Role of newly identified, thyroid-specific LincRNA in BRAFV600E thyroid carcinoma

新发现的甲状腺特异性 LincRNA 在 BRAFV600E 甲状腺癌中的作用

• 批准号: 10368959
• 负责人: Carmelo Nucera
• 金额: $ 39.23万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10368959
• 关键词: 3-Dimensional; ATAC-seq; Affect; Antisense RNA; Binding Sites; Biology; CDK4 gene; Cancer Patient; Cell Death; Cell Proliferation; Cell Survival; Cells; Chromatin; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Code; Combined Modality Therapy; Complex; DNA Binding; Data; Disease Progression; Down-Regulation; Drug resistance; E2F transcription factors; ERBB3 gene; EZH2 gene; Early Diagnosis; Enhancers; Enzymes; Epigenetic Process; Extracellular Matrix Proteins; FRAP1 gene; Genes; Genetic Transcription; Genomic Instability; Histone Acetylation; Histones; Human; Hydrogen Peroxide; In Vitro; Iodide Peroxidase; Iodine; Link; Malignant Neoplasms; Maps; Metabolism; Mutation; Neoplasm Metastasis; Normal tissue morphology; Oxidative Stress; Papillary thyroid carcinoma; Paracrine Communication; Pathway interactions; Patient-Focused Outcomes; Pericytes; Pharmacotherapy; Play; Proliferating; RNA; Recurrence; Regulation; Reporting; Resistance; Resistance development; Role; Sampling; Secondary to; Signal Transduction; Survival Rate; Technology; Therapeutic; Thrombospondin 1; Thyroid Gland; Thyroid carcinoma; Time; Tissues; Transcription Repressor; Untranslated RNA; Up-Regulation; XCL1 gene; cancer cell; clinically significant; density; design; drug development; epigenetic silencing; histone methylation; in vivo; inhibitor; mortality; mouse model; neoplastic cell; novel; novel therapeutics; overexpression; promoter; radioiodine therapy; restoration; screening; synergism; targeted treatment; transcriptome; transcriptome sequencing; translational impact; tumor; tumor growth; tumor microenvironment; tumor progression; uptake

BRAFV600E-papillary thyroid carcinoma (V600E-PTC) is particularly aggressive, and as it deregulates iodine metabolism, its victims are unresponsive to radioiodine treatment, and suffer high rates of metastasis and recurrence, and low survival rates. BRAFV600E inhibitors such as vemurafenib (vemu), which at first showed clinical promise, are the only therapeutic option, but resistance to these drugs is widely reported. Our premise is that the epigenetic silencing of the first identified thyroid-specific long intergenic non-coding RNA (lincRNA), Xloc001313 (Xloc13) deregulates iodine metabolism, sustains tumor cell survival, and promotes progression and drug resistance. Xloc13 is active downstream of the coding gene for TPO, a key enzyme for iodine metabolism, and its expression is far lower in V600E-PTC than normal tissue. Our project is designed to determine the cellular mechanisms behind these effects, and use these findings to better target clinical trials of new treatments. Vemu treatment induces Xloc13 expression, indicating it is silenced by BRAFV600E, and its inactivation by CRISPR in normal thyroid cells reduces TPO levels and iodine uptake, while increasing TSP-1 levels (extracellular matrix protein) and cell proliferation. Xloc13 RNA enhancer maps to DNA-binding motifs on the TSP-1 promoter, leading to down-regulation of TSP-1, which inactivates TGFβ1 to inhibit PTC cell and pericyte viability. We recently demonstrated that V600E-PTC samples are enriched in pericytes that co- express TSP-1 and TGFβ1 to sustain V600E-PTC cell survival. Xloc13 silencing correlates with high pericyte density, linking its action to pericyte biology. We also identified regulatory motifs in the Xloc13 promoter for EZH2 and E2F1 as transcriptional repressors up-regulated in vemu-resistant V600E-PTC cells and ChIP- enriched at these DNA-binding motifs. Critically, combining inhibitors of BRAFV600E+EZH2+CDK4/6 durably rescued Xloc13, and reduced PTC cell and pericyte viability. Xloc13 down-regulation therefore plays a key role in V600E-PTC, and our central objective is to determine its mechanisms of regulation and their impact on tumor progression and resistance. We hypothesize that BRAFV600E synergizes with EZH2 and the CDK4/6 (upstream of E2F1) pathway to down-regulate Xloc13, which deregulates iodine metabolism, enhances tumor viability, and increases invasion signaling through pericyte action, which leads to secondary drug resistance and tumor progression. Using cutting-edge technologies (CRISPR, ChIP, ATAC, RAP) and new cancer mouse model, our specific Aims are: 1) To identify the mechanisms regulating Xloc13 in V600E-PTC and their impact on tumor aggressiveness; 2) To determine the role of Xloc13 in the regulation of TPO in V600E-PTC and its impact on iodine metabolism and PTC cell survival; 3) To assess the efficacy of anti-BRAFV600E-EZH2-CDK4/6 combined therapy on human V600E-PTC in vivo. Our studies will have high clinical significance by supporting new clinical trials of trimodal combined therapies for advanced V600E-PTC patients, and identifying new mechanisms in PTC and its microenvironment which could serve as drug development targets for this tumor.

