Independent of ErbB1 gene copy number, EGF stimulates migration but is not associated with cell proliferation in non-small cell lung cancer

Abstract Background Lung cancer often exhibits molecular changes, such as the overexpression of the ErbB1 gene. ErbB1 encodes epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, involved mainly in cell proliferation and survival. EGFR overexpression has been associated with more aggressive disease, poor prognosis, low survival rate and low response to therapy. ErbB1 amplification and mutation are associated with tumor development and are implicated in ineffective treatment. The aim of the present study was to investigate whether the ErbB1 copy number affects EGFR expression, cell proliferation or cell migration by comparing two different cell lines. Methods The copies of ErbB1 gene was evaluated by FISH. Immunofluorescence and Western blotting were performed to determine location and expression of proteins mentioned in the present study. Proliferation was studied by flow cytometry and cell migration by wound healing assay and time lapse. Results We investigated the activation and function of EGFR in the A549 and HK2 lung cancer cell lines, which contain 3 and 6 copies of ErbB1, respectively. The expression of EGFR was lower in the HK2 cell line. EGFR was activated after stimulation with EGF in both cell lines, but this activation did not promote differences in cellular proliferation when compared to control cells. Inhibiting EGFR with AG1478 did not modify cellular proliferation, confirming previous data. However, we observed morphological alterations, changes in microfilament organization and increased cell migration upon EGF stimulation. However, these effects did not seem to be consequence of an epithelial-mesenchymal transition. Conclusion EGFR expression did not appear to be associated to the ErbB1 gene copy number, and neither of these aspects appeared to affect cell proliferation. However, EGFR activation by EGF resulted in cell migration stimulation in both cell lines.

Decreased expression of ADAMTS-1 in human breast tumors stimulates migration and invasion

Abstract Background ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a member of the ADAMTS family of metalloproteases. Here, we investigated mRNA and protein levels of ADAMTS-1 in normal and neoplastic tissues using qPCR, immunohistochemistry and immunoblot analyses, and we addressed the role of ADAMTS-1 in regulating migration, invasion and invadopodia formation in breast tumor cell lines. Results In a series of primary breast tumors, we observed variable levels of ADAMTS-1 mRNA expression but lower levels of ADAMTS-1 protein expression in human breast cancers as compared to normal tissue, with a striking decrease observed in high-malignancy cases (triple-negative for estrogen, progesterone and Her-2). This result prompted us to analyze the effect of ADAMTS-1 knockdown in breast cancer cells in vitro. MDA-MB-231 cells with depleted ADAMTS-1 expression demonstrated increased migration, invasion and invadopodia formation. The regulatory mechanisms underlying the effects of ADAMTS-1 may be related to VEGF, a growth factor involved in migration and invasion. MDA-MB-231 cells with depleted ADAMTS-1 showed increased VEGF concentrations in conditioned medium capable of inducing human endothelial cells (HUVEC) tubulogenesis. Furthermore, expression of the VEGF receptor (VEGFR2) was increased in MDA-MB-231 cells as compared to MCF7 cells. To further determine the relationship between ADAMTS-1 and VEGF regulating breast cancer cells, MDA-MB-231 cells with reduced expression of ADAMTS-1 were pretreated with a function-blocking antibody against VEGF and then tested in migration and invasion assays; both were partially rescued to control levels. Conclusions ADAMTS-1 expression was decreased in human breast tumors, and ADAMTS-1 knockdown stimulated migration, invasion and invadopodia formation in breast cancer cells in vitro. Therefore, this series of experiments suggests that VEGF is involved in the effects mediated by ADAMTS-1 in breast cancer cells.