TTF-1 regulates PPFP thyroid cell adipogenic differentiation 1 Adipogenic differentiation of thyroid cancer cells through the Pax 8-PPAR γ fusion protein is regulated by thyroid transcription factor 1 ( TTF-1 )

A subset of thyroid carcinomas contains a t(2;3)(q13;p25) chromosomal translocation that fuses paired box gene 8 (PAX8) with the peroxisome proliferator activated receptor gamma gene (PPARG), resulting in expression of a PAX8PPARγ fusion protein, PPFP. We previously generated a transgenic mouse model of PPFP thyroid carcinoma and showed that feeding the PPARγ agonist pioglitazone greatly decreased the size of the primary tumor and prevented metastatic disease in vivo. The antitumor effect correlates with the fact that pioglitazone turns PPFP into a strongly PPARγ-like molecule, resulting in transdifferentiation of the thyroid cancer cells into adipocyte-like cells that lose malignant character as they become more differentiated. To further study this process, we performed cell culture experiments with thyrocytes from the PPFP mouse thyroid cancers. Our data show that pioglitazone induced cellular lipid accumulation and the expression of adipocyte marker genes in the cultured cells, and shRNA knockdown of PPFP eliminated this pioglitazone effect. In addition, we found that PPFP and thyroid transcription factor 1 (TTF-1) physically interact, and that these transcription factors bind near each other on numerous target genes. TTF-1 knockdown and overexpression studies showed that TTF-1 inhibits PPFP target gene expression and impairs adipogenic transdifferentiation. Surprisingly, pioglitazone repressed TTF-1 expression in PPFPexpressing thyrocytes. Our data indicate that TTF1 interacts with PPFP to inhibit the pro-adipogenic response to pioglitazone, and that the ability of pioglitazone to decrease TTF-1 expression contributes to its pro-adipogenic action. Introduction Approximately 30% of follicular thyroid carcinomas, as well as small subsets of follicular variant papillary carcinomas and follicular adenomas, harbor a t(2,3)(q13;p25) chromosomal translocation that fuses paired box gene 8 (PAX8) with the peroxisome proliferator activated receptor gamma gene (PPARG), resulting in the production of a PAX8-PPARγ fusion protein (PPFP) (1). This fusion protein is composed of all but the very carboxyl terminal segment of PAX8 followed by fully intact PPARγ1, and its expression is driven by the strong PAX8 promoter. Both the PAX8 and the PPARγ DNA binding domains within PPFP are functional, as determined by chromatin immunoprecipitation http://www.jbc.org/cgi/doi/10.1074/jbc.M116.740324 The latest version is at JBC Papers in Press. Published on July 19, 2016 as Manuscript M116.740324 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. TTF-1 regulates PPFP thyroid cell adipogenic differentiation 2 deep sequencing (ChIP-seq) (2). The transcription factor PAX8 is a master regulator of thyroid development and function. PAX8 mutations in humans (3) and mice (4) result in failure of thyroid gland development. In the mature thyroid gland, PAX8 induces the expression of thyroid-specific genes such as those encoding thyroglobulin, thyroid peroxidase, and the sodium iodide symporter (5-7). PPARγ is a member of the nuclear receptor superfamily of ligand-activated transcription factors (8,9) and among its functions, it is essential for adipogenesis (10). Synthetic agonist ligands for PPARγ such as pioglitazone are insulin sensitizers and hence are used to treat type 2 diabetes mellitus. PPARγ ligands also are ligands for PPFP. PPARγ has no identified role in the normal thyroid and is expressed at extremely low levels in that organ. We have recently developed a transgenic mouse model of PPFP thyroid carcinoma (11). In this model, pioglitazone was highly therapeutic, greatly shrinking thyroid size and preventing metastatic disease. The most remarkable aspect of this therapeutic response is that pioglitazone converted PPFP into a strongly PPARγ-like transcription factor, resulting in the induction of numerous adipocyte PPARγ target genes and the accumulation of large amounts of intracellular lipid. This pro-differentiation effect likely underlies the therapeutic efficacy of pioglitazone, but the factors that regulate the adipogenic response to ligand-bound PPFP have not been investigated. Thyroid transcription factor 1 (TTF-1), formally denoted Nkx2-1, is a homeobox transcription factor essential for the development of the thyroid, lung and brain (12). In the mature thyroid, TTF-1 interacts physically with PAX8 to induce the expression of thyroglobulin and thyroid peroxidase (13,14). TTF-1 appears to have a complex role in cancer biology. In lung adenocarcinoma, both pro-oncogenic and antioncogenic effects have been described (reviewed in (15)). A germline missense mutation (A339V) of TTF-1 has been identified in families with multinodular goiter and papillary thyroid carcinoma (16). A genome-wide association study of thyroid cancer cases revealed the variant SNP rs944289 located on chromosome 14q13.3, which