Rac signaling in breast cancer: a tale of GEFs and GAPs

Rac GTPases, small G-proteins widely implicated in tumorigenesis and metastasis, transduce signals from tyrosine-kinase, G-protein-coupled receptors (GPCRs), and integrins, and control a number of essential cellular functions including motility, adhesion, and proliferation. Deregulation of Rac signaling in cancer is generally a consequence of enhanced upstream inputs from tyrosine-kinase receptors, PI3K or Guanine nucleotide Exchange Factors (GEFs), or reduced Rac inactivation by GTPase Activating Proteins (GAPs). In breast cancer cells Rac1 is a downstream effector of ErbB receptors and mediates migratory responses by ErbB1/EGFR ligands such as EGF or TGFα and ErbB3 ligands such as heregulins. Recent advances in the field led to the identification of the Rac-GEF P-Rex1 as an essential mediator of Rac1 responses in breast cancer cells. P-Rex1 is activated by the PI3K product PIP3 and Gβγ subunits, and integrates signals from ErbB receptors and GPCRs. Most notably, P-Rex1 is highly overexpressed in human luminal breast tumors, particularly those expressing ErbB2 and estrogen receptor (ER). The P-Rex1/Rac signaling pathway may represent an attractive target for breast cancer therapy.     

Stretch-induced fetal type II cell differentiation is mediated via ErbB1-ErbB4 interactions

Stretch-induced differentiation of lung fetal type II epithelial cells is mediated through EGFR (ErbB1) via release of HB-EGF and TGF-α ligands. Employing an EGFR knock-out mice model, we further investigated the role of the ErbB family of receptors in mechanotranduction during lung development. Deletion of EGFR prevented endogenous and mechanical stretch-induced type II cell differentiation via the ERK pathway, which was rescued by overexpression of a constitutively active MEK. Interestingly, the expression of ErbB4, the only ErbB receptor that EGFR co-precipitates in wild-type cells, was decreased in EGFR-deficient type II cells. Similar to EGFR, ErbB4 was activated by stretch and participated in ERK phosphorylation and type II cell differentiation. However, neuregulin (NRG) or stretch-induced ErbB4 activation were blunted in EGFR-deficient cells and not rescued after ErbB4 overexpression, suggesting that induction of ErbB4 phosphorylation is EGFR-dependent. Finally, we addressed how shedding of ligands is regulated by EGFR. In knock-out cells, TGF-α, a ligand for EGFR, was not released by stretch, while HB-EGF, a ligand for EGFR and ErbB4, was shed by stretch although to a lower magnitude than in normal cells. Release of these ligands was inhibited by blocking EGFR and ERK pathway. In conclusion, our studies show that EGFR and ErbB4 regulate stretch-induced type II cell differentiation via ERK pathway. Interactions between these two receptors are important for mechanical signals in lung fetal type II cells. These studies provide novel insights into the cell signaling mechanisms regulating ErbB family receptors in lung cell differentiation.     

Endocytic downregulation of ErbB receptors: mechanisms and relevance in cancer

ErbB receptors (EGFR (ErbB1), ErbB2, ErbB3, and ErbB4) are important regulators of normal growth and differentiation, and they are involved in the pathogenesis of cancer. Following ligand binding and receptor activation, EGFR is endocytosed and transported to lysosomes where the receptor is degraded. This downregulation of EGFR is a complex and tightly regulated process. The functions of ErbB2, ErbB3, and ErbB4 are also regulated by endocytosis to some extent, although the current knowledge of these processes is sparse. Impaired endocytic downregulation of signaling receptors is frequently associated with cancer, since it can lead to increased and uncontrolled receptor signaling. In this review we describe the current knowledge of ErbB receptor endocytic downregulation. In addition, we outline how ErbB receptors can escape endocytic downregulation in cancer, and we discuss how targeted anti-cancer therapy may induce endocytic downregulation of ErbB receptors.     

