Detection of 5-hmC as a Novel Screening Biomarker for Pancreatic Cancer

 描述（由申请人提供）：胰腺癌（PaC）在美国癌症死亡率中排名第四，在全球癌症死亡率中排名第八。早期诊断和预防是影响死亡率的唯一可行选择。然而，由于人群中PaC的患病率较低，大多数阳性筛查试验可能是假阳性。因此，寻找更准确的生物标志物用于早期筛查和预后预测仍然是一个巨大的挑战。考虑到PaC复杂的发病机制，PaC生物标志物的鉴定将受益于针对各种基因调控机制的系统搜索，考虑到基因表达失调是疾病发展的基础。最近的研究使用高通量表观基因组分析技术已经证明了异常修饰的CpG位点在PaC细胞系和肿瘤组织，这表明这些表观遗传标记的潜在应用，以早期检测这种可怕的疾病。特别地，最近重新发现5-羟甲基胞嘧啶（5-hmC）是各种人体组织中的稳定碱基，其可以通过泰特酶家族氧化5-甲基胞嘧啶（5-mC）产生，具有新兴的基因调节作用和对人类癌症的影响。我们推断，确定与PaC有关的胞嘧啶修饰（5-mC或5-hmC）的确切类型将为其发病机制提供新的见解，以及PaC早期检测的新表观遗传生物标志物。具体而言，将探索癌症基因组图谱（TCGA）数据，以指导选择PaC特异性CpG，以使用我们的同事和合作者Chuan He博士（HHMI研究者）开发的一种新型且经验证的技术，泰特辅助亚硫酸氢盐测序（TAB-Seq），在一组具有不同遗传复杂度的PaC细胞系中检测5-hmC的存在。我们还将使用TAB-Seq检测直接检测PaC肿瘤和周围正常组织样本中的5-hmC，这些样本已经在我们之前或正在进行的项目中从30名美国和30名中国患者中收集。我们的设计包括两个不同的人群将允许调查这些表观遗传标记在这种疾病的健康差异中的作用。为了研究5-hmC的临床意义，我们将使用一种新的生物光子技术，评估我们在30名美国和30名中国PaC患者以及30/30名匹配对照的内镜下和组织学上正常的十二指肠壶腹周围活检细胞中检测到的CpG标志物（5-mC和5-hmC）与致癌作用引起的纳米级细胞结构改变的关系。总之，该项目将首次定义哪些胞嘧啶修饰， hmC或5-mC或两者都涉及PaC。本研究结果将为应用包括5-hmC在内的CpG标记物作为PaC的早期筛查和预测性生物标志物奠定基础。最后，PI，Zhang博士和Hou博士组建了一支优秀的调查团队，在相关研究领域取得了重大成就，并拥有互补的专业知识，因此确保了这个多学科和高度创新的项目的成功。

 DESCRIPTION (provided by applicant): Pancreatic cancer (PaC) ranks the fourth in the US and eighth worldwide for cancer mortality. Early diagnosis and prevention is the only viable option for impacting the mortality rate. Due to the low prevalence of PaC in the population, however, most positive screening tests are likely false positives. Therefore, it remains a grand challenge to identify more accurate biomarkers for early screening and prognosis prediction. Given the complex pathogenesis of PaC, identification of biomarkers for PaC will benefit from a systematical search targeting various gene regulation mechanisms, considering that gene expression dysregulation underlies the disease development. Recent studies using high-throughput epigenomic profiling technologies have demonstrated aberrant modifications of CpG sites in PaC cell lines and tumor tissues, suggesting the potential applications of these epigenetic markers to early detection of this dreadful disease. In particular, 5- hydroxymethylcytosine (5-hmC), which can be generated by oxidation of 5-methylcytosine (5-mC) through the Tet family of enzymes, was recently re-discovered to be a stable base in various human tissues, with emerging gene regulatory role and implications for human cancers. We reasoned that determining the exact types of cytosine modifications (5-mC or 5-hmC) implicated in PaC will provide novel insights into its pathogenesis, as well as novel epigenetic biomarkers for PaC early detection. Specifically, The Cancer Genome Atlas (TCGA) data will be explored to guide the selection of PaC-specific CpGs to be tested for the existence of 5-hmC in a panel of PaC cell lines with varying degrees of genetic complexity using a novel and proven technique, Tet- assisted Bisulfite Sequencing (TAB-Seq), developed by our colleague and collaborator, Dr. Chuan He (HHMI Investigator). We will also use the TAB-Seq assay to examine 5-hmC directly in PaC tumor and surrounding normal tissue samples that have already been collected from 30 US and 30 Chinese patients in our previous or ongoing projects. Our design of including two diverse populations will allow investigation of the role of these epigeneti markers in the health disparities in this disease. To examine the clinical significance of the 5-hmC, we will evaluate our detected CpG markers (5-mC and 5-hmC) in cells from endoscopically- and histologically normal-appearing periampullary duodenal biopsies from 30 US and 30 Chinese PaC patients as well as 30/30 matched controls in relation to the nanoscale cellular architecture alterations due to field carcinogenesis using a novel biophotonic technology. In summary, this project will for the first time define which cytosine modifications, 5 hmC or 5-mC or both, are implicated in PaC. The results from this proposal will lay the foundation of applying CpG markers including 5-hmC as a novel class of early screening and predictive biomarkers for PaC. Finally, the PIs, Drs. Zhang and Hou have assembled an excellent investigative team with significant achievements in relevant research areas and complimentary expertise, therefore ensuring the success of this multidisciplinary and highly innovative project.