ErbB2 Stabilizes Epidermal Growth Factor Receptor (EGFR) Expression via Erk and Sprouty2 in Extracellular Matrix-detached Cells

Abstract

Epithelial cells are dependent on extracellular matrix (ECM) attachment for maintenance of metabolic activity and suppression of apoptosis. Here we show that loss of ECM attachment causes down-regulation of epidermal growth factor receptor (EGFR) and ␤1 integrin protein and mRNA expression and that ErbB2, which is amplified in 25% of breast tumors, reverses these effects of ECM deprivation. ErbB2 rescue of ␤1 integrin mRNA and protein in suspended cells is dependent on EGFR, however, the rescue of EGFR expression does not require ␤1 integrin. We show that there is a significant decrease in the stability of EGFR in ECM-detached cells that is reversed by ErbB2 overexpression. Rescue of both EGFR and ␤1 integrin protein by ErbB2 is dependent on Erk activity and induction of its downstream target Sprouty2, a protein known to regulate EGFR protein stability. Interestingly, expression of EGFR and ␤1 integrin protein is more dependent on Erk/ Sprouty2 in ECM-detached ErbB2-overexpressing cells when compared with ECM-attached cells. These results provide further insight into the ErbB2-driven anchorage independence of tumor cells and provide a new mechanism for regulation of EGFR and ␤1 integrin expression in ECM-detached cells.One of the hallmarks of tumor cells is the ability to survive without attachment to the extracellular matrix (ECM) 3 (1). Detachment of normal epithelial cells from ECM leads to metabolic impairment and induction of apoptotic death (anoikis) (2-5). However, tumor cells are generally able to survive without ECM attachment, a property known as "anchorage independence" (2, 3). This is believed to allow tumor cells to invade and proliferate outside their natural ECM niches. For example, one early feature of breast cancer is the proliferation of cells into the ECM-deficient hollow lumen of the glandular epithelium (6). These premalignant cells are able to survive despite the lack of contact to the basement membrane. Additionally, it is believed that anchorage independence is an important aspect of the metastatic process, both for survival in the vasculature and lymphatic system and also for survival in distant sites with altered matrix environments (2). Thus, elucidation of the mechanisms responsible for tumor cell anchorage independence is critical for understanding of the tumorigenic processes and may lead to identification of novel drug targets.ECM detachment of mammary epithelial cells results in a decrease of growth factor signaling through the Mek/Erk and PI3K/Akt pathways (2, 4, 5, 7). This leads to decreased cell viability, both through the induction of anoikis and through a caspase-independent metabolic impairment (2, 4). We have previously found that decreased Erk activation in ECM-detached cells leads to increased expression of the pro-apoptotic protein, Bim (7-9). In addition, the lack of Akt signaling in the ECM-detached cells results in a dramatic decrease in glucose uptake and ATP levels, causing a severe energy deficiency (4). Therefore, survival of mammary epithelial cells under ECM-de...

Erk regulation of pyruvate dehydrogenase flux through PDK4 modulates cell proliferation

“…4F,G; Grassian et al 2011). This is consistent with a previous report showing that glucose uptake is insensitive to Mek inhibition (Lazar et al 1995).…”

“…S3F), suggesting that the ErbB2 regulation of PDK4 is independent of PI3K/Akt signaling. ErbB2 also maintains activation of the Mek/Erk pathway in ECMdetached cells (Reginato et al 2003;Schafer et al 2009;Grassian et al 2011), and treatment of MCF-10A ErbB2 cells with either of two Mek inhibitors (UO126 and PD98059) increased PDK4 mRNA and protein levels under ECMdetached conditions (Figs. 3A,B; Supplemental Fig.…”

“…ATP levels were determined using the ATPlite assay (Perkin Elmer) as previously described (Grassian et al 2011) and as in the Supplemental Material.…”

“…qPCR was performed as previously described (Grassian et al 2011) and as in the Supplemental Material. Microarray analyses on MCF-10A cells during morphogenesis were performed as described previously ).…”

Anoikis evasion in inflammatory breast cancer cells is mediated by Bim-EL sequestration

“…[18][19][20] Interestingly, previous studies have shown that ErbB2 and EGFR, which are hyperactivated in a substantial percentage of IBC patients, 21 can effectively antagonize the anoikis program to facilitate anchorage-independent growth. [22][23][24][25][26][27][28] However, a detailed examination of the molecular mechanisms underlying anoikis inhibition in IBC cells has yet to be completed. In this study, we demonstrate that signaling from EGFR and ErbB2 through ERK/MAPK has a major role in the ability of IBC cells to survive in the absence of ECM attachment.…”