array-based screening for the expression and regulation of tumor supressor genes

描述(申请人提供)：涎腺腺样囊性癌(ACC)是一种罕见的恶性肿瘤，具有不寻常的临床行为，以侵袭性局部生长为特征，通常通过手术和术后放射治疗来控制，随后可能存在数年的休眠期。然而，多达50%的患者经历了远处转移，最常见的是肺或骨转移，这可能也会持续多年。对ACC的分子基础知之甚少，因此细胞毒性或靶向治疗一直是经验性的和不一致的。在ACC和大多数其他肿瘤模型中，启动子甲基化已成为肿瘤抑制基因沉默的重要表观遗传学机制。通过利用5-氮杂胞苷治疗产生全局去甲基化的能力，我们能够从药物上揭示以前在原发AC细胞中沉默的肿瘤抑制基因。这种暴露将导致基因上调和启动子去甲基化，基因上调可以通过表达微阵列检测到，启动子去甲基化可以通过新一代甲基化阵列检测到。虽然这种发现试验通常是在体外进行的，但目前有 没有可靠的ACC细胞系模型。因此，我们建议在原发患者的ACC异种移植瘤中实施5-氮胞苷治疗的新方法，并每天用中剂量的5-氮胞苷治疗这些裸鼠28天。一旦完成，就可以采集组织，运行阵列，完成生物信息学整合和分析。然后，将通过亚硫酸氢盐测序，验证基因清单在正常唾液腺的子集中的甲基化状态，这是一种主要的ACC样本。在这个项目结束时，我们将在启动子甲基化的控制下，对候选的肿瘤抑制基因进行全基因组的表征。与单独使用甲基化阵列相比，这种涉及跨阵列平台集成的强大技术允许更广泛的基因发现，并将为我们提供更精简的验证过程。确定的候选基因将为进一步研究ACC致癌的基本机制提供更有针对性的靶点。 公共卫生相关性：该项目的目标是对与ACC相关的甲基化肿瘤抑制基因进行全面调查。在原发肿瘤异种移植中使用一种新的全局去甲基化的方法和集成的阵列分析，我们打算验证和识别相关的靶点，然后可以进一步研究它们在癌症发生中的机制作用。

DESCRIPTION (provided by applicant): Adenoid cystic carcinoma of the salivary glands (ACC) is a rare malignancy with an unusual clinical behavior, marked by aggressive local growth which is often controlled with surgery and postoperative radiation therapy, followed by a dormant period that may exist for years. However, up to 50% of patients experience distant metastasis, most often to lung or bone, which may remain indolent for years as well. Not much is known about the molecular underpinnings of ACC and thus cytotoxic or targeted therapy has been empiric and inconsistent. Promoter methylation has emerged as a prominent epigenetic mechanism of tumor suppressor gene silencing in ACC and most other tumor models. By capitalizing on the ability of 5-aza-cytidine treatment to produce global demethylation, we are able to pharmacologically unmask tumor suppressor genes, previously silenced in primary ACCs. This exposure will lead to gene upregulation, which can be detected with expression microarrays, and promoter demethylation, which can be detected with newer generation methylation arrays. While this discovery assay is typically performed in vitro, there presently are no reliable ACC cell line models. We thus propose performing a novel approach of 5-aza-cytidine treatment in heterotransplanted ACC tumors from primary patients and treating these nude mice with daily, moderate-dose 5-aza-cytidine for 28 days. Once performed, the tissue may be harvested, arrays run, and the bioinformatic integration and analysis completed. The gene list will then be validated for its methylation status in a subset of normal salivary gland an primary ACC samples through bisulfite sequencing. At the conclusion of this project, we will have genome-wide characterization of candidate tumor suppressor genes under the control of promoter methylation. This powerful technique involving integration across array platforms allows for a wider range of gene discovery than by the use of methylation arrays alone, and will provide us with a more streamlined validation process. The candidate genes identified will then provide more focused targets for further investigation into the basic mechanisms of ACC carcinogenesis. PUBLIC HEALTH RELEVANCE: The goal of this project is to perform a comprehensive survey of the methylated tumor suppressor genes relevant in ACC. Using a novel method of global demethylation in primary tumor heterotransplants along with an integrated array analysis; we intend to validate and identify pertinent targets that may then be further investigated for their mechanistic role in carcinogenesis.