Guidelines on the Use of Systemic Therapy in Patients with Advanced Thyroid Cancer.

With increasing understanding of the molecular alterations leading to thyroid cancers in recent years we have seen a rapid increase in the number of effective targeted systemic therapies available for patients with advanced thyroid cancer; firstly with the advent of the multi-kinase inhibitors and more recently with more specific RET, BRAF, MEK, ALK and NTRK inhibitors. Although these developments are very welcome, they have resulted in a paradigm shift in the management of advanced thyroid cancer to which thyroid oncologists have had to rapidly adapt, learning how to supervise treatment safely with novel agents, the management of novel toxicities, when and how to arrange molecular genetic testing of cancers and, perhaps most importantly, determining when the optimum time is to start these treatments in what can often be a relatively indolent, if progressive, disease. We hope that these guidelines will support clinicians in making these decisions with their patients, as well as signposting and providing useful supporting information both for patients and clinicians.

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.

Prediction of cure and risk of hypothyroidism in patients receiving 131I for hyperthyroidism.

CONTEXT: There is little consensus regarding the most appropriate dose of radioiodine ((131)I) to be administered to patients with hyperthyroidism. OBJECTIVE: To compare the efficacy of fixed dose regimens of (131)I in curing hyperthyroidism and to define simple clinical and biochemical factors that predict outcome in individual patients. DESIGN: Consecutive series of hyperthyroid subjects treated with (131)I. SETTING: Single Secondary/Tertiary Care Hospital Clinic. PARTICIPANTS: A total of 1278 patients (1013 females and 262 males, mean age 49.7 years) presenting with hyperthyroidism between 1984 and 2006. INTERVENTION: Treatment with (131)I using a fixed dose regimen. MAIN OUTCOME MEASURES: Probability of cure and risk of development of hypothyroidism following a single dose of (131)I. RESULTS: Patients given a single dose of (131)I of 600 MBq (n = 485) had a higher cure rate (84.1%) compared with those receiving either 370 MBq (74.9%, P < 0.001) or those given 185 Bq (63%, P < 0.001). An increased incidence of hypothyroidism by 1 year was evident with higher doses (600 MBq: 60.4%; 370 MBq: 49.2%, P = 0.001; 185 Bq: 38.1%, P < 0.001). Binary logistic regression analysis identified a 600 Bq dose of (131)I [adjusted odds ratio, AOR 3.33 (2.28-4.85), P < 0.001], female gender [AOR 1.75 (1.23-2.47), P = 0.002], lower presenting serum free T4 concentration [AOR 1.01 (1.01-1.02), P < 0.001] and absence of a palpable goitre [AOR 3.33 (2.00-5.56), P < 0.001] to be independent predictors of cure. Similarly, a 600 MBq dose [AOR 3.79 (2.66-5.38), P < 0.001], female gender [AOR 1.46 (1.05-2.02), P = 0.02], younger age [AOR 1.03 (1.02-1.04), P < 0.001], absence of a palpable goitre [AOR 3.85 (2.38-5.88), P < 0.001] and presence of ophthalmopathy [AOR 1.57 (1.06-2.31), P = 0.02] were identified as independent factors predicting the probability of development of hypothyroidism at one year. Based on these findings, formulae to indicate probability of cure and risk of hypothyroidism for application to individual patients were derived. CONCLUSIONS: Simple clinical/biochemical criteria can be used to predict outcome after (131)I treatment. These factors determine that males, those with severe biochemical hyperthyroidism, and those with a palpable goitre require larger doses (600 MBq) in order to achieve cure.

PTTG and PBF repress the human sodium iodide symporter.

