title: The role of amphiregulin in exemestane-resistant breast cancer cells: evidence of an autocrine loop.
abstract: Exemestane-resistant breast cancer cell lines (i.e., ExeR), derived from MCF-7 cells expressing a high level of aromatase (MCF-7aro), were generated in our laboratory. The epidermal growth factor (EGF)-like protein amphiregulin (AREG) was highly expressed in ExeR cells based on cDNA microarray analysis. The high levels of AREG mRNA in ExeR cell lines were confirmed by real-time reverse transcription-PCR. The high levels of AREG protein in ExeR cell lysates and culture media were confirmed by Western blot analysis and ELISA, respectively. Furthermore, our Western blot analysis showed that whereas no AREG was detected in the DMSO control, overnight treatment of parental MCF-7aro cells with 1 micromol/L exemestane strongly induced the expression of AREG. This induction was totally blocked by 100 nmol/L of pure antiestrogen ICI 182,780, implying estrogen receptor (ER) dependence of exemestane-induced AREG expression. MCF-7aro cells were not able to proliferate in hormone-free medium, but were able to proliferate in conditioned medium from ExeR cells, similar to the treatment of recombinant human AREG. Small interference RNA targeting AREG inhibited ExeR proliferation, confirming that AREG is truly functioning as a growth factor of ExeR cells. The specific inhibitors to ER (ICI 182,780), EGF receptor (EGFR; AG1478), and mitogen-activated protein kinase (MAPK; U0126) all showed dose-dependent suppression of the proliferation of ExeR cells, indicating the involvement of the ER, EGFR, and MAPK pathways. Based on these findings, we propose a possible mechanism that underlies exemestane resistance: exemestane induces AREG in an ER-dependent manner. AREG then activates the EGFR pathway and leads to the activation of the MAPK pathway that drives cell proliferation.
authors.affiliation: Department of Surgical Research, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.forename: Selma
authors.initials: S
authors.lastname: Masri
authors.forename: Sheryl
authors.initials: S
authors.lastname: Phung
authors.forename: Shiuan
authors.initials: S
authors.lastname: Chen
mesh_terms: D066258:Amphiregulin; D000730:Androstadienes / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D048708:Cell Growth Processes / Q000187:drug effects; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D066255:EGF Family of Proteins; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D011960:Receptors, Estrogen / Q000037:antagonists & inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology
chemical_list: C587848:AREG protein, human; D066258:Amphiregulin; D000730:Androstadienes; D000970:Antineoplastic Agents; D066255:EGF Family of Proteins; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D011960:Receptors, Estrogen; D011994:Recombinant Proteins; D066246:ErbB Receptors; D020928:Mitogen-Activated Protein Kinases; C056516:exemestane