Validation of HB-EGF and amphiregulin as targets for human cancer therapy

Aberrant expression levels of epidermal growth factor receptor (EGFR) and its cognate ligands have been recognized as one of the causes of cancer progression. To investigate the validity of EGFR ligands as targets for cancer therapy, we examined the expression of EGFR ligands and in vitro anti-tumor effects of small interference RNA (siRNA) for EGFR ligands in various cancer cells. HB-EGF expression was dominantly elevated in ovarian, gastric, and breast cancer, melanoma and glioblastoma cells, whereas amphiregulin was primarily expressed in pancreatic, colon, and prostate cancer, renal cell carcinoma and cholangiocarcinoma cells. Transfection of siRNAs for HB-EGF or amphiregulin into these cells significantly increased the numbers of apoptotic cells with attenuation of EGFR and ERK activation. In lung cancer cells, any EGFR ligand was not recognized as a validated target for cancer therapy. These results suggest that HB-EGF and amphiregulin are promising targets for cancer therapy.     

TACE/ADAM-17 phosphorylation by PKC-epsilon mediates premalignant changes in tobacco smoke-exposed lung cells

Background

Tobacco smoke predisposes humans and animals to develop lung tumors, but the molecular events responsible for this are poorly understood. We recently showed that signaling mechanisms triggered by smoke in lung cells could lead to the activation of a growth factor signaling pathway, thereby promoting hyperproliferation of lung epithelial cells. Hyperproliferation is considered a premalignant change in the lung, in that increased rates of DNA synthesis are associated with an increased number of DNA copying errors, events that are exacerbated in the presence of tobacco smoke carcinogens. Despite the existence of DNA repair mechanisms, a small percentage of these errors go unrepaired and can lead to tumorigenic mutations. The results of our previous study showed that an early event following smoke exposure was the generation of oxygen radicals through the activation of NADPH oxidase. Although it was clear that these radicals transduced signals through the epidermal growth factor receptor (EGFR), and that this was mediated by TACE-dependent cleavage of amphiregulin, it remained uncertain how oxygen radicals were able to activate TACE.

Principal Findings

In the present study, we demonstrate for the first time that phosphorylation of TACE at serine/threonine residues by tobacco smoke induces amphiregulin release and EGFR activation. TACE phosphorylation is triggered in smoke-exposed lung cells by the ROS-induced activation of PKC through the action of SRC kinase. Furthermore, we identified PKCε as the PKC isoform involved in smoke-induced TACE activation and hyperproliferation of lung cells.

Conclusions

Our data elucidate new signaling paradigms by which tobacco smoke promotes TACE activation and hyperproliferation of lung cells.     

Epidermal growth factor receptor pathway analysis identifies amphiregulin as a key factor for cisplatin resistance of human breast cancer cells

The use of platinum complexes for the therapy of breast cancer is an emerging new treatment modality. To gain insight into the mechanisms underlying cisplatin resistance in breast cancer, we used estrogen receptor-positive MCF-7 cells as a model system. We generated cisplatin-resistant MCF-7 cells and determined the functional status of epidermal growth factor receptor (EGFR), MAPK, and AKT signaling pathways by phosphoreceptor tyrosine kinase and phospho-MAPK arrays. The cisplatin-resistant MCF-7 cells are characterized by increased EGFR phosphorylation, high levels of AKT1 kinase activity, and ERK1 phosphorylation. In contrast, the JNK and p38 MAPK modules of the MAPK signaling pathway were inactive. These conditions were associated with inactivation of the p53 pathway and increased BCL-2 expression. We investigated the expression of genes encoding the ligands for the ERBB signaling cascade and found a selective up-regulation of amphiregulin expression, which occurred at later stages of cisplatin resistance development. Amphiregulin is a specific ligand of the EGFR (ERBB1) and a potent mitogen for epithelial cells. After exposure to cisplatin, the resistant MCF-7 cells secreted amphiregulin protein over extended periods of time, and knockdown of amphiregulin expression by specific short interfering RNA resulted in a nearly complete reversion of the resistant phenotype. To demonstrate the generality and importance of our findings, we examined amphiregulin expression and cisplatin resistance in a variety of human breast cancer cell lines and found a highly significant correlation. In contrast, amphiregulin levels did not significantly correlate with cisplatin resistance in a panel of lung cancer cell lines. We have thus identified a novel function of amphiregulin for cisplatin resistance in human breast cancer cells.     

