Oncogene (2005) 24, 6902–6916 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc Gene expression in thyroid autonomous adenomas provides insight into their physiopathology Sandrine Wattel1, Hortensia Mircescu1, David Venet1, Agnes Burniat1, Brigitte Franc2, Sandra Frank1, Guy Andry3, Jacqueline Van Sande1, Pierre Rocmans1, Jacques E Dumont1, Vincent Detours1 and Carine Maenhaut*,1 1Institute of Interdisciplinary Research, School of Medicine, Free University of Brussels, Brussels, Belgium; 2Service d’Anatomie et de Cytologie Pathologiques, Hoˆpital A Pare´ (AP-HP), Universite´ de Versailles, St-Quentin-en-Yvelines, France; 3Department of Surgery, Institut Bordet, 1000 Brussels, Belgium The purpose of this study was to use the microarray Oncogene (2005) 24, 6902–6916. doi:10.1038/sj.onc.1208849; technology to define expression profiles characteristic of published online 18 July 2005 thyroid autonomous adenomas and relate these findings to physiological mechanisms. Experiments were performed Keywords: thyroid; autonomous adenoma; gene expres- on a series of separated adenomas and their normal sion; microarray counterparts on Micromax cDNA microarrays covering 2400 genes (analysis I), and on a pool of adenomatous tissues and their corresponding normal counterparts using microarrays of 18 000 spots (analysis II). Results for Introduction genes present on the two arrays corroborated and several gene regulations previously determined by Northern Autonomous thyroid adenomas are monoclonal encap- blotting or microarrays in similar lesions were confirmed. sulated benign tumors that grow, metabolize iodide, and Five overexpressed and 24underexpressed genes were also secrete thyroid hormones independently of the normal confirmed by real-time RT–PCR in some of the samples thyrotropin (TSH) control, the main proliferative and used for microarray analysis, and in additional tumor functional stimulus of the thyroid gland (Corvilain specimens. Our results show: (1) a change in the cell et al., 2001). They can be either solitary or part of a populations of the tumor, with a marked decrease in multinodular goiter, and grow rather slowly. Their lymphocytes and blood cells and an increase in endothelial frequency is an inverse function of the iodine dietary cells. The latter increase would correspond to the supply, and they are therefore uncommon in the USA or establishment of a close relation between thyrocytes and in Japan, but very common in Europe (Knudsen et al., endothelial cells and is related to increased N-cadherin 2000; Tonacchera et al., 2000; Dremier et al., 2002). expression. It explains the increased blood flow in the Their autonomous secretion of thyroid hormones tumor; (2) a homogeneity of tumor samples correlating decreases TSH secretion by the classical negative feed- with their common physiopathological mechanism: the back exerted on the pituitary thyrotrophs and on TRH constitutive activation of the thyrotropin (TSH)/cAMP secretion. This leads to functional quiescence of the cascade; (3) a low proportion of regulated genes consistent nonaffected tissue. If untreated, in the presence of with the concept of a minimal deviation tumor; (4) a sufficient iodine supply to allow the synthesis and higher expression of genes coding for specific functional secretion of excess thyroid hormones and once they proteins, consistent with the functional hyperactivity of reach a certain size, autonomous adenomas cause the tumors; (5) an increase of phosphodiesterase gene hyperthyroidism (Ermans and Camus, 1972; Corvilain expression which explains the relatively low cyclic AMP et al., 2001). They therefore represent a major medical levels measured in these tumors; (6) an overexpression of problem and account for about half of the cases of antiapoptotic genes and underexpression of proapoptotic hyperthyroidism in Europe (Russo et al., 1995; Bauch, genes compatible with their low apoptosis rate; (7) an 1998; Corvilain et al., 2001). Clinically, they are overexpression of N-cadherin and downregulation of diagnosed by the presence of hot nodules that take up 99m caveolins, which casts doubt about the use of these high amounts of radioiodide or Tc pertechnetate, and expressions as markers for malignancy. are surrounded by poorly radioactive quiescent tissue, and by low TSH serum levels, with normal or elevated thyroid hormone levels. Their histology represents a mixture in various proportions of micro- and macro- *Correspondence: C Maenhaut, IRIBHM, University of Brussels, follicles surrounded by a well-defined capsula. The Campus Erasme, Building C, 808 route de Lennik, B-1070 Brussels, Belgium; E-mail: [email protected] majority have a monoclonal origin (Namba et al., Received 1 March 2005; revised 3 May 2005; accepted 11 