29 previously uncharacterized genes, which had pronounced responses. The Egfr pathway is important for wing-vein patterning and using reverse genetic analysis we identified five genes that showed venation defects. Three of these genes are expressed in vein primordia and all showed transcriptional changes in response to altered Egfr activity consistent with being targets of the pathway. Genetic interactions with Egfr further linked two of the genes, Sulfated (Sulf1), an endosulfatase gene, and CG4096, an A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS) gene, to the pathway. Sulf1 showed a strong genetic interaction with the neuregulin-like ligand vein (vn) and may influence binding of Vn to heparan-sulfated proteoglycans (HSPGs). How Drosophila Egfr activity is modulated by CG4096 is unknown, but interestingly vertebrate EGF ligands are regulated by a related ADAMTS protein. We suggest Sulf1 and CG4096 are negative feedback regulators of Egfr signaling that function in the extracellular space to influence ligand activity.

Nuclear EGFRvIII‐STAT5b complex contributes to glioblastoma cell survival by direct activation of the Bcl‐XL promoter

Aberrant EGFR signaling strongly promotes glioma malignancy and treatment resistance. The most prevalent mutation, ΔEGFR/EGFRvIII, is an in-frame deletion of the extracellular domain, which occurs in more than 25% of glioblastomas and enhances growth and survival of tumor cells. Paradoxically, the signaling of the potent oncogene ΔEGFR is of low intensity, raising the question of whether it exhibits preferential signaling to key downstream targets. We have observed levels of phosphorylation of STAT5 at position Y699 in cells expressing ΔEGFR that are similar or higher than in cells that overexpress EGFR and are acutely stimulated with EGF, prompting us to investigate the role of STAT5 activation in glioblastoma. Here, we show that in human glioblastoma samples, pSTAT5 levels correlated positively with EGFR expression and were associated with reduced survival. Interestingly, the activation of STAT5b downstream of ΔEGFR was dependent on SFKs, while the signal from acutely EGF-stimulated EGFR to STAT5b involved other kinases. Phosphorylated STAT5b and ΔEGFR associated in the nucleus, bound DNA and were found on promoters known to be regulated by STAT5 including that of the Aurora A gene. ΔEGFR cooperated with STAT5b to regulate the Bcl-XL promoter and knockdown of STAT5b suppressed anchorage independent growth, reduced the levels of Bcl-XL and sensitized glioblastoma cells to cisplatin. Together these results delineate a novel association of nuclear ΔEGFR with STAT5b, which promotes oncogenesis and treatment resistance in glioblastoma by direct regulation of anti-apoptotic gene, Bcl-XL.

EGFR inhibitor BIBU induces apoptosis and defective autophagy in glioma cells

The importance of aberrant EGFR signaling in glioblastoma progression and the promise of EGFR-specific therapies, prompted us to determine the efficacy of novel EGFR inhibitor BIBU-1361 [(3-chloro-4-fluoro-phenyl)-[6-(4-diethylaminomethyl-piperidin-1-yl)-pyrimido [5,4-d]pyrimidin-4-yl]-amine] in affecting glioma survival. BIBU induced apoptosis in a caspase-dependent manner and induced cell cycle arrest in glioma cells. Apoptosis was accompanied by decreased EGFR levels and its increased distribution towards caveolin rich lipid raft microdomains. BIBU inhibited pro-survival pathways Akt/mTOR and gp130/JAK/STAT3; and decreased levels of pro-inflammatory cytokine IL-6. BIBU caused increased LC3-I to LC3-II conversion and triggered the internalization of EGFR within vacuoles along with its increased co-localization with LC3-II. BIBU caused accumulation of p62 and increased levels of cleaved forms of Beclin-1 in all the cell lines tested. Importantly, BIBU failed to initiate execution of autophagy as pharmacological inhibition of autophagy with 3-Methyladenine or Bafilomycin failed to rescue BIBU mediated death. The ability of BIBU to abrogate Akt and STAT3 activation, induce apoptosis and prevent execution of autophagy warrants its investigation as a potent anti-glioma target.

