Epidermal Growth Factor Receptor Crosstalk Research Articles

Modulating the Effect of β-Sitosterol Conjugated with Magnetic Nanocarriers to Inhibit EGFR and Met Receptor Cross Talk.

The cross-talk between the EGFR (Epidermal Growth Factor Receptor) and MET (Hepatocyte Growth Factor Receptor) poses a significant challenge in the field of molecular signaling. Their intricate interplay leads to dysregulation and contributes to cancer progression and therapeutic resistance. β-Sitosterol (BS), a plant sterol with promising anticancer properties, shows increased research on its potential as a chemopreventive agent. However, significant modifications are required to deliver BS in cancer cells due to its lower efficacy. The present work aims to design a carrier-mediated delivery system specifically targeting cancer cells with EGFR and MET receptor cross-talk. Surface modification of BS was performed with superparamagnetic iron oxide nanoparticles (SPIONs), polyethylene glycol (PEG), and poly(N-isopropylacrylamide) (PNIPAM) to enhance the delivery of BS at the target site. BS was conjugated with SPIONs (BS-S), PNIPAM (BS-SP), PEG, and PNIPAM (BS-SPP) polymers, respectively, and the conjugated complexes were characterized. Results showed an increase in size, stability, and monodispersity in the following order, BS-S, BS-SP, and BS-SPP. The drug encapsulation efficiency was observed to be highest in BS-SPP (82.5%), compared to BS-S (61%) and BS-SP (74.9%). Sustained drug release was achieved in both BS-SP (82.6%) and BS-SPP (83%). The IC 50 value of BS, BS-S, BS-SP, and BS-SPP towards MCF 7 was 242 µg/mL,197 µg/mL, 168 µg/mL, and 149 µg/mL, HEPG2 was 274 µg/mL, 261 µg/mL, 233 µg/mL and 207 µg/mL and NCIH 460 was 191 µg/mL, 185 µg/mL, 175 and 164 µg/mL, indicating highest inhibition towards NCIH 460 cells. Our results conclude that β-sitosterol conjugated with SPION, PEG, and PNIPAM could be a potential targeted therapy in inhibiting EGFR and MET receptor-expressing cancer cells.

Cell Behavior of Non-Small Cell Lung Cancer Is at EGFR and MicroRNAs Hands.

Lung cancer is a complex disease associated with gene mutations, particularly mutations of Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) and epidermal growth factor receptor (EGFR). Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) are the two major types of lung cancer. The former includes most lung cancers (85%) and are commonly associated with EGFR mutations. Several EGFR-tyrosine kinase inhibitors (EGFR-TKIs), including erlotinib, gefitinib, and osimertinib, are effective therapeutic agents in EGFR-mutated NSCLC. However, their effectiveness is limited by the development (acquired) or presence of intrinsic drug resistance. MicroRNAs (miRNAs) are key gene regulators that play a profound role in the development and outcomes for NSCLC via their role as oncogenes or oncosuppressors. The regulatory role of miRNA-dependent EGFR crosstalk depends on EGFR signaling pathway, including Rat Sarcoma/Rapidly Accelerated Fibrosarcoma/Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase 1/2 (Ras/Raf/MEK/ERK1/2), Signal Transducer and Activator of Transcription (STAT), Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NF-kB), phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), Janus kinase 1 (JAK1), and growth factor receptor-bound protein 2 (GRB2). Dysregulated expression of miRNAs affects sensitivity to treatment with EGFR-TKIs. Thus, abnormalities in miRNA-dependent EGFR crosstalk can be used as diagnostic and prognostic markers, as well as therapeutic targets in NSCLC. In this review, we present an overview of miRNA-dependent EGFR expression regulation, which modulates the behavior and progression of NSCLC.

The Role of p53 Expression in Patients with RAS/BRAF Wild-Type Metastatic Colorectal Cancer Receiving Irinotecan and Cetuximab as Later Line Treatment.

