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: 8504792
• 负责人: RONALD Jay KOENIG
• 金额: $ 29.4万
• 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 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8504792
• 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; public health relevance; 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）：长期目标是揭示一种独特的核受体转录因子PAX 8-PPARg融合蛋白（PPFP）的转录活性、代谢效应和致瘤性的潜在机制，该蛋白是滤泡性甲状腺癌中染色体易位的结果。PPFP几乎包含转录因子配对盒8（PAX 8）的全部序列以及核受体过氧化物酶体增殖物激活受体g1（PPARg 1）的全部序列。PPFP可以激活PPAR-responsive基因的启动子，尽管PPFP的活性与PPARg的活性明显不同。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PPFP癌症基因签名的鉴定。PPFP特征基因中最丰富的代谢途径是脂肪酸2氧化，其由PPARs生理调节。以强力霉素依赖性方式表达PPFP的非转化甲状腺细胞系PCCL 3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、肿瘤发生的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。在第一个特定目标中，染色质免疫沉淀-深度测序（ChIP-Seq）将用于在全基因组水平上鉴定PPFP结合位点。进一步的研究将评估与特定DNA位点的结合是否需要PPFP的PAX 8 DNA结合结构域、其PPARg DNA结合结构域或两者。将在DoxyPPFP细胞上进行Affyphin微阵列基因表达谱分析，以比较基因表达与全基因组DNA结合。这些数据也将与已经从PPFP癌症中获得的基因表达数据进行比较。将评价PPFP向其靶基因的共调节蛋白募集。假设PPFP的PAX 8部分招募不适当的共调节子到PPAR应答基因，反之亦然。这些研究将主要依靠染色质免疫沉淀-真实的时间PCR。具体目标2将检验PPFP表达细胞高度依赖于脂肪酸2氧化以促进细胞生长的假设;即，PPFP诱导2氧化产生生长优势。将使用Seahorse XF 24生物分析仪来评估通过糖酵解与TCA循环进行的能量代谢。将评价葡萄糖与脂肪酸作为能量来源的使用。将评估PPFP对从头脂肪酸合成、细胞增殖和凋亡的影响，以更好地理解PPFP带来的假定生长优势。在具体目标3中，将在NOD-SCID小鼠中评价PPARg配体（噻唑烷二酮）对异种移植肿瘤和转移形成的作用。PPFP功能结构域对异种移植肿瘤形成和转移的重要性将用表达突变PPFP（例如，PAX 8或PPARg DNA结合结构域的突变）的DoxyPPFP细胞系来评估。