Abstract
Thyroid cancer is the most frequent endocrine tumor. BRAF mutation is the most frequent alteration in thyroid cancer. The metabolic consequences of these mutations is unknown. Recent studies have suggested a pro-metastatic role of the metabolic processes in several solid malignancies. The aim of this study is to identify the metabolic pathways dysregulated in thyroid carcinogenesis with oncogenic mutations. BRAF(V600E)induced HEK293 cells underwent a metabolomic profiling via mass spectrometry, which showed an increase in glutamine metabolism. Glutaminolysis, a dysregulated metabolic pathway is initiated by glutaminase (GLS), which converts glutamine to glutamate. Analysis of GLS expression in thyroid cancer tissue revealed an increase of GLS within the tumor compared to normal tissue. GEO dataset analysis revealed higher GLS expression in undifferentiated thyroid tumors compared to differentiated cancer and normal tissue (p=0.0001, p<0.0001). TCGA analysis further bolstered the findings and showed high GLS level in BRAF mutated tumors compared to wild-type (p<0.0001), that was associated with more aggressive clinico-pathological features (tumor size (p<0.0001) and high stage (p=0.0029)). To analyze the role of BRAF(V600E) in regulating GLS expression, two BRAF-mutated cell lines (8505c and BCPAP) were treated with increasing concentration (5, 10 and 15 µM) of BRAF(V600E) inhibitor, PLX4720 that showed a reduction of GLS and p-MEK expression. To identify the role of glutaminolysis in cancer cell growth, we carried out cell growth assay in three cancer cell lines (BCPAP and 8505c with BRAF(V600E),TPC1 with BRAF(wild-type)) in the presence of increasing concentration of the pharmacological inhibitor of GLS CB-839 (10, 100, 500 and 2000 nM). CB-839 significantly reduced the cell growth at nanomolar concentrations. In order to test the metabolic consequences of glutaminase inhibition, the rate of mitochondrial respiration was assessed. CB-839 treated cells had significantly lower basal oxygen consumption rate and mitochondrial respiratory capacity. To further investigate the pathways mediating the effects of glutaminolysis in thyroid carcinogenesis we analyzed the expression profile of phospho-kinases and apoptosis related-proteins. Our findings showed that the treatment of BRAF mutated thyroid cancer cells with CB-839 for 48 hours reduced the phosphorylation of mTOR(S2448), ERK1/2(T202/Y204,T185/Y187),SRC(Y419)/FAK(Y397). These kinases regulate carcinogenesis and cell growth. In addition, CB-839 treatment increased the key cell cycle regulator p21,and the pro-apoptotic proteins Bad and Bcl2. Our data suggest a role of glutamine metabolism in thyroid carcinogenesis and its association with oncogenic activation. To validate our findings in vivo, CB-839 will be combined with PLX4720. This combination may be an effective strategy to inhibit thyroid cancer progression.