Multiple Epidermal Growth Factor Receptor Research Articles

Abstract 4908: Establishment of multiplexed ultra-sensitive detection of epidermal growth factor receptor mutations using picodroplet digital PCR

Abstract BACKGROUND: The epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of EGFR tyrosine kinase inhibitor (TKI) treatment. Three most common EGFR mutations (L858R, exon19 deletion, and T790M) are known to be major selection marker for EGFR-TKI therapy. Noninvasive techniques for tumor genotyping may be needed to fully realize the potential of genotype-directed cancer care as a “Liquid biopsy”. The low abundance of mutants and limited amount and quality of available cell-free DNA (cfDNA) in plasma samples render it difficult to reliably detect multiple mutations with current platforms and methods. Here, we develop a multiplex dPCR assay to detect three common EGFR mutations in one reaction using picodroplet digital PCR (ddPCR) technology, which enable us to provide 3-fold throughput and save the cost as well as DNA samples. METHODS: Positive and negative control plasmids for the EGFR assay were prepared by cloning DNA fragments containing wild-type or the EGFR mutations. The appropriate concentration of plasmid DNA was determined empirically to yield a mixture in which the number of copies of mutant DNA was ca. 0.01-1.00% of the number of wild-type EGFR fragments. Genomic DNA from the lung tumor cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and then used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels. RESULTS: Serial dilutions experiments using plasmid as well as genomic DNA harboring EGFR mutations revealed a linear performance with an analytical sensitivity of approximately 0.01% for each mutation. The regression analysis between duplex and multiplex assay demonstrated a correlation coefficient (R2) of 0.9983for L858R, 0.9911 for exon19 deletion, and 0.9781 for T790M. The limit of blank (LOB) was defined by the frequency of positive droplets measured in 400 ng of wild-type DNA control samples. The number of false events of mutant droplets detected per analysis is: 8 for L858R, 6 for Exon 19 deletion, 6 for T790M from human normal gDNA, and 9 for L858R, 4 for Exon 19 deletion, 2 for T790M from A549 gDNA. Thus, the LOB was determined as a 10 events/assay. CONCLUSIONS: Using ddPCR, we established multiplex and ultra-sensitive genotyping platform for three common EGFR mutations. Results of a proof-of-principle study using preclinical model indicated that the clinical utility of multiplex ddPCR to detect for multiple EGFR mutations. Further evaluation using clinical samples is warranted. Citation Format: Hiroaki Akamatsu, Yasuhiro Koh, Masaru Watanabe, Takashi Kikuchi, Masanori Nakanishi, Kazuto Matsunaga, Nobuyuki Yamamoto. Establishment of multiplexed ultra-sensitive detection of epidermal growth factor receptor mutations using picodroplet digital PCR. [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 4908. doi:10.1158/1538-7445.AM2015-4908

Intratumoral distribution of EGFR mutations and copy number in metastatic lung cancer, what impact on the initial molecular diagnosis?

BackgroundActivating epidermal growth factor receptor (EGFR) mutations characterize a subgroup of non-small-cell lung cancer that benefit from first line EGFR tyrosine kinase inhibitors (EGFR-TKI). However, the existence of polyclonal cell populations may hinder personalized-medicine strategies as patients’ screening often depends upon a single tumor-biopsy sample. The purpose of this study is to clarify and to validate in clinical testing conditions the accuracy of EGFR genotyping using different tumor sites and various types of samples (transthoracic, surgical or endoscopic biopsies and cytology specimens).MethodsWe conducted a retrospective review of 357 consecutive patients addressed for EGFR mutation screening in accordance with the directive of the European Medicines Agency (stage IV NSCLC). Fifty-seven samples were EGFR mutated and 40 had adequate tumor specimens for analysis on multiple spatially separated sites. Ten wild type samples were also analyzed. A total of 153 and 39 tumor fragments, from mutated and non-mutated cases respectively, were generated to analyze tumor heterogeneity or primary-metastatic discordances. After histological review of all fragments, EGFR genotyping was assessed using the routine diagnostic tools: fragment analysis for insertions and deletions and allele specific TaqMan probes for point mutations. EGFR copy number (CN) was evaluated by qPCR using TaqMan probes.ResultsThe identification of EGFR mutations was independent of localization within primary tumor, of specimen type and consistent between primary and metastases. At the opposite, for half of the samples, tumor loci showed different EGFR copy number that may affect mutation detection cut-off.ConclusionsThis is the largest series reporting multiple EGFR testing in Caucasians. It validates the accuracy of EGFR mutation screening from single tumor-biopsy samples before first line EGFR-TKI. The unpredictable variability in EGFR CN and therefore in EGFR wild type/mutant allelic ratio justifies the implementation of sensitive methods to identify patients with EGFR mutated tumors.

