Abstract
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 γ gene (PPARG), resulting in expression of a PAX8-PPARγ 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 trans-differentiation 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 trans-differentiation. Surprisingly, pioglitazone repressed TTF-1 expression in PPFP-expressing thyrocytes. Our data indicate that TTF-1 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.
Highlights
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)2 with the
We established a PPAR␥ fusion protein (PPFP) thyroid cell culture model system that replicates the pioglitazone-dependent trans-differentiation of thyroid carcinoma cells into adipocytelike cells, and we show this effect requires the expression of PPFP
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 (PPFPThy;PtenThyϪ/Ϫ mice) [11]
Summary
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 (PPFPThy;PtenThyϪ/Ϫ mice) [11]. For Crat, FIMO predicted a TTF-1 binding site 351 bp 3Ј to the PPFP peak This region gave a modest ChIP-qPCR signal (Fig. 5A), suggesting that it may or may not account for the effect of TTF-1 knockdown on gene expression. If this hypothesis is correct, TTF-1 with its DNA binding domain deleted might still give a moderately strong ChIP-qPCR signal on PPFP peaks that lack a TTF-1 motif To explore this hypothesis, PCCL3-PPFP cells were transfected with either HA-TTF-1 or HA-TTF-1HDD (deletion of the DNA binding domain), and ChIP was performed with an anti-HA tag antibody followed by qPCR for a negative control gene (Hprt1) and the PPFP peaks in Cidec, Ech, and Kpna (Fig. 5B). The expression of exogenous HA-TTF-1, which is driven by a non-native promoter, was not repressed by pioglitazone (Fig. 9E, lane 4 versus 3), suggesting that the effect of PPFP/pioglitazone on inhibiting endogenous TTF-1 is at the level of transcription
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