Pax8-PPARgamma regulation of transcription and metabolism in thyroid cancer

描述(申请人提供)：长期目标是揭示一种独特的核受体转录因子PAX8-PPARg融合蛋白(PPFP)的转录活性、代谢效应和致瘤性的潜在机制，它是滤泡性甲状腺癌染色体易位的结果。PPFP几乎包含转录因子配对框8(PAX8)的全序列以及核受体过氧化物酶体增殖物激活受体G1(PPARg1)的完整序列。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癌基因特征的识别。PPFP信号基因最丰富的代谢途径是脂肪酸2氧化，它受PPAR的生理调节。以强力霉素依赖的方式表达PPFP的未转化甲状腺细胞系PCCL3的稳定转基因克隆(DoxPPFP细胞)将用于了解脂肪酸代谢在PPFP癌症生物学中的重要性、肿瘤发生的机制以及潜在的治疗方法。在这些细胞中，PPFP诱导了许多在PPFP甲状腺癌中被诱导的基因，这些细胞形成了NOD-SCID小鼠的异种移植瘤和肺转移。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。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、PPARg DNA结合域，或者两者兼而有之。Affymetrix微阵列基因表达图谱将在DoxPPFP细胞上进行，以便将基因表达与全基因组DNA结合进行比较。这些数据还将与已经从PPFP癌症中获得的基因表达数据进行比较。将评估PPFP对其靶基因的共调节蛋白募集。假设是PPFP的PAX8部分为PPAR反应基因招募了不适当的协调制子，反之亦然。这些研究将主要依靠染色质免疫沉淀-实时聚合酶链式反应。特定目标2将验证PPFP表达细胞高度依赖于脂肪酸2氧化来促进细胞生长的假设，即PPFP诱导2氧化产生生长优势。一个海马XF24生物分析仪将被用来评估有多少能量代谢是通过糖酵解，而不是TCA循环。将对使用葡萄糖和脂肪酸作为能源进行评估。将评估PPFP对从头合成脂肪酸、细胞增殖和凋亡的影响，以更好地了解PPFP可能带来的生长优势。在具体目标3中，将评估PPARg配体(噻唑烷二酮)对NOD-SCID小鼠移植瘤和转移形成的影响。PPFP功能结构域在异种移植瘤形成和转移中的重要性将通过表达突变的PPFP的DoxPPFP细胞株来评估(例如，PAX8或PPARg DNA结合域的突变)。

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).