Targeting multiple ErbB family members by EGFR peptide immunization. (VAC12P.1016)

Abstract A major breakthrough in cancer treatment has been the development of monoclonal antibodies targeting tumor antigens. A majority of solid tumors overexpress the epidermal growth factor receptor (EGFR). Current treatments for EGFR overexpressing tumors include anti-EGFR monoclonal antibodies alone or in conjunction with standard chemotherapy or other EGFR inhibitors. While monoclonal antibody treatment is efficacious in some patients, drawbacks include the high cost of treatment, side effects associated with multiple drug infusions, and the development of antibody-resistant tumors. As an alternative to monoclonal antibody treatment, we have focused on peptide-based vaccination to trigger long lasting natural anti-tumor antibodies. Mice immunized with membrane-proximal epitopes in the extracellular domain of EGFR generate antibodies against EGFR as well homologous regions of the EGFR family members HER2 and HER3. HER2 and HER3 form heterodimers with EGFR and compensate for the lack of signaling through EGFR. EGFR peptide immune serum inhibits in vitro growth and promotes tumor killing of both human EGFR+ and HER2+ EGFR- tumor cells. A multi-specific tumor vaccine that targets multiple EGFR family members will provide greater tumor targeting than single agents alone while providing long-lasting immunity.

Pro-MMP-9 upregulation in HT1080 cells expressing CD9 is regulated by epidermal growth factor receptor

Degradation of the surrounding extracellular matrix (ECM) by matrix metalloproteinases (MMPs) drives invasion and metastasis of cancer cells. We previously demonstrated that tetraspanin CD9 expression upregulates pro-MMP-9 expression and release and promotes cellular invasion in a human fibrosarcoma cell line (HT1080). These events were dependent upon the highly functional second extracellular loop of CD9. We report here that the epidermal growth factor receptor (EGFR) tyrosine kinase expression and activity are involved in the CD9-mediated increase in pro-MMP-9 release and cellular invasion. Pro-MMP-9 expression was significantly decreased in a dose-dependent manner using first a broad spectrum receptor tyrosine kinase inhibitor and multiple specific EGFR inhibitors in CD9-HT1080 cells. Furthermore, gefitinib treatment of CD9-HT1080 cells reduced invasion through matrigel. EGFR knockdown using short interfering RNA resulted in decreased pro-MMP-9 expression and release into the media and subsequent cellular invasion without affecting CD9 expression or localization. Conclusively, this study points to EGFR as a key mediator between CD9-mediated pro-MMP-9 release and cellular invasion of HT1080 cells.

EGFR and IL-1 Signaling Synergistically Promote Keratinocyte Antimicrobial Defenses in a Differentiation-Dependent Manner

