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

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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.