Transmembrane helix-helix interactions involved in ErbB receptor signaling

Among the many transmembrane receptor classes, the receptor tyrosine kinases represent an important superfamily, involved in many cellular processes like embryogenesis, development and cell division. Deregulation and dysfunctions of these receptors can lead to various forms of cancer and other diseases. Mostly, only fragmented knowledge exists about functioning of the entire receptors, and many studies have been performed on isolated receptor domains. In this review we focus on the function of the ErbB family of receptor tyrosine kinases with a special emphasis on the role of the transmembrane domain and on the mechanisms underlying regulated and deregulated signaling. Many general aspects of ErbB receptor structure and function have been analyzed and described. All human ErbBs appear to form homo- and heterodimers within cellular membranes and the single transmembrane domain of the receptors is involved in dimerization. Additionally, only defined structures of the transmembrane helix dimer allows signaling of ErbB receptors.Key words: ErbB, EGFR, receptor, receptor-tyrosine kinase, transmembrane proteins, signaling, helix-helix interaction     

Epidermal growth factor receptor (EGFR) signaling in cancer

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTK). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Evidence suggests that the EGFR is involved in the pathogenesis and progression of different carcinoma types. The EGFR and EGF-like peptides are often over-expressed in human carcinomas, and in vivo and in vitro studies have shown that these proteins are able to induce cell transformation. Amplification of the EGFR gene and mutations of the EGFR tyrosine kinase domain have been recently demonstrated to occur in carcinoma patients. Interestingly, both these genetic alterations of the EGFR are correlated with high probability to respond to anti-EGFR agents. However, ErbB proteins and their ligands form a complex system in which the interactions occurring between receptors and ligands affect the type and the duration of the intracellular signals that derive from receptor activation. In fact, proteins of the ErbB family form either homo- or hetero-dimers following ligand binding, each dimer showing different affinity for ligands and different signaling properties. In this regard, evidence suggests that cooperation of multiple ErbB receptors and cognate ligands is necessary to induce cell transformation. In particular, the growth and the survival of carcinoma cells appear to be sustained by a network of receptors/ligands of the ErbB family. This phenomenon is also important for therapeutic approaches, since the response to anti-EGFR agents might depend on the total level of expression of ErbB receptors and ligands in tumor cells.     

Targeting EGFR resistance networks in head and neck cancer

A core set of oncoproteins is overexpressed or functionally activated in many types of cancer, and members of this group have attracted significant interest as subjects for development of targeted therapeutics. For some oncoproteins such as EGFR/ErbB1, both small molecule and antibody agents have been developed and applied in the clinic for over a decade. Analysis of clinical outcomes has revealed an initially unexpected complexity in the response of patients to these agents. Diverse factors, including developmental lineage of the tumor progenitor cell, co-mutation or epigenetic modulation of genes encoding proteins in an extended EGFR signaling network or regulating core survival responses in individual tumors, and environmental factors including inflammatory agents and viral infection, all have been identified as modulating response to treatment with EGFR-targeted drugs. Second and third generation therapeutic strategies increasingly incorporate knowledge of cancer type-specific signaling environments, in a more personalized treatment approach. This review takes squamous cell carcinoma of the head and neck (SCCHN) as a specific example of an EGFR-involved cancer with idiosyncratic biological features that influence design of treatment modalities, with particular emphasis on commonalities and differences with other cancer types.     

The role of epidermal growth factor and its receptors in mammalian CNS

Epidermal growth factor (EGF) is a common mitogenic factor that stimulates the proliferation of different types of cells, especially fibroblasts and epithelial cells. EGF activates the EGF receptor (EGFR/ErbB), which initiates, in turn, intracellular signaling. EGFR family is also expressed in neurons of the hippocampus, cerebellum, and cerebral cortex in addition to other regions of the central nervous system (CNS). EGF enhances the differentiation, maturation and survival of a variety of neurons. Transgenic mice lacking the EGFR developed neurodegenerative disease and die within the first month of birth. EGF acts not only on mitotic cells but also on postmitotic neurons, and many studies have indicated that EGF has neuromodulatory effect on various types of neurons in the CNS. This review highlights some of the major recent findings pertinent to the EGF and ErbB family with special references to elucidating their roles in the regulation of neurogenesis, signal transduction and trafficking and degradation.     

Targeting of solid tumors and blood malignancies by antibody-based therapies — EGFR-pathway as an example

A well-coordinated interaction between extracellular signals and intracellular response forms the basis of life within multicellular organisms, with growth factors playing a crucial role in these interactions. Discoveries in recent years have shown that components of the Epidermal Growth Factor (EGF) signaling system have frequently been used by cancer cells to autonomously provide survival and proliferation signals. The main focus of this review is the ErbB epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases including ErbB1/EGFR, ErbB2/HER2/neu, ErbB3/HER3, and ErbB4/HER4 as therapeutic targets. Since the ErbB receptor family regulates cell proliferation through the Ras-mitogen-activated protein kinase (RAS/MAPK) pathway, and cell survival and transformation through the phosphatidylinositol 3-kinase (PI3K/AKT) pathway, pharmacological targeting of these pathways is also discussed. We will also address the clinical studies that have been conducted to evaluate antibody-based therapies mostly on solid tumors and hematologic malignancies.     

