ErbB2 rescue of β1 integrin protein and mRNA levels is dependent on EGFR, but β1 integrin is not required for rescue of EGFR. A and B, MCF-10A ErbB2 cells were transfected with siRNAs targeting luciferase (Luc) or EGFR. 24 h later, cells were plated under attached or detached conditions, and after an additional 24 h, protein levels were assessed by immunoblotting (A), and mRNA levels were measured by qPCR (B). Quantification of average protein levels from three independent experiments is also shown (A, right panel). Lane A, ECM-attached; lane D, ECM-detached. C and D, MCF-10A ErbB2 cells were transfected with siRNAs targeting luciferase or β1 integrin. 24 h later, cells were plated under attached or detached conditions, and after an additional 24 h, protein levels were assessed by immunoblotting (C), and mRNA levels were measured by qPCR (D). Quantification of average protein levels from three independent experiments is also shown (C, right panel). E and F, MCF-10A cells were transfected with siRNAs targeting luciferase, EGFR, or β1 integrin. 48 h later, protein levels were analyzed by immunoblotting (E), and mRNA levels were measured by qPCR (F). Quantification of average protein levels from three independent experiments is also shown (E, right panel). G and H, MCF-10A ErbB2 cells were transfected with siRNAs targeting luciferase, EGFR (G), or β1 integrin (H). Cells were then plated under attached and detached conditions, and ATP levels were measured using the ATPlite assay.

ErbB2 increases EGFR protein stability in ECM-detached cells. A, MCF-10A or MCF-10A ErbB2 cells were plated under detached conditions and lysed after the indicated amount of time. Protein expression was analyzed by immunoblotting. B, MCF-10A or MCF-10A ErbB2 cells were detached from tissue culture plates using either trypsin or cell dissociation buffer (CDB, Invitrogen). Cells were then plated under detached conditions for 1 h, and protein levels were analyzed by immunoblotting. C, MCF-10A or MCF-10A ErbB2 cells were plated under detached conditions. After 3 h, cells were either lysed or treated with 10 μg/ml cycloheximide (CHX) for an additional 3 h. Protein levels were measured by immunoblotting, and data are presented as the percentage of EGFR remaining after the 3-h cycloheximide treatment. D, MCF-10A cells were plated under detached conditions. After 3 h, cells were either lysed or treated with 10 μg/ml cycloheximide or 10 μg/ml cycloheximide and 100 nm concanamycin A (CHX & CCA) for an additional 3 h. Protein levels were measured by immunoblotting, and data are presented as the amount of EGFR remaining after the 3-h cycloheximide treatment. E and F, MCF-10A cells were plated under detached conditions. After 3 h, 20 μm chloroquine diphosphate salt (E), 20 nm bafilomycin A1 (F), or vehicle was added. Cells were lysed 24 h after plating, and EGFR protein levels were measured by immunoblotting.

Model for regulation of EGFR and β1 integrin expression by ErbB2 and extracellular matrix contact. Detachment of MCF-10A cells from ECM prevents integrin engagement, leading to decreased Erk signaling and decreased Sprouty2; Cbl is thus active, and EGFR is degraded via the lysosome and can no longer maintain β1 integrin expression or ATP levels, or prevent apoptosis (left panel). Overexpression of ErbB2 maintains activation of Erk under ECM-detached conditions, leading to increased Sprouty2, which inhibits Cbl-induced degradation of EGFR and maintains β1 integrin expression and ATP levels and prevents apoptosis (right panel). The striped receptor represents any of the ErbB family members. Circled P, phosphorylation.

ErbB2 prevents the decrease of EGFR and β1 integrin protein and mRNA in ECM-detached cells. A, MCF-10A or MCF-10A ErbB2 cells were plated under ECM-attached (lane A) or ECM-detached (lane D) conditions for 24 h. Expression of the indicated proteins was determined by immunoblotting. pErk, phosphorylated Erk. B, quantification from three experiments performed as described in A. Data are presented as ratios of protein in detached cells to protein in attached cells. C, human mammary epithelial cells (HMEC) were plated and analyzed as described in A. D, MCF-10A or MCF-10A ErbB2 cells were processed as in A, and mRNA levels were measured by qPCR.

