Targeting the Epigenome for Lung Cancer Therapy

在已发表的研究中，我们已经证明，DAC和DP在恢复肿瘤抑制因子（如p16和RASSF1A）的表达的条件下，协同诱导肺癌细胞中多种C-T基因的表达，包括NY-ESO-1和MAGE-A1，这些抑制因子已被启动子超甲基化机制沉默。值得注意的是，这种药物治疗方案不足以介导这些基因在增殖的NHBE细胞中的诱导，可能是由于这些细胞的整体甲基化状态。最近，我们报道了生殖细胞限制性转录因子BORIS（印迹位点调控因子的兄弟）在肺癌发生过程中被激活，并且BORIS的表达与NY-ESO-1和MAGE-A1的去抑制一致。此外，我们已经表明，ctcf -to- boris -占据NY-ESO-1和MAGE-A1启动子的转变与肺癌细胞中这些C-T基因的去抑制相一致。最近，我们观察到BORIS招募Sp1来增加肺癌细胞中C-T基因的表达。总的来说，这些已发表的数据表明，与正常配子体发生类似，BORIS和CTCF之间的竞争可能是恶性转化过程中调节C-T基因的共同机制，并且BORIS的异常表达可能导致肺癌细胞中的DNA甲基化悖论。正在进行的研究致力于进一步确定BORIS在恶性转化过程中介导C-T和T-S基因表达的机制。我们已经发现了几种高水平表达BORIS的肺癌系，但没有MAGE-A1、MAGE-3或NY-ESO-1的表达。有趣的是，在连续的DAC/DP暴露后，这些细胞中可以诱导MAGE和NY-ESO-1的表达，这表明药物治疗可以诱导必要的辅助因子的表达，或者抑制boris介导的C-T基因激活的抑制因子的表达。目前正在使用多种技术进行实验，包括酵母-两种杂交，染色质免疫沉淀（ChIP），焦磷酸测序和蛋白质共诱导多能性，以确定boris介导的C-T基因去抑制需要哪些额外的辅助因子，相对于那些介导肺癌细胞中T-S基因异常沉默的辅助因子。与NIAID的Victor Lobanenkov博士合作，我们克隆了25个BORIS剪接变体，目前正在培养的肺癌细胞中表征这些变体的表达。我们正在启动与Lloyd Old博士（Ludwig癌症研究所）和Nasser Altorki博士（康奈尔大学医学中心）的合作，以全面检查BORIS剪接变体的表达是否与原发性肺癌中独特的C-T和T-S基因表达谱相关，以及这些肿瘤患者的预后。简而言之，这些研究将涉及使用定制的寡核苷酸和DNA甲基化阵列以及定量RT-PCR实验，对已知结果的肺癌患者的RNA和DNA样本进行盲法分析。这些研究有望深入了解肺癌细胞中表观遗传调控基因表达的整体模式，并确定BORIS激活在肺癌发生过程中的潜在临床意义。在相关研究中，我们试图探究BORIS是否能够启动和维持肺癌细胞的恶性表型。在正在进行的实验中，短期培养的正常人支气管上皮细胞（NHBE）、小气道上皮细胞（SAEC）、经cdk4和h-tert永生化的人支气管上皮细胞（有或没有过表达EGFR和k-ras (HBEC)）、SV40 t抗原永生化的支气管上皮细胞（BEAS）和几种肺癌细胞系（表现出不同的BORIS、C-T和T-S基因表达谱）暴露于NM、DAC、DP、或使用我们的标准治疗方案进行顺序DAC/DP。目前正在进行定量RT-PCR、DNA甲基化和长寡核苷酸阵列实验，以检测肺癌细胞中已知通过表观遗传机制诱导/抑制的各种印迹、C-T和T-S基因的表达水平。一些似乎在肺癌细胞中优先激活的BORIS剪接变异体正在被克隆到受测试调节的慢病毒载体中；这些载体将用于转导原代和永生化呼吸道上皮，以确定BORIS表达是否足以诱导恶性转化，通过体外增殖、软琼脂、基质和肿瘤异种移植试验进行评估。鲍里斯在维持恶性表型中的作用将通过在鲍里斯缺失的肺癌细胞中组成性过表达鲍里斯，在鲍里斯高水平表达的细胞中敲除鲍里斯，然后暴露于各种相关化疗药物和电离辐射来评估。总的来说，这些研究可能会进一步揭示BORIS在肺癌发生过程中激活的临床意义。尽管对新型癌症疗法的发展很重要，但对肿瘤细胞中表观遗传机制的分析提供了与恶性转化起始相关的染色质重塑事件的有限信息。令人惊讶的是，关于暴露于烟草烟雾的呼吸上皮的表观遗传反应的信息很少。最近，我们假设烟草烟雾会诱导培养的正常呼吸道上皮细胞的表观遗传改变，这是肺癌的特征，并且短期和永生呼吸道上皮细胞对烟草烟雾的相对敏感性将反映表观基因组的可塑性，这是对染色质重塑剂的反应所证明的。因此，我们试图开发一种体外模型系统来研究正常呼吸道上皮细胞中烟草介导的表观遗传事件。简而言之，NHBE、SAEC和HBEC都曾接触过TSC。微阵列、定量RT-PCR、甲基化特异性PCR （MSP）、焦磷酸测序、western blot和ChIP技术被用来确定TSC诱导/抑制基因表达是否与DNA甲基化和组蛋白编码的可重复性变化相一致。我们的初步数据表明，在生理暴露条件下，TSC诱导正常呼吸道上皮细胞的表观遗传改变，这是完全转化细胞的特征。这些具有挑衅性的发现支持进一步完善和验证这一创新模型，这可能对阐明与烟草诱发肺癌相关的表观遗传事件序列非常有用。我们正在进行的实验室实验数据为一系列临床方案提供了基本原理，这些方案评估了利用DNA去甲基化剂和HDAC抑制剂治疗胸部恶性肿瘤的可行性。在过去的几年中，大约有90名胸部恶性肿瘤（主要是肺癌）患者接受了各种染色质重塑剂的治疗。这些试验旨在概括我们发表的实验室实验中使用的临床环境药物暴露条件，并广泛依赖于使用免疫组化、MSP、定量RT-PCR、长寡核苷酸阵列和焦磷酸测序技术对治疗前和治疗后活检中的分子终点进行评估。总的来说，这些试验已经证实了在原发性胸部恶性肿瘤中使用染色质重塑剂来操纵基因表达的概念。这些阵列实验的结果提供了对体内药物活性新机制的见解，使染色质重塑剂更合理地开发用于癌症治疗

In published studies, we have demonstrated that DAC and DP synergistically induce expression of a variety of C-T genes including NY-ESO-1 and MAGE-A1 in lung cancer cells under conditions that restore expression of tumor suppressors such as p16 and RASSF1A that have been silenced by promoter hypermethylation mechanisms. Notably, this drug treatment regimen is insufficient to mediate induction of these genes in proliferating NHBE cells, possibly due to global methylation status in these cells. Recently, we reported that the germ cell-restricted transcription factor BORIS (brother of the regulator of imprinted sites) is activated during pulmonary carcinogenesis, and that BORIS expression coincides with de-repression of NY-ESO-1, as well as MAGE-A1. Furthermore, we have shown that a CTCF-to-BORIS-shift in occupancy of the NY-ESO-1 and MAGE-A1 promoters coincides with de-repression of these C-T genes in lung cancer cells. More recently, we have observed that BORIS recruits Sp1 to augment C-T