Ligands of the EGF family regulate autocrine keratinocyte proliferation, and IL-1 family cytokines orchestrate epithelial defense responses. Although members of both families are overexpressed in wound healing and psoriasis, their roles in regulating the innate immune functions of keratinocytes remain incompletely explored. Using sensitive assays, we found significant increases of heparin-binding EGF-like growth factor, transforming growth factor-α, and amphiregulin mRNA and protein in lesional psoriasis compared with uninvolved or control skin. In normal human keratinocyte (NHK) monolayers, EGFR ligands were ineffective in inducing DEFB4, S100A7, and CCL20 mRNAs and human β-defensin (hBD)-2 peptide. Combined with IL-1α, however, EGFR ligands provoked 250 × more DEFB4 and CCL20 and a 9-fold rise in S100A7 mRNA relative to the EGFR ligand alone. This synergy was also reflected in secreted hBD-2 protein, both from NHK and reconstituted human epidermis. Keratinocyte differentiation was critical for these responses, as postconfluent NHK yielded mRNA and protein levels an order of magnitude greater than subconfluent cells. Differentiation also influenced signal transduction, with subconfluent cells using NF-κB and postconfluent cells using EGFR, MEK1/2, and p38. We propose that EGFR ligands are important modifiers of IL-1 activity, synergizing with IL-1 to stimulate epidermal production of hBD-2, S100A7, and CCL20, three of the most upregulated transcripts in psoriatic plaques.

Abstract CN09-03: The EGFR conundrum: EGFR-based diagnostics in the era of precision medicine

Abstract CN09-03: The EGFR conundrum: EGFR-based diagnostics in the era of precision medicine

206 The cis/trans effect of the T790M drug resistant mutation in non-small cell lung cancer

The first-generation epidermal growth factor receptor tyrosine kinase inhibitors erlotinib and gefitinib have been incorporated into treatment paradigms for patients with advanced non-small cell lung cancer. These agents are particularly effective in a subset of patients whose tumors harbor activating epidermal growth factor receptor mutations. However, most patients do not respond to these tyrosine kinase inhibitors, and those who do will eventually acquire resistance that typically results from a secondary epidermal growth factor receptor mutation (e.g., T790M), mesenchymal–epithelial transition factor amplification, or activation of other signaling pathways. For patients whose tumors have wild-type epidermal growth factor receptor, there are several known mechanisms of initial resistance (e.g., Kirsten rat sarcoma viral oncogene homolog mutations) but these do not account for all cases, suggesting that unknown mechanisms also contribute. To potentially overcome the issue of resistance, next-generation tyrosine kinase inhibitors are being developed, which irreversibly block multiple epidermal growth factor receptor family members (e.g., afatinib [BIBW 2992] and PF-00299804) and/or vascular endothelial growth factor receptor pathways (e.g., BMS-690514 and XL647). In addition, drugs that block parallel signaling pathways or signaling molecules downstream of the epidermal growth factor receptor, such as the insulin-like growth factor-1 receptor and the mammalian target of rapamycin, are undergoing clinical evaluation. As drug resistance appears to be pleomorphic, combinations of drugs or drugs with multiple targets may be more effective in circumventing resistance.

Epidermal growth factor receptor inhibition in lung cancer: the evolving role of individualized therapy.

Non-small cell lung cancer (NSCLC) is the major cause of cancer-related deaths in the USA and worldwide. Most patients present with advanced disease, and treatment options for these patients are generally limited to platinum-based chemotherapy and a few targeted therapies. Targeted agents currently in use for NSCLC inhibit oncogenic receptor tyrosine kinase pathways, such as the epidermal growth factor receptor (EGFR) pathway. While current EGFR-targeted agents, including erlotinib and gefitinib, may result in dramatic responses, they demonstrate efficacy in only a fraction of patients, and resistance to these agents frequently develops. In order to select patients most likely to benefit from blockade of EGFR pathways, investigators have focused on identifying molecular correlates of response to anti-EGFR therapy. New strategies to minimize the risk of resistance to EGFR inhibition have been employed in the development of next-generation EGFR tyrosine kinase inhibitors, such as PF00299804 and BIBW 2992; these include irreversibility of target binding, inhibition of multiple EGFR family receptors, and/or simultaneous inhibition of EGFR and other oncogenic pathways.

