E2F1/IGF-R Loop Contributes to BRAF Inhibitor Resistance in Melanoma

Abstract

Background: BRAF inhibitors have successfully treated melanoma patients harboring BRAF mutation. However, nearly all these patients developed resistance to BRAF inhibitors. The molecular mechanisms of BRAF inhibitor resistance in melanoma are not fully understood. Methods: BRAF inhibitor acquired resistant melanoma cell lines were generated by continuous exposure to increasing concentration of PLX4032. E2F1, IGF-1R and AKT overexpression/knockdown were performed by lentivirus transfection. The binding activity of E2F1 on the promoter of IGF-1R were determined by CHIP and CHIP-qPCR. Findings: E2F1 was highly expressed in BRAF inhibitor resistant melanoma. The increases of E2F1 in resistant melanoma was partially caused by PI3K/AKT activation. Overexpressed E2F1 mediated resistance to BRAF inhibitor in melanoma, while knockdown of E2F1 reversed BRAF inhibitor resistance. E2F1 can bind to the gene promoter of IGF-1R. Then, IGF-1R knockdown completely overcome E2F1 overexpression-induced BRAF inhibitor resistance, while IGF-1R overexpression completely reversed E2F1 knockdown-induced response to BRAF inhibitor. Next, overexpression of IGF-1R markedly upregulated E2F1 in parental cells, while knockdown of IGF-1R notably downregulated E2F1 in resistant cells. In addition, HLM006474, an E2F inhibitor, was proved to potentiate the effects of BRAF inhibitor in resistant melanoma cells. AG1024, an IGF-1R inhibitor, further improved HLM006474-induced response of resistant melanoma cells to BRAF inhibitor. Interpretation: E2F1 and IGF-1R forms a positive feedback loop in melanoma, and this loop mediates BRAF inhibitor resistance. Furthermore, combined E2F1 inhibitor and IGF-1R inhibitor might be a novel strategy to enhance the therapeutic benefits of BRAF inhibitor in resistant melanoma. Funding Statement: This work is supported by grants from the National Natural Science Foundation of China (81673917). Declaration of Interests: The authors stated: No conflict of interests is needed to declare. Ethics Approval Statement: Not required.