Wood, MK; (2005) The role of transforming growth factor beta in mesothelioma collagen production and tumour growth. Doctoral thesis , UCL (University College London).

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Abstract

Malignant Mesothelioma (MM) is an aggressive, fibrotic tumour predominantly of pleural origin. It is poorly understood, insensitive to conventional therapy and its incidence is increasing with a peak predicted around 2020. Its fibrous nature is.due to abundant extracellular matrix (ECM) deposition with collagen as its major component. ECM has essential roles in many cancers as a scaffold for tumour growth and by interacting with tumour cells it is essential in tissue invasion, metastasis, angiogenesis and protection of tumours from chemotherapy. MMs produce growth factors, including TGFp, which is pro-angiogenic, immunosuppressive, and a potent stimulator of collagen production. TGFp signals predominantly through a unique signalling system - the Smad pathway. This involves Smad7, a negative feedback inhibitor of Smad signalling, which when over-expressed has been shown to inhibit TGFp activity. This thesis examined the role of TGFp and collagen in MM tumour growth, using 7 MM cell lines derived from 3 murine and 4 human tumours. In vra>, a murine model was used to assess tumour growth following subcutaneous inoculation with syngeneic MM cells. MM cell TGFp production was assessed using a mink lung reporter assay, which confirmed high levels of TGFp synthesis. High-pressure liquid chromatography (HPLC) was used to assess collagen production and MM cells found to synthesise large quantities of collagen, responding to TGFp stimulation by increasing production. Furthermore, tumours contained abundant collagen, assessed histologically and using HPLC. Two approaches were used to inhibit TGFp activity TGFp neutralising antibodies and transfection of Smad7. Assessment of collagen responses, at gene level (reporter assays) and protein level (HPLC), following inhibition of TGFp found that TGFp antibodies reduced the collagen response, but Smad7 transfection resulted in increased basal collagen production and no inhibition of TGFp. Tumour growth and collagen production were reduced in mice systemically treated with TGFp neutralising antibodies, but tumours derived from Smad7 transfected cells grew larger than controls. These paradoxical findings suggested the possibility of either alternative non-Smad signalling pathways or altered receptor expression. Smad and MAPK signalling were assessed using Western blotting and Smad found to be normally activated. MAPK signalling (ERK1/2 and p38 kinase) was found to be directly activated by TGFp, but inhibition using specific inhibitors found that these pathways may play a role in regulating basal collagen production but were not the signalling route for the TGFp response. Finally, MM tumour expression of TGFp type I receptors (TpRI) were examined and both the ALK1 and ALK5 isoforms found to be present. This novel finding of ALK1 was unexpected and its responses have previously been shown to oppose ALK5 effects. Conclusion: This work confirms the importance of TGFp and collagen in MM with inhibition of TGFp causing a reduction in tumour growth. Transfection of Smad7 resulted in paradoxical findings that might relate to changes in the expression of different isoforms of the TGFp type I receptor in MM. These novel findings warrant further investigation that may lead to new therapeutic targets.

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