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被上调，并已被确定为癌症治疗的新靶点。氯喹（CQ）衍生物的自噬抑制增强了许多抗癌疗法的疗效。许多临床试验正在测试各种抗癌药物的组合， 目前，研究人员已经发现了具有羟氯喹（HCQ）的药物，但目前没有生物标志物可以将HCQ组合或方案的开发集中在最有可能从该策略中受益的患者亚群中，所述HCQ组合或方案涉及新出现的自噬抑制剂。 为了鉴定对自噬抑制敏感性的候选生物标志物，使用微阵列分析差异表达的 在HCQ敏感性和HCQ抗性人癌细胞系中进行了基因表达的研究。HCQ敏感细胞中下调最多的基因是解旋酶样转录因子（HLTF），这是一种研究不足的肿瘤抑制基因，参与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表达被启动子甲基化沉默。 在一组大的细胞系中，HLTF基因沉默几乎仅在对HCQ敏感的细胞系中发现。 HLTF沉默细胞中HLTF的强制表达赋予了对HCQ的抗性。自噬抑制和HLTF之间的联系可能是通过HCQ处理后不久观察到的氧化DNA损伤。甲基化特异性PCR测定能够检测黑素瘤和乳腺癌患者血清样品中肿瘤的HLTF甲基化状态，表明HLTF基因沉默在多种恶性肿瘤中是常见的，并且血清测定能够将患者分类为HLTF基因沉默或表达。 该提案将检验以下假设：a）HLTF基因沉默通过允许氧化DNA损伤不被修复而赋予对自噬抑制剂的敏感性~ B）患者血清或肿瘤中HLTF启动子甲基化的临床级测定可以是HLTF基因沉默的敏感性和特异性测定c）HLTF基因沉默在许多恶性肿瘤中是常见的，并且预测用HCQ治疗的患者的临床应答。 从完成这些特定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，由于目前正在开发用于癌症患者临床试验的新型自噬抑制剂，这些目标的完成将为未来开发CLIA批准的预测分析提供必要的支持数据，该预测分析可用于招募最有可能受益于自噬抑制剂的患者。