Identification of epidermal growth factor-responsive genes in normal rat ovarian surface epithelial cells

Alteration in epidermal growth factor receptor (EGFR) family signaling is among the most frequently implicated effectors of human oncogenesis. Overexpression of members of this family of receptors has often been detected in many epithelial tumors and is believed to be associated with an overall poor prognosis in patients with cancer. Therefore, we hypothesized that identification of potential EGF target genes in normal cells will provide a basis for unbiased genetic analysis of this signaling pathway in cancer. We utilized Atlas Rat 1.2 nylon cDNA arrays (Clontech) to determine gene expression changes in normal rat ovarian surface epithelial (ROSE) cells following EGF treatment. The results indicate activation of genes involved in a wide variety of cellular mechanisms, including regulation of cell cycle and proliferation, apoptosis, and protein turnover. In addition, using an in vitro model of ovarian cancer, we demonstrated that malignant transformation of ROSE cells resulted in alteration of downstream effectors of the EGFR pathway, as exemplified by aberrant expression of p66Shc, c-Jun, c-Myc, c-Fos, Lot1, p21Cip/Waf, and cdc25A. These data suggest that knowledge of the downstream genetic lesions, which may result in loss of growth factor requirement of the affected cells, will be crucial for the selection of the EGFR pathway as an effective target for cancer therapy.     

靶向EGFR家族的抗肿瘤药物研究进展

表皮生长因子受体（EGFR,ErbB）家族在肿瘤的发生、发展中具有重要的作用。很多实体肿瘤中存在EGFR家族受体过表达或异常激活。靶向EGFR家族的抗肿瘤药物研发已经成为一个热点领域,并且成功地应用于临床。靶向EGFR家族的抗肿瘤药物可以分为单克隆抗体和小分子酪氨酸激酶抑制剂两大类。单克隆抗体与受体胞外区结合阻止配体．受体的结合或者阻止配体结合引起的受体活化;而小分子酪氨酸激酶抑制剂则结合于胞内激酶区,抑制激酶自磷酸化和下游信号通路激活。     

c-Src Associates with ErbB2 through an Interaction between Catalytic Domains and Confers Enhanced Transforming Potential

