Cavanna, T.C.L.; (2006) Microarray expression analysis of metastasising sarcoma cells implies a role for protein 4.1b in metastasis. Doctoral thesis , University of London.

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Abstract

The identification of the genes underlying the mechanisms of metastasis is of great interest, since metastatic disease is the major cause of death in cancer patients. To investigate the possible mechanisms of metastasis, I used an inbred rat sarcoma model and analysed four related cell populations with significant differences in their ability to shed metastases when subcutaneously injected into inbred rats (P < 0.01). I characterised the motility and morphology of the four cell populations and found that the metastatic cells had a stronger chemotactic response to PDGF/IGF, migrated faster, and had fewer stress fibres, than the non-metastastic cells. Next I performed microarray analysis to investigate the gene expression differences underlying the progression to the metastatic phenotype. I examined gene expression in the cultured cells, and in the tumours that formed after subcutaneous injection of the cells into rats. In this way, I was able to identify genes whose expression was significantly changed with metastatic potential, both in cultured cells and in primary tumours. Twenty-three genes were differentially expressed more than 2.5-fold in metastatic cells (P < 0.05). The gene encoding protein 4.IB was down-regulated in the metastatic cells. To investigate the possible function of 4.IB, I reduced its expression in non-metastatic cells by RNA interference (RNAi). Cells with reduced 4. IB expression displayed an altered F-actin morphology, with a loss of stress fibres compared to control cells. The stress fibre phenotype was rescued by transfection of an RNAi-resistant 4. IB cDNA, showing that the effect was specific. I also found that the 4. IB RNAi cells migrated at twice the speed of the wild-type cells. I conclude that the loss of 4.IB in the metastatic cells causes a significant loss of actin stress fibres and increase in cell speed, and thus plays a role in progression to metastatic phenotype.

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