In vitro studies of te novel protein Q7: role on hyaluronian regulation in breast tumor microenvironment

Cancer is a group of multifactorial diseases, which involves variations in multiple genes, often coupled with environmental causes. It is characterized by uncontrolled growth of cells that are able to divide continually and invade into surrounding tissues. In Italy, breast cancer is the most common cancer form among women, accounting for over 20% of the cancer cases and about 15% of the mortality (Jemal et al., 2011). Dysregulation of the composition of the extracellular matrix (ECM) is associated with cancer, by facilitating cell growth, survival and invasion. Among various ECM glycosaminoglycans, hyaluronan (HA) has a remarkable structural importance but also a role in regulating cellular processes through a binding with membrane receptors and activation of signalling pathways. The role of HA in tumour cells’ functions depends on its molar mass which is regulated by the enzymes that synthesize HA, i.e. hyaluronan synthases (HAS), and hyaluronidases (HYALs). Alterations of these metabolic enzymes are correlated with breast cancer progression. In this thesis, we aimed to explore the role of crosstalk between tumour cells and stroma, focusing our attention to the HA regulation. Specifically, we studied the mechanism by which proteins secreted by breast tumour cells alter HA metabolizing enzymes and its synthesis in the stromal cells. Recently, in our laboratory we discovered a new protein in the conditioned medium (CM) of a breast tumour cell line, called “Uncharacterized protein of c10orf118” or “Q7z3e2”. For simplicity, this protein is called “Q7”. Further studies on the two well-known breast cancer cell lines MCF-7 (low invasive cells) and MDA-MB231 (high invasive cells) demonstrated a higher expression and secretion of Q7 in tumour cells than in normal cells. Information obtained from bioinformatics databanks (the Ensemble website) showed that the gene for Q7 is located on human chromosome 10 in the region q25.3. The gene is composed of 18 exons and there are six splicing variants, but only four of them code for proteins. The secreted isoform found in the CM of breast cancer cell lines is the full-length isoform that consists of 898 aminoacids and has a molecular weight of 104 kDa. In the literature and in our data, it was shown that co-culture of breast cancer cells with fibroblasts results to an induction of HAS2 in fibroblasts and an increase of the secreted HA. Among the three HASes, HAS2 isoform was the most expressed and induced by breast tumour cells CM in fibroblasts, whereas HAS1 was not detected. When fibroblasts were treated with CM from MCF-7 cells, in the absence of Q7, the relative expression of HAS2 was significantly decreased. This last data was further confirmed when fibroblasts were treated with a recombinant protein of Q7 and a HAS2 induction and HA increase were observed. To sum up, the data of this thesis demonstrate that the novel protein Q7 may play a key role in the increment of pericellular HA and in the breast tumour progression.