Exploring mechanisms of therapeutic demethylation effects in HPV-associated head and neck cancer

探索 HPV 相关头颈癌去甲基化治疗作用的机制

• 批准号: 10192704
• 负责人: Karen S. Anderson
• 金额: $ 40.47万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10192704
• 关键词: Aftercare; Apoptosis; Azacitidine; Caspase; Cell Death; Cell Line; Cell Survival; Cells; Cervical; Characteristics; Chemotherapy and/or radiation; Clinical Data; Clinical Trials; Cytidine Deaminase; DNA; DNA Damage; DNA Double Strand Break; Data; Deglutition; Dental caries; Detection; Diagnosis; Disease; Dose; Double Strand Break Repair; Down-Regulation; Dysmyelopoietic Syndromes; Enrollment; Excision; FDA approved; Family member; Fibrosis; Functional disorder; Gene Expression; Genes; Genetic Transcription; Genomic Instability; Growth; Guidelines; Head and Neck Cancer; Head and Neck Neoplasms; Head and Neck Squamous Cell Carcinoma; Human; Human Papillomavirus; Human papillomavirus 16; Immune; In Vitro; Infection; Infiltration; Interferon Activation; Interferons; Interstitial Collagenase; Lead; Location; Lymph Node Dissections; Lymphedema; Malignant - descriptor; Malignant Epithelial Cell; Malignant Neoplasms; Matrix Metalloproteinases; Methods; Methylation; Modeling; Molecular; Molecular Biology; Mus; National Comprehensive Cancer Network; Neoplasm Circulating Cells; Neoplasm Metastasis; Oncogenes; Oncogenic; Operative Surgical Procedures; Oropharyngeal Squamous Cell Carcinoma; Pathologic; Pathway interactions; Patients; Pharmaceutical Preparations; Production; Prognosis; Prognostic Marker; Property; Radiation; Recurrence; Regulation; Retinoblastoma; Role; Site; Skeletal muscle structure of neck; Specimen; Speech; Survival Rate; T-Lymphocyte; TP53 gene; Therapeutic; Tobacco use; Toxic effect; Transcript; Tumor Suppressor Proteins; Tumor-Infiltrating Lymphocytes; United States; Uterus; Xerostomia; Yale Cancer Center; cancer cell; cancer therapy; carcinogenesis; cell transformation; cervical and uterine cancer; chemotherapeutic agent; clinically relevant; cytokine; cytotoxic; demethylation; design; experimental study; falls; human papilloma virus oncogene; improved; in vitro Assay; molecular modeling; mouse model; multimodality; novel therapeutics; overexpression; patient derived xenograft model; pre-clinical; prevent; promoter; public health relevance; response; response biomarker; restoration; side effect; targeted treatment; tumor; tumor growth; tumor xenograft; tumorigenesis

Abstract Oncogenic human papillomaviruses (HPV) are the causative agents of uterine cervical and an increasing portion of head and neck squamous cell carcinomas (HNSCC), but HNSCC is almost exclusively associated with HPV type 16. The oncogenic properties of HPV type 16 are largely attributed to two major HPV oncogenes, E6 and E7, that degrade p53 and retinoblastoma (RB) family members, respectively. Degradation of these tumor suppressors by E6 and E7 results in uncontrolled proliferation, diminished apoptosis and increased genomic instability that predisposes to malignant transformation. The crucial roles of E6/E7 in HPV-related carcinogenesis make them an attractive target for anti-cancer therapy, since methods for decreasing their expression or activities would restore p53 and RB activity in tumors driven by HPV. Our preliminary results indicate that treatment of HPV-positive (HPV+) HNSCC with the demethylating agent, 5-azacytidine (5-aza) at clinically relevant concentrations, resulted in remarkable downregulation of all HPV gene expression, including E6 and E7. 5-aza treatment restored p53 expression and activity in HPV+ head and neck cancer cells, which was partially responsible for the sensitivity of these cells to 5-aza. In addition to restoration of p53, 5-aza also was toxic to HPV+ HNSCC through creation of DNA double strand breaks (DSBs). Mechanistically, 5-aza-induced DNA DSBs in HPV+ HNSCC were dependent on transcription and replication and on overexpression of the cytidine deaminase, APOBEC3B (A3B). Experimental depletion of A3B inhibited 5-aza toxicity and diminished DSB formation, but also indicated that untreated HPV+ HNSCC depend on A3B for clonogenic growth. The observations that untreated HPV+ HNSCC dependent on A3B, but that A3B contributes to 5-aza toxicity and DSBs, suggests an A3B-dependent synthetic lethality upon treatment with 5-aza, and that A3B may serve as a biomarker of response. Treatment of mice bearing HPV+ tumors with 5-aza revealed significant tumor growth inhibition and prevented detection of circulating tumor cells. A window clinical trial in patients with HNSCC using standard dosing for 5 days achieved demethylation (LINE-1) similar to that seen in our in vitro experiments and confirmed that 5-aza treatment: 1) significantly decreased expression of HPV genes; 2) reactivated p53; 3) activated caspases, 4) and inhibited matrix metalloproteinase expression in HPV+ HNSCCs. This proposal is designed to determine molecular mechanisms of demethylation-induced downregulation of HPV oncogenes, elucidate the role of A3B in 5-aza-induced synthetic lethality and DNA DSBs formation, determine effect of demethylation on immune cell infiltration in HPV+ HNSCC, and explore the potential of 5-aza alone or in combination with chemotherapeutic agents to suppress HPV-associated HNSCC metastasis and inhibit growth using patient-derived xenografts. These studies will provide a basis for a new rational targeted therapy for HPV+ HNSCC, which is desperately needed to treat patients with recurrent or metastatic HPV+ HNSCC and to decrease the toxicity associated with current therapy.