Previous studies have demonstrated that c-Src tyrosine kinase interacts specifically with ErbB2, but not with other members of the epidermal growth factor receptor (EGFR) family. To identify the site of interaction, we recently used a chimeric EGFR/ErbB2 receptor approach to show that c-Src requires the kinase region of ErbB2 for binding. Here, we demonstrate that retention of a conserved amino acid motif surrounding tyrosine 877 (referred to here as EGFR^YHAD) is sufficient to confer binding to c-Src. Surprisingly the association of c-Src was not dependent on its SH2 or SH3 domain or on the phosphorylation or kinase activity of the receptor. We further show that the chimeric EGFRs that contain the Y877 motif are transforming in vitro and in vivo following ligand stimulation. Transformation was also partially dependent on sustained activation of Stat3. Finally, we demonstrate that EGFRs with mutations in the catalytic domain, originally identified in lung cancer and conferring increased sensitivity to gefitinib and erlotinib, two EGFR kinase inhibitors, gained the capacity to bind c-Src. Moreover, transformation by these EGFR mutants was inhibited by Src inhibitors regardless of their sensitivities to gefitinib and erlotinib. These observations have important implications for understanding the molecular basis for resistance to EGFR inhibitors and implicate c-Src as a critical signaling molecule in EGFR mutant-induced transformation.The epidermal growth factor receptor (EGFR) family is comprised of four members, EGFR, ErbB2, ErbB3, and ErbB4, with distinct ligand specificities, which, upon homo- or heterodimerization after ligand binding, autophosphorylate and recruit different effector proteins to specific tyrosine residues located in their cytoplasmic tails. These signaling molecules, which are either adapter molecules that recruit other kinases or kinases themselves, mediate diverse functions, such as proliferation, growth, and survival (27). There are now several pieces of evidence demonstrating that these growth factor receptors are mutated or overexpressed in a variety of different cancers, including salivary gland adenocarcinoma (44), breast cancer (47), esophageal squamous carcinoma (22), bladder cancer (58), and lung cancer (57). Accordingly, ErbB2 is overexpressed in 20 to 30% of all human breast cancer, which correlates with poor prognosis, and in 40 to 60% of ductal carcinoma in situ (19). ErbB2 is 100-fold more potent in its transforming ability than ErbB1/EGFR, although the two receptors are 85% homologous (14, 15). Breast carcinoma cells devoid of ErbB2, but not other ErbB receptor family members, are defective in cell invasion upon EGF ligand stimulation (49). In fact, ErbB2 could induce cell migration when overexpressed in cells devoid of any other ErbB receptors. In a three-dimensional cell culture system, overexpression of ErbB2, but not EGFR, disrupts mammary acinus structure by reinitiating cell proliferation, leading to an absence of lumen and disruption of tight junctions and of cell polarity, although the cells still lack invasive properties (31).Src is a nonreceptor tyrosine kinase implicated in signal transduction pathways downstream of multiple receptors, such as platelet-derived growth factor, insulin receptor, G-coupled receptors, and ErbB family receptors, where it regulates a wide variety of cellular functions that include proliferation, migration, and apoptosis (17). Src tyrosine kinase activity is sporadically increased in many cases of human cancer, including colon and breast cancer (10, 38, 52). Moreover, Src kinase activity is elevated in ErbB2-induced mammary tumors (33). Direct evidence supporting a role in mammary tumor progression derives from observations made in transgenic mice. Constitutive activation of c-Src in mammary epithelia led to frequent mammary epithelial hyperplasias, which occasionally developed into solid tumors (54). Conversely, deletion of c-Src in a mouse mammary tumor virus/polyomavirus middle T-antigen (PyMT) transgenic strain abrogates mammary tumor formation (21).c-Src is also an important player downstream of the EGFR family. Phosphorylation of several tyrosine residues within the EGFR has been demonstrated to be increased following c-Src overexpression both in vitro and in vivo, suggesting that c-Src is required for full biological response following EGF stimulation (29, 51). In addition to EGFR, c-Src specifically interacts with tyrosine-phosphorylated ErbB2 in ErbB2-induced mammary tumors. This association was further demonstrated to result in enhanced c-Src kinase activity (3, 28, 34, 35). More recently, using chimeric EGF/ErbB2 receptors, we demonstrated that c-Src specifically associates with ErbB2, but not with other ErbB family members. c-Src was demonstrated to specifically associate with the ErbB2 kinase domain (24). Moreover, the chimeric EGFR that contained the c-Src binding site was able to disrupt cell polarity and cell-cell junctions to induce epithelial cell scattering in a three-dimensional cell culture system in a MAPK-dependent manner (24).Here, we demonstrate that c-Src association with ErbB2 is conformation dependent and that the residues necessary for interaction are centered around Y877 in the kinase domain of ErbB2, an association that is further strengthened by residues located in the amino-terminal part of the kinase domain. This association was not dependent on the SH2 or SH3 domain or the kinase activity of c-Src or ErbB2. We further show that mammary epithelial cells expressing the EGFR/ErbB2 chimeric receptors that have regained the capacity to associate with c-Src have disrupted epithelial polarity that is correlated with enhanced transforming potential, an effect dependent on c-Src kinase activity and Stat3 activation. Finally, we show that mutant EGFRs isolated from lung adenocarcinomas have the capacity to associate with c-Src and that these EGFR mutants require Src kinase activity for transformation.     

Role of somatostatin receptor 1 and 5 on epidermal growth factor receptor mediated signaling

Epidermal growth factor (EGF) regulates normal and tumor cell proliferation via epidermal growth factor receptor (EGFR) phosphorylation, homo- or heterodimerization and activation of mitogen-activated protein kinases (MAPKs) and PI3K/AKT cell survival pathways. In contrast, SST via activation of five different receptor subtypes inhibits cell proliferation and has been potential target in tumor treatment. To gain further insight for the effect of SSTRs on EGFR activated signaling, we determine the role of SSTR1 and SSTR1/5 in human embryonic kidney (HEK) 293 cells. We here demonstrate that cells transfected with SSTR1 or SSTR1/5 negatively regulates EGF mediated effects attributed to the inhibition of EGFR phosphorylation, MAPKs as well as the cell survival signaling. Furthermore, SSTR effects were significantly enhanced in cells when EGFR was knock down using siRNA or treated with selective antagonist (AG1478). Most importantly, the presence of SSTR in addition to modulating signaling pathways leads to the dissociation of the constitutive and EGF induced heteromeric complex of EGFR/ErbB2. Furthermore, cells cotransfected with SSTR1/5 display pronounced effect of SST on the signaling and dissociation of the EGFR/ErbB2 heteromeric complex than the cells expressing SSTR1 alone. Taken together this study provides the first evidence that the presence of SSTR controls EGF mediated cell survival pathway via dissociation of ErbB heteromeric complex. We propose that the activation of SSTR and blockade of EGFR might serve novel therapeutic approach in inhibition of tumor proliferation.     

