The goal of this study is to determine the effects of expression of the N-terminal 16 kDa prolactin fragment on growth of human breast tumor cells in vitro and tumor development in vivo. Using a rat lymphoma cell line (Nb2), 16 kDa prolactin has been shown to be mitogenic but with only 60% of the potency of equivalent amounts of the 23 kDa full-length prolactin. 16 kDa prolactin also has unique properties when compared to the full-length 23 kDa prolactin. 23 kDa prolactin is pro-angiogenic while the 16 kDa prolactin has been shown to be anti-angiogenic both in vitro and in vivo. Although 16 kDa prolactin is mitogenic in the Nb2 bioassay, in animal models expression of 16 kDa prolactin has been shown to be inhibitory to tumor formation of both prostate and colon cancer cell lines, presumably through its anti-angiogenic effects. Therefore, the tumor formation properties of 16 kDa prolactin may be the converse of those of the full-length 23 kDa prolactin. In addition, the characteristics of 16 kDa prolactin may be quite different in an in vitro assay compared to an in vivo environment. For our studies, we hypothesize that 16 kDa prolactin will confer a growth advantage to breast cancer cells grown in vitro, however, when these same cells are injected into an immune-compromised animal, 16 kDa prolactin expression will be inhibitory to tumor formation. In vivo 16 kDa prolactin is an N-terminal cleavage product of full-length 23 kDa prolactin. To express 16 kDa prolactin in MDA-MB-435 cells, we created an expression construct containing the N-terminal sequence through a stop codon introduced at amino acid 151, one of the in vivo cleavage sites for 16 kDa prolactin. We have made a stable MDA-MB-435 human breast cancer cell line expressing 16 kDa prolactin (435-16K) and secreting biologically active 16 kDa prolactin into the media, as measured by the Nb2 bioassay. We have also characterized the growth properties of the 435-16K cells compared to MDA-MB-435 cells stably transfected with empty vector (435-EV). In normal growth media with 5% fetal bovine serum or in growth media with 1% charcoal-stripped serum, the 435-16K cells proliferate at a significantly higher rate than the 435-EV cells. In soft agar assays 435-16K cells exhibit a greater amount of colony formation than 435-EV cells. While our in vitro studies suggest that 16 kDa prolactin is pro-mitogenic and stimulates soft agar colony formation, ongoing studies are focused on whether these in vitro results translate into altered tumor growth characteristics in nude mice.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA