Advanced melanoma poses a major therapeutic challenge, and despite the development of recent promising therapeutic agents, resistance to treatment remains a problem. Until recently, despite low response rates, alkylating agents dacarbazine and temozolomide (TMZ) were the standard of care for the treatment of advanced melanoma. The cytotoxic effects of these agents relies upon the formation of alkylated base lesions such as O<sup>6</sup>-methylguanine (O<sup>6</sup>MeG), which is repaired by a protein implicated in TMZ resistance called O<sup>6</sup>-methylguanine-DNA methyltransferase (MGMT). Failure to resolve such alkylated bases results in DNA replication-associated double-strand breaks (DSBs). The type 1 insulin-like growth factor receptor (IGF-1R) mediates a number of characteristics common to cancers, including proliferation and survival, and evidence suggests it may also contribute to resistance to many anticancer agents. The aims of this study were to test whether melanoma cells could be sensitized to TMZ by small molecule IGF1R inhibitors, and to explore the mechanism of any chemosensitization. This study found an association between basal IGF1R phosphorylation and in vitro TMZ resistance in seven MGMT-proficient melanoma cell lines, suggesting that IGF1R activation may be linked with TMZ resistance. Furthermore, IGF1R inhibition caused dose-dependent sensitization of melanoma cells to TMZ, regardless of BRAF mutation status. This reduction in cell survival was not accompanied by an increase in apoptosis, but rather Chk2 activation and an accumulation of cells in the G2/M phase of the cell cycle, suggesting a possible effect of IGF1R inhibition on DNA repair. IGF1R depletion was found to increase MGMT protein levels and activity, but this effect was not seen in IGF1R inhibited cells. In addition, IGF1R inhibition was not epistatic with MGMT inhibition, and IGF1R inhibition reduced survival of TMZ treated MGMT-null cells. This suggested that TMZ sensitization by IGF1R inhibition was independent of MGMT. IGF1R inhibition did however cause an increase in the accumulation of TMZ-induced RPA foci, and delay in resolution of RAD51 foci. Together with the finding that IGF1R inhibition reduced survival in PARP inhibited melanoma cells, these results suggested that IGF1R inhibition influenced DSB repair by homologous recombination. Finally, the combination of IGF1R inhibition with TMZ was tested in a mouse model and was found to be tolerable. TMZ or IGF-1R inhibitor alone caused minor reduction in melanoma xenograft growth rates (rate reduction by 13% and 25% respectively), while combination treatment caused supra-additive growth delay (72%) that was significantly different from other treatment groups (p<0.05). The findings of this study suggest IGF1R inhibition as a possible option in overcoming alkylating drug resistance in melanoma.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:640032 |
| Date | January 2014 |
| Creators | Ramcharan, Roger Navine |
| Contributors | Macaulay, Valentine; Middleton, Mark |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:50578edf-5730-42ce-8305-0d52ff61538a |
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