The effects of adrenomedullin overexpression in breast tumor cells.

Abstract

Adrenomedullin is a secreted peptide hormone with multiple activities. Several reports have indicated that adrenomedullin may be involved in tumor survival, but this has not been directly shown. Here we evaluate the in vitro and in vivo effects of adrenomedullin overexpression in human breast cancer cells. The human breast cancer cell lines T47D and MCF7, both of which express low basal levels of adrenomedullin, were stably transfected with an expression construct that contained the coding region of the human adrenomedullin gene or with empty expression vector. Properties of the transfected cells were assessed by proliferation and apoptosis assays, in vitro and in vivo angiogenesis assays, cell migration experiments, and xenograft implants. The effect of synthetic adrenomedullin on human ovarian (ECV) cancer cell motility was also tested. Western blot analysis was used to compare expression levels of several genes whose products are associated with cell growth and regulation of apoptosis. T47D and MCF7 cells transfected with the adrenomedullin construct both expressed high levels of adrenomedullin mRNA and protein. Compared with cells transfected with empty vector, cells that overexpressed adrenomedullin displayed a more pleiotropic morphology, an increased angiogenic potential both in vitro and in vivo, and less apoptosis after serum deprivation. T47D and MCF7 cells did not display measurable motility, but ECV ovarian cancer cells treated with synthetic adrenomedullin were more motile than saline-treated ECV cells. Adrenomedullin-overexpressing T47D cells had higher levels of proteins involved in oncogenic signal transduction pathways (such as Ras, Raf, PKC, and MAPKp49) and lower levels of pro-apoptotic proteins (such as Bax, Bid, and caspase 8) than T47D cells transfected with empty vector. In a preliminary in vivo experiment, three of 10 nude mice injected with adrenomedullin-overexpressing T47D cells developed xenograft tumors, whereas none of the 10 nude mice injected with cells carrying the empty plasmid developed tumors. These results further support the role of adrenomedullin as a survival factor for tumors. Development of physiologically efficient inhibitors of adrenomedullin may prove useful in the clinical management of cancer.