City of Hope Comprehensive Cancer Center Duarte, CA
L. Yang, H. Feng, T. Cui, and T. M. Williams; Department of Radiation Oncology, City of Hope National Medical Center, Duarte, CA
Purpose/Objective(s): Anaplastic thyroid cancer (ATC) bears a dismal prognosis, with a 2-year survival rate of ~10% and is associated with major morbidity from local-regional disease. Management of ATC requires local and systemic control, including surgery, external beam radiation therapy (EBRT), and chemotherapy. BRAF activating mutations are the single most common oncogenic mutations in ATC (occurring in 30-40% of tumors). Our previous studies revealed that BRAF mutations promote therapeutic resistance to radiation and other genotoxic therapies by upregulating tumor-intrinsic nuclear DNA repair activity. In the current study, we further explored the mechanisms by which BRAF mutations promote DNA damage repair. Materials/
Methods: The regulation of BRAF mutations in the DNA damage response (DDR) genes was screened by analyzing publicly available data of the thyroid carcinoma (THCA) and skin cutaneous melanoma in The Cancer Genome Atlas (TCGA) database and then confirmed by chemical inhibition, BRAF genetic silencing, and overactivation in THCA cell lines. Putative transcriptional factors mediating oncogenic BRAF-driven DDR gene expression were predicted by the PROMOv3analysis and TCGA database analysis and confirmed by chemical and genetic silencing. The effect of DDR genes on THCA radiation sensitivity was evaluated by radiation clonogenic assay, immunoblotting, mitotic catastrophe, and functional DNA damage repair reporter assays. Results: TCGA database analysis revealed that FANCI gene is upregulated and associated with poor prognosis in the BRAF mutated subgroup of THCA patients. In cell lines harboring BRAFV600E, inhibition of BRAF/MEK/ERK signaling using the BRAFV600E inhibitor vemurafenib, MEK inhibitor trametinib, or BRAF siRNA suppressed FANCI mRNA and protein expression. Conversely, in BRAF wild type cells, ectopic expression of BRAFV600E enhanced FANCI expression. We further found that FANCI knockdown by siRNA enhanced radiotherapy sensitivity of THCA cells, accompanied by increased expression of the DNA damage marker g-H2AX and radiotherapy-induced mitotic catastrophe. Functional reporter assays demonstrated that the absence of FANCI suppressed both HR and NHEJ DNA damage repair activity. Transcriptional factor E2F1 is correlated with FANCI expression, and regulated by BRAF/MEK/ERK activity, and E2F1 knockdown by siRNA or inhibition by an E2F inhibitor suppressed FANCI expression. Conclusion: We identified FANCI, a critical component in DNA crosslink repair correlated with BRAF mutation and associated with poor prognosis in BRAF-mutated THCA. FANCI depletion sensitized thyroid cancer cells to radiation therapy. E2F1 is a putative transcriptional factor mediating BRAF regulation of FANCI expression. Our study revealed a novel BRAF-E2F1-FANCI axis that contributes to oncogenic BRAF-mediated radiation resistance and suggests that targeting this axis could be a novel therapeutic strategy to improve radiation therapy in BRAF-mutated thyroid cancers.