Autocrine Growth Loop of The Epidermal Growth Factor Receptor in Normal and Immortalized Human Bronchial Epithelial Cells

Epidermal growth factor (EGF) is a potent growth factor for human normal bronchial epithelial (HBE) cells and lung cancer cells, which often demonstrate an EGF receptor (EGFR) autocrine loop. We have found that HBE cells are capable of proliferating in basal medium without EGF supplementation, and this suggests the probable presence of an active EGFR autocrine loop in non-neoplastic HBE cells. Northern blot hybridization shows that the parental and immortalized HBE cells express comparable and high levels of mRNA for EGFR, transforming growth factor-alpha (TGF-α), and amphiregulin (AR), but not EGF. Incubation with neutralizing monoclonal antibodies (mAb) against EGFR partially inhibits the growth of these cells. Immunohistochemistry shows that HBE cells express the TGF-α peptidein vitroandin vivo,however, neutralizing mAbs against TGF-α fail to inhibit their proliferation. In contrast, AR stimulates the growth of HBE cells. Thus, several EGF-family ligands appear to be involved functionally in the EGFR autocrine growth loop in HBE cells.     

Gastrin-releasing peptide activates Akt through the epidermal growth factor receptor pathway and abrogates the effect of gefitinib

Gastrin-releasing peptide (GRP) is a mitogen for lung epithelial cells and initiates signaling through a G-protein-coupled receptor, gastrin-releasing peptide receptor (GRPR). Because GRPR transactivates the epidermal growth factor receptor (EGFR), we investigated induction by GRP of Akt, an EGFR-activated signaling pathway, and examined effects of GRP on viability of non-small cell lung carcinoma (NSCLC) cells exposed to the EGFR tyrosine kinase inhibitor gefitinib. GRP induced Akt activation primarily through c-Src-mediated transactivation of EGFR. Transfection of dominant-negative c-Src abolished GRP-induced EGFR and Akt activation. GRP induced release of amphiregulin, and pre-incubation with human amphiregulin neutralizing antibody eliminated GRP-induced Akt phosphorylation. Pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 completely blocked GRP-initiated Akt phosphorylation. These results suggest that GRP stimulates Akt activation primarily via c-Src activation, followed by extracellular release of the EGFR ligand amphiregulin, leading to the activation of EGFR and PI3K. Pretreatment of NSCLC cells with GRP resulted in an increase in the IC(50) of gefitinib of up to 9-fold; this protective effect was mimicked by the pretreatment of cells with amphiregulin and reversed by Akt or PI3K inhibition. GRP appears to rescue NSCLC cells exposed to gefitinib through release of amphiregulin and activation of the Akt pathway, suggesting GRPR and/or EGFR autocrine pathways in NSCLC cells may modulate therapeutic response to EGFR inhibitors.     

A Potent Anti-HB-EGF Monoclonal Antibody Inhibits Cancer Cell Proliferation and Multiple Angiogenic Activities of HB-EGF