BackgroundPreclinical and clinical data indicate that p53 expression might modulate the activity of the epidermal growth factor receptor (EGFR), influencing response/resistance to anti-EGFR monoclonal antibodies. However, the association between p53 status and clinical outcome has not been clarified yet.ObjectiveIn our study, we evaluated the role of p53 expression in patients with RAS/BRAF wild-type metastatic colorectal cancer (mCRC) receiving irinotecan/cetuximab in an exploratory and a validation cohort.Patients and Methodsp53 expression was analysed in patients with RAS/BRAF wild-type mCRC receiving second-line or third-line irinotecan/cetuximab. Survival distribution was assessed by the Kaplan–Meier method, while the log-rank test was used for survival comparison.ResultsAmong 120 patients with RAS/BRAF wild-type mCRC included in our analysis, 52 (59%) and 19 (59%) patients showed p53 overexpression in the exploratory and validation cohort, respectively. In the exploratory cohort, low p53 expression was correlated with better median progression-free survival (hazard ratio 0.39; p < 0.0001), median overall survival (hazard ratio: 0.23; p < 0.0001) and response rate (p < 0.0001). These results were confirmed by data of the validation cohort where we observed better median progression-free survival (hazard ratio: 0.48; p = 0.0399), median overall survival (hazard ratio: 0.26; p = 0.0027) and response rate (p =0.0007) in patients with p53 normal expression mCRC.ConclusionsIn our study, p53 overexpression was associated with anti-EGFR treatment resistance in patients with RAS/BRAF WT mCRC, as confirmed in a validation cohort. Larger studies are needed to validate the role of p53 and investigate EGFR cross-talk in these patients.

MiR-149 Suppresses Breast Cancer Metastasis by Blocking Paracrine Interactions with Macrophages.

Paracrine activation of cells contained in the tumor microenvironment promotes tumor progression and metastasis. In breast cancer, malignant cells recruit and educate macrophages into a M2 tumor-promoting phenotype that supports the metastatic spread of cancer cells. Here, we show that miR-149 functions as a metastasis-suppressing microRNA in breast cancer cells by limiting colony-stimulating factor-1 (CSF1)-dependent recruitment and M2 polarization of macrophages. In lymph node-positive, triple-negative breast cancer (TNBC) tissues, low miR-149 expression correlated with macrophage infiltration and reduced patient survival. By directly targeting CSF1, miR-149 expression in TNBC cell lines (MDA-MB-231 and BT-549) inhibited the recruitment of human monocytic THP-1 cells and primary human macrophages. Furthermore, in macrophages cocultured with MDA-MB-231 cells expressing miR-149, epidermal growth factor (EGF) and amphiregulin expression levels were strongly reduced, resulting in reduced EGF receptor activation in the cancer cells. In vivo, lung metastases developing from orthotopic MDA-MB-231 tumors were reduced by 75% by miR-149 expression, and this was associated with impaired M2 macrophage infiltration of the primary tumors. These data suggest that miR-149 downregulation functionally contributes to breast tumor progression by recruiting macrophages to the tumor and facilitating CSF1 and EGF receptor cross-talk between cancer cells and macrophages. SIGNIFICANCE: These findings contribute to the understanding of tumor-stroma interactions by showing that miR-149 downregulation in TNBC enhances reciprocal growth factor signaling between macrophages and cancer cells, which promotes tumor progression and metastasis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/6/1330/F1.large.jpg.

Antiplatelet Drug Ticagrelor Enhances Chemotherapeutic Efficacy by Targeting the Novel P2Y12-AKT Pathway in Pancreatic Cancer Cells.

Background: Extensive research has reported that extracellular ADP in the tumour microenvironment can stimulate platelets through interaction with the platelet receptor P2Y12. In turn, activated platelets release biological factors supporting cancer progression. Experimental data suggest that the tumour microenvironment components, of which platelets are integral, can promote chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC). Thus, overcoming chemoresistance requires combining multiple inhibitors that simultaneously target intrinsic pathways in cancer cells and extrinsic factors related to the tumour microenvironment. We aimed to determine whether ticagrelor, an inhibitor of the ADP–P2Y12 axis and a well-known antiplatelet drug, could be a therapeutic option for PDAC. Methods: We investigated a functional P2Y12 receptor and its downstream signalling in a panel of PDAC cell lines and non-cancer pancreatic cells termed hTERT-HPNE. We tested the synergistic effect of ticagrelor, a P2Y12 inhibitor, in combination with chemotherapeutic drugs (gemcitabine, paclitaxel and cisplatin), in vitro and in vivo. Results: Knockdown studies revealed that P2Y12 contributed to epidermal growth factor receptor (EGFR) activation and the expression of SLUG and ZEB1, which are transcriptional factors implicated in metastasis and chemoresistance. Studies using genetic and pharmacological inhibitors showed that the P2Y12–EGFR crosstalk enhanced cancer cell proliferation. Inhibition of P2Y12 signalling significantly reduced EGF-dependent AKT activation and promoted the anticancer activity of anti-EGFR treatment. Importantly, ticagrelor significantly decreased the proliferative capacity of cancer but not normal pancreatic cells. In vitro, synergism was observed when ticagrelor was combined with several chemodrugs. In vivo, a combination of ticagrelor with gemcitabine significantly reduced tumour growth, whereas gemcitabine or ticagrelor alone had a minimal effect. Conclusions: These findings uncover a novel effect and mechanism of action of the antiplatelet drug ticagrelor in PDAC cells and suggest a multi-functional role for ADP-P2Y12 signalling in the tumour microenvironment.

