Abstract Prolactin (PRL) is critical for mammary development and lactation. Epidemiological studies also support a role for PRL in breast cancer. In order to study the contributions of PRL to tumor development and progression, we developed a transgenic mouse model that expresses PRL in the mammary epithelial cells (NRL-PRL), mimicking the mammary PRL production in women. Loss of function of the tumor suppressor p53 is a common occurrence in many human cancers including breast tumors. Interactions between PRL and p53 were examined by crossing the NRL-PRL mouse to p53 knockout mice which were made congenic on the FVB/N background. To circumvent the problem that p53−/− mice are prone to multiple nonmammary tumors, mammary cells from 10–12 week old wild-type, NRL-PRL, p53−/−, and NRL-PRL/ p53−/− were transplanted to wild type mammary glands. Histological analysis of the donor glands showed that NRL-PRL glands displayed marked epithelial proliferation and focally dilated ducts, whereas p53−/− glands exhibited irregular ductal epithelium with increased stromal density compared to wild type mice. In contrast, glands of donor PRL/ p53−/− females showed widespread epithelial hyperplasias and highly irregular ductal epithelium often surrounded by dense stroma. By one year of age, no tumors had developed from either wild-type or NRL-PRL transplanted epithelium. However, recipients of either p53−/− or NRL-PRL/ p53−/− mammary epithelium developed histologically similar anaplastic carcinomas. The presence of transgenic PRL decreased tumor latency (205 vs 244 days) and significantly increased tumor cell proliferation. In addition, these carcinomas appeared to be more aggressive: 5/16 (32%) of the NRL-PRL/ p53−/− tumors invaded into the peritoneal cavity, compared to 0/10 p53−/− tumors. Expression of matrix metalloproteinase 9 (MMP-9), but not MMP-2, was found to be significantly higher in the NRL-PRL/ p53−/−, compared to p53−/− tumors. MMP-9 is regulated by the transcription factor, AP-1, and PRL can signal through AP-1. NRL-PRL/ p53−/− tumors displayed significantly increased expression of the AP-1 family members, c-Jun and FosL, but not JunD or c-Fos compared to tumors from p53−/− mice. In addition, levels of c-Jun and FosL proteins increased in a similar pattern. In summary, interactions between PRL and loss of p53 promote breast cancer by increasing proliferation and invasiveness, potentially via AP-1 target genes. Supported by CDMRP BC053412. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-04-04.