Antisense expression for amphiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line

The epidermal growth factor (EGF) family of receptors and their respective ligands play a major role in breast cancer progression and are the targets of new therapeutic approaches. Following immortalization with SV40 T antige n of normal human breast epithelial cells, a transformed variant cell line (NS2T2A1) was selected for its increased tumorigenicity in nude mice. This cell line was shown to have a higher expression of EGF receptors (EGFR) and amphiregulin (AR) when compared to their normal counterparts or less aggre ssive transformed cells. Dual staining of EGFR and AR was observed in 50-60 % of NS2T2A1 cells, while 30-40% cells expressed AR only, To explore the po tential tumorigenic role of AR, a 1.1 kb AR cDNA in an antisense orientatio n was transfected in NS2T2A1 cells. Three clones, selected by hygromycin B, expressed AR antisense RNA (AR AS1, AR AS2 and AR AS3 cell lines) in which AR protein expression was reduced (ranging from about 50 to <5%), The anch orage-independent growth of AR AS cell lines was reduced to levels ranging from 32.4-6.8% relative to the control cell line transfected with the vecto r alone. The clones expressing AR antisense RNA showed a reversion of the m alignant phenotype when injected in nude mice, since a significant reductio n of tumor intake was observed coincident with a significant tumor mass red uction (>96%), Moreover, intra-tumoral vascularization decreased significan tly in tumors derived from AR AS cells (26.7, 70.7 and 50.4% of control). T hese in vitro and in vivo data reveal the oncogenic nature of AR in transfo rmed breast epithelial cells and imply a role for AR in tumor angiogenesis.