Abstract 5906: EGFR and SOX2 crosstalk determines EGFR-TKI

Abstract Activating mutations in the epidermal growth factor receptor (EGFR), while initiating lung tumorigenesis, render tumors susceptible to EGFR-tyrosine kinase inhibitors (TKIs). SOX2 functions as a cell fate determination factor in lung progenitor cells. However, how SOX2 expression affects TKI treatment and tumor invasion in lung cancer is obscure. Here we report that EGFR mutations crosstalk with SOX2 to maintain cell proliferation and barrier properties in lung cancer cells, some of which generate heterogeneity by switching off SOX2 expression, thus promoting TKI resistance and invasiveness. Downregulation of SOX2 expression by shRNA against SOX2 or TGF-beta treatment induced epithelial-to-mesenchymal transition (EMT) and endowed cell resistant to TKIs. TKI treatment selected EGFR-mutated lung cancer cells harboring low SOX2 expression with the EMT signature. TKI naïve EGFR-mutated lung cancer cells harboring low SOX2 expression exhibits intrinsic TKI resistance. Conversely, enhancing endogenous SOX2 expression reverses EMT and increases sensitivity to TKIs. Immunohistochemistry analysis revealed that low SOX2 expression predicts a poor survival in patients harboring EGFR mutations. Taken together, our findings show how cancer plasticity elicited by SOX2 expression and TKI selection generates distinct oncogenic properties and TKI sensitivity, providing critical insights into tumor progression in EGFR-mutated lung cancer. Citation Format: An-Chun Lee, Chia-Cherng Yu, Yuan-Hung Wang, Yu-Ting Chou. EGFR and SOX2 crosstalk determines EGFR-TKI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5906.

Dysregulation and detection methods of EGFR in oral cancer. A narrative review.

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein, with an intracellular domain and tyrosine kinase function (TK) involved in cell proliferation. Dysfunctions in EGFR signaling pathways have been associated with oral malignant tumors such as oral squamous cell carcinoma (OSCC). Dysfunctions of EGFR may result from: increased EGF ligand; EGFR overexpression and copy number gain of the EGFR gene (EGFR CNG); EGFR mutations; failure in the downregulation of EGFR; and EGFR crosstalk. Of these alterations, overexpression of EGFR is by far the most studied dysfunction in OSCC. Clinicians should identify possible alterations of EGFR in the oral mucosa of patients, as EGFR can act as a biomarker for the diagnosis and prognosis of OSCC. Currently, there are several methods and techniques for detecting EGFR. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR), are used to identify overexpression of EGFR, EGFR CNG and EGFR mutations, respectively. Detection of EGFR as a biomarker is key to identify any oral malignant transformation. Consequently, it becomes imperative to implement a non-invasive and inexpensive method of early diagnosis for OSCC in clinical practice.

The renin-angiotensin system mediates EGF receptor-vitamin d receptor cross-talk in colitis-associated colon cancer.

We previously showed that EGF receptor (EGFR) promotes tumorigenesis in the azoxymethane/dextran sulfate sodium (AOM/DSS) model, whereas vitamin D suppresses tumorigenesis. EGFR-vitamin D receptor (VDR) interactions, however, are incompletely understood. Vitamin D inhibits the renin-angiotensin system (RAS), whereas RAS can activate EGFR. We aimed to elucidate EGFR-VDR cross-talk in colorectal carcinogenesis. To examine VDR-RAS interactions, we treated Vdr(+/+) and Vdr(-/-) mice with AOM/DSS. Effects of VDR on RAS and EGFR were examined by Western blotting, immunostaining, and real-time PCR. We also examined the effect of vitamin D3 on colonic RAS in Vdr(+/+) mice. EGFR regulation of VDR was examined in hypomorphic Egfr(Waved2) (Wa2) and Egfr(wild-type) mice. Angiotensin II (Ang II)-induced EGFR activation was studied in cell culture. Vdr deletion significantly increased tumorigenesis, activated EGFR and β-catenin signaling, and increased colonic RAS components, including renin and angiotensin II. Dietary VD3 supplementation suppressed colonic renin. Renin was increased in human colon cancers. In studies in vitro, Ang II activated EGFR and stimulated colon cancer cell proliferation by an EGFR-mediated mechanism. Ang II also activated macrophages and colonic fibroblasts. Compared with tumors from Egfr(Waved2) mice, tumors from Egfr(wild-type) mice showed upregulated Snail1, a suppressor of VDR, and downregulated VDR. VDR suppresses the colonic RAS cascade, limits EGFR signals, and inhibits colitis-associated tumorigenesis, whereas EGFR increases Snail1 and downregulates VDR in colonic tumors. Taken together, these results uncover a RAS-dependent mechanism mediating EGFR and VDR cross-talk in colon cancer.

