HLTF gene silencing: a novel determinant of sensitivity to autophagy inhibition

HLTF 基因沉默：自噬抑制敏感性的新决定因素

• 批准号: 8664818
• 负责人: RAVI K AMARAVADI
• 金额: $ 33.24万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8664818
• 关键词: Advanced Malignant Neoplasm; Antineoplastic Agents; Autophagocytosis; Biological Assay; Biological Markers; Blood; Cancer Patient; Cancer cell line; Cell Line; Cells; Cessation of life; Characteristics; Chloroquine; Clinical; Clinical Treatment; Clinical Trials; Colon Carcinoma; DNA Damage; DNA Methylation; DNA Repair; Data; Development; Disease; Enrollment; Frequencies; Future; Gene Silencing; Genes; Genome; Genomics; Goals; Human; Hydroxychloroquine; Institution; Knowledge; Light; Link; Lysosomes; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of pancreas; Measures; Mediating; Metabolic; Methylation; Microarray Analysis; Molecular; Outcome; Pathway interactions; Patients; Performance; Prevalence; Process; Prognostic Marker; Reactive Oxygen Species; Refractory; Regimen; Research Personnel; Resistance; SMARCA3 gene; Sampling; Serum; Specific qualifier value; Stable Disease; Stomach Carcinoma; Stress; Testing; Therapeutic; Tissue Microarray; Tumor Suppressor Genes; Tumor Tissue; Validation; Work; assay development; base; cancer cell; cancer therapy; cohort; colon cancer cell line; demethylation; helicase; improved; inhibition of autophagy; inhibitor/antagonist; lung Carcinoma; mRNA Expression; malignant breast neoplasm; malignant stomach neoplasm; melanoma; new therapeutic target; novel; overexpression; oxidative DNA damage; pre-clinical; promoter; public health relevance; repaired; response; transcription factor; tumor

DESCRIPTION (provided by applicant): Autophagy is a lysosome-dependent degradative process that protects cancer cells from metabolic and therapeutic stress. Autophagy is up regulated in most advanced cancers and has identified as a new target for cancer therapy. Autophagy inhibition with chloroquine (CQ) derivatives augments the efficacy of many anticancer therapies. Numerous clinical trials are testing the combination of variety of anticancer agents with hydroxychloroquine (HCQ), but there currently is no biomarker that can focus the development of HCQ combinations or regimens involving novel emerging autophagy inhibitors into patient subsets that will most likely benefit from this strategy. To identify a candidate biomarker of sensitivity to autophagy inhibition, a microarray analysis of differentially expressed genes in HCQ-sensitive and HCQ-resistant human cancer cell lines was conducted. The most down regulated gene in HCQ-sensitive cells was helicase- like transcription factor (HLTF), an understudied tumor suppressor gene involved in multiple aspects of maintaining genomic integrity during replication stress. HLTF expression is silenced by promoter methylation in 20-40% of lung, colon, and gastric carcinomas. In a large panel of cell lines HLTF gene silencing was found almost exclusively in cell lines that were sensitive to HCQ. Forced expression of HLTF in HLTF silenced cells conferred resistance to HCQ. The link between autophagy inhibition and HLTF may be through oxidative DNA damage that was observed soon after HCQ treatment. A methylation-specific PCR assay was able to detect HLTF methylation status of tumors in the serum samples of patients with melanoma and breast cancer indicating that HLTF gene silencing is common across multiple malignancies and a serum assay may be able to classify patients as HLTF gene silenced or expressed. This proposal will test the hypotheses that a) HLTF gene silencing confers sensitivity to autophagy inhibitors by allowing oxidative DNA damage to go unrepaired~ b) A clinical grade assay for HLTF promoter methylation in the serum or tumors of patients can be a sensitive and specific assay for HLTF gene silencing c) HLTF gene silencing is common in a number of malignancies and predicts of clinical response in patients treated with HCQ. Knowledge gained from completion of these specific aims will establish a new mechanistic framework that links autophagy inhibition to the DNA damage response. This work will shed light on the functions of commonly silenced tumor suppressor gene HLTF and will determine which malignancies should be a target for the development of autophagy inhibitors. Finally, as novel autophagy inhibitors are currently being developed for clinical trials in cancer patients, completion of these aims will provide the supporting data necessary for future development of a CLIA approved predictive assay that could be used to enroll the patients most likely to benefit from autophagy inhibitors.

描述（由申请人提供）：自噬是一种溶酶体依赖性降解过程，可保护癌细胞免受代谢和治疗应激。自噬在大多数晚期癌症中表达上调，并已被确定为癌症治疗的新靶点。使用氯喹 (CQ) 衍生物抑制自噬可增强许多抗癌疗法的疗效。大量临床试验正在测试多种抗癌药物的组合 羟氯喹 (HCQ) 药物，但目前没有生物标志物可以将 HCQ 组合或涉及新型自噬抑制剂的治疗方案的开发集中到最有可能从该策略中受益的患者亚群中。 为了确定对自噬抑制敏感的候选生物标志物，对差异表达的微阵列分析 对 HCQ 敏感和 HCQ 抗性人类癌细胞系中的基因进行了研究。 HCQ 敏感细胞中下调最多的基因是解旋酶样转录因子 (HLTF)，这是一种正在研究的肿瘤抑制基因，涉及复制应激期间维持基因组完整性的多个方面。在 20-40% 的肺癌、结肠癌和胃癌中，HLTF 表达因启动子甲基化而沉默。 在一大组细胞系中，HLTF 基因沉默几乎只出现在对 HCQ 敏感的细胞系中。 HLTF 沉默细胞中 HLTF 的强制表达赋予了对 HCQ 的抗性。自噬抑制与 HLTF 之间的联系可能是通过 HCQ 治疗后不久观察到的氧化 DNA 损伤而产生的。甲基化特异性 PCR 检测能够检测黑色素瘤和乳腺癌患者血清样本中肿瘤的 HLTF 甲基化状态，表明 HLTF 基因沉默在多种恶性肿瘤中很常见，血清检测可能能够将患者分类为 HLTF 基因沉默或表达。 该提案将测试以下假设：a) HLTF 基因沉默通过允许氧化 DNA 损伤无法修复来赋予对自噬抑制剂的敏感性 ~ b) 对患者血清或肿瘤中 HLTF 启动子甲基化进行临床级检测可能是一种敏感且特异的检测HLTF 基因沉默 c) HLTF 基因沉默在许多恶性肿瘤中很常见，可预测接受 HCQ 治疗的患者的临床反应。 从完成这些具体目标中获得的知识将建立一个新的机制框架，将自噬抑制与 DNA 损伤反应联系起来。这项工作将揭示通常沉默的肿瘤抑制基因 HLTF 的功能，并将确定哪些恶性肿瘤应该成为自噬抑制剂开发的目标。 最后，由于目前正在开发新型自噬抑制剂用于癌症患者的临床试验，这些目标的完成将为未来开发 CLIA 批准的预测测定提供必要的支持数据，该预测测定可用于招募最有可能从自噬中受益的患者抑制剂。