BRAFV 600 E-乳头状甲状腺癌（V600 E-PTC）特别具有侵袭性，并且由于其碘代谢失调，其患者对放射性碘治疗无反应，并且具有高转移率和复发率，以及低存活率。BRAFV 600 E抑制剂如维罗非尼（vemu）最初显示出临床前景，是唯一的治疗选择，但对这些药物的耐药性被广泛报道。我们的前提是，第一个确定的甲状腺特异性长基因间非编码RNA（lincRNA）Xloc 001313（Xloc 13）的表观遗传沉默使碘代谢失调，维持肿瘤细胞存活，并促进进展和耐药性。Xloc 13是TPO（碘代谢的关键酶）编码基因的活性下游，其在V600 E-PTC中的表达远低于正常组织。我们的项目旨在确定这些效应背后的细胞机制，并利用这些发现更好地针对新疗法的临床试验。Vemu处理诱导Xloc 13表达，表明它被BRAFV 600 E沉默，并且其在正常甲状腺细胞中被CRISPR灭活降低了TPO水平和碘摄取，同时增加了TSP-1水平（细胞外基质蛋白）和细胞增殖。Xloc 13 RNA增强子定位于TSP-1启动子上的DNA结合基序，导致TSP-1下调，从而使TGFβ1失活以抑制PTC细胞和周细胞活力。我们最近证明，V600 E-PTC样品富含共表达TSP-1和TGFβ1的周细胞，以维持V600 E-PTC细胞存活。Xloc 13沉默与高周细胞 密度，将其作用与周细胞生物学联系起来。我们还鉴定了EZH 2和E2 F1的Xloc 13启动子中的调控基序作为在vemu抗性V600 E-PTC细胞中上调的转录抑制物，并且ChIP在这些DNA结合基序处富集。重要的是，BRAFV 600 E + EZH 2 + CDK 4/6的组合抑制剂持久地拯救了Xloc 13，并降低了PTC细胞和周细胞的活力。因此，Xloc 13下调在V600 E-PTC中起着关键作用，我们的中心目标是确定其调节机制及其对肿瘤进展和耐药性的影响。我们假设BRAFV 600 E与EZH 2和CDK 4/6（E2 F1的上游）通路协同作用，下调Xloc 13，Xloc 13使碘代谢失调，增强肿瘤活力，并通过周细胞作用增加侵袭信号，导致继发性耐药性和肿瘤进展。使用尖端技术（CRISPR、ChIP、ATAC、RAP）和新的癌症小鼠模型，我们的具体目的是：1）确定Xloc 13在V600 E-PTC中的调节机制及其对肿瘤侵袭性的影响; 2）确定Xloc 13在V600 E-PTC中TPO调节中的作用及其对碘代谢和PTC细胞存活的影响; 3）评估抗BRAFV 600 E-EZH 2-CDK 4/6联合治疗对体内人V600 E-PTC的疗效。我们的研究将通过支持晚期V600 E-PTC患者的三模式联合治疗的新临床试验，并确定PTC及其微环境中的新机制，可作为该肿瘤的药物开发靶点，具有很高的临床意义。