The ability of the thyroid to accumulate iodide provides the basis for radioiodine ablation of differentiated thyroid cancers and their metastases. Most thyroid tumours exhibit reduced iodide uptake, although the mechanisms accounting for this remain poorly understood. Pituitary tumour transforming gene (PTTG) is a proto-oncogene implicated in the pathogenesis of thyroid tumours. We now show that PTTG and its binding factor PBF repress expression of sodium iodide symporter (NIS) messenger RNA (mRNA), and inhibit iodide uptake. This process is mediated at least in part through fibroblast growth factor-2. In detailed studies of the NIS promoter in rat FRTL-5 cells, PTTG and PBF demonstrated specific inhibition of promoter activity via the human upstream enhancer element (hNUE). Within this approximately 1 kb element, a complex PAX8-upstream stimulating factor 1 (USF1) response element proved critical both to basal promoter activity and to PTTG and PBF repression of NIS. In particular, repression by PTTG was contingent upon the USF1, but not the PAX8, site. Finally, in human primary thyroid cells, PTTG and PBF similarly repressed the NIS promoter via hNUE. Taken together, our data suggest that the reported overexpression of PTTG and PBF in differentiated thyroid cancer has profound implications for activity of the NIS gene, and hence significantly impacts upon the efficacy of radioiodine treatment.

Functional consequences of the first reported mutations of the proto-oncogene PTTG1IP/PBF.

Pituitary tumor-transforming gene 1-binding factor (PTTG1IP; PBF) is a multifunctional glycoprotein, which is overexpressed in a wide range of tumours, and significantly associated with poorer oncological outcomes, such as early tumour recurrence, distant metastasis, extramural vascular invasion and decreased disease-specific survival. PBF transforms NIH 3T3 fibroblasts and induces tumours in nude mice, while mice harbouring transgenic thyroidal PBF expression show hyperplasia and macrofollicular lesions. Our assumption that PBF becomes an oncogene purely through increased expression has been challenged by the recent report of mutations in PBF within the Catalogue of Somatic Mutations in Cancer (COSMIC) database. We therefore sought to determine whether the first 10 PBF missense substitutions in human cancer might be oncogenic. Anisomycin half-life studies revealed that most mutations were associated with reduced protein stability compared to wild-type (WT) PBF. Proliferation assays narrowed our interest to two mutational events which significantly altered cell turnover: C51R and R140W. C51R was mainly confined to the endoplasmic reticulum while R140W was apparent in the Golgi apparatus. Both C51R and R140W lost the capacity to induce cellular migration and significantly reduced cell invasion. Colony formation and soft agar assays demonstrated that, in contrast to WT PBF, both mutants were unable to elicit significant colony formation or anchorage-independent growth. However, C51R and R140W retained the ability to repress radioiodide uptake, a functional hallmark of PBF. Our data reveal new insight into PBF function and confirm that, rather than being oncogenic, mutations in PBF are likely to be passenger effects, with overexpression of PBF the more important aetiological event in human cancer.

Elevated PTTG and PBF predicts poor patient outcome and modulates DNA damage response genes in thyroid cancer.

The proto-oncogene PTTG and its binding partner PBF have been widely studied in multiple cancer types, particularly thyroid and colorectal, but their combined role in tumourigenesis is uncharacterised. Here, we show for the first time that together PTTG and PBF significantly modulate DNA damage response (DDR) genes, including p53 target genes, required to maintain genomic integrity in thyroid cells. Critically, DDR genes were extensively repressed in primary thyrocytes from a bitransgenic murine model (Bi-Tg) of thyroid-specific PBF and PTTG overexpression. Irradiation exposure to amplify p53 levels further induced significant repression of DDR genes in Bi-Tg thyrocytes (P=2.4 × 10-4) compared with either PBF- (P=1.5 × 10-3) or PTTG-expressing thyrocytes (P=NS). Consistent with this, genetic instability was greatest in Bi-Tg thyrocytes with a mean genetic instability (GI) index of 35.8±2.6%, as well as significant induction of gross chromosomal aberrations in thyroidal TPC-1 cells following overexpression of PBF and PTTG. We extended our findings to human thyroid cancer using TCGA data sets (n=322) and found striking correlations with PBF and PTTG expression in well-characterised DDR gene panel RNA-seq data. In addition, genetic associations and transient transfection identified PBF as a downstream target of the receptor tyrosine kinase-BRAF signalling pathway, emphasising a role for PBF as a novel component in a pathway well described to drive neoplastic growth. We also showed that overall survival (P=1.91 × 10-5) and disease-free survival (P=4.9 × 10-5) was poorer for TCGA patients with elevated tumoural PBF/PTTG expression and mutationally activated BRAF. Together our findings indicate that PBF and PTTG have a critical role in promoting thyroid cancer that is predictive of poorer patient outcome.

