Gene expression in human thyrocytes and autonomous adenomas reveals suppression of negative feedbacks in tumorigenesis

Abstract

The cAMP signaling pathway regulates growth of many cell types, including somatotrophs, thyrocytes, melanocytes, ovarian follicular granulosa cells, adrenocortical cells, and keratinocytes. Mutations of partners from the cAMP signaling cascade are involved in tumor formation. Thyroid-stimulating hormone (TSH) receptor and Gs␣ activating mutations have been detected in thyroid autonomous adenomas, Gs␣ mutations in growth hormone-secreting pituitary adenomas, and PKAR1A mutations in Carney complex, a multiple neoplasia syndrome. To gain more insight into the role of cAMP signaling in tumor formation, human primary cultures of thyrocytes were treated for different times (1.5, 3, 16, 24, and 48 h) with TSH to characterize modulations in gene expression using cDNA microarrays. This kinetic study showed a clear difference in expression, early (1.5 and 3 h) and late (16 -48 h) after the onset of TSH stimulation. This result suggests a progressive sequential process leading to a change of cell program. The gene expression profile of the long-term stimulated cultures resembled the autonomous adenomas, but not papillary carcinomas. The molecular phenotype of the adenomas thus confirms the role of long-term stimulation of the TSH-cAMP cascade in the pathology. TSH induced a striking up-regulation of different negative feedback modulators of the cAMP cascade, presumably insuring the oneshot effect of the stimulus. Some were down-or nonregulated in adenomas, suggesting a loss of negative feedback control in the tumors. These results suggest that in tumorigenesis, activation of proliferation pathways may be complemented by suppression of multiple corresponding negative feedbacks, i.e., specific tumor suppressors.cyclic AMP ͉ microarrays ͉ papillary tumors ͉ thyrotropin T ight regulation of the second messenger cAMP is of crucial importance for cells because it regulates function, differentiation, and proliferation (1). In cells in which cAMP stimulates growth, activating mutations in partners of this pathway induce uncontrolled growth. In most benign thyroid autonomous adenomas, activating mutations have been found in the thyroidstimulating hormone (TSH) receptor (TSHR) (2) and, to a lesser extent, in the Gs␣ protein, an activator of the cAMP-producing adenylyl cyclase (1, 3). These mutations result in a TSHindependent growth and lead to hyperfunction (1). In addition, activating mutations of the Gs␣ protein have been detected in growth hormone-secreting pituitary adenomas (4) and inactivating mutations in the type I-␣ regulatory subunit inhibiting protein kinase A (PKAR1A) in Carney complex, a multiple neoplasia syndrome (5).Our knowledge of the genes regulated by the cAMP-protein kinase A cascade and its uncontrolled activation is still sketchy (6). To gain more insight into the cAMP-activated signal transduction cascade in tumors, human primary cultures of thyrocytes treated for different times with the TSH growth and differentiation stimulus were used as a model. Thyrocytes in primary culture are expected to be better mod...

Human Thyroid Tumor Cell Lines Derived from Different Tumor Types Present a Common Dedifferentiated Phenotype

“…For the investigation of gene expression profiles of a panel of benign and malignant thyroid tumors, paired samples of nontumor and tumor thyroid tissues were obtained from patients undergoing surgery for thyroid disease. Diagnoses and preparation of a pool of hyperfunctioning autonomous adenomas (n = 5) was done as previously described (16). PTCs were obtained from the Ambroise Paré Hospital (Boulogne, France; sporadic PTCs, n = 10) and from the Institute of Oncology and Metabolism (Kiev, Ukraine; post-Chernobyl PTCs, n = 6), which also provided one FTC (n = 1) and one follicular adenoma (n = 1).…”

“…Five micrograms of total RNA from the cultures and tissues were used for microarray analysis. Samples were prepared as described previously (16). Scanning of in-house cDNA microarrays containing 23,232 spots, preprocessing and normalization of raw data, and replicate averaging were done as previously described (16) with the bioconductor 1.9 bioinformatics software (18) for the R programming language 2.4.0.…”

Perchlorate and ethylenethiourea induce different histological and molecular alterations in a non-mammalian vertebrate model of thyroid goitrogenesis

“…Goitrogen treatment resulted in altered expression of many genes involved in TH synthesis and iodine metabolism. Notably, well-established TSH-responsive genes in mammalian systems such as slc5a5, tpo, tg, dio2, dehal1, and ctsb (Calebiro et al, 2006;De Felice et al, 2004;van Staveren et al, 2006a;Yamada et al, 2006) consistently showed increased expression in thyroids of goitrogen-treated tadpoles. The most marked increase was detected for slc5a5 mRNA (encoding for NIS) being increased in PER-treated tadpoles more than 60-fold over control level.…”

TSH induces metallothionein 1 in thyrocytes via Gq/11- and PKC-dependent signaling

“…Even primary cells, which are usually obtained from patients with goiter or GD, exhibit some genetic alterations or modified protein expression patterns when compared with normal thyrocytes (Xu et al 2007, Davies et al 2010, Park et al 2005. However, these cell culture models still have some advantages to study the principal mechanisms of TSHR signaling when compared with in vivo models as discussed previously (van Staveren et al 2006). Particularly, the relative homogeneity of conditions in cell culture and the possibility to exactly control the dosage of and time of exposure to TSH and to refer to untreated controls are considerable advantages of cell culture when compared with in vivo models (van Staveren et al 2006).…”

“…However, these cell culture models still have some advantages to study the principal mechanisms of TSHR signaling when compared with in vivo models as discussed previously (van Staveren et al 2006). Particularly, the relative homogeneity of conditions in cell culture and the possibility to exactly control the dosage of and time of exposure to TSH and to refer to untreated controls are considerable advantages of cell culture when compared with in vivo models (van Staveren et al 2006). Of note, the TSHR is a prime example of a receptor possessing a very complex signaling by coupling to G s , G q/11 , G i/o , and G 12/13 proteins (Laugwitz et al 1996).…”