The role of ATBF1 nuclear translocation in gastric and intestinal phenotype and chemosensitivity of gastric cancer

背景与目的：MUC 5AC属于分泌型粘蛋白家族，表达于胃小凹细胞。胃癌组织中粘蛋白的表达发生了改变。已有研究表明，胃癌组织中MUC 5AC的表达与预后不良有关。然而，MUC 5AC的转录调控机制尚未得到很好的阐明。AT基序结合因子1（AT motif binding factor 1，ATBF 1）是一种同源异型转录因子，负调控甲胎蛋白（AFP）和癌蛋白Myb。我们之前已经证明ATBF 1负调节刷状缘酶基因（氨基肽酶-N）的转录，并且ATBF 1的缺失是产生AFP的胃癌细胞的显著特征，其特征在于极高的恶性度（Oncogene 2001）。ATBF 1最近已被鉴定为前列腺癌的肿瘤抑制因子的候选者（Nat.Genet. 2005年）。本研究旨在探讨ATBF 1对MUC 5AC的转录调控作用。我们 ...更多信息 应用免疫组化方法检测41例胃癌标本中123个癌灶ATBF 1和MUC 5AC的表达。2.我们分析了MUC 5AC启动子区是否存在AT基序。3.为了分析ATBF 1对MUC 5AC的转录调控，我们在MKN 45细胞（胃癌细胞）中进行了瞬时转染和双报告基因检测。4.为了从蛋白水平分析ATBF 1对MUC 5AC的转录调控作用，我们利用共聚焦显微镜检测了过表达ATBF 1的MKN 45细胞中MUC 5AC的蛋白表达。5.采用染色质免疫沉淀法（ChIP）检测ATBF 1与MUC 5AC启动子区AT基序的结合情况。在ATBF 1阳性（核染色）的胃癌中，MUC 5AC阳性率仅为9%（3/33），而在ATBF 1阴性（胞浆染色或不染色）的胃癌中，MUC 5AC阳性率为76%（68/90）。ATBF 1核染色倾向于在MUC 5AC阴性的胃癌细胞中观察到（p<.0001）。2.在MUC 5AC启动子区（-1646-1634）检测到AT基序。3.瞬时转染和双酶报告基因检测表明ATBF 1抑制MUC 5AC启动子的活性（35 ± 1.1%）。4. 70.用对照转染的MKN 45细胞中7 ± 1.2%为MUC 5AC阳性，而用ATBF 1转染的MKN 45细胞中仅11.3 ± 3.1%为MUC 5AC阳性（p<0.0001）。过表达ATBF 1抑制胃癌细胞中MUC 5AC内源性蛋白。5.结论：胃癌组织中ATBF 1与MUC 5AC启动子AT基序结合，负调控MUC 5AC基因的转录。少

Background and aims: MUC5AC belongs to the family of secreted mucins and expresses in foveolar cells of the stomach. Alterations of mucin expression take place in gastric cancer. It was reported that MUC5AC expression in gastric cancer correlates with poor prognosis. However, the transcriptional regulation mechanism of MUC5AC has not been well elucidated. AT motif binding factor 1 (ATBF1) is a homeotic transcription factor which negatively regulates Alpha-fetoprotein (AFP) and oncoprotein Myb. We have showed previously that ATBF1 negatively regulates transcription of brush-border enzyme gene, aminopeptidase-N and that the absence of ATBF1 is a distinct feature of AFP-producing gastric cancer cells, which are characterized by extremely high malignancy (Oncogene 2001). ATBF1 has recently been identified as a candidate of tumor suppressor for prostate cancer (Nat. Genet. 2005). In this study, we investigated the transcriptional regulation of MUC5AC by ATBF1.Materials and Methods: 1. We an … More alyzed ATBF1 and MUC5AC expression by immunohistochemistry in 123 area of 41 gastric cancers specimen. 2. We analyzed whether there is an AT motif in MUC5AC promoter region or not. 3. To analyze the transcriptional regulation of MUC5AC by ATBF1, we performed transient transfection and dual luciferase-reporter assay in MKN45 cells (gastric cancer cells). 4. To analyze the transcriptional regulation of MUC5AC by ATBF1 in protein level, we examined the protein expression of MUC5AC in MKN45 cells which was overexpressed ATBF1 using confocal microscopy. 5. To assess the binding of ATBF1 to the AT motif in MUC5AC promoter region, we performed chromatin immunoprecipitation (ChIP).Results: 1. Only 9% (3/33) of ATBF1-positive(nuclear staining) gastric cancers were MUC5AC positive, and, 76% (68/90) of ATBF1-negative (cytoplasm staining or no staining )gastric cancers were MUC5AC positive. ATBF1 nuclear staining tends to be observed in gastric cancer cells negative for MUC5AC (p<.0001). 2. We detected the AT motif in MUC5AC promoter region (-1646-1634). 3. Transient transfection and dual luciferase-reporter assay demonstrated that ATBF1 suppressed the activity of MUC5AC promoter (35 plus minus 1.1%). 4. 70. 7 plus minus 1.2% of MKN45 cells transfected with control were MUC5AC positice, however only 11.3 plus minus 3.1% of MKN45 cells transfected with ATBF1 were MUC5AC positive (p<.0001). Overexpressed ATBF1 suppressed MUC5AC endogeneous protein in gastric cancer cells. 5. ChIP revealed that ATBF1 binds AT motif of MUC5AC promoter.Conclusion: ATBF1 binds to AT motif of MUC5AC promoter and negatively regulates transcription of the MUC5AC gene in gastric cancer. Less