Molecular pathways involved in mediating effects of type 1 insulin-like growth factor receptor (IGF1R) signalling on sensitivity of melanoma cells to Temozolomide (TMZ)


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Roger Ramcharan, Nicholas Thomas, Johan Riedemann, Anthony Yeh, Mark Middleton, Valentine Macaulay

Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK

Abstract

The DNA methylating agent TMZ has limited activity in metastatic malignant melanoma, and TMZ resistance of melanoma cells is associated with high levels of the repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT). The IGF1R is a potent growth and survival factor, and may also influence DNA repair. We are interested in the role of the IGF1R as a potential mediator of chemo-resistance, and showed previously that IGF1R depletion enhances sensitivity of melanoma cells to TMZ. The aim of this study is to investigate the molecular pathways linking IGF signaling to regulation of TMZ sensitivity in human tumour cells. By antibody array screen and multiplex immunoblotting, we identified MGMT as a protein that is significantly up-regulated following IGF1R depletion in breast cancer cells. Conventional immunoblotting confirmed a similar effect in human CHL-1 melanoma cells, where MGMT levels were up-regulated 2-fold by IGF1R depletion. This effect was induced by different IGF1R siRNAs at low ‘nM’ concentration, rendering ‘off-target’ effects of siRNA unlikely. However this increase in MGMT appears paradoxical, given that it is associated with sensitisation, rather than resistance, to TMZ. We are currently investigating the hypothesis that IGF signaling may influence the methylation, subcellular localisation and/or stability of MGMT protein. We will also assess IGF effects on the expression and function of other proteins, including Poly ADP Ribose Polymerase (PARP), that influence TMZ sensitivity. These studies may shed light on the role of the IGF1R in repair of TMZ damage, an effect that we aim to exploit in the treatment of advanced melanoma.

The DNA methylating agent TMZ has limited activity in metastatic malignant melanoma, and TMZ resistance of melanoma cells is associated with high levels of the repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT). The IGF1R is a potent growth and survival factor, and may also influence DNA repair. We are interested in the role of the IGF1R as a potential mediator of chemo-resistance, and showed previously that IGF1R depletion enhances sensitivity of melanoma cells to TMZ. The aim of this study is to investigate the molecular pathways linking IGF signaling to regulation of TMZ sensitivity in human tumour cells. By antibody array screen and multiplex immunoblotting, we identified MGMT as a protein that is significantly up-regulated following IGF1R depletion in breast cancer cells. Conventional immunoblotting confirmed a similar effect in human CHL-1 melanoma cells, where MGMT levels were up-regulated 2-fold by IGF1R depletion. This effect was induced by different IGF1R siRNAs at low ‘nM’ concentration, rendering ‘off-target’ effects of siRNA unlikely. However this increase in MGMT appears paradoxical, given that it is associated with sensitisation, rather than resistance, to TMZ. We are currently investigating the hypothesis that IGF signaling may influence the methylation, subcellular localisation and/or stability of MGMT protein. We will also assess IGF effects on the expression and function of other proteins, including Poly ADP Ribose Polymerase (PARP), that influence TMZ sensitivity. These studies may shed light on the role of the IGF1R in repair of TMZ damage, an effect that we aim to exploit in the treatment of advanced melanoma.