S1993 Tumor Suppressor At Motif Binding Factor 1 (ATBF1) Negatively Regulates Cell Growth Through the Nuclear Translocation with Runt Domain Transcription Factor 3 (RUNx3) in Cooperation with TGF-β Signal Transduction

Background and aims: AT motif binding factor 1 (ATBF1), a homeotic transcription factor, was identified as a tumor suppressor (Nat. Genet., 2005) and frequent loss of heterozygosity at ATBF1 locus in gastric cancer was recently reported (Clin Cancer Res., 2007). We previously showed that ATBF1 expression inversely correlated with the malignant character of gastric cancer (Oncogene, 2001) and ATBF1 enhanced the promoter activity of the p21 (Cancer Res., 2003). We also found that ATBF1 translocates between cytoplasm and nucleus (Int. J. Cancer, 2007), but its precise mechanism has not been fully elucidated. In this study, we investigated the mechanism of nuclear translocation of ATBF1 with runt domain transcription factor 3 (RUNX3) in cooperation with TGF-β signal transduction. Materials and Methods: 1. To analyze the expression of ATBF1 in gastric cancer cells, we performed immunohistochemistry of 123 resected gastric cancer tissues. 2. ATBF1 and RUNX3 were over expressed by transfecting myc-tagged ATBF1 expression vector and flag-tagged RUNX3 expression vector. Immunoprecipitation (IP) between ATBF1 and RUNX3 was performed. 3. To investigate the nuclear translocation of endogenous ATBF1 and RUNX3, we examined the subcellular localization of ATBF1 and RUNX3 in SNU-16 gastric cancer cells treated with from 0.2 to 2.0 ng/ml of recombinant TGF-β1 by confocal mirosope. 4. We confirmed ATBF1 and RUNX3 protein expression in cytoplasm and nucleus extracted fraction by western blot. 5. Cell cycle analyses by using FACScan were performed. 6. Dual-luciferase assays were performed by transfecting ATBF1 and RUNX3 with p21 reporter vector. Results: 1. The nuclear localization of ATBF1 was observed in 33 (26.8%) gastric cancers and the cytoplasm localization of ATBF1 was observed in 61 (49.6%) gastric cancers. ATBF1 expression was not observed in 29 (23.6%) gastric cancers. 2. IP revealed that the physical interaction of ATBF1-RUNX3. 3. In SNU-16 gastric cancer cells, ATBF1 and RUNX3 existed in the nucleus before recombinant TGF-β1 stimulation, but after 24 hours of TGF-β1 stimulation, endogenous ATBF1 and RUNX3 translocated to the nucleus by confocal microscopic observation. 4. Western blot also revealed ATBF1 and RUNX3 protein in the nucleus was increased after TGF-β1 stimulation. 5.ATBF1 induced the accumulation in the G0/G1 phase. 6. ATBF1 and RUNX3 synergistically up-regulated p21 promoter activity. Conclusion: ATBF1 associates with RUNX3 and translocates to the nucleus with RUNX3 by TGF-β signal transduction and works as tumor suppressor and transcription regulator in the nucleus.