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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, particularly in ER-/HER2- (basal-like) and high grade tumors. We now describe the phenotypic and molecular changes induced in MDA-MB-231 breast carcinoma cells that have the lowest endogenous NET-6 mRNA levels by stable NET-6 transfection. Subcellular localization was examined by confocal microscopy. The growth rate of parental, mock transfected, and NET-6 transfected cells was determined by MTT assay in vitro, as well as in SCID mice. Growth in soft agar and migration in a Boyden chamber assay was measured as well. Cell cycle analysis was performed by flow cytometry. Western blot analysis was performed for a large number of proteins involved in cell cycle control, apoptosis and matrix invasion. Differential gene expression in NET-6 and mock transfected MDA-MB-231 cells was determined in duplicate using the CodeLink Human Whole Genome Arrays from Amersham. A twofold difference in mRNA level was considered significant. Ectopic expression of NET-6 led to loss of filo- and lamellopodia. The protein was localized to the cytoplasm and cell membrane. We observed inhibition of anchorage independent growth, invasion and substrate adhesion. These effects were associated with downregulation of the matrix metalloprotease MMP-3. NET-6 had marked antiproliferative activity, both in vitro and in SCID mice. This effect was largely due to increased apoptosis. In addition, there was a minor increase in the G1 cell cycle fraction. By Western blotting, we identified upregulation of the pro-apoptotic molecules p53, bak and cleaved caspase 3. The levels of cell cycle regulatory proteins were not significantly altered. A gene expression profiling experiment revealed upregulation of 111, and downregulation of 223 transcripts in NET-6 transfected versus mock transfected MDA-MB-231 cells. The downregulated genes included candidate oncogenes, transcription factors, cell membrane markers, intermediate filaments, telomerase, and MMP-1. Our data provide compelling evidence that NET-6 is a potent new breast cancer suppressor gene. NET-6 downregulation appears to have an anti-apoptotic effect.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]