The progression from preinvasive lesion to invasive carcinoma is a critical step contributing to breast cancer lethality. We identified downregulation of milk fat globule-EGF factor 8 (MFG-E8) as a contributor to breast cancer progression using microarray analysis of laser capture microdissected (LCM) tissues. We first identified MFG-E8 downregulation in invasive lesions in transgenic mammary tumor models, which were confirmed in LCM-isolated human invasive ductal carcinomas compared with patient-matched normal tissues. In situ analyses of MFG-E8 expression in estrogen receptor (ER) positive cases confirmed its downregulation during breast cancer progression and small inhibitory MFG-E8 RNAs accelerated ER þ breast cancer cell proliferation. MFG-E8 also decreased in erbB2 þ human cancers and erbB2 transgenic mice lacking MFG-E8 showed accelerated tumor formation. In contrast, MFG-E8 expression was present at high levels in triple-negative (ER À , PgR À , erbB2 À ) breast cancers, cell lines, and patient sera. Knockdown, chromatin immunoprecipitation, and reporter assays all showed that p63 regulates MFG-E8 expression, and MFG-E8 knockdowns sensitized triple-negative breast cancers to cisplatin treatment. Taken together, our results show that MFG-E8 is expressed in triple-negative breast cancers as a target gene of the p63 pathway, but may serve a suppressive function in ER þ and erbB2 þ breast cancers. Its potential use as a serum biomarker that contributes to the pathogenesis of triple-negative breast cancers urges continued evaluation of its differential functions. Cancer Res; 71(3); 937-45. Ó2010 AACR.

Estrogen and progesterone are the defining hormones of normal female development, and both play critical roles in breast carcinogenesis. Cyclin D1 is a breast cancer oncogene whose amplification is linked to poor prognosis in estrogen and progesterone receptor-positive breast cancers. Here we report that cyclin D1 regulates progesterone receptor expression, consequently enhancing responses to estrogen and progesterone. Estrogen treatment of cyclin D1 transgenic mice increased progesterone receptor expression and induced mammary hyperplasias that were stimulated by progesterone and blocked by a progesterone antagonist. Progesterone receptor levels decreased in cyclin D1 knockout mice. Cyclin D1 regulated progesterone receptor expression through a novel estrogen-and cyclin D1-responsive enhancer in DNA encoding part of the 3 untranslated region of the progesterone receptor gene. Small inhibitory RNAs for cyclin D1 decreased progesterone receptor expression and estrogen receptor binding to the 3 enhancer region in human breast cancer cells. Since estrogen and progesterone regulate cyclin D1, our results suggest that cyclin D1's participation in a feed-forward loop could contribute to increased breast cancer risks associated with estrogen and progesterone combinations. Additionally, its regulation of the progesterone receptor identifies a novel role for cyclin D1 in ovarian hormone control of breast development and breast carcinogenesis.We have long known that ovarian hormones control normal breast development (13). Likewise, the connection between ovarian hormones and breast cancer was first therapeutically exploited over 100 years ago (3). While estrogen and progesterone (Pgr) combine to regulate normal breast development during pregnancy, the Women's Health Initiative study of hormone replacement has clearly demonstrated an increased breast cancer risk when progestins are combined with estrogen (7). This result reinvigorated the view that combinations of progesterone with estrogen can be carcinogenic.Cyclin D1 (CCND1) is a multifunctional G 1 -phase cyclin whose regulatory effects are particularly important in breast development and cancer (44). The cloning of cyclin D1 at 11q13 translocation breakpoints in parathyroid and mantle cell tumors was strong evidence of its oncogenic function, and evidence indicating that D1 was a G 1 cyclin was a key finding that linked cancer to perturbations of the cell cycle. Extensive evidence links cyclin D1 to cyclin-dependent kinase regulation of the cell cycle (27), although a recent report demonstrates that it also acts in transcriptional control (4). 11q13 amplification has been repeatedly demonstrated in breast cancers (22) where CCND1 overexpression has been linked to estrogen and progesterone receptor (PR) status. This linkage was first attributed to cyclin D1's regulation by estrogen and progesterone (12, 37). In this model, CCND1 is proposed to contribute to poor treatment response of estrogen receptor (ER)-positive tumors by acting downstream to promote hormone agonist...