Receptor-targeted cancer therapy

Insight into the molecular mechanisms of malignant transformation is changing the way cancer is being treated. Conventional treatment strategies target the DNA of all dividing cells, resulting in a significantly increased risk of collateral toxicity. In addition, the accumulation of multiple mutations leads to drug resistance in many cancer cells. Targeted strategies have now been developed that specifically disrupt oncogenically active cell surface receptors and endogenous signaling molecules. These agents have a much greater selectivity for tumor tissue and decreased risk of side effects. Increased signaling through ErbB receptors via gene amplification, overexpression, and mutation has been implicated in many human cancers and associated with poor prognosis. Interruption of this process has been shown to cause antitumor effects. Downregulation of the ErbB receptors, HER-2/neu, and later EGFR, with monoclonal antibodies was the first demonstration of targeted therapy. Subsequently, the ErbB tyrosine kinase domain has been successfully targeted with small molecule inhibitors. The development of novel ErbB-directed entities is ongoing, with particular promise being shown by strategies targeting receptor interaction in oligomeric complexes.     

The ErbB receptors and their role in cancer progression

The involvement of the ErbB receptor tyrosine kinases in human cancer, as well as their essential role in a variety of physiological events during normal development, have motivated the interest in this receptor family. Approaches taken to block the activity of ErbB receptors in cancer cells have not only proven that they drive in vitro tumor cell proliferation, but have also become clinically relevant for targeting tumors with deregulated ErbB signaling. The mechanisms and downstream effectors through which the ErbB receptors influence processes linked to malignant development, including proliferation, cell survival, angiogenesis, migration, and invasion, are, however, only now becoming apparent. Our particular emphasis in this review will be on how ErbB receptors, in particular ErbB1 and ErbB2, contribute to processes linked to cancer progression. Importantly, in keeping with the emerging theme that ErbB receptors do not function in isolation, we will focus on receptor cooperativity, i.e., ErbB1 cooperates with other classes of receptors, and the ligand-less ErbB2 functions as a heterodimer with other ErbBs.     

Mammary Cells with Active Wnt Signaling Resist ErbB2-Induced Tumorigenesis

Aberrant activation of Wnt signaling is frequent in human malignancies. In normal epithelial tissues, including the breast, Wnt signaling is active only in a subset of cells, but it is unknown whether this subset of Wnt signaling-active cells is at increased risk of carcinogenesis. We created transgenic mice (TOP-tva) in which the synthetic Wnt-responsive promoter TOP controlled the gene encoding TVA, which confers susceptibility to infection by the retroviral vector RCAS. Thus, only cells in which Wnt signaling is active will express tva and be targeted by RCAS. Surprisingly, we found that RCAS-mediated delivery of cDNA encoding a constitutively activated version of ErbB2 (HER2/Neu) into the small number of TVA+ mammary epithelial cells in TOP-tva mice failed to induce tumor, while the same virus readily induced mammary tumors after it was delivered into a comparable number of cells in our previously reported mouse line MMTV-tva, whose tva is broadly expressed in mammary epithelium. Furthermore, we could not even detect any early lesions or infected cells in TOP-tva mice at the time of necropsy. Therefore, we conclude that the Wnt pathway-active cell subset in the normal mammary epithelium does not evolve into tumors following ErbB2 activation–rather, they apparently die due to apoptosis, an anticancer “barrier” that we have reported to be erected in some mammary cells followed ErbB2 activation. In accord with these mouse model data, we found that unlike the basal subtype, ErbB2+ human breast cancers rarely involve aberrant activation of Wnt signaling. This is the first report of a defined sub-population of mammalian cells that is “protected” from tumorigenesis by a potent oncogene, and provides direct in vivo evidence that mammary epithelial cells are not equal in their response to oncogene-initiated transformation.     

