Purpose: NET-6 is a largely uncharacterized member of the tetraspanin superfamily. We have recently shown that its expression level was lowest in breast carcinomas with aggressive characteristics. We now describe the phenotypic and molecular changes induced in MDA-MB-231 breast carcinoma cells by ectopic NET-6 expression.
Methods: A GFP-NET-6 construct was transfected into very low expressing MDA-MB-231 cells. The subcellular distribution was studied by confocal microscopy. Cell proliferation in vitro was measured by MTT assay. Subcutaneous tumor formation in SCID mice was also studied. Other phenotypic parameters measured included growth in soft agar and extracellular matrix invasion. The effect of NET-6 transfection on the cell cycle was interrogated by flow cytometry. Deregulation of metalloproteinase expression was investigated by RT-PCR. Deregulation of proteins involved in apoptosis and cell cycle control was studied by Western blotting.
Results: Ectopic expression of NET-6 inhibited anchorage independent growth and invasion in a Boyden chamber assay. These effects were associated with downregulation of the matrix metalloproteinases MMP-1 and MMP-3. NET-6 had marked antiproliferative activity, both in vitro and in SCID mice. This effect was largely due to increased apoptosis. We identified upregulation of the pro-apoptotic molecules p53, bax, bak and caspase 3.
Conclusions: Our data provide novel and compelling evidence that NET-6 is a potent new breast cancer suppressor gene.