Abstract 1188: BRCA1 regulates EGFR expression through microRNAs

Abstract Germline mutations in the tumor suppressor BRCA1 (breast cancer 1, early onset) are associated with a substantially higher risk of developing basal-like breast cancer (BLBC). Many sporadic breast cancers express decreased levels of BRCA1 and show a strong resemblance to BRCA1-hereditary tumors. BRCA1-associated tumors are triple-negative (TN), basal-like high grade ductal carcinomas that display an undifferentiated phenotype and frequently overexpress epidermal growth factor receptor (EGFR/ERBB1/HER1) and basal cytokeratins (CK5, CK6, CK14 and CK17) and confer a poor clinical prognosis. Although upregulation of EGFR is commonly observed in BRCA1-associated breast cancer, the mechanism of EGFR regulation by BRCA1 is not understood. EGFR upregulation could occur at multiple transcriptional and/or post-transcriptional levels. MicroRNAs (miRNAs) are an evolutionarily conserved class of small non-coding RNAs that regulate gene expression post-transcriptionally and/or translationally. Several miRNAs are found to be differentially expressed in breast cancer and studies show their involvement in stem cell differentiation, epithelial-to-mesenchymal transition (EMT), invasion and metastasis. Identifying the functional relationship between loss of BRCA1 and miRNAs will help us understand the post-transcriptional mechanism by which BRCA1 deficiency leads to aberrant EGFR expression. In the current study, we show for the first time, a direct inverse correlation between BRCA1 and EGFR protein expression levels in breast cancer cell lines representing the different intrinsic subtypes and in primary mammary epithelial cells. By overexpression and knockdown of BRCA1, we demonstrate that BRCA1 loss results in increased stability of EGFR protein. Using our in vitro system of immortalized primary mammary epithelial cells, we evaluated the effect of functional loss of BRCA1 on the expression of miRNAs in secondary mammospheres which are enriched for stem-like cells and in cells differentiated in vitro. We observed that BRCA1 deficiency leads to dysregulation of miRNAs linked to stem cell renewal, EGFR signaling and EMT. miRNA gain-of-function experiments using Pre-miR™ miRNA precursors and loss-of-function experiments using miRCURY LNA™ miRNA inhibitors were performed with the selected miRNAs to further confirm their effect on EGFR expression. Results suggest that miRNA mediated EGFR deregulation could be a critical step in tumor initiation and progression in BRCA1 deficiency. Since EGFR upregulation is an early event in breast cancer, additional functional studies will provide a novel molecular mechanism by which BRCA1 deficiency leads to the development of BLBC with aberrant EGFR expression mediated by miRNAs. The study will offer new therapeutic approaches for BLBC, a highly aggressive disease with limited therapeutic options. 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 1188. doi:10.1158/1538-7445.AM2011-1188

Simultaneous Assessment of Cyclin D1 and Epidermal Growth Factor Receptor Gene Copy Number for Prognostic Factor in Oral Squamous Cell Carcinomas

Cyclin D1 gene (CCND1) numerical aberrations are independent prognostic indicators of head and neck squamous cell carcinomas (HNSCCs). High epidermal growth factor receptor gene (EGFR) copy number is associated with poor prognosis in lung cancer, but such findings are controversial in oral SCCs (OSCCs). We analyzed copy number status in CCND1 and EGFR in OSCC patients and its association with clinical outcome.EGFR and CCND1 statuses were analyzed in 85 OSCC patients by fluorescence in situ hybridization (FISH) of specimens obtained by fine-needle aspiration biopsy.CCND1 numerical aberration was found in 35 of 85 tumors (41%), and aberrant EGFR copy number was observed in 36 (42%). Gene amplification (GA) was dominant among CCND1 copy number changes (14/35:40%). Balanced trisomy (BT) was the most frequently observed EGFR aberration (17/36:47%). In a multivariate Cox's proportional hazards analysis, CCND1 GA was correlated with disease-free survival (P < 0.001), whereas EGFR BT was significantly correlated with overall survival (P = 0.001). Patients with a combination of CCND1 GA and/or EGFR BT had significantly poorer clinical outcome.CCND1 and EGFR copy number changes were frequent in OSCC and had differing aberration patterns. CCND1 GA and EGFR BT statuses by dual-color FISH were the predominant predictors of clinical outcome. Further investigation is needed to determine the implications for EGFR inhibitor therapy in OSCC.

2648

Oncogene activation not only plays a major role in tumor development and progression, but is also an important determinant in the response of tumor cells to anticancer therapies. Consistent with this notion, in head and neck squamous cell carcinomas (HNSCC), aberrant expression or activation of EGFR and of NF-κB signaling has been implicated not only in tumor development but also in chemo- and/or radiation resistance. In this study we used immortalized but non-tumorigenic human keratinocytes (HaCaT cells) over-expressing EGFR (HaCaTEGFR) and NF-κBp65 (HaCaTp65), respectively, to study the function of aberrant EGFR and NF-κB signaling in epithelial tumor development and radiation resistance.