Pituitary tumor transforming gene (PTTG) stimulates thyroid cell proliferation via a vascular endothelial growth factor/kinase insert domain receptor/inhibitor of DNA binding-3 autocrine pathway.

CONTEXT: Vascular endothelial growth factor (VEGF) exerts its biological effects by binding to the tyrosine kinase receptors VEGF receptor type 1 (VEGFR1/Flt-1) and VEGFR2 (Flk-1/KDR). Kinase insert domain receptor (KDR) is the critical receptor controlling proliferation and migration of endothelial cells and has been shown to be expressed in some nonendothelial cells. We recently reported that the proangiogenic pituitary tumor transforming gene (PTTG) stimulates VEGF and up-regulates inhibitor of DNA binding-3 (ID3), an important gene in VEGF-dependent angiogenesis. OBJECTIVE: Our objective was to test whether VEGF, ID3, and KDR confer a PTTG-mediated effect on thyroid cell growth. DESIGN: Gene expression, MAPK stimulation, and cell proliferation were assessed in follicular thyroid cancer FTC133 cells. Gene expression and clinical associations were determined in 21 normal and 38 tumorous thyroid specimens (nine follicular and 29 papillary). RESULTS: ID3 correlated with VEGF mRNA expression in our series of thyroid cancers, which also showed up-regulated KDR mRNA. Stimulation of FTC133 cells with exogenous VEGF enhanced ID3 expression, which could be abrogated by the KDR-specific inhibitor ZM323881, suggesting that VEGF regulation of ID3 is KDR dependent. PTTG significantly correlated with KDR mRNA expression in our thyroid cancer cohort and up-regulated KDR and VEGF expression in FTC133 cells. Finally, cells transfected with PTTG demonstrated increased cell proliferation and phosphorylation of MAPK, which was abrogated by ZM323881. CONCLUSIONS: We report the presence of a VEGF/KDR/ID3-dependent autocrine pathway in FTC133 thyroid cells. By up-regulating both VEGF and KDR expression, we propose a novel PTTG-mediated proliferative pathway that may be critical to thyroid cancer growth and progression.

Serum thyrotropin concentration as a novel predictor of malignancy in thyroid nodules investigated by fine-needle aspiration.