Canonical Transforming Growth Factor-β Signaling Regulates Disintegrin Metalloprotease Expression in Experimental Renal Fibrosis via miR-29

Fibrosis pathophysiology is critically regulated by Smad 2- and Smad 3-mediated transforming growth factor-β (TGF-β) signaling. Disintegrin metalloproteases (Adam) can manipulate the signaling environment, however, the role and regulation of ADAMs in renal fibrosis remain unclear. TGF-β stimulation of renal cells results in a significant up-regulation of Adams 10, 17, 12, and 19. The selective Smad2/3 inhibitor SB 525334 reversed these TGF-β-induced changes. In vivo, using ureteral obstruction to model renal fibrosis, we observed increased Adams gene expression that was blocked by oral administration of SB 525334. Similar increases in Adam gene expression also occurred in preclinical models of hypertension-induced renal damage and glomerulonephritis. miRNAs are a recently discovered second level of regulation of gene expression. Analysis of 3' untranslated regions of Adam12 and Adam19 mRNAs showed multiple binding sites for miR-29a, miR-29b, and miR-29c. We show that miR-29 family expression is decreased after unilateral ureter obstruction and this significant decrease in miR-29 family expression was observed consistently in preclinical models of renal dysfunction and correlated with an increase in Adam12 and Adam19 expression. Exogenous overexpression of the miR-29 family blocked TGF-β-mediated up-regulation of Adam12 and Adam19 gene expression. This study shows that Adams are involved in renal fibrosis and are regulated by canonical TGF-β signaling and miR-29. Therefore, both Adams and the miR-29 family represent therapeutic targets for renal fibrosis.

Epidermal growth factor receptor signaling impairs the antiviral activity of interferon-alpha

Interferon-alpha (IFN-α) exhibits its antiviral activity through signal transducer and activator of transcription protein (STAT) signaling and the expression of IFN response genes (IRGs). Viral infection has been shown to result in activation of epidermal growth factor receptor (EGFR)-a host cell entry factor used by several viruses, including hepatitis C virus. However, the effect of EGFR activation for cellular antiviral responses is unknown. Here, we uncover cross-talk between EGFR and IFN-α signaling that has a therapeutic effect on IFN-α-based therapies and functional relevance for viral evasion and IFN resistance. We show that combining IFN-α with the EGFR inhibitor, erlotinib, potentiates the antiviral effect of each compound in a highly synergistic manner. The extent of the synergy correlated with reduced STAT3 phosphorylation in the presence of erlotinib, whereas STAT1 phosphorylation was not affected. Furthermore, reduced STAT3 phosphorylation correlated with enhanced expression of suppressors of cytokine signaling 3 (SOCS3) in the presence of erlotinib and enhanced expression of the IRGs, radical S-adenosyl methionine domain containing 2 and myxovirus resistance protein 1. Moreover, EGFR stimulation reduced STAT1 dimerization, but not phosphorylation, indicating that EGFR cross-talk with IFN signaling acts on the STATs at the level of binding DNA. Our results support a model where inhibition of EGFR signaling impairs STAT3 phosphorylation, leading to enhanced IRG expression and antiviral activity. These data uncover a novel role of EGFR signaling in the antiviral activity of IFN-α and open new avenues of improving the efficacy of IFN-α-based antiviral therapies.

Sustained Activation of EGFR Triggers Renal Fibrogenesis after Acute Kidney Injury

Severe acute kidney injury (AKI) is frequently accompanied by maladaptive repair and renal fibrogenesis; however, the molecular mechanisms that mediate these acute and chronic consequences of AKI remain poorly understood. In this study, we examined the role of epidermal growth factor receptor (EGFR) in these processes using waved-2 (Wa-2) mice, which have reduced EGFR activity, and their wild-type (WT) littermates after renal ischemia. Renal EGFR phosphorylation was induced within 2 days after ischemia, increased over time, and remained elevated at 28 days in WT mice, but this was diminished in Wa-2 mice. At the early stage of postischemia (2 days), Wa-2 mice developed more severe acute renal tubular damage with less reparative responses as indicated by enhanced tubular cell apoptosis, and reduced dedifferentiation and proliferation as compared to WT animals. At the late stage of postischemia (28 days), Wa-2 mice exhibited a less severe renal interstitial fibrosis as shown by reduced activation/proliferation of renal myofibroblasts and decreased deposition of extracellular matrix proteins. EGFR activation also contributed to cell cycle arrest at the G2/M phase, a cellular event associated with production of profibrogenetic factors, in the injured kidney. Collectively, these results indicate that severe AKI results in sustained activation of EGFR, which is required for reparative response of renal tubular cells initially, but eventually leads to fibrogenesis.

