title: Knock-down of amphiregulin inhibits cellular invasion in inflammatory breast cancer.
abstract: We have previously shown that SUM-149 human breast cancer cells require an amphiregulin (AREG) autocrine loop for cell proliferation. We also demonstrated that AREG can increase epidermal growth factor receptor (EGFR) stability and promote EGFR localization to the plasma membrane. In the present studies we successfully knocked-down AREG expression in SUM-149 cells by lentiviral infection of AREG shRNA. In the absence of AREG expression, SUM-149 cell growth was slowed, but not completely inhibited. Furthermore, cells infected with AREG shRNA constructs showed an increase in EGFR protein expression by Western blot. Immunofluorescence and confocal microscopy showed that following AREG knock-down, EGFR continued to localize to the cell surface. Soft agar assays demonstrated that AREG knock-down cells retain anchorage-independent growth capacity. Additionally mammosphere forming assays and Adefluor staining analysis showed that knock-down of AREG expression did not affect the expression of stem cell phenotypes. However, following AREG knock-down, SUM-149 cells demonstrated a dramatic decrease in their ability to invade a Matrigel matrix. Consistent with this observation, microarray analysis comparing cells infected with a non-silencing vector to the AREG knock-down cells, identified genes associated with the invasive phenotype such as RHOB and DKK1, and networks associated with cell motility such as integrin-linked kinase signaling, and focal adhesion kinase signaling. AREG was also found to modulate WNT and Notch signaling in these cells. Thus, AREG functions in regulating the invasive phenotype, and we propose that this regulation may be through altered signaling that occurs when AREG activates plasma membrane localized EGFR.
authors.affiliation: Department of Oncology, Wayne State University, Detroit, Michigan, USA.
authors.forename: Andrea
authors.initials: A
authors.lastname: Baillo
authors.forename: Craig
authors.initials: C
authors.lastname: Giroux
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Ethier
mesh_terms: D066258:Amphiregulin; D045744:Cell Line, Tumor; D066255:EGF Family of Proteins; D066246:ErbB Receptors / Q000502:physiology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D055785:Gene Knockdown Techniques / Q000379:methods; D006023:Glycoproteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000502:physiology; D006801:Humans; D058922:Inflammatory Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000502:physiology; D009361:Neoplasm Invasiveness / Q000473:pathology; D010641:Phenotype; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology
chemical_list: C587848:AREG protein, human; D066258:Amphiregulin; D066255:EGF Family of Proteins; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D066246:ErbB Receptors