P2Y2 Nucleotide Receptors Mediate Metalloprotease-dependent Phosphorylation of Epidermal Growth Factor Receptor and ErbB3 in Human Salivary Gland Cells

The G protein-coupled receptor P2Y₂ nucleotide receptor (P2Y₂R) has been shown to be up-regulated in a variety of tissues in response to stress or injury. Recent studies have suggested that P2Y₂Rs may play a role in immune responses, wound healing, and tissue regeneration via their ability to activate multiple signaling pathways, including activation of growth factor receptors. Here, we demonstrate that in human salivary gland (HSG) cells, activation of the P2Y₂R by its agonist induces phosphorylation of ERK1/2 via two distinct mechanisms, a rapid, protein kinase C-dependent pathway and a slower and prolonged, epidermal growth factor receptor (EGFR)-dependent pathway. The EGFR-dependent stimulation of UTP-induced ERK1/2 phosphorylation in HSG cells is inhibited by the adamalysin inhibitor tumor necrosis factor-α protease inhibitor or by small interfering RNA that selectively silences ADAM10 and ADAM17 expression, suggesting that ADAM metalloproteases are required for P2Y₂R-mediated activation of the EGFR. G protein-coupled receptors have been shown to promote proteolytic release of EGFR ligands; however, neutralizing antibodies to known ligands of the EGFR did not inhibit UTP-induced EGFR phosphorylation. Immunoprecipitation experiments indicated that UTP causes association of the EGFR with another member of the EGF receptor family, ErbB3. Furthermore, stimulation of HSG cells with UTP induced phosphorylation of ErbB3, and silencing of ErbB3 expression inhibited UTP-induced phosphorylation of both ErbB3 and EGFR. UTP-induced phosphorylation of ErbB3 and EGFR was also inhibited by silencing the expression of the ErbB3 ligand neuregulin 1 (NRG1). These results suggest that P2Y₂R activation in salivary gland cells promotes the formation of EGFR/ErbB3 heterodimers and metalloprotease-dependent neuregulin 1 release, resulting in the activation of both EGFR and ErbB3.     

Constitutively active ErbB4 and ErbB2 mutants exhibit distinct biological activities.

ErbB4 is a member of the epidermal growth factor receptor(EGFR) family of tyrosine kinases, which includes EGFR/ErbB1, ErbB2/HER2/Neu, and ErbB3/HER3. These receptors play important roles both in normal development and in neoplasia. For example, deregulated signaling by ErbB1 and ErbB2 is observed in many human malignancies. In contrast, the roles that ErbB4 plays in tumorigenesis and normal biological processes have not been clearly defined. To identify the biological responses that are coupled to ErbB4, we have constructed three constitutively active ErbB4 mutants. Unlike a constitutively active ErbB2 mutant, the ErbB4 mutants are not coupled to increased cell proliferation, loss of contact inhibition, or anchorage independence in a rodent fibroblast cell line. This suggests that ErbB2 and ErbB4 may play distinct roles in tumorigenesis in vivo.     

EGF receptor inhibitors increase ErbB3 mRNA and protein levels in breast cancer cells

The potential benefits of drugs directly targeting the ErbB receptors for cancer therapy have led to an extensive development within this field. However, the clinical effects of ErbB receptor-targeting drugs in cancer treatment are limited due to a high frequency of resistance. It has been reported that, when inhibiting the epidermal growth factor receptor (EGFR) with the tyrosine kinase inhibitor gefitinib, increased activation of ErbB3 via MET, or by re-localization of ErbB3 mediates cell survival. Here we show further evidence that members of the ErbB receptor family facilitate resistance to EGFR inhibitor treatment in ErbB2 overexpressing breast cancer cells. We found that gefitinib treatment increased ErbB3 expression, both at protein and mRNA levels. ErbB3 expression was upregulated not only by gefitinib but also by a panel of different EGFR inhibitors, suggesting that inhibition of EGFR in general affects ErbB3 expression. In addition, we found that gefitinib treatment increased ErbB2 expression levels while EGFR inhibitors decreased the activity of ErbB2. Concentrations of gefitinib that decreased phospho-ErbB2 reversely increased ErbB3 levels. We further examined changes induced by gefitinib treatment on mRNA levels of the most common genes known to be involved in breast cancer. As expected, we found that gefitinib downregulated genes whose functions were linked to cellular proliferation, such as Ki-67, topoisomerase II alpha and cyclins, and surprisingly downregulated gene expression of FAS which is involved in apoptotic signaling. Together, our data strongly suggest that resistance to EGFR inhibitors may result from the compensation of other family members and that combinations of anti-cancer drugs are required to increase the sensitivity of these treatments.