gene expression in lung cancer cells. Collectively, these publised data indicate that similar to normal gametogenesis, competition between BORIS and CTCF may be a common mechanism regulating C-T genes during malignant transformation, and that aberrant BORIS expression may contribute to the DNA methylation paradox in lung cancer cells. Ongoing studies have been devoted to further defining the mechanisms by which BORIS mediates C-T and T-S gene expression during malignant transformation. We have identified several lung cancer lines with high level BORIS expression but no MAGE-A1, MAGE-3, or NY-ESO-1 expression. Interestingly, MAGE as well as NY-ESO-1 expression can be induced in these cells following sequential DAC/DP exposure, suggesting that drug treatment either induces expression of a requisite co-factor, or inhibits expression of a repressor of BORIS-mediated activation of C-T genes. Experiments using a variety of techniques including yeast-two hybrid, chromatin immunoprecipitation (ChIP), pyrosequencing, and protein co-IPs are presently underway to identify which additional co-factors are required for BORIS-mediated de-repression of C-T genes, relative to those mediating aberrant silencing of T-S genes in lung cancer cells. In collaboration with Dr. Victor Lobanenkov from NIAID, we have cloned 25 BORIS splice variants, and are presently characterizing expression of these variants in cultured lung cancer cells. We are initiating collaborations with Dr. Lloyd Old (Ludwig Cancer Institute) and Dr. Nasser Altorki (Cornell University Medical Center) in order to comprehensively examine if expression of BORIS splice variants correlates with unique C-T and T-S gene expression profiles in primary lung cancers, and prognosis in patients with these neoplasms. Briefly, these studies will involve blinded analysis of RNA and DNA samples from lung cancer patients with known outcomes utilizing customized oligo and DNA methylation arrays as well quantitative RT-PCR experiments. These studies are expected to yield insight regarding global patterns of epigenetically-regulated gene expression in lung cancer cells, and define the potential clinical relevance of BORIS activation during pulmonary carcinogenesis. In related studies, we have sought to investigate if BORIS can initiate and maintain the malignant phenotype of lung cancer cells. In ongoing experiments, short term cultures of normal human bronchial epithelia (NHBE), small airway epithelial cells (SAEC), human bronchial epithelial cells that have been immortalized by cdk4 and h-tert with or without over-expression of EGFR and k-ras (HBEC), SV40 T-antigen-immortalized bronchial epithelial cells (BEAS), and several lung cancer cell lines, which exhibit different BORIS, C-T, and T-S gene expression profiles have been exposed to NM, DAC, DP, or sequential DAC/DP using our standard treatment regimen. Quantitative RT-PCR, DNA methylation, and long-oligo array experiments are presently underway to examine expression levels of a variety of imprinted, C-T, and T-S genes that are known to be induced/repressed via epigenetic mechanisms in lung cancer cells. Several BORIS splice variants that appear to be preferentially activated in lung cancer cells are being cloned into tet-regulated lentiviral vectors; these vectors will be used to transduce primary as well as immortalized respiratory epithelia to ascertain if BORIS expression is