| Summary: | Over-expression of parathyroid hormone-related protein (PTHrP) is commonly described in a number of different forms of cancer and it has been suggested that this over-expression leads to tissue-specific metastasis whereby primary tumours have a propensity to metastasise to one particular organ e.g. breast tumours metastasise to bone whereas gastrointestinal tumours favour the liver. The aim of my PhD was to examine the role PTHrP plays in cancer cell adhesion to the extracellular matrix (ECM), to explore a mechanism of action and to elucidate any tissue-specific differences to explain the apparent partiality during metastasis. In order to do this a small interfering RNA was used to silence PTHrP gene expression and expression vectors containing cDNA for PTHrP were used to create several stable PTHrP over-expressing cell lines. Analysis of cell adhesion revealed that regulating PTHrP expression caused changes in adhesion to the ECM proteins collagen type I, fibronectin and laminin in breast cancer cell lines. However although cell adhesion of gastrointestinal cancer cell lines to collagen type I and fibronectin was similarly affected, adhesion to laminin was unchanged by variations in PTHrP expression. The cell adhesion molecules integrins were subsequently investigated for their role in PTHrP mediated cell adhesion. Integrins are heterodimers composed of an α and β subunit and to date 24 different subunits have been identified. Each unique combination of subunits results in a different ligand specificity, which includes collagens, fibronectin and laminin. Analysis of integrin gene and protein expression in over-expressing cell lines and in cells where PTHrP had been silenced it was possible to demonstrate a link between expression of PTHrP and a number of different integrin subunits. One of the more significant findings was the observation that the integrin subunit α6 changed in parallel with PTHrP in breast but not gastrointestinal cancer cell lines. As laminin is a ligand for this subunit these results correlate with the results previously described regarding this ECM protein. The effects of PTHrP appeared to be mediated at the transcriptional rather than the translational level so integrin transcriptional activity was investigated using a reporter vector containing the coding region for firefly luciferase. The integrin subunit α5 had shown a link to PTHrP expression in both breast and gastrointestinal cell lines so the promoter region for this subunit was inserted into the reporter vector, which was then transiently transfected into PTHrP over-expressing cell lines. Subsequent examination of luciferase activity revealed a significant increase in promoter activity compared with control thus implicating PTHrP as a key factor in integrin gene transcription. The results described here suggest that PTHrP increases cell adhesion by inducing integrin gene transcription and is able to regulate expression of different subunits in different tissues thereby encouraging tissue-specific metastasis.
|
|---|
