Pax8-PPARgamma regulation of transcription and metabolism in thyroid cancer

Pax8-PPARgamma 对甲状腺癌转录和代谢的调节

• 批准号: 8683123
• 负责人: RONALD Jay KOENIG
• 金额: $ 29.58万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8683123
• 关键词: 2,4-thiazolidinedione; Apoptosis; Benign; Binding; Binding Sites; Biology; Boxing; Cell Line; Cell Proliferation; Cells; ChIP-seq; Chimeric Proteins; Chromosomal translocation; Citrates; Citric Acid Cycle; DNA; DNA Binding; DNA Binding Domain; Data; Diabetes Mellitus; Doxycycline; Energy Metabolism; Energy-Generating Resources; Fatty Acids; Follicular thyroid carcinoma; Gene Activation; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Targeting; Genes; Genus Hippocampus; Glucose; Glycolysis; Growth; Human; Ligands; Lung; Lung Neoplasms; Malignant Neoplasms; Malignant neoplasm of thyroid; Metabolic; Metabolic Pathway; Metabolism; Microarray Analysis; Molecular Profiling; Mutation; NOD/SCID mouse; Neoplasm Metastasis; Nuclear Receptors; Oncogenes; PPAR gamma; Pathway Analysis; Patients; Peroxisome Proliferator-Activated Receptors; Pharmaceutical Preparations; Primary Neoplasm; Protein Binding; Proteins; Public Health; RXR; Recruitment Activity; Response Elements; Role; Site; Testing; Thiazolidinediones; Thyroid Gland; Time; Transcriptional Regulation; Transfection; Tumorigenicity; cancer cell; cell growth; chromatin immunoprecipitation; deep sequencing; diabetes mellitus therapy; fatty acid metabolism; genome-wide; genome-wide analysis; inhibitor/antagonist; mutant; novel strategies; oxidation; promoter; research study; transcription factor; tumor growth; tumor xenograft; tumorigenesis

DESCRIPTION (provided by applicant): The long term objective is to reveal the mechanisms underlying the transcriptional activity, metabolic effects and tumorigenicity of a unique nuclear receptor transcription factor, PAX8-PPARg Fusion Protein (PPFP) that is produced as a consequence of a chromosomal translocation in follicular thyroid carcinomas. PPFP contains nearly the full sequence of the transcription factor paired box 8 (PAX8) plus the entirety of the nuclear receptor peroxisome proliferator-activated receptor g1 (PPARg1). PPFP can activate the promoters of PPAR-responsive genes, although the activity of PPFP is clearly distinct from that of PPARg. Gene expression profiling of PPFP thyroid cancers (compared to all other benign and malignant thyroid neoplasms) resulted in the identification of a PPFP cancer gene signature. The metabolic pathway most enriched in PPFP signature genes is fatty acid 2 oxidation, which is physiologically regulated by PPARs. Stably transfected clones of the non-transformed thyroid cell line PCCL3 that express PPFP in a doxycycline-dependent manner (DoxyPPFP cells) will be used to understand the importance of fatty acid metabolism in the biology of PPFP cancers, the mechanism of oncogenesis, and potential approaches to therapy. In these cells, PPFP induces many genes that are induced in PPFP thyroid cancers, and these cells form xenograft tumors and lung metastases in NOD-SCID mice. In the first Specific Aim, chromatin immunoprecipitation - deep sequencing (ChIP-Seq) will be used to identify PPFP binding sites on a genome wide level. Additional studies will evaluate whether binding to specific DNA sites requires PPFP's PAX8 DNA binding domain, its PPARg DNA binding domain, or both. Affymetrix microarray gene expression profiling will be performed on DoxyPPFP cells to allow a comparison of gene expression with genome wide DNA binding. These data also will be compared with gene expression data already obtained from PPFP cancers. Coregulatory protein recruitment by PPFP to its target genes will be evaluated. The hypothesis is that the PAX8 portion of PPFP recruits inappropriate coregulators to PPAR responsive genes, and vice versa. These studies will rely primarily on chromatin immunoprecipitation - real time PCR. Specific Aim 2 will test the hypothesis that PPFP-expressing cells are highly dependent on fatty acid 2 oxidation for cell growth; i.e., that PPFP induction of 2 oxidation creates a growth advantage. A Seahorse XF24 bioanalyzer will be used to evaluate how much energy metabolism is via glycolysis versus the TCA cycle. The use of glucose versus fatty acids as energy sources will be evaluated. The effects of PPFP on de novo fatty acid synthesis, cell proliferation and apoptosis will be assessed, to better understand the putative growth advantage brought about by PPFP. In Specific Aim 3, the effects of PPARg ligands (thiazolidinediones) on xenograft tumor and metastasis formation will be evaluated in NOD-SCID mice. The importance of PPFP functional domains on xenograft tumor formation and metastases will be assessed with DoxyPPFP cell lines expressing mutant PPFPs (for example, mutation of either the PAX8 or PPARg DNA binding domain).

