Molecular Intervention in Thoracic Malignancies

目前的治疗方式无法对肺癌和食道癌的自然病史产生影响，加上这些恶性肿瘤的流行，突出表明需要更基本地了解空气中消化道癌症发生的分子机制。在以前的研究中，我们已经利用重组病毒载体来证明恢复肺癌和食道癌细胞中细胞周期蛋白D1、p16和p53表达的影响。这些破坏G1限制点控制的突变已知发生在多步骤呼吸道癌变的早期，因此它们是干预已建立的恶性肿瘤及其前体病变的合适靶点。最近，与胸部肿瘤科高级研究员Dao Nguyen博士一起，最近的努力集中在评估药物方面，这些药物可能实现分子终点，而不需要依赖病毒载体的基因治疗策略的复杂性和局限性。我们观察到，无论组织学或抑癌基因状态如何，合成的黄酮类化合物都能诱导食道癌细胞发生深刻的细胞周期停滞和细胞凋亡，而且黄烷醇对食管癌细胞的预处理能显著增强其对紫杉醇的敏感性。这些数据为最近批准的一项方案提供了理论基础，该方案评估了食道癌患者单独使用黄烷醇与黄烷醇联合紫杉醇的疗效。对已建立的癌症疗效的文献可能支持对黄吡醇在高危人群中化学预防呼吸消化道癌症的评估。大约30%的肺癌和食道癌过度表达erb-2/p185癌基因，据信该癌基因介导了对顺铂或紫杉醇等化疗药物的耐药性。下调erbB-2/pl85基因功能可以通过反义技术、酪氨酸激酶抑制剂或与pl85蛋白胞外区结合的单抗来实现。最近，我们观察到阿萨霉素类似物17-烯丙氨基格尔达那霉素(17-AA GA)作用于肺癌和食道癌细胞后，pl85蛋白迅速耗竭，且呈剂量依赖关系。17-AA GA抑制p185的表达显著增强了紫杉醇的细胞毒性；特别是，17-AA GA/紫杉醇处理的细胞对紫杉醇的敏感性比单独暴露于紫杉醇的细胞高3~40倍。这种增强效应在17-AA GA的纳摩尔浓度下达到。流式细胞仪分析显示，联合用药后72小时内，细胞发生明显的G2/M期阻滞和细胞凋亡。这些临床前数据支持17-AA、GA和紫杉醇在胸部肿瘤患者中的评估。在其他研究中，我们试图使用去甲基化药物和组蛋白脱乙酰酶抑制剂来诱导肿瘤抑制基因如p16和ARF的表达，这些基因在胸部肿瘤中经常被启动子超甲基化失活。有趣的是，这些实验与我们实验室的平行研究一致，证明了5-氮-2脱氧胞苷(地他滨)诱导肺癌细胞NY-ESO-1和MAGE-3肿瘤-睾丸抗原的表达。我们的研究表明，相对于正常的人支气管上皮细胞、成纤维细胞或EBV转化的淋巴细胞，肺癌细胞中癌症睾丸抗原的表达更容易诱导--可能是由于已知在恶性转化过程中发生的全基因组去甲基化。一项旨在评估地西他滨诱导肺癌患者肿瘤抗原和肿瘤抑制基因表达的I期试验目前正在向患者开放。一项旨在评估肺癌患者中Depsi多肽介导的基因诱导和细胞凋亡的第二阶段研究将在不久的将来开放。近20%的癌症患者死于孤立的肺转移，本部分的额外努力已被用于评估局部给药到肺部的效果。建立了绵羊离体肺灌流模型，并完成了紫杉醇的药代动力学研究。通过非病毒技术提供的其他药理学药物和基因构建将使用该模型进行评估。这些动物实验为利用紫杉醇和中等温度治疗不能切除的肺部恶性肿瘤患者的隔离肺灌注分析提供了理论基础。由NCI-IRB胸部肿瘤科提交的以下临床方案已经由NCI-IRB审查，并有望在不久的将来向患者开放：1)连续三天静脉滴注与联合三小时紫杉醇灌注治疗食道癌患者的随机第二阶段研究2)地西他滨介导的肺癌患者肿瘤抗原和肿瘤抑制基因表达的第一阶段研究。3)FR901228/NSC630176四小时静脉滴注多肽对肺癌患者基因诱导的第二阶段研究。4)紫杉醇隔离肺灌注和中温热疗对不能切除的肺部恶性肿瘤患者的第一阶段研究。

The inability of current treatment modalities to impact on the natural history of lung and esophageal cancers, together with the prevalence of these malignancies, underscores the need for more fundamental appreciation of molecular mechanisms of aerodigestive tract carcinogenesis. In previous studies we have utilized recombinant viral vectors to demonstrate the impact of restoration of cyclin D1, p16, and p53 expression in lung and esophageal cancer cells. These mutations, which disrupt G1 Restriction Point control are known to occur early during multistep aerodigestive tract carcinogenesis, hence they are appropriate targets for intervention in established malignancies as well as their precursor lesions.In conjunction with Dr. Dao Nguyen, Senior Investigator, Thoracic Oncology Section, recent efforts have focused on the evaluation of pharmacologic agents which might achieve molecular end points without the complexities and limitations of gene therapy strategies dependent upon viral vectors. We have observed that the synthetic flavone, flavopiridol, mediates profound cell cycle arrest and apoptosis in esophageal cancer cells irrespective of histology or tumor suppressor gene status, and that pretreatment of esophageal cancer cells with flavopiridol markedly enhances their sensitivity to paclitaxel. These data have provided the rationale for a recently-approved protocol evaluating flavopiridol alone versus flavopiridol with paclitaxel in esophageal cancer patients. Documentation of efficacy in established cancers may support the evaluation of flavopiridol for the chemoprevention of aerodigestive tract cancers in high risk individuals.Approximately 30% of lung and esophageal cancers overexpress the erbB-2/p185 oncogene which is believed to mediate resistance to chemotherapeutic agents such as cisplatin or paclitaxel. Downregulation of erbB-2/pl85 gene function can be achieved by