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).
This project is relevant to the public health because it will reveal how PAX8-PPARg Fusion Protein (PPFP) causes thyroid cancer and will suggest new approaches to therapy. In addition, many patients with diabetes take a class of drugs called thiazolidinediones that activates PPARg, but these drugs also activate PPFP and the consequences of this are not known. This study will help determine whether this diabetes therapy is beneficial or harmful to patients who also have PPFP thyroid cancers.
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