Abstract 183: Inhibition of BRAF kinase alone in BRAF V600E-mutated undifferentiated thyroid carcinoma results in no growth arrest

Abstract

Abstract Background: Undifferentiated thyroid carcinoma is the most aggressive type of thyroid carcinoma. It is refractory to most of the conventional therapies and its median overall survival is less than six months. Mutation in BRAF gene was found around 25% of undifferentiated thyroid carcinoma. Mutated BRAF particularly the V600E mutated BRAF auto-activates the BRAF-MEK proliferation-survival cell signalling pathway. The objectives of our current study were to inhibit the BRAF kinase and investigate its effect on undifferentiated thyroid carcinoma proliferation. Materials and methods: 8050C, a homozygous BRAF V600E mutate undifferentiated thyroid carcinoma cell line, was used to develop anti-BRAF shRNA stable cell line. Quantitative real time polymerase chain reaction (qPCR) and western blot were performed for ratifying the BRAF inhibition efficiency of stable cell line. Proliferation and colony formation assay were done to see the effect of BRAF inhibition on undifferentiated thyroid carcinoma proliferation. In addition, impact of BRAF inhibition on proliferation cell signalling pathways were checked through western blot analysis. Results: Efficiency of BRAF knockdown was around 87% which resulted in approximately 83% inhibition of BRAF kinase (p < 0.05). About 54% proliferation inhibition (p < 0.05) was observed in the BRAF inhibited group compare to control group in the first week. But the proliferation rate in the BRAF inhibited group increased over the subsequent weeks and we did not observed any sign of cell growth arrest. In addition, colony formation assay revealed approximately 13% reduction of colony formation ability (p < 0.05) in BRAF inhibited group compare to control group. Western blot analysis of signalling kinases discovered reduction of around 72% MEK kinase activity after BRAF kinase inhibition. As we observed increasing trend of proliferation after an initial sharp drop in cell proliferation after BRAF kinase inhibition, we hypothesized and analysed the alternative PI3K-AKT proliferation pathway. Surprisingly we found about 63% increase of AKT kinase activity in the BRAF inhibited group compare to control group (p < 0.05). Conclusions: Inhibition of BRAF kinase reduced cell proliferation initially, but this proliferation inhibition did not lead to cell growth arrest at the end. Increased AKT kinase activity counteracted the effect of BRAF kinase inhibition as AKT kinase has the ability to contribute in overall cell proliferation. Therefore, both kinases need to be inhibited simultaneously for cell growth arrest in undifferentiated thyroid carcinoma. Citation Format: Md. Atiqur Rahman, Ali Salajegheh, Haleh Vosgha, Hamidreza Maroof, Robert A. Smith, Alfred KY Lam. Inhibition of BRAF kinase alone in BRAF V600E-mutated undifferentiated thyroid carcinoma results in no growth arrest. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 183.