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family and has a variety of physiological and pathological functions. Modulation of HB-EGF activity might have a therapeutic potential in the oncology area. We explored the therapeutic possibilities by characterizing the in vitro biological activity of anti-HB-EGF monoclonal antibody Y-142. EGF receptor (EGFR) ligand and species specificities of Y-142 were tested. Neutralizing activities of Y-142 against HB-EGF were evaluated in EGFR and ERBB4 signaling. Biological activities of Y-142 were assessed in cancer cell proliferation and angiogenesis assays and compared with the anti-EGFR antibody cetuximab, the HB-EGF inhibitor CRM197, and the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab. The binding epitope was determined with alanine scanning. Y-142 recognized HB-EGF as well as the EGFR ligand amphiregulin, and bound specifically to human HB-EGF, but not to rodent HB-EGF. In addition, Y-142 neutralized HB-EGF-induced phosphorylation of EGFR and ERBB4, and blocked their downstream ERK1/2 and AKT signaling. We also found that Y-142 inhibited HB-EGF-induced cancer cell proliferation, endothelial cell proliferation, tube formation, and VEGF production more effectively than cetuximab and CRM197 and that Y-142 was superior to bevacizumab in the inhibition of HB-EGF-induced tube formation. Six amino acids in the EGF-like domain were identified as the Y-142 binding epitope. Among the six amino acids, the combination of F115 and Y123 determined the amphiregulin cross-reactivity and that F115 accounted for the species selectivity. Furthermore, it was suggested that the potent neutralizing activity of Y-142 was derived from its recognition of R142 and Y123 and its high affinity to HB-EGF. Y-142 has a potent HB-EGF neutralizing activity that modulates multiple biological activities of HB-EGF including cancer cell proliferation and angiogenic activities. Y-142 may have a potential to be developed into a therapeutic agent for the treatment of HB-EGF-dependent cancers.     

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.     

Decreased autocrine EGFR signaling in metastatic breast cancer cells inhibits tumor growth in bone and mammary fat pad

Breast cancer metastasis to bone triggers a vicious cycle of tumor growth linked to osteolysis. Breast cancer cells and osteoblasts express the epidermal growth factor receptor (EGFR) and produce ErbB family ligands, suggesting participation of these growth factors in autocrine and paracrine signaling within the bone microenvironment. EGFR ligand expression was profiled in the bone metastatic MDA-MB-231 cells (MDA-231), and agonist-induced signaling was examined in both breast cancer and osteoblast-like cells. Both paracrine and autocrine EGFR signaling were inhibited with a neutralizing amphiregulin antibody, PAR34, whereas shRNA to the EGFR was used to specifically block autocrine signaling in MDA-231 cells. The impact of these was evaluated with proliferation, migration and gene expression assays. Breast cancer metastasis to bone was modeled in female athymic nude mice with intratibial inoculation of MDA-231 cells, and cancer cell-bone marrow co-cultures. EGFR knockdown, but not PAR34 treatment, decreased osteoclasts formed in vitro (p<0.01), reduced osteolytic lesion tumor volume (p<0.01), increased survivorship in vivo (p<0.001), and resulted in decreased MDA-231 growth in the fat pad (p<0.01). Fat pad shEGFR-MDA-231 tumors produced in nude mice had increased necrotic areas and decreased CD31-positive vasculature. shEGFR-MDA-231 cells also produced decreased levels of the proangiogenic molecules macrophage colony stimulating factor-1 (MCSF-1) and matrix metalloproteinase 9 (MMP9), both of which were decreased by EGFR inhibitors in a panel of EGFR-positive breast cancer cells. Thus, inhibiting autocrine EGFR signaling in breast cancer cells may provide a means for reducing paracrine factor production that facilitates microenvironment support in the bone and mammary gland.     

Combination therapy with KRAS siRNA and EGFR inhibitor AZD8931 suppresses lung cancer cell growth in vitro

