The ErbB3 receptor and the downstream signaling kinase Akt are implicated in proliferation of lung adenocarcinoma cells. Inhibition by siRNAs to ErbB3 and Akt isoforms 1, 2 and 3 was utilized to investigate the contribution of these molecules to tumor survival, spreading and invasiveness, and the roles of specific Akt isoforms. ErbB3 siRNA stably and dose-dependently suppressed ErbB3 protein for 2 days or more, and reduced cell numbers, by both suppressing cell cycle and causing apoptosis and necrosis. It also inhibited soft agar growth, cell motility and migration, and invasiveness. Akt1, 2 and 3 siRNAs had similar suppressive effects on cell number, apoptosis/ necrosis and soft agar growth. However, although Akt1 siRNA had no effect on cell migration or invasion, Akt2 siRNA effectively suppressed both activities, and Akt3 siRNA had moderate effectiveness. In A549 cells, ErbB3 is indicated as having major effects on cell division, survival, motility, migration and invasiveness. All three Akt isoforms are to varying degrees involved in these cell behaviors, with Akt2 especially implicated in migration and invasion. ErbB3 and the Akts are promising targets for therapy, and siRNAs may be useful for this purpose.
Although ErbB3, a member of the epidermal growth factor receptor family, has been implicated in mammary tumorigenesis, investigation of its role in lung tumorigenesis has been limited. We found that ErbB3 was present at high levels in five of seven human lung adenocarcinoma cell lines examined, along with its ligands, heregulins alpha and beta, whereas ErbB3 was absent from HPL1D, a non- transformed cell line from human pulmonary peripheral epithelium. Interactions and effects of ErbB3 were studied in detail in adenocarcinoma lines H441 and H1373. Complexes containing phosphorylated ErbB2, phosphorylated ErbB3 and the p85 regulatory subunit of phosphoinositidyl 3-kinase were detected by co-immunoprecipitation experiments and were present constitutively even in the absence of serum-stimulated cell division. Serum treatment increased the pErbB3/p85 complexes and also stimulated phosphorylation of Akt and GSK3beta, increase in cyclin D1 and cell cycle progression, and these events were blocked by the Akt activation inhibitor LY294002. An ErbB3-specific antisense oligonucleotide reduced amounts of ErbB3 protein and p85 complex in both cell lines, and significantly suppressed cell proliferation. These results together suggest involvement of ErbB3 in growth of lung adenocarcinomas, through activation of phosphoinositidyl 3 kinase and Akt, inactivation of GSK3beta and stabilization of cyclin D1 for cell cycle maintenance. It could be a useful therapeutic target.
In many human lung adenocarcinoma cell lines, a pathway involving epidermal growth factor receptor (EGFR), ErbB2 and ErbB3 receptors, phosphatidyl inositol 3-kinase (PI3K), Akt, glycogen synthase kinase 3- (GSK3-), and cyclin D1 controls cell growth, survival, and invasiveness. We have investigated this pathway in paired transformed/ nontransformed cell lines from murine peripheral lung epithelium, E9/E10 and A5/C10. The E9 and A5 carcinoma lines expressed ErbB3 and transforming growth factor-␣ (TGF-␣) and responded to TGF-␣ stimulation with protein complex formation including the p85 regulatory subunit of PI3K, activation of Akt, phosphorylation of GSK3-, and increased cyclin D1 protein and the cell cycle. ErbB3 and TGF-␣ were not detected in the nontransformed E10 and C10 cell lines. Nevertheless, exposure of E10 or C10 cells to TGF-␣ activated PI3K and Akt and increased cyclin D1 and cell growth. The effector pathway from the EGFR to PI3K in these nontransformed cells included the adaptor Grb2, the docking protein Gab1, and the phosphatase Shp2. Gab1 was highly expressed in E10 and C10 cells but not in the malignant E9 and A5 sister lines. Complexes of EGFR/Grb2/ Gab1/Shp2 after TGF-␣ stimulation were prominent only in E10 and C10 cells. Thus, alternate pathways downstream of EGFR regulate mitosis in these paired malignant versus nontransformed lung cell lines.Keywords: EGFR; ErbB3; Gab1; Akt; lung epithelial cells Complex interactions of growth factors, receptors, and signaling pathways regulate normal cell division of lung epithelial cells and, in their derangement, contribute to the development of malignancy. Systematic distinction of essential from secondary changes is facilitated by the availability of two pairs of cell lines derived from cells of the peripheral lung epithelium of BALB/c female mice, which grew out after explanting the lungs (1, 2). Each of these pairs was derived from a single immortalized cell line, one subline of which underwent spontaneous malignant transformation to an adenocarcinoma line showing growth in soft agar and as nude mouse xenografts. These pairs are designated E9/E10 and A5/C10, where E9 and A5 are the malignant lines.The presence of lamellar bodies and cytoskeletal features indicated that type II cells may have been the origin of these lines (1, 2), confirmed by specific staining with antibodies to cytokeratins (3). We have confirmed lamellar body-like struc- tures in E10 and C10 cells by electron microscopy (unpublished data). E10 and C10 present 2-5% S-phase cells at confluence, compared with 20-30% of E9 and A5 cells (3). The two nontransformed cell lines, compared with the two transformed lines, have higher levels of fibronectin, laminin, and vitronectin; more organized cytoskeletons; more numerous gap junctions; and more glucocorticoid-and platelet-derived growth factor receptors (3).We have used these paired lines to investigate mechanisms of regulation of cell growth via the epidermal growth factor receptor (EGFR) ErbB1 in malignant compared with nontransforme...
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