Cellular and molecular basis of mammary microcalcifications
Mammary microcalcifications represent one of the most reliable mammographic features of non-palpable breast cancer and are often the sole indicator of the disease. However, it is unknown whether these microcalcifications are a sign of degeneration or an active cellular process. The aims of this project were to establish and characterise an in vitro model of mammary mineralisation in monolayer, 3D scaffolds and in vivo and to investigate the molecular mechanisms involved in this process, focusing on the potential roles bone matrix proteins and microRNAs. Several mammary cell lines were capable of depositing hydroxyapatite in vitro when treated with an osteogenic cocktail (10mM p-glycerophosphate and 50pglml ascorbic acid). Mineralisation potential was found to be associated with more aggressive mammary cell phenotypes. Increasing concentrations of p-glycerophosphate, calcium and exogenous hydroxyapatite enhanced 4T1 cell migration. In contrast, exogenous calcium oxalate had no effect. Real-time RTPCR detected changes in the expression of alkaline phosphatase (ALP), bone sialoprotein, osteopontin (OPN) and collagen type I in mineralising 4T1 cells. These are all known markers of osteoblast mineralisation. 4T1 cells were also capable of mineralising within 3D collagen scaffolds when treated with the osteogenic cocktail. In contrast, no mineralisation was detected following BMP217 treatment of the 4T1 cells in the scaffolds. Further studies found that BMP2 enhances the mineralisation of 4T1 cells in vitro, however an exogenous source of phosphate is essential to this process. The in vivo mineralisation potential of 4T1 and 4T1.2 cells was also examined. These cells were implanted in the mammary fat pad of BALBIc mice and mineralisation was confirmed in 6 out of 11 primary tumours. Inhibition of ALP and sodium-phosphate cotransporter pumps by treatment with levamisole and phosphonoformic acid respectively, inhibited rnineralisation of 4T1 cells. Exogenous ALP was found to enhanced mineralisation. Exogenous OPN and pyrophosphate had no effect on mineralisation of 4T1 cells, however this may be due to high endogenous levels of ALP which would counteract the potential inhibitory effects of OPN and pyrophosphate. The 4T1 cells were found to have greater ALP activity in comparison to the non-mineralising MCFlOa cell line. The potential role of microRNAs in mammary mineralisation was examined using microRNA microarrays. 25 differentially regulated microRNAs were identified in mineralising of 4T1 cell. Many of the predicted targets of these microRNAs are regulators of osteoblast mineralisation. Of particular interest are miR-20-5p, which was upregulated and mmu-miR- 133a, which was downregulated as the same pattern of expression has been reported in the literature for physiological osteogenesis. This is the first time that mammary cell lines have been shown to be capable of mineralising in vitro. The results presented here indicate that the formation mammary microcalcifications may be an active regulated process and several markers of osteoblast mineralisation have been identified that may play a key role in this process.