Lung cancer is a leading cause of cancer-related deaths worldwide, with less than a 5-year survival rate for both men and women. Epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma oncogene (KRAS) signaling pathways play a critical role in the proliferation and progression of various cancers, including lung cancer. Genetic studies have shown that amplification, over-expression, or mutation of EGFR is an early and major molecular event in many human tumors. KRAS mutation is a negative factor in various cancer, including non-small-cell lung cancer, and complicates therapeutic approaches with adjuvant chemotherapy and anti-EGFR directed therapies. This article is dedicated to evaluating the synergistic effect of a novel EGFR inhibitor AZD8931 and KRAS small interfering RNA (siRNA) on the proliferation and apoptosis of lung adenocarcinoma cancer cells. A549 lung cancer cells were treated with KRAS siRNA and the EGFR inhibitor alone or in combination. The cytotoxic effects of KRAS siRNA and te EGFR inhibitor were determined usingMTT assay, and induction of apoptosis was determined by FACS analysis. Suppression of KRAS, Her-2, and EGFR expression by treatments was measured by qRT-PCR and western blotting. KRAS siRNA and the EGFR inhibitor significantly reduced the proliferation of A549 cells as well as KRAS and EGFR mRNA levels 24 hr after treatment. The results also indicated that the silencing of KRAS and EGFR has synergistic effects on the induction of apoptosis on the A549 cells. These results indicated that KRAS and EGFR might play important roles in the progression of lung cancer and could be potential therapeutic targets for treatment of lung cancer.     

Association of epidermal growth factor receptor (EGFR) gene polymorphism with lung cancer risk: a systematic review

Abstract

Epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family, which is thought to be involved in the development of cancer, as the EGFR gene is often amplified, and/or mutated in cancer cells. Lung cancer remains one of the most major causes of morbidity and mortality worldwide, accounting for more deaths than any other cancer cause. Gene polymorphism factor has been reported to be an important factor which increases the susceptibility of lung cancer. There lacks a well-documented diagnostic approach for the lung cancer risk, and the etiology of lung cancer is not clear. The current systematic review was performed to explore the association of EGFR gene polymorphism with lung cancer risk. In this review, association of EGFR 181946C?>?T, 8227G?>?A gene polymorphism with lung cancer was found, and EGFR Short genotype of cytosine adenine repeat number polymorphism was significantly associated with an increased risk of lung cancer.     

MUC1 contributes to BPDE-induced human bronchial epithelial cell transformation through facilitating EGFR activation

Although it is well known that epidermal growth factor receptor (EGFR) is involved in lung cancer progression, whether EGFR contributes to lung epithelial cell transformation is less clear. Mucin 1 (MUC1 in human and Muc1 in animals), a glycoprotein component of airway mucus, is overexpressed in lung tumors; however, its role and underlying mechanisms in early stage lung carcinogenesis is still elusive. This study provides strong evidence demonstrating that EGFR and MUC1 are involved in bronchial epithelial cell transformation. Knockdown of MUC1 expression significantly reduced transformation of immortalized human bronchial epithelial cells induced by benzo[a]pyrene diol epoxide (BPDE), the active form of the cigarette smoke (CS) carcinogen benzo(a)pyrene (BaP)s. BPDE exposure robustly activated a pathway consisting of EGFR, Akt and ERK, and blocking this pathway significantly increased BPDE-induced cell death and inhibited cell transformation. Suppression of MUC1 expression resulted in EGFR destabilization and inhibition of the BPDE-induced activation of Akt and ERK and increase of cytotoxicity. These results strongly suggest an important role for EGFR in BPDE-induced transformation, and substantiate that MUC1 is involved in lung cancer development, at least partly through mediating carcinogen-induced activation of the EGFR-mediated cell survival pathway that facilitates cell transformation.     

Epidermal growth factor receptor-dependent stimulation of amphiregulin expression in androgen-stimulated human prostate cancer cells.

Amphiregulin is a heparin-binding epidermal growth factor (EGF)-related peptide that binds to the EGF receptor (EGF-R) with high affinity. In this study, we report a role for amphiregulin in androgen-stimulated regulation of prostate cancer cell growth. Androgen is known to enhance EGF-R expression in the androgen-sensitive LNCaP human prostate carcinoma cell line, and it has been suggested that androgenic stimuli may regulate proliferation, in part, through autocrine mechanisms involving the EGF-R. In this study, we demonstrate that LNCaP cells express amphiregulin mRNA and peptide and that this expression is elevated by androgenic stimulation. We also show that ligand-dependent EGF-R stimulation induces amphiregulin expression and that androgenic effects on amphiregulin synthesis are mediated through this EGF-R pathway. Parallel studies using the estrogen-responsive breast carcinoma cell line, MCF-7, suggest that regulation of amphiregulin by estrogen may also be mediated via an EGF-R pathway. In addition, heparin treatment of LNCaP cells inhibits androgen-stimulated cell growth further suggesting that amphiregulin can mediate androgen-stimulated LNCaP proliferation. Together, these results implicate an androgen-regulated autocrine loop composed of amphiregulin and its receptor in prostate cancer cell growth and suggest that the mechanism of steroid hormone regulation of amphiregulin synthesis may occur through androgen upregulation of the EGF-R and subsequent receptor-dependent pathways.     