描述（由申请人提供）：长期目标是揭示独特核受体转录因子 PAX8-PPARg 融合蛋白（PPFP）的转录活性、代谢效应和致瘤性的机制，该蛋白是滤泡性甲状腺癌中染色体易位的结果。 PPFP 包含转录因子配对盒 8 (PAX8) 的几乎完整序列以及核受体过氧化物酶体增殖物激活受体 g1 (PPARg1) 的全部序列。 PPFP 可以激活 PPAR 反应基因的启动子，尽管 PPFP 的活性明显不同于 PPARg。 PPFP 甲状腺癌的基因表达谱（与所有其他良性和恶性甲状腺肿瘤相比）导致了 PPFP 癌症基因特征的鉴定。 PPFP 特征基因中最丰富的代谢途径是脂肪酸 2 氧化，该途径在生理上受 PPAR 调节。以强力霉素依赖性方式表达 PPFP 的非转化甲状腺细胞系 PCCL3 的稳定转染克隆（DoxyPPFP 细胞）将用于了解脂肪酸代谢在 PPFP 癌症生物学中的重要性、肿瘤发生机制和潜在的治疗方法。在这些细胞中，PPFP 诱导许多在 PPFP 甲状腺癌中诱导的基因，这些细胞在 NOD-SCID 小鼠中形成异种移植肿瘤和肺转移。在第一个具体目标中，染色质免疫沉淀 - 深度测序 (ChIP-Seq) 将用于在全基因组水平上识别 PPFP 结合位点。其他研究将评估与特定 DNA 位点的结合是否需要 PPFP 的 PAX8 DNA 结合域、其 PPARg DNA 结合域或两者。 Affymetrix 微阵列基因表达谱分析将在 DoxyPPFP 细胞上进行，以便将基因表达与全基因组 DNA 结合进行比较。这些数据还将与已从 PPFP 癌症获得的基因表达数据进行比较。将评估 PPFP 对其靶基因的辅助调节蛋白招募。假设是 PPFP 的 PAX8 部分招募不合适的辅助调节因子到 PPAR 响应基因，反之亦然。这些研究将主要依靠染色质免疫沉淀——实时 PCR。具体目标 2 将检验以下假设：表达 PPFP 的细胞高度依赖脂肪酸 2 氧化来促进细胞生长；即，PPFP 诱导 2 氧化创造了生长优势。 Seahorse XF24 生物分析仪将用于评估通过糖酵解与 TCA 循环进行的能量代谢。将评估葡萄糖与脂肪酸作为能源的使用。将评估 PPFP 对脂肪酸从头合成、细胞增殖和凋亡的影响，以更好地了解 PPFP 带来的假定生长优势。在具体目标 3 中，将在 NOD-SCID 小鼠中评估 PPARg 配体（噻唑烷二酮）对异种移植肿瘤和转移形成的影响。 PPFP 功能域对异种移植肿瘤形成和转移的重要性将使用表达突变 PPFP（例如 PAX8 或 PPARg DNA 结合域的突变）的 DoxyPPFP 细胞系进行评估。