maps close to TTF-1, was associated with an increased risk of thyroid cancer (17). Concurrent overexpression of RET/PTC1 and TTF-1 confers tumorigenicity to thyrocytes in nude mice (18). In the present study, we established a PPFP thyroid cell culture model system that replicates the pioglitazone-dependent transdifferentiation of thyroid carcinoma cells into adipocyte-like cells, and we show this effect requires the expression of PPFP. We show that TTF-1 physically interacts with PPFP, that these transcription factors bind near each other on numerous target genes, and that TTF-1 has an inhibitory effect on PPFP/pioglitazone-mediated adipogenic function. Importantly, PPFP down regulates endogenous TTF-1 expression in a pioglitazone-dependent manner, suggesting that this effect contributes to the pro-adipogenic transdifferentiation and anti-tumor activities of pioglitazone in PPFP thyroid carcinomas. Results The pioglitazone-dependent adipogenic differentiation of thyroid cancer cells is mediated through PPFP−Thyroid specific expression of PPFP combined with thyroid-specific deletion of Pten is an established murine model of PPFP thyroid carcinoma (PPFP;Pten mice) (11). The PPARγ agonist pioglitazone has a profound therapeutic effect in these mice, and results in broad up regulation of adipocyte PPARγ target genes as the thyroid cancer cells adopt an adipocyte-like phenotype. To elucidate the molecular mechanisms underlying this process, we created a thyroid cell line from the PPFP;Pten mice. These cells highly express PPFP (Figure 1A, lane 1) but there is no detectable expression of PPARγ protein. Nine days of treatment with pioglitazone resulted in the accumulation of intracellular lipid as assessed by Oil Red O staining and quantification (Figure 1B), as well as the induction of adipocyte PPARγ target genes such as Fabp4, Plin1, Cd36 and Lpl at the RNA level (Figure 1C). The induction of Fabp4, Plin1 and Cd36 was confirmed at the protein level, but there was still no detectable PPARγ protein (Figure 1D). These data support the hypothesis that pioglitazone-induced adipogenic differentiation reflects the PPARγ-like function of PPFP. TTF-1 regulates PPFP thyroid cell adipogenic differentiation 3 To further demonstrate the central role of PPFP in the pioglitazone proadipogenic effect, we performed a series of PPFP knockdown experiments. Two shRNAs targeting the PPARγ portion of PPFP effectively knocked down PPFP expression in this cell line (Figure 2A). As expected, silencing of PPFP inhibited the proadipogenic effect of pioglitazone as assessed by Oil Red O staining (Figure 2B) and quantification (Figure 2C), as well as by decreased expression of adipocyte genes (Figures 2D, E). Although there is no detectable PPARγ protein expression in these cells, the possibility remains that a very low level of PPARγ protein is playing a role and that the shRNA is acting by depletion of this putative low level PPARγ. To eliminate this possibility, we also knocked down PPFP using an shRNA against the PAX8 potion of the protein (Figure 2F). This also impaired the ability of pioglitazone to induce an adipogenic response as assessed by Oil Red O staining (Figure 2G) and quantification (Figure 2H), as well as by decreased expression of adipocyte genes (Figure 2I). These data indicate that PPFP is responsible for the adipogenic differentiation of PPFP thyroid cells when treated with pioglitazone. Thyroid transcription factor 1 (TTF-1) interacts with PPFP and inhibits PPFP target gene expression in a pioglitazone-dependent manner−We recently identified PPFP genomic binding sites by ChIP-seq and characterized PPFPdependent gene expression by mRNA deep sequencing (RNA-seq) in PCCL3-PPFP rat thyroid cells (2). We tested for transcription factor motifs overrepresented in the proximal regions surrounding the PPFP peaks from the ChIP-seq data using Genomatix Genome Analyzer, to identify candidate transcription factors that interact with PPFP. As shown in Supplemental Table 1, Nkx2 family proteins were prominently over represented, with the motif matrix for TTF-1 (Nkx2-1) having the fifth highest Z-score in promoters and the ninth highest in the genome. We decided to focus our attention on TTF-1, since as noted above, it interacts physically and functionally with PAX8 and plays a role in carcinogenesis. We hypothesized that PPFP binds to TTF1 and that this interaction modulates PPFP target gene expression. We cotransfected Myc-PPFP and HA-TTF1 into JEG3 cells, and found that they coimmunoprecipitate (Figure 3A). Furthermore, Myc-PPFP also pulled down endogenous TTF-1 in the mouse PPFP thyroid cell line (Figure 3B). We next investigated the functional importance of the PPFP-TTF-1 interaction by knockdown of endogenous TTF-1 in PCCL3-PPFP cells or PCCL3-EV (empty vector) control cells. Immunoblotting indicated that two different targeting shRNAs (shTTF-1-1 and shTTF-1-2) knocked down endogenous TTF-1 protein expression (Figure 3C). The effectiveness of this knockdown was confirmed by showing that both shRNAs down regulated the e