Aberrant O-linked glycosylation is seen in many cancers and in 90% of breast carcinomas. Breast cancers and especially those expressing the estrogen receptor overexpress two sialyltransferases, ST6GalNAc-II and ST3Gal-I. This results in these tumours carrying core 1 rather than the core 2 glycans observed in the normal mammary gland. We will: 1.Determine how over-expression of ST3Gal-I contributes to tumorigenesis. Expressing this sialyltransferase in the mammary gland results in earlier development of tumours when mice transgenic for ST3Gal-I are crossed with a model of mammary cancer (PyMT mice). We will determine if expression of ST3Gal-I in the virgin gland alters their development by microscopically comparing morphology to control glands. Mammary glands from ST3Gal-I mice x PyMT mice will then be examined for morphology, glycan expression and immune infiltration using histochemistry, applying the proximity ligation assay when necessary. Changes in signalling will be analysed by co-immunprecipitations and Western blots looking at pathways activated in PyMT tumours, many of which are also activated in human cancers. 2.Investigate how the O-linked glycosylation of two glycoproteins influences tumour growth. Using a breast cancer line engineered to express MUC1 carrying core 2 or core 1 glycans, we will study the effect of these glycans on its interaction with EGFR after EGF stimulation using immunofluorescence microscopy. The transcriptome of the cells lines carrying MUC1 with core 1 and core 2 glycans will also be established. Osteopontin (OPN) acts as an instigator stimulating growth of tumour cells. We will determine the glycosyltransferase expression (by qRT-PCR) and glycomic profile (by mass spectrometry) of cells expressing OPN that can stimulate cell growth and engineer the glycosylation of another cell type by shRNA and/or transfection of glycosylatransferases to mimic this glycosylation. The activity of OPN will be tested in murine models.