Angio-associated migratory cell protein interacts with epidermal growth factor receptor and enhances proliferation and drug resistance in human non-small cell lung cancer cells

Angio-associated migratory cell protein (AAMP) is expressed in some human cancer cells. Previous studies have shown AAMP high expression predicted poor prognosis. But its biological role in non-small cell lung cancer (NSCLC) cells is still unknown. In our present study, we attempted to explore the functions of AAMP in NSCLC cells. According to our findings, AAMP knockdown inhibited lung cancer cell proliferation and inhibited lung cancer cell tumorigenesis in the mouse xenograft model. Epidermal growth factor receptor (EGFR) is a primary receptor tyrosine kinase (RTK) that promotes proliferation and plays an important role in cancer pathology. We found AAMP interacted with EGFR and enhanced its dimerization and phosphorylation at tyrosine 1173 which activated ERK1/2 in NSCLC cells. In addition, we showed AAMP conferred the lung cancer cells resistance to chemotherapeutic agents such as icotinib and doxorubicin. Taken together, our data indicate that loss of AAMP from NSCLC inhibits tumor growth and elevates drug sensitivity, and these findings have clinical implications to treat NSCLC cancers.     

Interaction between epidermal growth factor receptor and interleukin-6 receptor in NSCLC progression

Even though the interaction between epithelial growth factor receptor (EGFR) and interleukin-6 receptor (IL-6R) has been found in many tumors, there is a lack of relevant in-depth study of lung cancer. The following study investigates the interaction of EGFR and IL-6R in lung cancer. In the current study, EGFR, IL-6, and glycoprotein 130 (GP130) were highly expressed in non–small cell lung cancer (NSCLC) tissue samples and were associated with clinicopathological features and poor prognosis of patients with NSCLC. Furthermore, the effect of EGF and IL-6 on biological behavior of lung cancer cells (cell proliferation, invasion, cycle, and apoptosis) and the expression of EGFR, GP130, p-protein kinase B (p-AKT), and p-p44/42 mitogen-activated protein kinase (p-p44/42 MAPK) was significantly stronger compared with other treatment groups (all P < 0.05). These results suggest that EGFR and IL-6R have synergistic effects on NSCLC progression. This could help to solve the problem of EGFR inhibitors in the treatment of lung cancer resistance and improve the efficacy of current treatment for lung cancer through a combination of EGFR and IL-6R signaling pathways.     

Lung tumor-associated dendritic cell-derived amphiregulin increased cancer progression

The interaction of cancer within a microenvironment is an important factor determining cancer development. This study analyzed the soluble factors secreted by tumor-associated dendritic cells (TADCs), which are responsible for increasing lung cancer growth, migration, invasion, and epithelial-to-mesenchymal transition. Addition of amphiregulin, present in large amounts in TADC-conditioned medium (CM), mimicked the inductive effect of TADC-CM on lung cancer progression, supported by the enhancement of cell proliferation, migration, and invasion as well as osteolytic bone metastases phenotypes. In contrast, neutralization of amphiregulin from TADC-CM decreased the advanced malignancy-inductive properties of TADC-CM. Significant upregulation of amphiregulin has been seen in tumor-infiltrating CD11c(+) DCs in human lung cancer samples and patients' sera. The enhancement of amphiregulin in TADCs has also been noted in mice transplanted with lung cancer cells. Induction of lung cancer progression by TADC-derived amphiregulin is associated with increased STAT3 and AKT activation, which subsequently increases the expression of cyclin D, Twist, and Snail. Blocking AKT significantly decreases TADC-CM and amphiregulin-mediated migration by decreasing the upregulation of Snail, whereas inhibition of STAT3 reduced the modulation of TADC-derived amphiregulin on Twist and cyclin D expression, suggesting that cooperation of STAT3 and AKT plays a critical role in TADC-mediated cancer progression. Moreover, mice treated with anti-amphiregulin Abs showed decreased incidence of cancer development and increased survival rates. Our study suggests that inhibition of amphiregulin or amphiregulin-related signaling is an attractive therapeutic target in lung cancer patients.     

