Epigenetic Mechanisms of Gene Expression in Lung Cancer Cells

我们已发表的实验表明，DNA去甲基化试剂5-氮-2-脱氧胞苷(DAC)可在培养的肺癌细胞中显著诱导CT-X基因，如NY-ESO-1、MAGE-A1和MAGE-A3，但不能诱导正常的人支气管上皮细胞(NHBE)。此外，我们还证明了HDAC抑制剂Depsi肽FK228(DP)增强了DAC介导的CT-X基因在肺癌细胞中的诱导。顺序性DAC/DP治疗诱导CT-X基因表达的同时，肺癌细胞中Boris基因的表达显著增加。甲基化特异性聚合酶链式反应(MSP)和亚硫酸氢钠测序实验表明，在培养的肺癌细胞中，Boris的激活与Boris启动子中CpG岛的去甲基化一致。另外的MSP实验显示，在12例原发肺癌标本中，有6例发现Boris的表达下调；在几名患者中，在肺癌以及这些肿瘤附近的组织正常组织中检测到Boris的下调，这增加了Boris的激活是肺癌发生过程中早期表观遗传学事件的可能性。随后的凝胶迁移率改变分析(EMSA)和染色质免疫沉淀(CHIP)实验表明，Boris与NY-ESO-1启动子在物理上相互作用，并且NY-ESO-1在肺癌细胞中的抑制与NY-ESO-1启动子内CTCF到Boris的转移相一致。肺癌细胞中MAGE-A1的激活也有类似的现象。还进行了其他研究，以进一步研究Boris对NY-ESO-1转录调控的作用机制。对NY-ESO-1启动子区域的软件引导分析揭示了各种潜在的转录因子结合位点，包括几个SP1基序。Western印迹实验表明，DAC/DP序贯处理可增强SP1在肺癌细胞中的核转位。利用缺失突变型启动子-报告基因构建的瞬时转染实验，以及EMSA和芯片实验表明，NY-ESO-1启动子的活性与肺癌细胞中SP1近端结合位点的占位一致。SP1识别序列中的突变在体外消除了SP1与该基序的结合，并在体内显著降低了NY-ESO-1启动子的活性(图1)。进一步使用siRNA技术的实验表明，抑制SP1的表达降低了NY-ESO-1启动子的活性，而下调CTCF则增强了该CT-X基因在肺癌细胞中的表达。免疫沉淀实验表明，SP1在体内与Boris发生物理相互作用，但不与CTCF发生相互作用。总而言之，这些数据表明，鲍里斯招募SP1来降低肺癌细胞中NY-ESO-1(可能还有其他CT-X基因)的抑制。由于组织学上的正常上皮和癌前病变表现出Boris的下调，因此可以想象，这种生殖细胞转录因子的下调有助于肺癌细胞转化表型的启动和维持。因此，我们开展了一系列实验，以全面研究Boris基因表达缺失的SV40或CDK4/h-tert永生化人支气管上皮细胞(BEAS，HBEC)以及表达极低水平Boris和CT-X基因的肺癌细胞CALU-6(P53 Null)和A549(P53wt)中Boris表达的影响。有趣的是，与对照细胞相比，转导Boris的BEAS细胞表现出接触抑制的解除，增殖和侵袭的增加，以及四环素诱导的克隆形成能力的增强。这种现象似乎与E-钙粘附素的沉默相一致，E-钙粘附素是一种已知的调节上皮-间充质转化的基因，它与癌症转移有关。其他研究正在进行中，以进一步评估Boris表达对肺癌细胞增殖、致瘤性和化疗/XRT敏感性的影响。这些研究可能揭示肺癌患者预后的新的表观遗传学生物标志物。

Our published experiments have indicated that the DNA demethylating agent 5 aza 2 deoxycytidine (DAC) mediates dramatic induction of CT-X genes such as NY-ESO-1, MAGE-A1, and MAGE-A3 in cultured lung cancer cells, but not normal human bronchial epithelia (NHBE). Furthermore we have demonstrated that the HDAC inhibitor Depsipeptide FK228 (DP) enhances DAC-mediated induction of CT-X genes in lung cancer cells. Induction of CT-X gene expression by sequential DAC/DP treatment coincided with a dramatic increase in BORIS expression in lung cancer cells. Methylation specific PCR (MSP) and sodium bisulfite sequencing experiments demonstrated that activation of BORIS in cultured lung cancer cells coincided with demethylation of a CpG island within the BORIS promoter. Additional MSP experiments revealed de-repression of BORIS in 6 of 12 primary lung cancer specimens; in several patients, de-repression of BORIS was detected in lung cancers as well as histologically normal tissue adjacent to these neoplasms, raising the possibility that activation of BORIS is an early epigenetic event during pulmonary carcinogenesis. Subsequent electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) experiments demonstrated that BORIS physically interacts with the NY-ESO-1 promoter, and that de-repression of NY-ESO-1 in lung cancer cells coincides with a CTCF-to-BORIS shift in occupancy within the NY-ESO-1 promoter. A similar phenomenon was observed for MAGE-A1 activation in lung cancer cells. Additional studies were undertaken to further examine the mechanisms by which BORIS contributes to transcriptional regulation of NY-ESO-1. Software guided analysis of the NY-ESO-1 promoter region revealed a variety of potential transcription factor binding sites including several SP1 motifs. Western blot experiments demonstrated that sequential DAC/DP treatment enhanced nuclear translocation of SP1 in lung cancer cells. Transient transfection assays using deletion mutant promoter-reporter constructs, as well as EMSA and ChIP experiments demonstrated that NY-ESO-1 promoter activity coincided with occupancy of the proximal SP1 binding site in lung cancer cells. Mutations within the SP1 recognition sequence eliminated binding of SP1 to this motif in vitro, and markedly diminished NY-ESO-1 promoter activity in vivo (Figure 1). Additional experiments using siRNA techniques revealed that inhibition of SP1 expression decreased NY-ESO-1 promoter activity, whereas knock-down of CTCF augmented expression of this CT-X gene in lung cancer cells. Immunoprecipitation experiments demonstrated that SP1 physically interacts with BORIS, but not with CTCF in vivo. Collectively these data suggested that BORIS recruits SP1 to de-repress NY-ESO-1 (and possibly other CT-X genes) in lung cancer cells. Because histologically normal epithelia as well as premalignant lesions adjacent to established lung cancers exhibit de-repression of BORIS, it is conceivable that de-repression of this germ cell transcription factor contributes to initiation and maintenance of the transformed phenotype of lung cancer cells. As such, a series of experiments were initiated to comprehensively investigate the effects of BORIS expression in SV40- or cdk4/h-tert- immortalized human bronchial epithelial cells (BEAS, and HBEC, respectively), which are deficient for BORIS and CT-X gene expression, as well as Calu-6 (p53 null) and A549 (p53 wt) lung cancer cells exhibiting extremely low level BORIS and CT-X gene expression. Interestingly, BEAS cells transduced with BORIS exhibited loss of contact inhibition, increased proliferation and invasion, as well as enhanced clonogenic potential following tetracycline exposure relative to control cells. This phenomenon appeared to coincide with silencing of E-cadherin, a gene known to regulate epithelial-mesenchymal transition, which has been implicated in cancer metastasis. Additional studies are in progress to further evaluate the effects of BORIS expression regarding proliferation, tumorigenicity, and chemo/XRT sensitivity of lung cancer cells. These studied could reveal novel epigenetic biomarkers of prognosis in lung cancer patients.