Erk signaling is necessary for the ErbB2 maintenance of EGFR and β1 integrin protein expression in ECM-detached cells. A, MCF-10A cells or MCF-10A ErbB2 cells treated with 20 μm PD98059 (+) or vehicle (−) were plated and analyzed as described in the legend for Fig. 1. Lane A, ECM-attached; lane D, ECM-detached. B, quantification from three experiments as described in A. Data are presented as a ratio of protein in detached cells to protein in attached cells (left panel) or ratio of protein in Erk inhibitor-treated cells to control cells (right panel). Mek Inhib., Mek inhibitor. C, MCF-10A ErbB2 cells were transfected with siRNA targeting luciferase (−), ERK1, ERK2, or ERK1 and ERK2. Cells were plated under attached and detached conditions and analyzed as in A. D, MCF-10A cells treated with 10 μm UO126 (+) or vehicle (−) were plated under attached or detached conditions for 24 h, and protein levels were analyzed by immunoblotting. E, MCF-10A, MCF-10A ErbB2, or MCF-10A MekDD was plated and analyzed as in A. F, SK-BR-3 cells were treated with 10 μm UO126 (+) or vehicle (−) and plated under attached or detached conditions for 24 h. Protein levels were analyzed by immunoblotting. G, MCF-10A or MCF-10A ErbB2 cells were treated with 10 μm UO126 (+) or vehicle (−) and plated under attached or detached conditions for 24 h. mRNA levels were then measured by qPCR. H, MCF-10A cells, or MCF-10A ErbB2 cells treated with 20 μm LY29402 (+) or vehicle (−) were plated and analyzed as in A. I, MCF-10A cells or MCF-10A ErbB2 cells treated with 10 μm LY29402 or 10 μm LY29402 and 20 μm PD98059 (+) or vehicle (−) were plated under detached conditions for 24 h, and mRNA levels were measured by qPCR.

ErbB2 regulates EGFR and β1 integrin via Erk-dependent regulation of Sprouty2. A, MCF-10A or MCF-10A ErbB2 cells treated with 20 μm PD98059(+) or vehicle (−) were plated under attached or detached conditions for 24 h. mRNA levels were measured by qPCR. Column A, ECM-attached; column D, ECM-detached. B, MCF-10A, MCF-10A ErbB2, or MCF-10A MekDD cells were plated under attached or detached conditions for 24 h, and mRNA levels were measured by qPCR. C, MCF-10A cells infected with pMSCV-IRES-PURO or pMSCV-SPRY2-IRES-PURO were plated under attached or detached conditions for 24 h, and protein levels were analyzed by immunoblotting. D, MCF-10A or MCF-10A ErbB2 cells infected with pMSCV-SPRY2-IRES-PURO were plated under attached or detached conditions with 10 μm UO126 or 20 μm PD98059 (+) or vehicle (−). 24 h later, protein levels were analyzed by immunoblotting. pErk1/2, phosphorylated Erk1/2. E, cells were plated and analyzed as described in C. F, MCF-10A ErbB2 cells were infected with shRNA vectors targeting Sprouty2, and mRNA levels were measured by qPCR to confirm knockdown. G and H, MCF-10A ErbB2 cells infected with shRNAs targeting Sprouty2 were plated under detached (G) or attached (H) conditions for 24 h, and protein levels were measured by immunoblotting. I, SK-BR-3 cells were infected with shRNA vectors targeting Sprouty2, and mRNA levels were measured by qPCR to confirm knockdown. J and K, SK-BR-3 cells infected with shRNA vectors targeting Sprouty2 were plated under detached (J) or attached (K) conditions for 24 h, and protein levels were measured by immunoblotting.

Sprouty2 mRNA levels correlate with sensitivity to Mek inhibitors. Box plots depicting the level of Sprouty2 mRNA from two publicly available microarray study datasets are shown. Sprouty2 mRNA expression levels are in normalized expression units downloaded from Oncomine.