A disintegrin and metalloproteinase 17 (ADAM17) mediates epidermal growth factor receptor transactivation by angiotensin II on hepatic stellate cells

Aims

Epidermal growth factor receptor (EGFR) transactivation induced by angiotensin II (Ang II) participates in the progression of various diseases. A disintegrin and metalloproteinase 17 (ADAM17) is thought to promote renal fibrosis, cardiac hypertrophy with fibrosis and atherosclerosis by activation of the EGFR through secretion of EGFR ligands. The purpose of this study was to investigate whether Ang II-induced EGFR transactivation occurs on hepatic stellate cells (HSCs) and whether the reaction is mediated via ADAM17.

Main methods

Ang II-induced EGFR transactivation and cellular proliferation of the human HSC line LI90 were investigated using Western blotting and ATP assay, respectively. Ang II-induced secretion of mature amphiregulin into the cell culture medium was evaluated by enzyme-linked immunosorbent assay (ELISA).

Key findings

An inhibitor of ADAM17, TAPI-1, as well as antagonists of EGFR and angiotensin II type-1 receptor (AT1), attenuated Ang II-induced EGFR transactivation and proliferation of LI90 cells. Furthermore, silencing of ADAM17 inhibited Ang II-induced secretion of mature amphiregulin in addition to EGFR transactivation.

Significance

These results indicate that ADAM17 mediates Ang II-induced EGFR transactivation on HSCs, and that this process may participate in the progression of liver fibrosis.     

Altered EGFR localization and degradation in human breast cancer cells with an amphiregulin/EGFR autocrine loop

The epidermal growth factor receptor (EGFR) and its ligand amphiregulin (AR) have been shown to be co-over expressed in breast cancer. We have previously shown that an AR/EGFR autocrine loop is required for SUM149 human breast cancer cell proliferation, motility and invasion. We also demonstrated that AR can induce these altered phenotypes when expressed in the normal mammary epithelial cell line MCF10A, or by exposure of these cells to AR in the medium. In the present studies, we demonstrate that SUM149 cells and immortalized human mammary epithelial MCF10A cells that over express AR (MCF10A AR) or are cultured in the presence of exogenous AR, express higher levels of EGFR protein than MCF10A cells cultured in EGF. Pulse-chase analysis showed that EGFR protein remained stable in the presence of AR, yet was degraded in the presence of EGF. Consistent with this observation, tyrosine 1045 on the EGFR, the c-cbl binding site, exhibited less phosphorylation following stimulation with AR than following stimulation with EGF. Ubiquitination of the receptor was also dramatically less following stimulation with AR than following stimulation with EGF. Flow cytometry analysis showed that EGFR remained on the cell surface following stimulation with AR but was rapidly internalized following stimulation with EGF. Immunofluorescence and confocal microscopy confirmed the flow cytometry results. EGFR in MCF10A cells cultured in the presence of EGF exhibited a predominantly intracellular, punctate localization. In stark contrast, SUM149 cells and MCF10A cells growing in the presence of AR expressed EGFR predominantly on the membrane and at cell-cell junctions. We propose that AR alters EGFR internalization and degradation in a way that favors accumulation of EGFR at the cell surface and ultimately leads to changes in EGFR signaling.