Identification of innate drug resistance against raf inhibitors in patients with papillary thyroid cancer

Abstract

Recently, several novel targeted agents have been developed for treatment of BRAFV600E-positive cancers. However, de novo and acquired resistance to these agents has since emerged as a therapeutic obstacle. Two major mechanisms, based on their dependence on RAF dimerization, drive tumor resistance to RAF inhibitors – mutations in NRAS such as NRAS Q61, the p61BRAFV600E splice variant, and C-RAF overexpression are involved in a mechanism that is dependent on RAF dimerization; in contrast, aberrant expression of Cancer Osaka Thyroid Oncogene mitogen-activated protein kinase kinase kinase 8 (COT) or mitogen-activated protein kinase kinase (MEK) mutation function independently of RAF dimerization. The aim of this study was to identify the molecular basis for innate drug resistance against RAF inhibitors in patients with papillary thyroid cancer (PTC). 167 PTC patients undergoing total thyroidectomy were enrolled. Patient information and clinicopathological parameters were analyzed. BRAFV600E mutation was included in this study. Exome sequencing for detection of mutation in NRAS and MEK was performed. For the analysis of p61BRAFV600E splice variant, DNA sequencing, Western blot and mass spectrometry were performed. CRAF overexpression and aberrant expression of COT were analyzed by quantitative polymerase chain reaction (qPCR) and immunohistochemical staining. In the results, NRAS and MEK mutation, and the p61BRAFV600E splice variant were not detected in PTC. However, qPCR data showed that the relative expression of CRAF and COT mRNA in PTC was higher than in normal tissues ( p<0.01). Furthermore, COT mRNA expression in PTC correlated positively with CRAF expression (r=0.5954, p<0.001). Immunohistochemical analysis showed that the staining intensities of COT were higher in PTC than in normal thyroid tissues (p <0.001). Aberrant expression of COT was more frequently detected in BRAFV600E-positive PTC (p=0.013). These results suggest that COT expression may be associated with innate drug resistance against RAF inhibitors in PTC.