A140
The Effect of type 1 insulin-like growth factor receptor (IGF1R) signalling on sensitivity of melanoma cells to Temozolomide (TMZ)
Roger Ramcharan1, Nicholas Thomas1, Johann Riedemann1, Anthony Yeh1, Amanda Watson2, Geoff Margison2, Mark Middleton3, Valentine Macaulay1
1Weatherall Institute of Molecular Medicine, Oxford, UK, 2Paterson Institute for Cancer Research, Manchester, UK, 3Churchill Hospital, CRUK, Oxford, UK
Background
The IGF1R is a potent survival factor and is upregulated in malignant melanoma compared with benign naevi. Metastatic melanoma is highly chemoresistant, although shows some response to TMZ, a DNA methylating agent. TMZ resistance can be associated with high levels of the DNA repair protein, O6methylguanine-DNA methyltransferase (MGMT). Previously we showed that IGF1R depletion using small interfering RNA (siRNA) is able to induce apoptosis, reduce melanoma cell survival, and enhance sensitivity to TMZ.
Method
We conducted a screen for proteins that are regulated by the IGF1R, using antibody arrays and multiplex and conventional immunoblotting. The initial screen was performed using siRNA to deplete IGF1R in human breast cancer cells. The consequences for melanoma biology were pursued in human melanoma cells by measuring MGMT expression and activity, using immunoblotting, real time PCR, and oligonucleotide-based activity assays.
Results
MGMT was identified as one of the few proteins up-regulated by IGF1R depletion in breast cancer cells. Similarly, two-fold up-regulation of MGMT was induced in CHL1 human melanoma cells, following transfection of two different IGF1R siRNAs (siRNA1, p<0.02; siRNA2, p<0.05 compared with non-silencing control siRNA). This effect was shown to be unrelated to changes in cell confluence and was reproduced in three other human melanoma cell lines. The reverse effect was demonstrated following IGF1 stimulation, which caused a reduction in levels of MGMT protein. IGF1R depletion also induced modest (1.5-fold) increase in MGMT activity in whole cell extracts (siRNA1, p=0.054; siRNA2, p=0.028 compared with control).
Conclusion
IGF1R depletion sensitises to TMZ, but paradoxically up-regulates MGMT at the protein level. We are testing effects of IGF1R depletion on base adduct formation and removal, and on the transcription, stability and subcellular localisation of MGMT. These studies may inform the evaluation of IGF1R inhibitors in melanoma treatment.
Acknoledgements
Supported by CR-UK and the NIHR BRC programme.