sufficient to induce malignant transformation, as assessed by in-vitro proliferation, soft agar, matrigel, and tumor xenograft assays. The role of BORIS in maintaining the malignant phenotype will be assessed by constitutive over-expression of BORIS in BORIS-deficient lung cancer cells, and knock-down of BORIS in cells exhibiting high level BORIS expression, followed by exposure to a variety of relevant chemotherapeutic agents and ionizing radiation. Collectively, these studies may provide further insight regarding the clinical significance of BORIS activation during pulmonary carcinogenesis. Although important with regard to the development of novel cancer therapies, analysis of epigenetic mechanisms in tumor cells provides limited information pertaining to chromatin remodeling events associated with initiation of malignant transformation. Surprisingly little information is available concerning epigenetic response in respiratory epithelia exposed to tobacco smoke. Recently, we hypothesized that tobacco smoke will induce epigenetic alterations in cultured normal respiratory epithelia that are characteristic of established lung cancers, and that the relative sensitivity of short-term as well as immortalized respiratory epithelial cells to tobacco smoke will reflect the plasticity of the epigenome as evidenced by response to chromatin remodeling agents. As such, we have sought to develop an in-vitro model system for investigating tobacco-mediated epigenetic events in normal respiratory epithelial cells. Briefly, NHBE, SAEC, and HBEC have been exposed to TSC. Micro-array, quantitative RT-PCR, methylation-specific PCR (MSP), pyrosequencing, western blot, and ChIP techniques have been utilized to ascertain if induction/repression of gene expression by TSC coincides with reproducible changes in DNA methylation and the histone code. Our preliminary data suggest that under physiologic exposure conditions, TSC induces epigenetic alterations in normal respiratory epithelia that are characteristic of fully-transformed cells. These provocative findings support further refinement and validation of this innovative model, which could prove considerably useful for elucidating the sequence of epigenetic events associated with tobacco-induced lung cancers. Data from our ongoing laboratory experiments have provided the rationale for a series of clinical protocols evaluating the feasibility of utilizing DNA demethylating agents and HDAC inhibitors for the treatment of thoracic malignancies. During the past several years, approximately 90 patients with thoracic malignancies (predominantly lung cancer) have been treated with a variety of chromatin remodeling agents on protocols conducted in our Section. These trials have been designed to recapitulate in clinical settings drug exposure conditions used in our published laboratory experiments, and have relied extensively on the evaluation of molecular endpoints in pre and post- treatment biopsies using IHC, MSP, quantitative RT-PCR, long-oligo array, and pyrosequencing techniques. Collectively, these trials have established proof of concept pertaining to the use of chromatin remodeling agents for manipulating gene expression in primary thoracic malignancies. Results of the arrays experiments have provided insight into novel mechanisms of drug activity in vivo, enabling more rational development of chromatin remodeling agents for cancer therapy