Metagenomic profiling of oral polymicrobial flora in head and neck cancers

描述(申请人提供)：当胃中的幽门螺杆菌与胃癌联系在一起，其根除与回归相关时，建立了癌症发生的范式转换模型。许多细菌种类通常存在于口咽粘膜的不同区域，并可能随宿主的年龄和饮食消费而变化。有趣的是，头颈部鳞状细胞癌(HNSCC)的发病率已被证明与牙齿卫生有关，这可能表明细菌与这种特殊恶性肿瘤的发病机制有关。一些细菌会导致炎症，这可能会导致甲基化增加。利用基因组技术，我们已经证明了HNSCC和基于它们的元基因组图谱的特定细菌种群之间可能存在的联系。值得注意的是，我们在每个代谢组谱中存在特定细菌亚群与HNSCC中MDR1甲基化状态之间存在原则上的相关性。因此，我们假设，如元基因组学所揭示的那样，特定的细菌亚群可以触发靶向甲基化事件，从而启动HNSCC的发生和/或进展。我们计划通过3个广泛的目标来解决这一假说：(1)利用最新的高通量测序技术来确定口咽粘膜中与HNSCC肿瘤的启动、进展和/或预防相关的细菌亚群，以创新的亚目标来探索特定的微生物组特征是否是HNSCC中场效应的一部分；(2)询问影响炎症的特定微生物“联合体”，从而导致HNSCC中目标肿瘤抑制基因的表观遗传学改变；(3)从功能上验证MAD2和/或MDR1在HNSCC肿瘤抑制中的参与。将询问由后基因组特征所反映的由此产生的微生物种群与暴露和临床信息以及靶向甲基化的相关性。最后，我们将从功能上验证靶向甲基化是否与HNSCC肿瘤的发生有关，或者是炎症反应中的随机甲基化效应。为了解决这一问题，我们将在HNSCC细胞系中过表达或下调mdr1，并检测其对生长、凋亡、细胞形态或分化的影响。这项研究承诺揭示特定的头部和颈部细菌菌群与癌症的发生和发展的相关性。有趣的未来方向包括临床筛查特定细菌指示物的存在，以便提供早期癌症或癌症前诊断，并可能基于益生菌或抗生素进行预防。通过代谢组谱能够根据先前的放射暴露或化疗区分样本，我们将对难治性疾病患者实施有针对性的筛查和/或去甲基化治疗。这种类型的数据还将指出，早期发现、意识教育以及卫生员和护理人员的预防将发挥更大的作用。 与公共卫生相关：全世界每年诊断出超过500,000例新的头颈部癌症(HNSCC)，仅在美国每年就有超过11,000人死于这种侵袭性的恶性肿瘤；尽管在这一主题的研究成果方面取得了巨大进展，但大多数晚期患者的总体结果在30多年内保持不变。接受积极手术切除癌症的患者有严重的毁容和生活质量下降，吞咽、嗅觉和听力能力受损。如果成功，那么我们目前的提议有望对口腔中特定细菌亚群的作用有一个新的理解，这些细菌亚群会导致炎症，从而关闭癌症抑制基因，从而导致HNSCC。如果是真的，那么我们可以利用我们的发现进行最早的诊断，甚至用益生菌治疗进行预防。重要的是，即使对于晚期HNSCC或已经扩散的HNSCC，我们的发现也将有助于指出抗生素和药物可以重新启动抑制癌症的基因，从而使治疗发生革命性变化。

DESCRIPTION (provided by applicant): When H. pylori in the stomach was linked with gastric cancer, and its eradication associated with regression, a paradigm-shifting model of carcinogenesis was created. Many bacterial species normally reside in various regions of the oropharyngeal mucosa, and may fluctuate depending on the host's age and dietary consumption. Interestingly, the incidence of head and neck squamous cell carcinoma (HNSCC) has been shown to correlate with dental hygiene, which may suggest the relevance of bacteria in the pathogenesis of this particular malignancy. Some bacteria can result in inflammation, which can cause increased methylation. Using genomic technology, we have demonstrated a possible link between HNSCC and specific bacterial populations based on their metagenomic profiles. Notably, we have proof-of-principle association between the presence of particular bacterial subpopulations per metabiomic profiling and MDR1 methylation status in HNSCC. We therefore hypothesize that specific bacterial subpopulations, as revealed by metagenomic profiling, can trigger targeted methylation events which then initiate HNSCC genesis and/or progression. We plan to address this hypothesis via 3 broad aims: (1) To utilize most recent high throughput sequencing technologies to identify the bacterial subpopulations in the oropharyngeal mucosa associated with HNSCC tumor initiation, progression, and/or prevention, with the innovative sub-aim to explore if specific microbiomic profiles are part of the field effect seen in HNSCC; (2) To interrogate the specific microbial "consortia" influencing inflammation with consequent epigenetic alterations of target tumor suppressor genes in HNSCC; (3) To functionally validate the involvement of MAD2 and/or MDR1 in HNSCC tumor suppression. The resulting microbial populations as reflected by metagenomic profiles will be interrogated for correlation with exposure and clinical information and targeted methylation. Finally, we will functionally validate whether the targeted methylation is relevant in HNSCC tumorigenesis or a random methylation effect in response to inflammation. To address this, we will overexpress or knock-down MDR1 in HNSCC cell lines and examine any effect on growth, apoptosis, cell morphology, or differentiation. This study promises to reveal the relevance of specific head and neck bacterial flora to cancer initiation and progression. Interesting future directions include clinical screening for the presence of specific bacterial indicators in order to provide early cancer or pre-cancer detection and perhaps prevention, based on probiotics or antibiotics. With metabiomic profiles capable of distinguishing samples based on prior radiation exposure or chemotherapy, we will implement targeted screening and/or demethylation treatment for patients with refractory disease. This type of data will also point to larger roles for early detection, awareness education, and prevention from hygienists and nursing. PUBLIC HEALTH RELEVANCE: Over 500,000 new cases of head and neck cancers (HNSCC) are diagnosed worldwide annually, with more than 11,000 individuals/year succumbing to this aggressive malignancy in the United States alone; despite huge strides in research achievements in this topic, overall outcome has remained unchanged for over 3 decades for the majority of patients with advanced stages of this disease. Patients who receive aggressive surgery to remove the cancer have significant disfigurement and diminished quality of life, with impaired ability to swallow, smell, and hear. If successful, then our current proposal promises a new understanding of the role of specific subpopulations of bacteria in the mouth leading to inflammation which shuts off cancer-suppressing genes, thus leading to HNSCC. If true, then we can take advantage of our findings for the earliest diagnosis and even prevention with probiotic therapy. Importantly, even for advanced HNSCC or that which has spread, our findings will help point to antibiotics and agents that can turn back on the cancer-suppressing genes to revolutionize treatment.