CONTEXT: Thyroid nodules and goiter are common, and fine-needle aspiration biopsy (FNAB) is the first investigation of choice in distinguishing benign from malignant disease. OBJECTIVE: The objective of the study was to assess whether simple clinical and biochemical parameters can predict the likelihood of thyroid malignancy in subjects undergoing FNAB. DESIGN: The design was a prospective cohort. SETTING: The study was conducted at a single secondary/tertiary care clinic. PARTICIPANTS: One thousand five hundred consecutive patients without overt thyroid dysfunction (1304 females and 196 males, mean age 47.8 yr) presenting with palpable thyroid enlargement between 1984 and 2002 were evaluated by FNAB of the thyroid. INTERVENTION(S): There were no interventions. MAIN OUTCOME MEASURES: Goiter type was assessed clinically and classified as diffuse in 183, multinodular in 456, or solitary nodule in 861 cases. Serum TSH concentration at presentation was measured in a sensitive assay in patients presenting after 1988 (n = 1183). The final cytological or histological diagnosis was determined after surgery (n = 553) or a minimum 2-yr clinical follow-up period (mean 9.5 yr, range 2-18 yr). RESULTS: The overall sensitivity and specificity of FNAB in predicting malignancy were 88 and 84%, respectively. The risk of diagnosis of malignancy rose in parallel with the serum TSH at presentation, with significant increases evident in patients with serum TSH greater than 0.9 mU/liter, compared with those with lower TSH. Binary logistic regression analysis revealed significantly increased adjusted odds ratios (AORs) for the diagnosis of malignancy in subjects with serum TSH 1.0-1.7 mU/liter, compared with TSH less than 0.4 mU/liter [AOR 2.72, 95% confidence interval (CI) 1.02-7.27, P = 0.046], with further increases evident in those with TSH 1.8-5.5 mU/liter (AOR 3.88, 95% CI 1.48-10.19, P = 0.006, compared with TSH < 0.4 mU/liter) and greater than 5.5 mU/liter (AOR 11.18, 95% CI 3.23-8.63, P < 0.001, compared with TSH < 0.4 mU/liter). Males (AOR 1.8, 95% CI 1.04-3.1, P = 0.04), younger patients (AOR 1.1, 95% CI 1.01-1.15, P = 0.025), and those with clinically solitary nodules (AOR 2.53, 95% CI 1.5-4.28, P = 0.001) were also at increased risk. Based on these findings, a formula to predict the risk of the diagnosis of thyroid malignancy in individual patients, taking into account their gender, age, goiter type determined clinically, and serum TSH, was calculated. CONCLUSIONS: The risk of malignancy in a thyroid nodule increases with serum TSH concentrations within the normal range. In addition to patient's gender, age, and goiter type, the serum TSH concentration at presentation is an independent predictor of the presence of thyroid malignancy. We propose that these simple clinical and biochemical factors can serve as an adjunct to FNAB in predicting risk of malignancy.

Pituitary tumor-transforming gene regulates multiple downstream angiogenic genes in thyroid cancer.

CONTEXT: Pituitary tumor-transforming gene (PTTG) is a multifunctional protein involved in several tumorigenic mechanisms, including angiogenesis. PTTG has been shown to promote angiogenesis, a key rate-limiting step in tumor progression, by up-regulation of fibroblast growth factor-2 and vascular endothelial growth factor. OBJECTIVE: To investigate whether PTTG regulates other angiogenic genes in thyroid cells, we performed angiogenesis-specific cDNA arrays after PTTG transfection. Two of the genes [inhibitor of DNA binding-3 (ID3) and thrombospondin-1 (TSP-1)] which showed differential expression in primary thyroid cells were validated in vitro and in vivo. RESULTS: TSP-1 showed a 2.5-fold reduction and ID3 showed a 3.5-fold induction in expression in response to PTTG overexpression in vitro. Conversely, suppression of PTTG with small interfering RNA was associated with a 2-fold induction of TSP-1 and a 2.2-fold reduction in ID3 expression. When we examined TSP-1 and ID3 expression in 34 differentiated thyroid cancers, ID3 was significantly increased in tumors compared with normal thyroid tissue. Furthermore, ID3 expression was significantly higher in follicular thyroid tumors than in papillary tumors. Although mean TSP-1 expression was not altered in cancers compared with normal thyroids, we observed a significant independent association between TSP-1 expression and early tumor recurrence, with recurrent tumors demonstrating 4.2-fold lower TSP-1 expression than normal thyroid tissues. CONCLUSION: We have identified ID3 and TSP-1 as two new downstream targets of PTTG in thyroid cancer. We propose that PTTG may promote angiogenesis by regulating the expression of multiple genes with both pro- and antiangiogenic properties and may thus be a key gene in triggering the angiogenic switch in thyroid tumorigenesis.

Influences of age, gender, smoking, and family history on autoimmune thyroid disease phenotype.