抽象的 致癌性人乳头瘤病毒 (HPV) 是宫颈癌的病原体，并且日益增多。 头颈鳞状细胞癌 (HNSCC) 的一部分，但 HNSCC 几乎完全与 HPV 16 型。HPV 16 型的致癌特性很大程度上归因于两个主要的 HPV 致癌基因：E6 和 E7，分别降解 p53 和视网膜母细胞瘤 (RB) 家族成员。这些肿瘤的降解 E6 和 E7 抑制因子导致增殖失控、细胞凋亡减少和基因组增加 不稳定，容易发生恶变。 E6/E7 在 HPV 相关癌发生中的关键作用 使它们成为抗癌治疗的有吸引力的目标，因为减少它们表达或活性的方法 将恢复 HPV 驱动的肿瘤中的 p53 和 RB 活性。我们的初步结果表明，治疗 使用去甲基化剂 5-氮杂胞苷 (5-aza) 治疗 HPV 阳性 (HPV+) HNSCC 具有临床相关性 浓度，导致所有 HPV 基因表达显着下调，包括 E6 和 E7。 5-氮杂 治疗恢复了 HPV+ 头颈癌细胞中的 p53 表达和活性，部分恢复了 负责这些细胞对 5-aza 的敏感性。除了恢复 p53 之外，5-aza 还对 通过 DNA 双链断裂 (DSB) 产生 HPV+ HNSCC。从机制上讲，5-氮杂诱导 DNA HPV+ HNSCC 中的 DSB 依赖于转录和复制以及胞苷的过度表达 脱氨酶，APOBEC3B (A3B)。 A3B 的实验消耗抑制了 5-aza 毒性并减少了 DSB 形成，但也表明未经治疗的 HPV+ HNSCC 依赖于 A3B 进行克隆生长。这 观察结果表明，未经治疗的 HPV+ HNSCC 依赖于 A3B，但 A3B 会导致 5-aza 毒性，并且 DSBs 表明，用 5-aza 处理后，A3B 依赖性合成致死率，并且 A3B 可以作为 反应的生物标志物。用 5-aza 治疗携带 HPV+ 肿瘤的小鼠发现肿瘤显着生长 抑制并阻止循环肿瘤细胞的检测。使用 HNSCC 患者进行的窗口临床试验 5 天的标准剂量实现了去甲基化 (LINE-1)，与我们的体外实验中看到的相似， 证实5-aza治疗：1）显着降低HPV基因的表达； 2)重新激活p53； 3） 激活半胱天冬酶，4) 并抑制 HPV+ HNSCC 中的基质金属蛋白酶表达。这个提议是 旨在确定去甲基化诱导的 HPV 癌基因下调的分子机制， 阐明 A3B 在 5-aza 诱导的合成致死性和 DNA DSB 形成中的作用，确定 去甲基化对 HPV+ HNSCC 免疫细胞浸润的影响，并探索 5-aza 单独使用或联合使用的潜力 与化疗药物联合抑制 HPV 相关的 HNSCC 转移并抑制生长 使用患者来源的异种移植物。这些研究将为HPV+的新的合理靶向治疗提供基础 HNSCC，迫切需要治疗复发性或转移性 HPV+ HNSCC 患者，并 减少与当前治疗相关的毒性。