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 AREG/EGFR autocrine loop for cell proliferation. We also demonstrated that AREG can increase 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 lenti-viral 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.

Perspectives on Epidermal Growth Factor Receptor Regulation in Triple-Negative Breast Cancer

“…As illustrated in Fig. 2, it was previously shown that three of the seven canonical ligands: HB-EGF, BTC, and EGF will promote receptor degradation whereas AREG, EREG, and TGFA binding will preferentially result in recycling (Baillo, Giroux, & Ethier, 2011). Interestingly, ligands including AREG and TGFA that preferentially drive EGFR recycling have also been implicated in autocrine/paracrine signaling in TN and inflammatory breast cancers (LeJeune et al, 1993;Ma et al, 2001;Panico et al, 1996;Streicher et al, 2007).…”

“…However, these effects were not seen when cells were stimulated with EGF (Willmarth & Ethier, 2006). Likewise, impairing AREG by shRNA knockdown in the SUM-149PT cell line inhibited invasion (Baillo et al, 2011).…”

“…SUM-149PT cells were originally isolated from a patient with TNBC and found to have constitutively active EGFR, while being EGF independent for growth (Baillo et al, 2011). It was determined that the EGFR activity in these cells was due to overexpression of AREG mRNA and protein (Berquin et al, 2001).…”

“…It was determined that the EGFR activity in these cells was due to overexpression of AREG mRNA and protein (Berquin et al, 2001). The SUM-149PT cells display a type of self-sustaining autocrine loop between AREG and EGFR (Willmarth & Ethier, 2006), where the binding of AREG to EGFR causes increased transcription and secretion of AREG itself, allowing for more AREG to bind to other EGFR receptors, thus causing EGFR to be constitutively active (Baillo et al, 2011).…”

Role of epidermal growth factor receptor in breast cancer

“…An in vitro study showed that gefitinib, an EGFR-TKI, suppressed the growth of SUM149 cells, which overexpress EGFR and lack ER expression and are widely used as a model of aggressive IBC [50]. Another in vitro study showed that treatment with neutralizing antibody against amphiregulin, one of the ligands of EGFR, decreased EGFR activity and reduced cell proliferation in SUM149 cells [51]. These findings led to an ongoing study of panitumumab (an anti-EGFR antibody), albumin-bound paclitaxel (Abraxane), and carboplatin in IBC (NCT01036087).…”