Combined assessment of EGFR pathway-related molecular markers and prognosis of NSCLC patients

The purpose of this study is to evaluate the prognostic value of the combined assessment of multiple molecular markers related to the epidermal growth factor receptor (EGFR) pathway in resected non-small cell lung cancer (NSCLC) patients. Tumour specimens of 178 NSCLC patients were collected and analysed for EGFR and KRAS mutation status by DNA sequencing, and for EGFR copy number by fluorescent in situ hybridisation. Tissue microarrays were generated and used to determine the expression of multiple EGFR pathway-related proteins by immunohistochemistry. We analysed the association between each marker and patient prognosis. Univariate analyses for each clinical variable and each molecular marker were performed using Kaplan–Meier curves and log-rank tests. From these results, we selected the variables KRAS mutations and expression of cytoplasmic EGFR, granular pERK, nuclear pSTAT3, cytoplasmic E-cadherin and cytoplasmic pCMET to enter into a Cox proportional hazards model, along with stage as the strongest clinical variable related with prognosis. Of the EGFR-related markers evaluated here, the markers EGFR, pERK, pSTAT3, E-cadherin, pCMET and mutations in KRAS were associated with survival when analysed in combination in our patient cohort, with P=0.00015 as the P-value for a test of the additional impact of markers on prognosis, after taking stage into consideration. Confirmation of the impact of these markers in independent studies will be necessary.

Multiple Mechanisms Are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells

The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.

Determinants of RASistance to Anti–Epidermal Growth Factor Receptor Agents

Determinants of <i>RAS</i>istance to Anti–Epidermal Growth Factor Receptor Agents

Syk Is a New Target for AML Differentiation.

One of the challenges of phenotype-based small molecule screening has been the difficulty of protein target identification for newly discovered compounds. For example, we previously performed a gene expression-based small molecule library screen, which identified gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, as an inducer of acute myeloid leukemia (AML) differentiation in cell lines and primary patient cells. Neither EGFR transcript nor protein is expressed in the tested AML cell lines, thus precluding inhibition of this kinase as the mechanism of AML differentiation. However, because multiple EGFR inhibitors induce this phenotype, we hypothesize that a shared off-target kinase is the target in AML differentiation. In order to identify candidate gefitinib targets of AML differentiation, we utilized a proteomics method: peptide immunoprecipitation-HPLC-mass spectrometry. This approach enriches for phospho-tyrosine peptides with immunoprecipitation (IP) after enzyme digestion. In contrast to IP at the protein level, the final mixture following peptide-IP contains primarily phospho-tyrosine-containing peptides. We treated the AML cell line HL-60 with gefitinib versus vehicle and then identified peptide sites with loss of phosphorylation with gefitinib treatment. Spleen tyrosine kinase (Syk) was identified as one of the only kinases with loss of phosphorylation post treatment. Syk is a nonreceptor tyrosine kinase, important in normal B-cell differentiation, and implicated in malignancies such as myelodysplastic syndrome and lymphoma. We first confirmed with IP western immunoblotting the inhibition of Syk phosphorylation with gefitinib treatment in HL-60 cells. We next confirmed with both pharmacological inhibition and genetic loss of Syk the induction of differentiation in the AML cell lines HL-60 and U937. Numerous determinants of myeloid maturation were tested: a complex differentiation gene expression signature, cellular morphology, cell surface proteins CD11b and CD14 expression, and nitro-blue tetrazolium (NBT) reduction, a functional assay for myeloid differentiation. Two reported pharmacological inhibitors of Syk scored positive on all measurements of differentiation. Furthermore, the shRNA construct inducing the most complete loss of Syk also scored the highest on all measurements of differentiation. The most convincing data that Syk is the true target of these inhibitors would be the identification of an inhibitor resistant Syk mutant, analogous to BCR-ABL or c-Kit mutants that confer imatinib resistance. To this end, a Syk random mutagenesis screen is ongoing. In summary, these data identify Syk as a strong candidate target of gefitinib, demonstrate that inhibition of Syk can induce AML differentiation, and identify Syk as a potential target for AML differentiation therapy.

