Figure 5.

Functional consequences of ERBB2 L869R mutation. (A,B) Expression of ERBB2 WT and ERBB2 L869R mutant in MCF10A cells results in increased relative phosphorylation of ERBB2 L869R. Cell lysates of MCF10A cells expressing vector control, ERBB2 WT, and ERBB2 L869R were resolved in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The immunoblots were probed with indicated antibodies (A). Quantitation of the western blots is represented by the bar graphs (B). (C,D) Increased proliferation of cells expressing ERBB2 L869R mutant. MCF10A cells expressing the vector control, ERBB2 WT, and ERBB2 L869R were grown in complete growth medium (C) or serum-free medium (D) in 96-well plates and counted every 2 d by a cell-titer glow assay. The data represents ±SD of five wells for each data point. (E,F) ERBB2 L869R mutant increases colony formation. MCF10A cells expressing vector control, ERBB2 WT, and ERBB2 L869R were grown in six-well plates (500 cells per well) for a colony formation assay. The bar graph (F) shows the number of colonies obtained. The data are representative of three independent experiments. (G,H) ERBB2 L869R mutant increases Matrigel invasion. MCF10A cells expressing vector control, ERBB2 WT, and ERBB2 L869R were grown on Matrigel-coated transwell chambers. In each well, 10⁵ cells were seeded. The bar graph (H) shows the number of cells that invaded through the Matrigel. The data are an average of triplicate wells. (I) The ERBB2 L869R mutant increases proliferation and reduces apoptosis in 3D cultures of MCF10A cells. MCF10 cells expressing ERBB2 WT and ERBB2 L869R were grown in basement membrane extract for 12 d. 3D colonies were fixed and stained for immunofluorescence with Ki67 and caspase-3-specific antibodies as indicated. DAPI was used for nuclear staining. (J,K) ERBB2 L869R mutant increases migration in a wound-healing assay. MCF10A cells grown in monolayer expressing vector control, ERBB2 WT, and ERBB2 L869R were subjected to a scratch-induced wound healing assay. The bar graph (K) represents quantitation of migration from three independent experiments. (L) Expression of ERBB2 L869R mutant results in increased expression of mesenchymal markers, N-cadherin and fibronectin and reduced expression of epithelial marker, E-cadherin. Immunofluorescence stainings with E-cadherin (green)-, N-cadherin (red)-, and fibronectin (green)-specific antibodies were performed, and DAPI was used for nuclear counterstaining. (M) GI₅₀ determination of lapatinib-treated MCF10A cells expressing ERBB2 WT or ERBB2 L869R first grown in 3D culture in the presence of dimethyl sulfoxide (DMSO) (left) and lapatinib (right). (N) Expression of EMT markers in MCF10A cells expressing vector control, ERBB2 WT, and ERBB2 L869R treated with DMSO or rendered lapatinib resistant in 3D culture. (O–R) MCF10A cells expressing ERBB2 L869R that are sensitive or resistant to lapatinib form xenograft tumors in nude mice. The lapatinib-resistant ERBB2 L869R-expressing cells form larger tumors (O,P). There are increases in Ki67-positive proliferating cells and angiogenesis as evidenced by increased CD34 staining in lapatinib-resistant ERBB2 mutant tumors (Q). Lapatinib-resistant ERBB2 mutant cells form significantly increased metastases in a tail-vein injection metastases assay (R). (***) P < 0.001, (*) P < 0.05.