Gregory Lewy

PTTG-Binding Factor (PBF) Is a Novel Regulator of the Thyroid Hormone Transporter MCT8

Within the basolateral membrane of thyroid follicular epithelial cells, two transporter proteins are central to thyroid hormone (TH) biosynthesis and secretion. The sodium iodide symporter (NIS) delivers iodide from the bloodstream into the thyroid, and after TH biosynthesis, monocarboxylate transporter 8 (MCT8) mediates TH secretion from the thyroid gland. Pituitary tumor-transforming gene-binding factor (PBF; PTTG1IP) is a protooncogene that is up-regulated in thyroid cancer and that binds NIS and modulates its subcellular localization and function. We now show that PBF binds MCT8 in vitro, eliciting a marked shift in MCT8 subcellular localization and resulting in a significant reduction in the amount of MCT8 at the plasma membrane as determined by cell surface biotinylation assays. Colocalization and interaction between PBF and Mct8 was also observed in vivo in a mouse model of thyroid-specific PBF overexpression driven by a bovine thyroglobulin (Tg) promoter (PBF-Tg). Thyroidal Mct8 mRNA and protein expression levels were similar to wild-type mice. Critically, however, PBF-Tg mice demonstrated significantly enhanced thyroidal TH accumulation and reduced TH secretion upon TSH stimulation. Importantly, Mct8-knockout mice share this phenotype. These data show that PBF binds and alters the subcellular localization of MCT8 in vitro, with PBF overexpression leading to an accumulation of TH within the thyroid in vivo. Overall, these studies identify PBF as the first protein to interact with the critical TH transporter MCT8 and modulate its function in vivo. Furthermore, alongside NIS repression, PBF may thus represent a new regulator of TH biosynthesis and secretion.

The pituitary tumor transforming gene in thyroid cancer

The pituitary tumor transforming gene (PTTG) is a multifunctional proto-oncogene that is over-expressed in various tumors including thyroid carcinomas, where it is a prognostic indicator of tumor recurrence. PTTG has potent transforming capabilities in vitro and in vivo, and many studies have investigated the potential mechanisms by which PTTG contributes to tumorigenesis. As the human securin, PTTG is involved in critical mechanisms of cell cycle regulation, whereby aberrant expression induces aneuploidy. PTTG may further contribute to tumorigenesis through its role in DNA damage response pathways and via complex interactions with hormones and growth factors. Furthermore, PTTG over-expression negatively impacts upon the efficacy of radioiodine therapy in thyroid cancer, through repression of expression and function of the sodium iodide symporter. Given its various roles at all disease stages, PTTG appears to be an important oncogene in thyroid cancer. This review discusses the current knowledge of PTTG with particular focus on its role in thyroid cancer.

Regulation of pituitary tumor transforming gene (PTTG) expression and phosphorylation in thyroid cells.

Human pituitary tumor transforming gene (hPTTG) is a multifunctional proto-oncogene implicated in the initiation and progression of several tumors. Phosphorylation of hPTTG is mediated by cyclin-dependent kinase 2 (CDC2), whereas cellular expression is regulated by specificity protein 1 (SP1). The mechanisms underlying hPTTG propagation of aberrant thyroid cell growth have not been fully defined. We set out to investigate the interplay between hPTTG and growth factors, as well as the effects of phosphorylation and SP1 regulation on hPTTG expression and function. In our study, epidermal growth factor (EGF), TGFα, and IGF-1 induced hPTTG expression and phosphorylation in thyroid cells, which was associated with activation of MAPK and phosphoinositide 3-kinase. Growth factors induced hPTTG independently of CDC2 and SP1 in thyroid carcinoma cells. Strikingly, CDC2 depletion in TPC-1 cells resulted in enhanced expression and phosphorylation of hPTTG and reduced cellular proliferation. In reciprocal experiments, hPTTG overexpression induced EGF, IGF-1, and TGFα mRNAs in primary human thyrocytes. Treatment of primary human thyrocytes with conditioned media derived from hPTTG-transfected cells resulted in autocrine upregulation of hPTTG protein, which was ameliorated by growth factor depletion or growth factor receptor tyrosine kinase inhibitors. A transgenic murine model of thyroid targeted hPTTG overexpression (hPTTG-Tg) (FVB/N strain, both sexes) demonstrated smaller thyroids with reduced cellular proliferation and enhanced secretion of Egf. In contrast, Pttg(-/-) knockout mice (c57BL6 strain, both sexes) showed reduced thyroidal Egf mRNA expression. These results define hPTTG as having a central role in thyroid autocrine signaling mechanisms via growth factors, with profound implications for promotion of transformed cell growth.

Abstract 493: A novel mechanism of colorectal tumorigenesis based on the functional inactivation of p53 by the pituitary tumor transforming gene binding factor (PBF)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC PBF is the poorly characterized binding partner of the pituitary tumor transforming gene (PTTG). PTTG is a multifunctional proto-oncogene overexpressed in colorectal cancer, which regulates the function of the tumor suppressor protein p53. Recent data indicates that PBF can transform cells independently of PTTG and that subcutaneous expression of PBF elicits large tumors in nude mice. Given that PBF is both transforming in vitro and tumorigenic in vivo, our present studies aim to investigate whether PBF has a role in colorectal tumorigenesis. Initially we demonstrated that PBF represses p53-mediated gene regulation through Hdm2 promoter assays in p53-null H1299 cells. Transfection of p53 elicited a 30.7 ± 2.6-fold stimulation of promoter activity. However, co-transfection of PBF significantly repressed p53 transcriptional activity (16.3 ± 1.1-fold; p<0.0003). Exposure of HCT116 cells to gamma-irradiation resulted in significantly increased endogenous PBF protein expression from 4 hours after treatment and reached maximal levels after 24 hours, in keeping with the observed increase in p53 protein stability. Furthermore, co-immunoprecipitation assays revealed a direct interaction between PBF and p53 in vivo, with a significant increase in binding following gamma-irradiation. In immunofluorescence studies, HCT116 and COS-7 cells treated with gamma-irradiation displayed increased nuclear localisation of PBF compared to untreated controls, resulting in an enhanced degree of colocalisation of both PBF and p53 within the nucleus. Transient overexpression of PBF in HCT116 cells resulted in substantially elevated nuclear p53 protein expression from as early as 2 hours after treatment, with increased p53 stabilization persisting for 24 hours compared to mock-transfected controls. Finally, in a cohort of matched cancer versus normal colorectal tissue samples we observed a significant increase in PBF expression at both the mRNA (2.4-fold, n=24, p=0.009) and protein level in tumors, which correlated with a significant increase in p53 protein expression. Taken together, these results highlight a potential role for PBF within the DNA damage response, whereby PBF stabilizes in response to genotoxic stress, binds directly to p53 and inhibits p53 mediated responses. From these findings we propose that aberrant regulation of PBF leads to the functional inactivation of p53, and thus represents a potential novel mechanism for colorectal tumorigenesis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 493.