Therapies Directed Against Epidermal Growth Factor Receptor in Aerodigestive Carcinomas

Malignancies arising from the aerodigestive epithelium, including lung, head and neck, and esophageal carcinomas, are the leading causes of cancer-related mortality worldwide. Given the biological importance of epidermal growth factor receptor (EGFR) in cancer development and progression, EGFR inhibitors have emerged as promising novel therapies. To summarize the current status of EGFR inhibitors in aerodigestive carcinomas (ADCs), highlight ongoing research designed to optimize their therapeutic effectiveness, and consider the future role of these agents. Systematic MEDLINE search of English-language literature (1966-April 2007) performed using the terms EGFR, EGFR inhibitors, monoclonal antibodies, tyrosine kinase inhibitors, lung cancer, head and neck cancer, esophageal cancer, and EGFR predictive factors. Quality assessment of selected studies included clinical pertinence, with an emphasis on controlled study design, publication in peer-reviewed journals, adequate number of enrolled patients, objectivity of measurements, and techniques used to minimize bias. The role of EGFR in ADC pathogenesis has been extensively studied, and multiple EGFR inhibition strategies are under evaluation. Erlotinib, an EGFR tyrosine kinase inhibitor used as a single agent, and cetuximab, an anti-EGFR monoclonal antibody used in combination with radiation, have conferred survival benefit in 1 trial of patients with advanced non-small cell lung cancer (median survival, 6.7 vs 4.7 months; hazard ratio, 0.70; 95% confidence interval, 0.58-0.87; P < .001) and in 1 trial of patients with locally advanced head and neck squamous cell carcinoma (median survival, 49 vs 29.3 months; hazard ratio, 0.74; 95% confidence interval, 0.57-0.97; P = .03), respectively. However, other trials have not shown these degrees of improvement. EGFR inhibitors toxicities include rash, diarrhea, and hypomagnesemia. Somatic mutations and other molecular tumoral characteristics offer opportunities for treatment individualization and optimal patient selection for anti-EGFR therapy. EGFR is a promising therapeutic target in ADC. Further translational research is needed to optimize ways of inhibiting EGFR using single-agent or combination regimens and to identify patients who benefit the most from these therapies.

Epidermal Growth Factor Receptor Inhibitors in Development for the Treatment of Non–Small Cell Lung Cancer

The epidermal growth factor receptor (EGFR) inhibitors erlotinib, gefitinib, and cetuximab have undergone extensive clinical testing and have established clinical activity in non-small cell lung cancer and other types of solid tumors. A number of newer inhibitors are currently in clinical development with different spectra of activity or mechanisms of receptor inhibition. These include monoclonal antibodies, such as panitumumab and matuzumab; dual inhibitors of EGFR and vascular endothelial growth factor receptor, such as ZD6474 and AEE788; inhibitors of multiple EGFR family members, such as lapatinib; and irreversible inhibitors, such as canertinib and HKI272. Preclinical studies suggest that several of these agents may have activity in tumors refractory to erlotinib or gefitinib. Among these agents, ZD6474 has undergone the most extensive clinical testing. The antitumor activity of ZD6474 in these two randomized phase II clinical trials in patients with non-small cell lung cancer was felt to be sufficiently promising to warrant phase III clinical testing. Several of the other EGFR inhibitors are also undergoing advanced clinical testing, either alone or in combination with other agents. EGFR has now been validated as a clinically relevant target, and several different types of agents inhibiting this receptor are currently in development. Future research will be needed to elucidate the role of these agents in patients with EGFR inhibitor-naive and EGFR inhibitor-refractory disease, to define the molecular characteristics that predict response, and to determine whether these drugs should be used in combination with other targeted agents or chemotherapy.