Energy Balance Modulates Mouse Skin Tumor Promotion through Altered IGF-1R and EGFR Crosstalk

Obesity, an established risk factor for epithelial cancers, remains prevalent in the United States and many other countries. In contrast to positive energy balance states (overweight, obesity), calorie restriction (CR) has been shown to act as a universal inhibitor of tumorigenesis in multiple animal models of human cancer. Unfortunately, the mechanisms underlying the enhancing effects of obesity or the inhibitory effects of CR on cancer etiology remain elusive. Here, we evaluated the impact of dietary energy balance manipulation on epithelial carcinogenesis and identified several potential mechanisms that may account for the differential effects of obesity and CR on cancer. Obesity enhanced tumor promotion during epithelial carcinogenesis, in part, due to altered insulin-like growth factor-1 receptor (IGF-1R)/EGF receptor (EGFR) crosstalk and downstream signaling to effectors such as Akt/mTOR. Obesity-induced changes in cellular signaling subsequently led to altered levels of cell-cycle proteins that favored enhanced epidermal proliferation during tumor promotion. In contrast, CR reduced susceptibility to tumor promotion, attenuated IGF-1R/EGFR crosstalk and downstream signaling, and altered levels of cell-cycle proteins that favored reduced epidermal proliferation during tumor promotion. Collectively, these findings suggest potential targets for the prevention of epithelial cancers, as well as for reversal of obesity-mediated cancer development and progression. Cancer Prev Res; 5(10); 1236-46. ©2012 AACR.

EGFR and c-Met Cross Talk in Glioblastoma and Its Regulation by Human Cord Blood Stem Cells

Receptor tyrosine kinases (RTK) and their ligands control critical biologic processes, such as cell proliferation, migration, and differentiation. Aberrant expression of these receptor kinases in tumor cells alters multiple downstream signaling cascades that ultimately drive the malignant phenotype by enhancing tumor cell proliferation, invasion, metastasis, and angiogenesis. As observed in human glioblastoma (hGBM) and other cancers, this dysregulation of RTK networks correlates with poor patient survival. Epidermal growth factor receptor (EGFR) and c-Met, two well-known receptor kinases, are coexpressed in multiple cancers including hGBM, corroborating that their downstream signaling pathways enhance a malignant phenotype. The integration of c-Met and EGFR signaling in cancer cells indicates that treatment regimens designed to target both receptor pathways simultaneously could prove effective, though resistance to tyrosine kinase inhibitors continues to be a substantial obstacle. In the present study, we analyzed the antitumor efficacy of EGFR inhibitors erlotinib and gefitinib and c-Met inhibitor PHA-665752, along with their respective small hairpin RNAs (shRNAs) alone or in combination with human umbilical cord blood stem cells (hUCBSCs), in glioma cell lines and in animal xenograft models. We also measured the effect of dual inhibition of EGFR/c-Met pathways on invasion and wound healing. Combination treatments of hUCBSC with tyrosine kinase inhibitors significantly inhibited invasion and wound healing in U251 and 5310 cell lines, thereby indicating the role of hUCBSC in inhibition of RTK-driven cell behavior. Further, the EGFR and c-Met localization in glioma cells and hGBM clinical specimens indicated that a possible cross talk exists between EGFR and c-Met signaling pathway.

Antitumor Mechanisms of Targeting the PDK1 Pathway in Head and Neck Cancer

G-protein-coupled receptors (GPCR) activate the epidermal growth factor receptor (EGFR) and mediate EGFR-independent signaling pathways to promote the growth of a variety of cancers, including head and neck squamous cell carcinoma (HNSCC). Identification of the common signaling mechanisms involved in GPCR-induced EGFR-dependent and EGFR-independent processes will facilitate the development of more therapeutic strategies. In this study, we hypothesized that phosphoinositide-dependent kinase 1 (PDK1) contributes to GPCR-EGFR cross-talk and signaling in the absence of EGFR and suggests that inhibition of the PDK1 pathway may be effective in the treatment of HNSCC. The contribution of PDK1 to the EGFR-dependent and EGFR-independent signaling in HNSCC was determined using RNA interference, a kinase-dead mutant, and pharmacologic inhibition. In vivo xenografts studies were also carried out to determine the efficacy of targeting PDK1 alone or in combination with the U.S. Food and Drug Administration-approved EGFR inhibitor cetuximab. PDK1 contributed to both GPCR-induced EGFR activation and cell growth. PDK1 also mediated activation of p70S6K in the absence of EGFR. Blockade of PDK1 with a small molecule inhibitor (AR-12) abrogated HNSCC growth, induced apoptosis, and enhanced the antiproliferative effects of EGFR tyrosine kinase inhibitors in vitro. HNSCC xenografts expressing kinase-dead PDK1 showed increased sensitivity to cetuximab compared with vector-transfected controls. Administration of AR-12 substantially decreased HNSCC tumor growth in vivo. These cumulative results show that PDK1 is a common signaling intermediate in GPCR-EGFR cross-talk and EGFR-independent signaling, and in which targeting the PDK1 pathway may represent a rational therapeutic strategy to enhance clinical responses to EGFR inhibitors in HNSCC.

