Abstract P1-01-09: BMP4 suppresses the progression of breast cancer through altered expression of metastasis regulating genes

Abstract

Abstract Metastasis is a lethal manifestation of cancer, the development of which is the major cause of death in cancer patients. During a search for metastasis-regulating elements, an inverse correlation was identified between the in vivo tumor expression of bone morphogenetic protein-4 (BMP4) and spontaneous metastasis in a panel of isogenic mammary tumors of varying metastatic capacity. BMP4 is an essential morphogen in development, regulating cellular mechanisms akin to those in metastasis, including cellular differentiation, pluripotency and apoptosis. We therefore initiated an investigation of the impact of BMP4 expression on the metastatic process. We studied the effect of enforced expression of BMP4 in a highly metastatic mammary tumour model called 4T1.2, comparing in vitro properties and tumour progression in mice. There were no differences in proliferation in vitro or when implanted into the mammary gland of immunocompetent mice. In contrast, mice bearing equivalent-sized 4T1.2-BMP4 tumors revealed dramatically reduced metastasis to lung, lymph node and bone. In a parallel study where the established orthotopic primary tumor was resected, survival was significantly extended in mice bearing 4T1.2-BMP4 tumors. Enforced BMP4 expression in tumor cells introduced intravenously resulted in a 2.5-fold decrease in lung metastatic burden, consistent with the impaired capacity of tumor cells to survive in circulation and colonize the lung. Conversely, silencing BMP4 expression in separate weakly metastatic tumours enhanced their ability to colonize the lung and shortened the survival of the mice. No changes were found in the ability of tumor cells expressing BMP4 or treated with recombinant BMP4 to migrate or invade through Matrigel in chemotactic assays but BMP4 enhanced anoikis in both mouse and human breast cancer cells, indicating that BMP4 sensitizes disseminated cells to anoikic stresses induced by cell-substrate detachment and shear flow during systemic transit. BMP4 activated canonical BMP-SMAD signaling in our mammary tumours, leading to altered expression of known metastasis-regulating genes, including SMAD7. SMAD7 depletion in metastasis-deficient 4T1.2-BMP4 tumors accelerated the onset of metastatic disease. In a meta-analysis of 3,587 breast cancer patients in publically available datasets, low BMP4 mRNA expression was significantly associated with reduced relapse-free survival (RFS) (HR = 0.85, P = 0.01). In an independent analysis using the BreastMark algorithm, low levels of BMP4 mRNA were associated with reduced RFS (HR = 0.88, P = 0.035), distant metastasis-free survival (HR = 0.83, P = 0.035) and overall survival (HR = 0.78, P = 0.006). At the protein level, in a tissue microarray from 415 treatment naïve patients, improved overall survival was observed in multivariate analysis for both BMP4 (HR = 0.66, P = 0.037) and SMAD7 expression (HR = 0.64, P = 0.035) individually. Expression of both proteins compared to neither further improved OS (HR = 0.55, P = 0.005). In summary, we found strong evidence that BMP4 is a metastasis suppressor correlating inversely with metastatic potential in preclinical breast cancer models and predicting improved relapse-free and overall survival in breast cancer patients. Citation Format: Redfern AD, Eckhardt BL, Cao Y, Sloan EK, Parker BS, Loi S, Ueno NT, Lau PK, Latham B, Anderson RL. BMP4 suppresses the progression of breast cancer through altered expression of metastasis regulating genes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-09.