CONTEXT: Both genetic and environmental factors contribute to susceptibility to Graves' disease (GD) and Hashimoto's thyroiditis (HT), as well as disease manifestations. OBJECTIVE: The objective of the study was to define how endogenous/environmental factors contribute to variation in phenotype. DESIGN/SETTING: This was a multicenter cohort study. PATIENTS/OUTCOME MEASURES: We prospectively collected clinical/biochemical data as part of the protocol for a United Kingdom DNA collection for GD and HT. We investigated, in 2805 Caucasian subjects, whether age at diagnosis, gender, family history (FH), smoking history, and presence of goiter influenced disease manifestations. RESULTS: For 2405 subjects with GD, the presence of goiter was independently associated with disease severity (serum free T4 at diagnosis) (P < 0.001). Free T4 (P < 0.05) and current smoking (P < 0.001) were both independent predictors of the presence of ophthalmopathy. Approximately half of those with GD (47.4% of females, 40.0% of males) and HT (n = 400) (56.4% of females, 51.7% of males) reported a FH of thyroid dysfunction. In GD, a FH of hyperthyroidism in any relative was more frequent than hypothyroidism (30.1 vs. 24.4% in affected females, P < 0.001). In HT, a FH of hypothyroidism was more common than hyperthyroidism (42.1 vs. 22.8% in affected females, P < 0.001). For GD (P < 0.001) and HT (P < 0.05), a FH was more common in maternal than paternal relatives. The reporting of a parent with thyroid dysfunction (hyper or hypo) was associated with lower median age at diagnosis of both GD (mother with hyperthyroidism, P < 0.001) and HT (father with hypothyroidism, P < 0.05). In GD and HT, there was an inverse relationship between the number of relatives with thyroid dysfunction and age at diagnosis (P < 0.01). CONCLUSIONS: Marked associations among age at diagnosis, disease severity, goiter, ophthalmopathy, smoking, and FH provide evidence for interactions between genetic and environmental/endogenous factors; understanding these may allow preventive measures or better tailoring of therapies.

Abnormal expression of 11 beta-hydroxysteroid dehydrogenase type 2 in human pituitary adenomas: a prereceptor determinant of pituitary cell proliferation.

The physiological effects of glucocorticoids (GCs) are, at least in part, mediated by inhibition of cell proliferation. Two isozymes of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) interconvert cortisol (F) and inactive cortisone (E), and are thus able to modulate GC action at an autocrine level. Previously, we have demonstrated absent expression of 11 beta-HSD2 in normal pituitaries; however, in a small number of pituitary tumors analysed, 11 beta-HSD2 was readily demonstrable. Here we have used real-time RT-PCR to quantify expression of mRNA for 11 beta-HSD1 and 2 in 105 human pituitary tumors and have performed enzyme expression and activity studies in primary pituitary cultures. Overall, pituitary tumors expressed lower levels of 11 beta-HSDl mRNA compared with normals (0.2-fold, P<0.05). In contrast, expression of 11 beta-HSD2 mRNA was 9.8-fold greater in tumors than in normals (P<0.001). Enzyme assays showed significant 11 beta-HSD2 activity (71.9+/-22.3 pmol/h/mg protein (mean+/-s.d.)) but no detectable 11 beta-HSDl activity. Proliferation assays showed that addition of glycyrrhetinic acid (an 11 beta-HSD2 inhibitor) resulted in a 30.3+/-7.7% inhibition of cell proliferation. In summary, we describe a switch in expression from 11 beta-HSDl to 11 beta-HSD2 in neoplastic pituitary tissue. We propose that abnormal expression of 11 beta-HSD2 acts as a proproliferative prereceptor determinant of pituitary cell growth, and may provide a novel target for future tumor therapy.

Thyroid hormone in health and disease.