Integrated effect of EGFR and PAR-1 signaling crosstalk on airway hyperresponsiveness

Among previous genetic studies for asthma, inconsistent findings were often reported. We used a new approach to examine an integrated effect of haplotype blocks, newly termed 'haplotype cluster' over two different types of receptors which share common intracellular pathways on airway hyperresponsiveness (AHR). We recruited 165 young atopic adults without respiratory symptoms and measured airway responsiveness to methacholine and classified them into two groups, those with AHR (PC20<8.0mg/ml) and without AHR. In addition, we identified haplotype blocks or cluster tagging single nucleotide polymorphisms (SNPs) through two genes, epidermal growth factor receptor (EGFR) and protease activated receptor (PAR)-1, in all subjects, and compared the frequencies of haplotype block or cluster between subject groups. Significant differences in the frequencies were observed in the haplotype blocks within the EGFR gene (rs4947972, rs12718945 and rs2072454; rs4947972, rs12718945 and rs2227983; and rs4947972, rs12718945 and rs2293347) and the PAR-1 gene (rs37243 and rs253072). An integrated effect was also observed in one haplotype cluster consisting of both regions of the EGFR gene (rs2293347) and the PAR-1 gene (rs253072) (P=0.0426). Our results suggest the possibility that the integrated effect of functionally-related EGFR and PAR-1 genes (haplotype cluster) is associated with susceptibility to AHR.

β3 Integrin–EGF receptor cross-talk activates p190RhoGAP in mouse mammary gland epithelial cells

Active RhoA localizes to plasma membrane, where it stimulates formation of focal adhesions and stress fibers. RhoA activity is inhibited by p190RhoGAP following integrin-mediated cell attachment to allow sampling of new adhesive environments. p190RhoGAP is itself activated by Src-dependent tyrosine phosphorylation, which facilitates complex formation with p120RasGAP. This complex then translocates to the cell surface, where p190RhoGAP down-regulates RhoA. Here we demonstrate that the epidermal growth factor receptor (EGFR) cooperates with β3 integrin to regulate p190RhoGAP activity in mouse mammary gland epithelial cells. Adhesion to fibronectin stimulates tyrosine phosphorylation of the EGFR in the absence of receptor ligands. Use of a dominant inhibitory EGFR mutant demonstrates that fibronectin-activated EGFR recruits p120RasGAP to the cell periphery. Expression of an inactive β3 integrin subunit abolishes p190RhoGAP tyrosine phosphorylation, demonstrating a mechanistic link between β3 integrin-activated Src and EGFR regulation of the RhoA inhibitor. The β3 integrin/EGFR pathway also has a positive role in formation of filopodia. Together our data suggest that EGFR constitutes an important intrinsic migratory cue since fibronectin is a key component of the microenvironment in normal mammary gland development and breast cancer. Our data also suggest that EGFR expressed at high levels has a role in eliciting cell shape changes associated with epithelial-to-mesenchymal transition.

Abstract 815: Dietary energy balance modulates skin tumor promotion through altered IGF-1R and EGFR crosstalk

