Epigenetic Profiling of Oral Cancer Cells

描述（由申请人提供）：拟议的工作将评估一种用于分析癌症起始细胞（也称为癌症干细胞）的创新技术，以鉴定可用于诊断的标记物，并鉴定可指导药物开发计划的分子靶点。口腔鳞状细胞癌（OSCC）在美国每年诊断为> 25，000例患者，在过去5年中一直稳步增加。口腔鳞状细胞癌在手术切除和放射治疗后会导致严重的言语和吞咽困难。此外，复发性和转移性OSCC对细胞毒化疗反应较差。因此，仍然迫切需要有助于早期检测的策略和具有更高治疗功效的新药物疗法的开发。口腔癌的发展和进展依赖于一小群癌症干细胞，这些干细胞不仅可以在局部治疗后再生肿瘤块，而且还通过对标准化疗特别难治而导致癌症复发。这项工作将应用一种新的肽阵列技术来分析口腔上皮细胞的裂解物， 细胞、OSCC细胞和已经经历EMT的OSCC细胞，以鉴定可能是CSC表型基础的脱乙酰酶活性模式。该技术基于SAMDI质谱技术，该技术使用基质辅助激光解吸-电离质谱来分析固定到自组装单层的肽阵列。SAMDI质谱法检测肽-烷硫醇盐缀合物的质量，因此可以鉴定肽乙酰化的变化。拟议的工作将应用这种方法来配置文件中的细胞培养物中的脱乙酰酶活性，并确定口腔角质形成细胞，OSCC细胞，OSCC细胞，已经经历了EMT，并在CSC之间的活性曲线的差异。第一个目标将开发用于分析细胞裂解物中乙酰化活性的肽阵列，并将验证裂解物实验的重现性，并将使用选择性抑制剂解析活性，以了解哪些特定酶对活性谱有贡献。第二个目标将应用阵列来比较OSCC细胞中的乙酰化活性与CSC群体中存在的乙酰化活性。这项R21提案的目标是验证SAMDI技术用于识别癌症干细胞生成和维持的分子活性的用途。这项工作有一个更广泛的目标，即引入一种理解癌症干细胞调控的创新策略，并可能导致新的诊断方法和药物开发目标。

DESCRIPTION (provided by applicant): The proposed work will evaluate an innovative technology for profiling cancer initiator cells (also known as cancer stem cells) to identify markers that can be used in diagnostics and to identify molecular targets that can guide drug development programs. Oral squamous cell carcinoma (OSCC), which is diagnosed in > 25,000 patients in the US each year, has been steadily increasing over the past 5 years. OSCC causes significant morbidity as a result of speech and swallowing difficulties following surgical resectio and radiation treatment. Moreover, recurrent and metastatic OSCC is poorly responsive to cytotoxic chemotherapy. Hence, there remains a pressing need for strategies that aid earlier detection and for the development of new drug therapies that have higher treatment efficacy. The development and progression of oral cancers (among others) relies on a small population of cancer stem cells which can not only regenerate the tumor bulk following local treatment but also contribute to cancer recurrence by being particularly refractory to standard chemotherapy. The proposed work will apply a novel peptide array technology to profile lysates of oral epithelial cells, OSCC cells and OSCC cells that have undergone EMT to identify patterns of deacetylase activity that may underlie the CSC phenotype. The technology is based on the SAMDI mass spectrometry technique that uses matrix- assisted laser desorption-ionization mass spectrometry to analyze an array of peptides immobilized to self- assembled monolayers. SAMDI mass spectrometry detects the mass of the peptide-alkanethiolate conjugate and therefore can identify changes in acetylation of the peptides. The proposed work will apply this method to profile deacetylase activities in cell cultures and identify differences in the activity profiles between oral keratinocytes, OSCC cells, OSCC cells that have undergone EMT, and in CSCs. The first Aim will develop peptide arrays for profiling acetylation activities in cell lysate and will validate the reproducibility of the experiment for the lysate and will use selective inhibitors to parse the activities to understand which specific enzymes contribute to the activity profiles. The second aim will apply the arrays to compare the acetylation activities in OSCC cells to those present in the CSC population. The goal of this R21 proposal is to validate the use of the SAMDI technology for identifying molecular activities that underlie the generation and maintenance of cancer stem cells. The work has the broader goal of introducing an innovative strategy for understanding the regulation of cancer stem cells and may lead to new diagnostic approaches and targets for drug development.