Sprouty in breast cancer

Abstract

Inappropriate activation of the Ras-mitogen activated protein kinase (MAPK) signalling pathway has long been implicated in many types of human cancers, implying that strict regulation of Ras-MAPK signalling is important for regulating the growth and survival of cells. Sprouty (Spry) proteins are endogeneous inhibitors of Ras/MAP kinase pathway that play an important role in the remodeling of branching tissues such as in development of the lung, kidney tubules and vascular system. This study evaluates the potential role of Sprouty proteins in cancer. The expression of Sprys in various types of cancer has been analyzed and Spry 1 and 2 were found to be significantly down-regulated in more than 90% of breast cancers. Spry 2 was also found to be down-regulated in liver cancers. Analysis of the possible genetic and epigenetic mechanisms causing the down-regulation of Spry genes indicates that methylation is not responsible for the down-regulation observed in breast and liver cancer. In liver cancers, no loss of heterozygosity was observed in the microstatellite markers flanking the hSpry2 gene locus. In this study, the suppressed expression and therefore function of Spry was found to potentiate aberrant signalling in tumourigenesis. Overexpression of the putative dominant negative hSpry2Y55F mutant that quenches the function of endogenous wild-type Spry in MCF-7 cells caused cells to proliferate faster, exhibit anchorage-independent growth and loss of contact inhibition. As a result, hSpry2Y55F over-expression in MCF-7 cells also caused the formation of larger tumours in nude mice assay compared to control MCF-7 cells. The knock down of Spry2 expression in NIH3T3 cells was also found to cause NIH3T3 cells to be more susceptible to growth factor induced transformation. The study subsequently demonstrates that Sprys were able to inhibit colony formation induced by Polyoma Middle T antigen (PyMT) oncogene via the inhibition of the activation of the Ras/ERK signalling pathway. NIH3T3-hSpry2 stable cells were more resistant to transformation by PyMT as compared with NIH3T3 control cells. NIH3T3-hSpry2 developed less colonies upon transformation, indicating that the over-expression of Spry2 in NIH3T3 cells inhibited colony formation. In contrast, NIH3T3-hSpry2Y55F stable cells were not as resistant to transformation, indicating that the Tyr55 residue in Spry2 was important for Spry2's ability to inhibit transformation downstream of PyMT. Overall, this thesis shows that Spry proteins are down-regulated in cancers and that they may play a crucial role in preventing cell transformation. Down-regulation of these genes may reduce the threshold for cells to become malignant.