antisense techniques, tyrosine kinase inhibitors, or monoclonal antibodies that bind to the extracellular domain of the pl85 protein. Recently, we have observed rapid, dose-dependent depletion of pl85 protein in lung and esophageal cancer cells treated with the ansamycin analogue 17-allylamino geldanamycin (17-AA GA). Reduction of p185 expression by 17-AA GA markedly enhanced the cytotoxicity of taxol in these cells; specifically, a 3 to over 40-fold increase in paclitaxel sensitivity was observed in cells treated with 17-AA GA/paclitaxel as compared to cells exposed to paclitaxel alone. This enhancement effect was achieved at nanomolar concentrations of 17-AA GA. Flow cytometry analysis revealed that combination drug treatment induced pronounced G2/M arrest and subsequent apoptosis within 72 hours of drug exposure. These preclinical data support the evaluation of 17-AA GA and paclitaxel in patients with thoracic neoplasms.In additional studies we have attempted to utilize demethylating agents and histone deacetylase inhibitors to induce expression of tumor suppressor genes such as p16 and ARF which are frequently inactivated by promoter hypermethylation in thoracic neoplasms. Interestingly, these experiments have converged with parallel studies in our laboratory demonstrating induction of NY-ESO-1 and MAGE-3 cancer-testis antigen expression in lung cancer cells by 5 Aza-2 deoxycytidine (decitabine). Our studies have shown that cancer testis antigen expression is more readily induced in lung cancer cells relative to normal human bronchial epithelial cells, fibroblasts, or EBV transformed lymphocytes--perhaps due to genome-wide demethylation which is known to occur during malignant transformation. A phase I trial designed to evaluate decitabine-mediated induction of tumor antigen and tumor suppressor gene expression in lung cancer patients is currently open for patient accrual. A Phase II study designed to evaluate Depsipeptide mediated gene induction and apoptosis in lung cancer patients will be open in the near future.Nearly 20% of all cancer patients succumb to isolated pulmonary metastases, and additional efforts in our section have been directed toward the evaluation of regional drug delivery to the lungs. A sheep model of isolated lung perfusion has been established , and pharmacokinetic studies using paclitaxel have been completed. Additional pharmacologic agents and gene constructs delivered via nonviral techniques will be evaluated using this model. These animal experiments have provided the the rationale for analysis of isolated lung perfusion utilizing paclitaxel and moderate hyperthermia in patients with unresectable pulmonary malignancies.The following clinical protocols submitted by the Thoracic Oncology Section have been reviewed by the NCI-IRB and are expected to be open for patient accrual in the near future:1)Randomized Phase II Study of Continuous Three Day Flavopiridol Infusion Alone vs. Flavopiridol In Combination With Three Hour Taxol Infusion inEsophageal Cancer Patients.2)Phase I Study of Decitabine Mediated Induction of Tumor Antigen and Tumor Supressor Gene Expression in Lung Cancer Patients. 3) Phase II Study of Gene Induction Mediated by Four-Hour Intravenous Depsipeptide (FR901228/NSC630176) Infusion in Lung Cancer Patients.4)Phase I Study of Isolated Lung Perfusion with Paclitaxel and Moderate Hyperthermia in Patients with Unresectable Pulmonary Malignancies.