Thyroid disease is common, affecting around 2% of women and 0.2% of men in the UK. Our understanding of the effects of thyroid hormones under physiological circumstances, as well as in pathological conditions, has increased dramatically during the last two centuries and it has become clear that overt thyroid dysfunction is associated with significant morbidity and mortality. Both hypo-and hyperthyroidism and their treatments have been linked with increased risk from cardiovascular disease and the adverse effects of thyrotoxicosis in terms of osteoporosis risk are well established. Although the evidence suggests that successful treatment of overt thyroid dysfunction significantly improves overall survival, the issue of treating mild or subclinical hyper- and hypothyroidism remains controversial. Furthermore, the now well-established effects of thyroid hormones on neurodevelopment have sparked a whole new debate regarding the need to screen pregnant women for thyroid function abnormalities. This review describes the current evidence of the effects of thyroid hormone on the cardiovascular, skeletal and neurological systems, as well as the influence of thyroid diseases and their treatments on the development of malignancy. Furthermore we will describe some recent developments in our understanding of the relationship between thyroid status and health.

A potential role for PTTG/securin in the developing human fetal brain.

Human securin, known also as PTTG, has established oncogenic and cell cycle regulatory functions. PTTG/securin transforms cells in vitro, inhibits sister chromatid separation, and regulates secretion of fibroblast growth factor-2. FGF-2 is a key regulator of CNS development and PTTG/securin expression has been reported in murine fetal brain. We examined the expression and function of securin and FGF-2 in the developing human fetal brain and in a fetal neuronal cell line (NT 2). Securin expression was significantly reduced in first and second trimester fetal cerebral cortex compared with adult cerebral cortex, where immunocytochemistry revealed intense securin staining in neuronal cell bodies. FGF-2 protein was concordantly lower in fetal cortex, whereas pretranslational expression of PTTG binding factor (PBF) was not significantly altered in fetal brain compared with adult. PCNA expression demonstrated that high securin levels in adult cortex were associated with absent cell proliferation. In NT-2 cells, securin stimulated FGF-2 expression, which could be abrogated by a carboxyl-terminal mutation. Low transient expression of securin resulted in a significant proliferative effect, whereas high levels of securin expression inhibited cell turnover. We propose a potential role for human PTTG/securin in modulating cell proliferation and FGF-2 expression during human neurogenesis.

Pituitary tumor transforming gene and fibroblast growth factor-2 expression: potential prognostic indicators in differentiated thyroid cancer.

Differentiated thyroid cancers are the most common endocrine cancers, but there are no reliable molecular markers of prognosis. Pituitary tumor transforming gene (PTTG) plays several potential roles in tumor initiation and progression, including regulating mitosis and stimulating expression of fibroblast growth factor (FGF)-2. Increased expression of PTTG has been demonstrated in follicular thyroid lesions, and expression of this oncogene has been identified as a potential prognostic marker in pituitary adenomas and colon carcinomas. We assessed the expression of PTTG and FGF-2 and its receptor FGF-R-1 in 27 differentiated thyroid cancers, and we compared this with expression in 11 normal thyroids, 25 multinodular goiters, and 13 Graves' disease specimens. We also examined the relationship between gene expression and clinical markers of tumor behavior. PTTG and FGF-2 were overexpressed in thyroid carcinomas (9.5-fold increase, P = 0.003, and 5.0-fold increase, P < 0.001, respectively) compared with normal thyroid. Increased FGF-2 mRNA expression was independently associated with the findings of lymph node invasion (R(2) = 0.71; P < 0.001) and distant metastasis (R(2) = 0.55; P = 0.009) at tumor presentation, after taking into account known prognostic factors such as age and gender of the patient and size and type of the tumor. High PTTG expression was independently associated with tumor recurrence (R(2) = 0.64; P = 0.003). We conclude that PTTG and FGF-2 expression are potential prognostic markers (and perhaps therapeutic targets) for differentiated thyroid cancer.