Multiple EGFR ligands participate in guiding migrating border cells

Cell migration is an important feature of embryonic development as well as tumor metastasis. Border cells in the Drosophila ovary have emerged as a useful in vivo model for uncovering the molecular mechanisms that control many aspects of cell migration including guidance. It was previously shown that two receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and PDGF- and VEGF-related receptor (PVR), together contribute to border cell migration. Whereas the ligand for PVR, PVF1, is known to guide border cells, it is unclear which of the four activating EGFR ligands function in this process. We developed an assay to detect the ability of secreted factors to reroute migrating border cells in vivo and tested the activity of EGFR ligands compared to PVF1. Two ligands, Keren and Spitz, guided border cells whereas the other ligands, Gurken and Vein, did not. In addition, only Keren and Spitz were expressed at the appropriate stage in the oocyte, the target of border cell migration. Therefore, a complex combination of EGFR and PVR ligands together guide border cells to the oocyte.

Palmitoylation of the EGFR Ligand Spitz by Rasp Increases Spitz Activity by Restricting Its Diffusion

Lipid modifications such as palmitoylation or myristoylation target intracellular proteins to cell membranes. Secreted ligands of the Hedgehog and Wnt families are also palmitoylated; this modification, which requires the related transmembrane acyltransferases Rasp and Porcupine, can enhance their secretion, transport, or activity. We show here that rasp is also essential for the developmental functions of Spitz, a ligand for the Drosophila epidermal growth factor receptor (EGFR). In cultured cells, Rasp promotes palmitate addition to the N-terminal cysteine residue of Spitz, and this cysteine is required for Spitz activity in vivo. Palmitoylation reduces Spitz secretion and enhances its plasma membrane association, but does not alter its ability to activate the EGFR in vitro. In vivo, overexpressed unpalmitoylated Spitz has an increased range of action but reduced activity. These data suggest a role for palmitoylation in restricting Spitz diffusion, allowing its local concentration to reach the threshold required for biological function.

Therapeutic Potential of EGFR-Related Protein, a Universal EGFR Family Antagonist

Interference with the activation of growth factor receptors, specifically epidermal growth factor receptor (EGFR) and/or other member(s) of its family (human epidermal growth factor [HER]-2, -3 and -4) represents a promising strategy for development of novel and selective anticancer therapies. Indeed, a number of inhibitors that target either EGFR or HER-2, but not both, have been developed for treatment of epithelial cancers. However, since most solid tumors express different EGFRs, identification of inhibitor(s) targeting multiple EGFR family members may provide a therapeutic benefit to a broader patient population. To this end, the author proposes that EGFR-related protein (ERRP), a recently isolated negative regulator of EGFR that possesses a substantial homology to the extracellular ligand-binding domain of EGFR and its family members, is a pan-ErbB inhibitor that targets multiple members of the EGFR family. This review discusses the significance of EbB [corrected] family of receptors in epithelial cancers, and describes isolation, characterization and the mechanisms of action of ERRP as well as its potential application as a therapeutic agent for a wide variety of epithelial cancers.

Epidermal growth factor receptor (EGFR)–related protein inhibits multiple members of the EGFR family in colon and breast cancer cells

Inactivation of epidermal growth factor receptor (EGFR) family members represents a promising strategy for the development of selective therapies against epithelial cancers. Current anti-EGFR therapies, such as cetuximab (Erbitux), gefitinib (Iressa), or trastuzumab (Herceptin), target EGFR or HER-2 but not both. Because solid tumors express different EGFRs, identification of inhibitor(s), targeting multiple EGFR family members may provide a therapeutic benefit to a broader patient population. We have identified a natural inhibitor of EGFRs called EGFR-related protein (ERRP), a 53 to 55 kDa protein that is present in most, if not all, normal human epithelial cells. The growth of colon (HCT-116, Caco2, and HT-29) and breast (MDA-MB-468 and SKBR-3) cancer cells expressing varying levels of EGFR, HER-2, and/or HER-4 was inhibited by recombinant ERRP in a dose-dependent manner. In contrast, ERRP caused no inhibition of growth of normal mouse fibroblast cell lines (NIH-3T3, NIH-3T3/P67), and the growth of nontransformed rat small intestinal IEC-6 cells expressing relatively low levels of EGFRs was inhibited only at high doses of ERRP. Transforming growth factor-alpha or heparin-binding epidermal growth factor-induced activation of EGFR and HER-2 was inhibited by ERRP in colon and breast cancer cells expressing high levels of EGFR or HER-2. In contrast, cetuximab inhibited the growth- and ligand-induced activation of EGFR in cell lines expressing high levels of EGFR, whereas trastuzumab was effective only in HER-2-overexpressing cells. ERRP and trastuzumab, but not cetuximab, attenuated heregulin-alpha-induced activation of colon and breast cancer cells that expressed high levels of HER-2. Furthermore, ERRP, but not cetuximab or trastuzumab, significantly induced apoptosis of colon and breast cancer cells. None of these agents induced apoptosis of either NIH-3T3 mouse fibroblast or normal rat small intestinal IEC cells. Our results suggest that ERRP is an effective pan-erbB inhibitor and, thus, may be a potential therapeutic agent for a wide variety of epithelial cancers expressing different levels and subclasses of EGFRs.

