Association of adenine nucleotide translocase 2 (ANT2) with [18F]fluoro-2-deoxy-D-glucose accumulation in various cancers

Abstract

Introduction: [18F]Fluoro-2-deoxy-D-glucose (FDG)-positron emission tomography (PET) has been widely used to detect cancer. Although glucose transporter 1 (GLUT1) and hexokinase 2 (HK2) are mainly considered to be associated with FDG uptake in cancer cells, the biological mechanism that determines FDG uptake remains poorly understood. In phosphorylation process of HK2, it preferentially uses ATP derived from the mitochondria by binding to voltage-dependent anion channels (VDAC). VDAC also can bind to adenine nucleotide translocases (ANT) in the mitochondria inner membrane, which transports ATP/ADP to the cytosol and is involved in cellular energy metabolism. Among the ANT isoforms, ANT2 is inducible in proliferative cells and has been associated with glycolytic metabolism. However, the expression of ANT2 with regards to FDG uptake has not yet been reported. Therefore, we evaluated the correlation between FDG accumulation and ANT2 expression in various cancers. Additionally, we investigated the association of ANT2 in the mechanism of FDG accumulation regarding to HK2-VDAC complex.<br/> Methods: Human thyroid, lung, liver, prostate, breast, and brain cancer cell lines were used. Protein levels of GLUT1, HK2, and mitochondrial proteins such as VDAC, ANT1, ANT2, and ANT3 were measured by western blotting. ANT2 siRNAs and pcDNA3.1-ANT2 vectors were used to modulate ANT2 expression. A gamma counter was used to measure FDG uptake. Luciferase-expressing FRO cells were subcutaneously grafted into BALB/c nude mice, and siRNA was directly injected into the tumors. Positron emission tomography (PET) and bioluminescence imaging were conducted. Immunostaining was performed in tumor tissues of xenograft models. Human lung, liver, and thyroid cancer tissue-microarrays were assessed for histological differentiation, types, and TNM staging. ANT2 expression was evaluated by immunostaining. The intensity of positive antibody uptake in the tissue was scored from 1 to 5. FDG-PET imaging for thyroid cancer (ten patients) was performed with a Siemens PET scanner. All PET images were reviewed by an experienced nuclear medicine physician using commercial imaging software. To visualize the complex of HK2-VDAC-ANT isoforms by bimolecular fluorescence complementation (BiFC)-based fluorescence resonance energy transfer (FRET) assay, we established fluorescence vectors inserted with each protein and transfected each vector into 293FT cells.<br/> Results: In each set of various cancer cell lines, ANT2 expression was correlated with FDG uptake which was not explained by GLUT1 and HK2. Down-regulated cells treated with ANT2 siRNA showed 0.55-fold decreased FDG uptake. In contrast, up-regulated cells treated with pcDNA3.1-ANT2 increased FDG uptake by 1.7-fold. In xenograft models, FDG-PET images showed 0.75-fold decreased FDG uptake after ANT2 siRNA injection. Immunostaining of tumor-injected ANT2 siRNA confirmed the decreased ANT2 expression without any changes in the expression of GLUT1, HK2, or other mitochondrial proteins. In thyroid, lung, and liver tissues, ANT2 expression was increased according to the differentiation grade and malignant type. In patients subjected to FDG-PET scans, a high maximum standardized uptake value was significantly related to ANT2 expression rather than GLUT1 or HK2 expression. Finally, 293FT cells transfected HK2, VDAC, and ANT2 constructs showed the highest FDG uptake. Additionally, FRET signals were only observed in this combination (HK2-VDAC-ANT2). Compared to ANT1 or ANT3, BiFC and FRET signals in cells expressing ANT2 and FDG accumulation were highest in these cells.<br/> Conclusion: ANT2 expression was significantly associated with FDG accumulation by forming complexes with HK2-VDAC in phosphorylation process of FDG. This study indicated that ANT2 is an important protein affecting FDG accumulation in various cancers that cannot be explained by GLUT1 and HK2 expression.<br/>