Placental iodothyronine deiodinase expression in normal and growth-restricted human pregnancies.

We have described the expression of specific iodothyronine deiodinase mRNAs (using quantitative RT-PCR) and activities in normal human placentas throughout gestation and compared our findings to those in placentas from pregnancies affected by intrauterine growth restriction (IUGR). The predominant deiodinase expressed in placenta was type III (D3); type II (D2) was also present. In general terms, the activities of the enzymes D2 and D3 (and mRNAs encoding these enzymes) were higher earlier in gestation (<28 wk) than at term and displayed an inverse relationship with the duration of gestation (P < 0.05). Comparison of the relative expressions of mRNAs encoding D2 and D3 as well as their activities in placentas associated with IUGR (early and late gestational groups) with findings from normal placentas of similar gestational ages revealed no significant differences. Immunolocalization of D2 and D3 in syncytiotrophoblast (including syncytial sprouts) and cytotrophoblast of human placentas was demonstrated at both early and late gestation. Treatment of primary cultures of term cytotrophoblast cells in vitro with increasing doses of T(3) (1, 10, and 100 nM) resulted in increased expression of mRNAs encoding both D2 and D3 at 100-nM concentrations (P < 0.01) compared with control. Experiments with JEG-3 choriocarcinoma cells demonstrated a similar effect on D3 mRNA at 10 and 100 nM T(3) (P < 0.01). The demonstrated changes in iodothyronine deiodinase expression in the placenta across pregnancy are likely to contribute to regulation of the thyroid hormone supply to the developing fetus. The lack of difference in deiodinase expression in normal placentas and those found in IUGR argues against placental deiodinases being responsible for the hypothyroxemia in circulating fetal thyroid hormones observed in this condition.

Vascular endothelial growth factor, its receptor KDR/Flk-1, and pituitary tumor transforming gene in pituitary tumors.

Pituitary tumorigenesis is a poorly understood process involving dysregulation of the cell cycle, proliferation, and angiogenesis. The novel securin pituitary tumor transforming gene (PTTG) disrupts cell division and stimulates fibroblast growth factor (FGF)-2-mediated angiogenesis. We investigated expression of the angiogenic vascular endothelial growth factor (VEGF) and its receptor KDR/Flk-1 in 103 human pituitary tumors, and we assessed functional relationships between these genes in vitro. Nonfunctioning tumors (n = 81) demonstrated markedly raised VEGF mRNA (3.2-fold, P < 0.05) and protein concentrations, compared with normal pituitaries (n = 10). KDR was also highly induced in nonfunctioning tumors (14-fold, P < 0.001, n = 78) as well as in the whole cohort of pituitary tumors, compared with normal pituitary samples (14-fold, P < 0.0001, n = 100). In vitro, PTTG induced VEGF, but not KDR, expression in fetal neuronal NT2 cells (2.7-fold, P < 0.001, n = 8), MCF-7 breast carcinoma cells (1.9-fold, P = 0.03, n = 10), and choriocarcinoma JEG-3 cells (P = 0.0002, n = 8). A mutated PTTG construct that cannot be phosphorylated showed identical VEGF up-regulation (2.9-fold, P < 0.001, n = 8) in NT2 cells, compared with wild-type PTTG, but a further mutated construct with abrogation of the key protein:protein interaction domain of PTTG resulted in a significant reduction in VEGF stimulation, compared with wild-type (0.37-fold reduction, P < 0.001, n = 8). FGF-2 findings mirrored those of VEGF, although antibody depletion of secreted FGF-2 in the cell medium failed to influence VEGF up-regulation by PTTG. Overall, our findings implicate altered VEGF and KDR signaling in pituitary tumorigenesis, and we propose that PTTG stimulation of FGF-2 and VEGF expression in the presence of up-regulated growth factor receptors may account for angiogenic growth and progression of human pituitary tumors.

PTTG's C-terminal PXXP motifs modulate critical cellular processes in vitro.