Quantitative Analysis of HER2-mediated Effects on HER2 and Epidermal Growth Factor Receptor Endocytosis: DISTRIBUTION OF HOMO- AND HETERODIMERS DEPENDS ON RELATIVE HER2 LEVELS

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR) family. Many cell types express multiple EGFR family members (including EGFR, HER2, HER3, and/or HER4) that interact to form an array of homo- and heterodimers. Differential trafficking of these receptors should strongly affect signaling through this system by changing substrate access and heterodimerization efficiency. Because of the complexity of these dynamic processes, we used a quantitative and computational model to understand their integrated operation. Parameters characterizing EGFR and HER2 interactions were determined using experimental data obtained from mammary epithelial cells constructed to express different levels of HER2, enabling us to estimate receptor-specific internalization rate constants and dimer uncoupling rate constants. Significant novel results obtained from this work are as follows: first, that EGFR homodimerization and EGFR/HER2 heterodimerization occur with comparable affinities; second, that EGFR/HER2 heterodimers traffic as single entities. Furthermore, model predictions of the relationship of HER2 expression levels to consequent distribution of EGFR homodimers and EGFR/HER2 heterodimers suggest that the levels of HER2 found on normal cells are barely at the threshold necessary to drive efficient heterodimerization. Thus, altering HER2 concentrations, either overall or local, could provide an effective mechanism for regulating EGFR/HER2 heterodimerization and may explain why HER2 overexpression found in some cancers has such a profound effect on cell physiology.

Altered expression of epidermal growth factor receptor ligands in tumor promoter–treated mouse epidermis and in primary mouse skin tumors induced by an initiation‐promotion protocol

Multiple epidermal growth factor receptor (EGFr) ligands have been identified, including transforming growth factor α (TGFα), heparin-binding epidermal growth factor (HB-EGF), amphiregulin (AR), and betacellulin (BTC). Previous work from our laboratory demonstrated that TGFα mRNA and protein are upregulated in epidermis during tumor-promoter treatment of mouse skin and in skin tumors produced by initiation-promotion regimens. The purpose of the study described here was to explore the role of other EGFr ligands in multistage skin carcinogenesis. A single topical treatment of either 12-O-tetradecanoylphorbol-13-acetate (TPA) or chrysarobin or a single full-thickness wound induced the expression of HB-EGF and AR in mRNA samples isolated from whole mouse skin. However, only full-thickness wounding significantly elevated expression of the BTC transcript. The levels of HB-EGF and AR transcripts were significantly elevated in skin tumors (both papillomas and squamous cell carcinomas) induced by initiation-promotion protocols. BTC transcript levels were low and barely detectable in all skin tumors examined. The level of keratinocyte growth factor (KGF) mRNA was also examined as a possible mechanism for upregulation of EGFr ligands. Only full-thickness wounding significantly elevated KGF transcript levels in whole-skin RNA samples. Furthermore, no evidence for upregulation of KGF mRNA in skin tumors was obtained. The results are discussed in terms of the role of EGFr activation in skin carcinogenesis and the mechanisms for altered regulation of EGFr ligands. Mol. Carcinog. 22:73–83, 1998. © 1998 Wiley-Liss, Inc.