Abstract Negative energy balance (calorie restriction, CR) inhibits, while positive energy balance enhances tumor promotion using the two-stage skin carcinogenesis model. Biochemical studies have demonstrated that CR reduced, while diet-induced obesity (DIO) increased insulin-like growth factor 1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) activation and downstream signaling (i.e., Akt and mTOR) following TPA treatment. Additional studies performed using the liver IGF-1 deficient (LID) mouse model (i.e., 75% reduction in circulating IGF-1) demonstrated reduced susceptibility to epithelial carcinogenesis and attenuated IGF-1R and EGFR signaling during tumor promotion, similar to CR mice. Taken together, these findings suggest that dietary energy balance, primarily through its effects on levels of circulating IGF-1, modulates epithelial carcinogenesis and tumor promotion through diet-induced changes in IGF-1R and EGFR signaling and receptor crosstalk. To determine the impact of IGF-1 on IGF-1R and EGFR signaling and receptor crosstalk, Western blot analyses, immunoprecipiation and qPCR analyses were performed using C50 cells (a nontumorigenic keratinocyte cell line) stimulated with IGF-1. IGF-1 treatment of serum starved cells induced rapid phosphorylation (within 5 minutes) of the IGF-1R, as well as the EGFR and ErbB2. Additional experiments were conducted to evaluate mechanisms underlying the effects of IGF-1 on EGFR activation. Immunoprecipitation experiments demonstrated that IGF-1 induced an association between the IGF-1R and the EGFR. Furthermore, qPCR analysis demonstrated that IGF-1 induced HB-EGF and amphiregulin mRNA expression. Current experiments are exploring the possibility that IGF-1 treatment also leads to increased ectodomain shedding of membrane bound EGFR ligands. Further studies were conducted to examine the impact of caloric consumption on IGF-1R and EGFR crosstalk in vivo. For these experiments, ICR female mice were maintained on either a 30% CR or 60Kcal% fat (DIO) regimen for 15 weeks, and then treated with a single application of either acetone or 3.4 nmol TPA. CR reduced, while DIO increased the interaction between the IGF-1R and the EGFR in the epidermis of TPA treated mice. Furthermore, CR reduced, while DIO increased mRNA expression of EGFR ligands both in the presence and absence of TPA treatment. Additional experiments are in progress to confirm that IGF-1 levels, per se, are directly responsible for these diet-induced changes in IGF-1R/EGFR receptor crosstalk. Collectively, the current data suggest that IGF-1 levels and the activation status of the IGF-1R modulate IGF-1R and EGFR receptor crosstalk. Furthermore, diet-induced changes in IGF-1R/EGFR receptor crosstalk subsequently modulate downstream signaling to Akt and mTOR, thus contributing, at least in part, to the effect of dietary energy balance on skin tumor promotion. 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 815. doi:10.1158/1538-7445.AM2011-815

Abstract 2911: EGFR and c-Src crosstalk in breast cancer cell migration

Abstract Patients with breast cancer do not respond to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in the clinic, although 56% of basal breast cancers overexpress EGFR. These observations suggest that inhibiting EGFR kinase activity is insufficient to abrogate tumor growth. Therefore, the contribution of EGFR overexpression in breast cancer remains unknown. Recently, the increased activation of other tyrosine kinases, such as Met and c-Src, has been shown to contribute to cellular responses to EGFR inhibitors. The non-receptor tyrosine kinase c-Src is overexpressed in 70% of breast cancers, yet like EGFR, its role in breast cancer tumorigenesis has not fully been elucidated. Using cell culture models, co-overexpression of EGFR and c-Src has been shown to enhance proliferation, transformation, and tumorigenesis. In addition, we have shown that inhibiting EGFR and c-Src kinase activities in combination results in a synergistic decrease in cell viability. Therefore, EGFR cooperativity with other kinases may mediate continued tumor growth and viability in breast cancer. The role of EGFR and c-Src co-overexpression in breast cancer metastasis remains unknown. Metastasis is a multi-step process that involves migration of tumor cells away from the primary tumor site. Here we present data demonstrating that when EGFR and c-Src are co-overexpressed in breast cancer cells (either endogenously or by inducible overexpression) there is an increase in EGF driven chemotactic cell migration. Interestingly, this EGF-induced increase in cell migration is dependent on the kinase activity of EGFR and c-Src, such that inhibition of either EGFR or c-Src kinase activity abrogates EGF-induced migration. This is in contrast to viability assays using the same cell lines where inhibiting EGFR kinase activity is insufficient to abrogate viability in EGFR/c-Src co-expressing cells. EGFR and c-Src crosstalk has been shown to result in the phosphorylation of EGFR on tyrosine 845. Tyrosine 845 on EGFR is not an autophosphorylation site and unlike other tyrosine residues found in the activation loops of tyrosine kinases, tyrosine 845 phosphorylation is not required for kinase activity, although it is required for EGFR/c-Src induced proliferation. In the EGFR and c-Src co-overexpressing breast cancer cell lines, treatment with either EGFR or c-Src kinase inhibitors led to a reduction in the phosphorylation on EGFR at tyrosine 845, correlating with the loss of EGF-mediated migration. In addition, mutation of tyrosine 845 on EGFR to a phenylalanine inhibited EGF-induced cell migration. These data suggest a role for Y845-EGFR phosphorylation in EGF-induced cell migration in EGFR/c-Src co-overexpressing breast cancer cells. In summary, we demonstrate that inhibiting either EGFR or c-Src kinase activity may be an effective way reduced breast cancer cell migration and abrogate metastasis. 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 2911. doi:10.1158/1538-7445.AM2011-2911