Human pituitary tumor-transforming gene (PTTG), known also as securin, is a multifunctional protein implicated in the control of mitosis and the pathogenesis of thyroid, colon, oesophageal and other tumour types. Critical to PTTG function is a C-terminal double PXXP motif, forming a putative SH3-interacting domain and housing the gene's sole reported phosphorylation site. The exact role of phosphorylation and PXXP structure in the modulation of PTTG action in vitro remains poorly understood. We therefore examined the mitotic, transformation, proliferation and transactivation function of the C-terminal PXXP motifs of human PTTG. Live-cell imaging studies using an EGFP-PTTG construct indicated that PTTG's regulation of mitosis is retained regardless of phosphorylation status. Colony-formation assays demonstrated that phosphorylation of PTTG may act as a potent inhibitor of cell transformation. In proliferation assays, NIH-3T3 cells stable transfected and overexpressing mutations preventing PTTG phosphorylation (Phos-) showed significantly increased [3H]thymidine incorporation compared with WT, whereas mutants mimicking constitutive phosphorylation of PTTG (Phos+) exhibited reduced cell proliferation. We demonstrated that PTTG transactivation of FGF-2 in primary thyroid and PTTG-null cell lines was not affected by PTTG phosphorylation but was prevented by a mutant disrupting the PXXP motifs (SH3-). Taken together, our data suggest that PXXP structure and phosphorylation are likely to exert independent and critical influences upon PTTG's diverse actions in vitro.

Radiotherapy for non-functioning pituitary adenomas.

The initial management of large non-functioning pituitary adenomas is surgical debulking. In some cases, postoperative radiotherapy (RT) is administered in order to reduce the likelihood of tumour regrowth. Historically, there have been concerns surrounding a number of potentially significant complications of pituitary RT. Recent contributions to the literature, however, suggest that pituitary RT may be less hazardous than was originally thought. This article reviews the evidence relating to the potential side-effects of RT and weighs these risks against the clinically beneficial effects of preventing pituitary tumour regrowth.

Manipulation of PBF/PTTG1IP phosphorylation status; a potential new therapeutic strategy for improving radioiodine uptake in thyroid and other tumors.

CONTEXT: The clinical effectiveness of ablative radioiodine treatment of thyroid tumors is limited by the availability of the sodium iodide symporter (NIS) at the plasma membrane (PM) for uptake of ¹³¹I. A significant proportion of well-differentiated thyroid tumors are unable to concentrate sufficient radioiodine for effective therapy, and in other tumor models such as breast tumors, where radioiodine uptake would be an attractive therapeutic option, uptake is insufficient. OBJECTIVE: Pituitary tumor-transforming gene-binding factor (PBF; PTTG1IP) is overexpressed in multiple cancers and significantly decreases NIS expression at the PM. The goal of this study was to identify a method by which PBF repression of NIS may be overcome in human tumors. RESULTS: Here, we identify PBF as a tyrosine phosphoprotein that specifically binds the proto-oncogene tyrosine protein kinase Src in mass spectrometry, glutathione S-transferase pulldown and coimmunoprecipitation assays. Src induction leads to phosphorylation at PBF residue Y174. Abrogation of this residue results in PM retention and a markedly reduced ability to bind NIS. The Src inhibitor PP1 inhibits PBF phosphorylation in multiple cell lines in vitro, including human primary thyroid cells. Of direct clinical importance to the treatment of thyroid cancer, PP1 stimulates iodide uptake by transfected NIS in TPC1 thyroid carcinoma cells and entirely overcomes PBF repression of iodide uptake in human primary thyroid cells. CONCLUSIONS: We propose that targeting PBF phosphorylation at residue Y174 via tyrosine kinase inhibitors may be a novel therapeutic strategy to enhance the efficacy of ablative radioiodine treatment in thyroid and other endocrine and endocrine-related tumors.

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.