Date of Award

2013

Degree Name

Master of Philosophy (MPhil)

Schools and Centres

Health Sciences

First Supervisor

Steven E. Mutsaers

Second Supervisor

Cecilia Prêle

Third Supervisor

Bahareh Badrian

Abstract

Malignant mesothelioma (MM) is an aggressive cancer, the consequence of which is neoplastic transformation of mesothelial cells and is strongly associated with previous asbestos exposure. The disease is associated with poor patient survival due to difficulties in diagnosis and treatment options currently available. Therefore, it is essential to identify novel molecular targets that can be used to develop new treatment opportunities. Hedgehog (Hh) and transforming growth factor-beta (TGF-β) signalling pathways are involved in numerous overlapping developmental processes as well as tissue repair and tumorigenesis. Recently, TGF-β has been shown to induce the expression of Hh signalling effector molecules, the Gli transcriptional factors in various cancer cell types. Our laboratory has been interested in assessing the interaction between the TGF-β and Hh signalling pathways and how this interaction may contribute to tumour growth and metastasis in MM.

This study will test the hypothesis that TGF-β induces Gli1 and Gli2 expression in MM cells through activation of the SMAD signalling pathway. To test the hypothesis, this study addresses three aims. Aim 1, to examine the effect of TGF-β on Gli expression. Aim 2, to determine if TGF-β-induced Gli mRNA is independent of the Hh signalling pathway and to examine the effects of blocking TGF-β on Gli mRNA levels and aim 3, to determine the signalling pathway through which TGF-β regulates Gli expression.

MM and control mesothelial cells all expressed variable amounts of basal TGF-β1 and TGF-β2 mRNA with most MM cells expressing quantitatively higher TGF-β1 but lower TGF-β2 mRNA than controls. Basal levels of Gli1 and Gli2 mRNA were variable across the cell lines but Gli2 was upregulated in all cells upon TGF-β2 stimulation. TGF-β1 had no effect on Gli1 or Gli2 mRNA levels and TGF-β2 had no effect on Gli1 mRNA levels. These findings are different to previous studies in different cancers where TGF-β1 has been shown to upregulate early expression of Gli2 mRNA and subsequent upregulation of Gli1. This is the first time the effect of TGF-β2 has been examined in any cell line.

The mechanism of TGF-β2-mediated Gli2 mRNA expression was also examined using pharmacological inhibitors to the type I TGF-β receptor ALK5, and siRNA against key mediators of the TGF-β SMAD signalling pathway, SMAD2 and SMAD3. Inhibition of ALK5 and SMAD2 and SMAD3 inhibited TGF-β2-induced Gli2 expression, confirming that TGF-β2-mediated Gli2 expression is SMAD2/3 dependent. Further studies are required to determine if TGF-β2-mediated Gli2 expression is independent of the classical Hh signalling Patched/Smoothened (Ptch/SMO) receptor complex.

In conclusion, TGF-β2 was identified as a potent transcriptional inducer of Gli2 transcription factor. These findings may be valuable in providing new therapeutic opportunities to treat MM using targeted TGF-β and Gli therapies. Further studies are now required to examine the functional importance of TGF-β2-mediated Gli2 expression in MM cell function and tumour growth.

In conclusion, TGF-β2 was identified as a potent transcriptional inducer of Gli2 transcriptional factor. These findings may be valuable in providing new therapeutic opportunities to treat MM using targeted TGF-β2 and Gli2 therapies. However, to make a definitive conclusion about this aspect, further studies need to be completed examining their functional importance in MM tumour growth.

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