May 2005; 1990; Krohn et al., 1998). A very minor proportion may published online 18 July 2005 degenerate into malignant follicular carcinomas, Gene expression in thyroid autonomous adenomas S Wattel et al 6903 whereas a much higher proportion of cold (i.e. non- Adenomas were identified by scintigraphy and in iodide trapping) follicular adenomas do. some cases by a higher in vitro iodide uptake than the TSH exerts most of its effects via a seven-transmem- control quiescent tissue. The analysed tissue RNAs were brane domain receptor positively coupled to adenylate validated for untreated patients by a NIS RNA cyclase, leading to a rise in intracellular cAMP levels expression higher than in control tissues, as evaluated (Dremier et al., 2002). In Europe and Japan, one of the by RT–PCR (see below). For control purposes, one main mechanisms responsible for the hyperfunction and multinodular goiter with no hyperfunctioning adenoma growth of thyroid autonomous adenomas is the (v6) was compared to normal adjacent tissue. For serial constitutive activation of the cAMP-dependent mito- real-time RT–PCR analysis, 10 samples from 10 new genic cascade, through mutations conferring constitu- patients, all of them untreated, were analysed. tive activity of the TSH receptor (50–80%) or an activating mutation of Gsa (8%) (Russo et al., 1995; Analysis of separated samples (analysis I) Van Sande et al., 1995; Fuhrer et al., 1997; Tonacchera et al., 1999; Vanvooren et al., 2002). Similar mutations Reproducibility of the TSA method in gene expression account for hyperfunctioning nodules in multinodular analysis Tyramide signal amplification in microarray goiters (Tonacchera et al., 1999, 2000; Krohn et al., gene expression is a very useful method when limiting 2000). Their phenotype is reproduced in transgenic mice amounts of RNA are available. To assess its reprodu- expressing the constitutive adenosine A2 receptor cibility, we labeled reverse-transcribed total RNA from (Ledent et al., 1992), constitutive Gsa (Michiels et al., the adenomatous tissue with biotin and total RNA from 1994) or the activator of Gsa choleratoxin (Zeiger et al., the control tissue with fluorescein. We performed 1997). Autonomous adenomas are thus a well-defined hybridization with the reverse combination of dyes as example of the results of long-term stimulation by the well (dye swap), and calculated the averages of the physiological TSH receptor/cAMP-dependent cascade intensity ratios obtained with the two opposite dye of human thyroid tissue in vivo (Dumont et al., 1989). As combinations (tumor/control) (Taniguchi et al., 2001; such, they illustrate the concept of minimal deviation Karsten et al., 2002). A high correlation (96%) between tumors: they are constituted of hyperfunctioning thyroid the ratios of the intensity values was obtained for cells whose operation is uncoupled from their normal duplicate spots on the same array. When comparing the physiological control (Van Sande et al., 1988; Weber, same spot on two different slides, correlation ranged 2002). However, data are scarce about specific gene between 70 and 80%. With microarray data generated expression modifications in these tumors and especially by two independent RNA preparations from the same about their physiopathological consequences. tumor sample (a11/v3), a correlation of 73% was Numerous studies in different tumor types have obtained (data not shown). We thus concluded that shown that gene expression profiling of clinical samples tyramide signal amplification is a consistent and by microarray offers unprecedented opportunities to reproducible method, adequate for our analyses. obtain molecular signatures of the state of the tumors. In this study, we have analysed the pattern of gene Gene expression analysis shows high homogeneity between expression in hyperfunctioning autonomous thyroid different autonomous adenomas For each adenoma/ adenomas and validated the relative expression of control paired sample, experiments were duplicated with selected genes by real-time RT–PCR. The data show dye switching, and the averages of the intensity ratios that several physiological and morphological character- (tumor/control) resulting from each dye combination istics of these adenomas can be explained by their were calculated. A total of 14 tumor samples were transcriptional program. analysed (Table 1). We performed unsupervised hier- archical clustering of the samples to analyse the data. As depicted in Figure 1, a set of up- or downregulated genes Results could be identified, with similar expression in the majority of the tumors, suggesting that thyroid adeno- Two types of analyses were performed: an analysis of mas are relatively homogeneous tissues, based on gene gene expression in