The integrin α v β 3-5 ligand MFG-E8 is a p63/p73 target gene in triple negative breast cancers but exhibits suppressive functions in ER+ and erbB2+ breast cancers.

The progression from preinvasive lesion to invasive carcinoma is a critical step contributing to breast cancer lethality. We identified down-regulation 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 down-regulation 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 down-regulation during breast cancer progression and small inhibitory MFGE8 RNAs accelerated ER+ breast cancer cell proliferation. MFG-E8 also decreased in erbB2+ 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-, PgR-, erbB2-) breast cancers, cell lines and patient sera. Knockdown, ChIP 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+ and erbB2+ 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. p53/p63/p73 binding sites in the MFGE8 promoter, which control transcriptional responses to p63/p73 in skin (18). Here we show that MFG-E8 is a potential biomarker for triple negative breast cancers due to its up-regulation by p63/p73, which contrasts with its down-regulation in ER+ and erbB2+ breast cancers. anti-proliferative of MFG-E8-integrin β 3/ β 5 anti-proliferative we showed this is functionally significant since a MFG-E8 siRNA increased cisplatin sensitivity. These results suggest that antagonists of MFG-E8-integrin signaling should be investigated in triple negative breast cancers and further suggest that circulating MFG-E8 levels might be used to monitor the clinical response of some patients where p63 dysregulation is a major feature of their triple negative breast cancer. MFG-E8 levels vastly exceeded those seen in any other tumor patient in three patients (3,000, 5,000, and 50,000 pg/ml). While these patients represent only 30% of the triple negative patients tested, markers like alpha fetoprotein and carcinoembryonic antigen are useful when found in similar proportions of patients with other cancers. One erbB2 tumor patient had a serum level of 3,670 pg/ml. This level could be due to misclassification, to a mixed tumor phenotype, to co-occurrence of another disease with elevated MFG-E8 levels like systemic lupus erythematosus (25) or to secretion by inflammatory cells in the tumor itself. Importantly, healthy donors exhibited no MFG-E8 levels higher than 366 pg/ml, again suggesting that further explorations of MFG-E8 levels as a serum tumor marker are warranted. Taken together, our results show that MFG-E8 joins a large group of ligands with context-specific functions in cancer. For example, these dual roles are not unlike the dual functions of transforming growth factor- β . Our results provide additional evidence that triple negative breast cancers are phenotypically distinct from other breast cancers and these interactions suggest that integrins might be a druggable target whose ligands need to be evaluated in context as integrin-related therapies are advanced in the clinic. positive), 2+ (moderate positive), and 3+ (strong positive). Comparing specimens staining 2+ or 3+ to those staining 0 or 1+, one ER+ tumor was among the top 13 MFG-E8 expressing tumors, and one ER negative tumors was among the bottom 12 expressing samples. MFG-E8 expression was highly correlated with the ER+ status of breast cancers across all staining intensities (p < 0.001 by Chi square analysis). performed ChIP-PCR for p63 binding as described in supplementary materials. anti-p63 ChIP-PCR signals are plotted as a percentage of the total input DNA for each reaction. We measured α -p63 ChIP DNA in triple negative MDA-MB468 and HCC1937 cells. Shown are the mean and standard deviation for signals at the actin promoter (negative control) versus reported p63 binding sites. Binding at the PUMA promoter was used as positive control (not shown). transgenic mice that express the non-mutated form of erbB2 in their mammary tissues. Resulting MFG-E8 +/- mice were then back-crossed to the MFG-E8 -/- mice and mice of four genotypes were identified using PCR-based genotyping: non-transgenic-MFG-E8 +/- , erbB2 transgenic-MFG-E8 +/- , non-transgenic-MFG-E8 -/- , and erbB2 transgenic-MFG-E8 -/- . Mice were evaluated weekly for palpable masses and scored as upon of a stained tumors arose compound