Abstract 966: Dietary energy balance modulates IGF-1R and EGFR signaling and crosstalk during tumor promotion

Abstract Negative energy balance (calorie restriction, CR) inhibits, while positive energy balance enhances tumor promotion during two-stage skin carcinogenesis. Biochemical studies have demonstrated that CR reduced, while diet-induced obesity (DIO) increased activation of the insulin-like growth factor-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR), as well as downstream signaling (e.g., Akt and mTOR) during tumor promotion. Additional studies performed using the liver IGF-1 deficient (LID) mouse model demonstrated that a 75% reduction in circulating IGF-1 attenuated growth factor signaling through the IGF-1R and the EGFR similar to mice on CR diets. These findings suggest that dietary energy balance, primarily through its effects on levels of circulating IGF-1, modulates epithelial carcinogenesis and tumor promotion through diet-induced changes in signaling and crosstalk between the IGF-1R and the EGFR, which subsequently alters signaling to downstream effectors. To determine the impact of IGF-1 on IGF-1R and EGFR signaling and crosstalk, Western blot analyses, immunoprecipiation and qPCR analyses were performed on C50 cells (nontumorigenic keratincyte cell line) stimulated with IGF-1. IGF-1 increased activation of the IGF-1R and the EGFR, as well as signaling to downstream effectors (i.e., Akt and mTOR). Immunoprecipitation experiments demonstrated an increase in association between the IGF-1R and the EGFR following IGF-1 stimulation. Furthermore, IGF-1 induced changes in expression levels of the EGFR, as well as EGFR ligands (i.e., HB-EGF, amphiregulin, TGF-α). Additional experiments performed using C50 cells stably transfected with EGFR shRNA or cells pretreated with PD153035 (EGFR inhibitor) demonstrated reduced IGF-1 mediated activation of the EGFR as well as reduced downstream signaling to Akt and mTOR. In vivo studies were performed to evaluate the effect of dietary energy balance on IGF-1R and EGFR crosstalk. For these experiments, ICR mice were maintained on a CR and DIO regimen for 15 weeks, after which they were treated with a single application of 3.4 nmol TPA. CR reduced, while DIO increased the interaction between the IGF-1R and the EGFR in the epidermis of TPA treated mice. Furthermore, dietary energy balance modulated EGFR and EGFR ligand expression levels. Taken together, these data suggest that levels of IGF-1 can modulate signaling through both the IGF-1R and the EGFR. Furthermore, diet-induced changes in IGF-1R/EGFR crosstalk subsequently modulate activation of downstream signaling to Akt and mTOR, thus contributing, at least in part, to the effect of dietary energy balance on skin tumor promotion. Supported by NIH grants CA37111 and CA129409. 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 966.

Cross-Talk between Estrogen Receptor and Epidermal Growth Factor Receptor in Head and Neck Squamous Cell Carcinoma

This study aimed to characterize estrogen receptor expression and signaling in head and neck squamous cell carcinoma (HNSCC) cell lines and patient tissues, and to evaluate estrogen receptor and epidermal growth factor (EGF) receptor (EGFR) cross-activation in HNSCC. Estrogen receptor expression and signaling in HNSCC cell lines were assessed by immunoblotting. In vitro proliferation and invasion were evaluated in HNSCC cell lines in response to estrogen receptor and EGFR ligands or inhibitors. Estrogen receptor and EGFR protein expression in patient tissues was assessed by immunohistochemical staining. Phospho-mitogen-activated protein kinase (P-MAPK) levels were significantly increased following combined estrogen and EGF treatment. Treatment of HNSCC cells with estrogen and EGF significantly increased cell invasion compared with either treatment alone, whereas inhibiting these two pathways resulted in reduced invasion compared with inhibiting either pathway alone. EGFR (P = 0.008) and nuclear estrogen receptor alpha (ER alpha(nuc); P < 0.001) levels were significantly increased in HNSCC tumors (n = 56) compared with adjacent mucosa (n = 30), whereas nuclear estrogen receptor beta (ER beta(nuc)) levels did not differ (P = 0.67). Patients with high ER alpha(nuc) and EGFR tumor levels had significantly reduced progression-free survival compared with patients with low tumor ER alpha(nuc) and EGFR levels (hazards ratio, 4.09; P = 0.01; Cox proportional hazards). In contrast, high ER beta(nuc) tumor levels were not associated with reduced progression-free survival alone or when combined with EGFR. ER alpha and ER beta were expressed in HNSCC, and stimulation with estrogen receptor ligands resulted in both cytoplasmic signal transduction and transcriptional activation. Estrogen receptor and EGFR cross-talk was observed. Collectively, these studies indicate that estrogen receptor and EGFR together may contribute to HNSCC development and disease progression.