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.

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...

BackgroundRibosomal RNA (rRNA) is a central regulator of cell growth and may control cancer development. A cis noncoding rRNA (nc-rRNA) upstream from the 45S rRNA transcription start site has recently been implicated in control of rRNA transcription in mouse fibroblasts. We investigated whether a similar nc-rRNA might be expressed in human cancer epithelial cells, and related to any genomic characteristics.Methodology/Principal FindingsUsing quantitative rRNA measurement, we demonstrated that a nc-rRNA is transcribed in human lung epithelial and lung cancer cells, starting from approximately −1000 nucleotides upstream of the rRNA transcription start site (+1) and extending at least to +203. This nc-rRNA was significantly more abundant in the majority of lung cancer cell lines, relative to a nontransformed lung epithelial cell line. Its abundance correlated negatively with total 45S rRNA in 12 of 13 cell lines (P = 0.014). During sequence analysis from −388 to +306, we observed diverse, frequent intercopy single nucleotide polymorphisms (SNPs) in rRNA, with a frequency greater than predicted by chance at 12 sites. A SNP at +139 (U/C) in the 5′ leader sequence varied among the cell lines and correlated negatively with level of the nc-rRNA (P = 0.014). Modelling of the secondary structure of the rRNA 5′-leader sequence indicated a small increase in structural stability due to the +139 U/C SNP and a minor shift in local configuration occurrences.Conclusions/SignificanceThe results demonstrate occurrence of a sense nc-rRNA in human lung epithelial and cancer cells, and imply a role in regulation of the rRNA gene, which may be affected by a +139 SNP in the 5′ leader sequence of the primary rRNA transcript.

No abstract