Abstract 1007: Cross-talk between the Prolactin receptor and HER2/neu in breast cancer is dependent upon tumor stromal cells

Abstract

Abstract Background: Prolactin (PRL), a polypeptide hormone, has been implicated in breast tumorigenesis via promoting cell proliferation and survival. One of the mechanisms by which PRL is linked to breast cancer is through cross-talk with other oncogenic growth factors including human epidermal growth factor receptor 2 (HER2/neu). Studies have demonstrated that PRL is able to cross-activate HER2/neu and that a PRL receptor (PRLR) antagonist, G129R, is able to inhibit HER2/neu phosphorylation (pHER2/neu) in established breast cancer cell lines. In this study, we investigate the role of tumor associated fibroblasts (stromal cells) in this molecular cross-talk process, using natural tumors and tumor epithelial and fibroblast cell lines derived from female MMTV/neu transgenic mice. Methods: (1) Primary tumor studies. Mammary tumors from MMTV/neu transgenic mice were surgically removed and divided into two portions. One portion was cut into small chunks (3 mm3 cubes) and cultured three-dimensionally. The other portion was minced and digested into a single cell suspension and cultured as a monolayer. (2) Direct co-culture of tumor epithelial and fibroblasts studies. A tumor epithelial cell line, McNeuA, and tumor associated fibroblast cell line, N202Fb3, derived from a MMTV/neu mammary tumor (kindly provided by Dr Campbell, UC San Francisco) were cultured alone or mix-cultured at different ratios. A normal mouse fibroblast cell line was used as control. (3) Transwell indirect co-culture studies. McNeuA and N202Fb3 were co-cultured indirectly using a transwell system to allow exposure to soluble factors secreted by both cell types. Tumor cells in all three experimental conditions were treated with various concentrations of either PRL or G129R for 1-, 6-, 24-, or 48 hours. The effects of the treatments on pHER2/neu and downstream signaling molecules were evaluated by immunoblotting. Results: (1) G129R inhibited the pHER2/neu in a dose-dependent manner (IC50=10μg/ml) after 24hr treatment in the tumor chunks (three-dimensional model), but had no effect in the monolayer primary culture; (2) Direct co-culture of McNeuA and N202Fb3 restored the response of McNeuA to G129R similar to that of tumor chunks with a best result at 4 (McNeuA) : 1 (N202Fb3) ratio. The inhibitory effect of G129R was reversed by addition of PRL. Furthermore, the inhibitory effect of G129R in McNeuA was vanished when N202Fb3 was replaced with normal fibroblasts. (3) Similarly, G129R had no effect upon McNeuA when N202Fb3 were separated in the transwell co-culture system. Conclusions: In this study we have demonstrated that the tumor stromal cells, in particular, the tumor associated fibroblasts, play an important role in bridging the cross-talk between PRLR and HER2/neu. The inhibitory effect of G129R on pHER2/neu in tumor epithelial cells is dependent upon physical contact with tumor associated fibroblasts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1007.