Both external and internal exposure to ionizing radiation are strong risk factors for the development of thyroid tumors. Until now, the diagnosis of radiation-induced thyroid tumors has been deduced from a network of arguments taken together with the individual history of radiation exposure. Neither the histological features nor the genetic alterations observed in these tumors have been shown to be specific fingerprints of an exposure to radiation. The aim of our work is to define ionizing radiation-related molecular specificities in a series of secondary thyroid tumors developed in the radiation field of patients treated by radiotherapy. To identify molecular markers that could represent a radiation-induction signature, we compared 25K microarray transcriptome profiles of a learning set of 28 thyroid tumors, which comprised 14 follicular thyroid adenomas (FTA) and 14 papillary thyroid carcinomas (PTC), either sporadic or consecutive to external radiotherapy in childhood. We identified a signature composed of 322 genes which discriminates radiation-induced tumors (FTA and PTC) from their sporadic counterparts. The robustness of this signature was further confirmed by blind case-by-case classification of an independent set of 29 tumors (16 FTA and 13 PTC). After the histology code break by the clinicians, 26/29 tumors were well classified regarding tumor etiology, 1 was undetermined, and 2 were misclassified. Our results help shed light on radiation-induced thyroid carcinogenesis, since specific molecular pathways are deregulated in radiation-induced tumors.
Catherine Ory, Nicolas Ugolin, Céline Levalois, Ludovic Lacroix, Bernard Caillou, Jean-Michel Bidart, Martin Schlumberger, Ibrahima Diallo, Florent de Vathaire, Paul Hofman, José Santini, Bernard Malfoy and Sylvie Chevillard
Nabahet Ameur, Ludovic Lacroix, Sophie Roucan, Véronique Roux, Sophie Broutin, Monique Talbot, Corinne Dupuy, Bernard Caillou, Martin Schlumberger and Jean-Michel Bidart
RET oncogene mutations are found in familial medullary thyroid carcinomas (MTC) and in one-third of sporadic cases. Oncogenic mechanisms involved in non-RET mutated sporadic MTC remain unclear. To study alterations associated with the development of both inherited and sporadic MTC, pangenomic DNA microarrays were used to analyze the transcriptome of 13 MTCs (four familial and nine sporadic). By using an ANOVA test, a list of 173 gene sequences with at least a twofold change expression was obtained. A subset of differentially expressed genes was controlled by real-time quantitative PCR and immunohistochemistry on a larger collection of MTCs. The expression pattern of those genes allowed us to distinguish two groups of sporadic tumors. The first group displays an expression profile similar to that expressed by inherited RET634 tumors. The second presents an expression profile close to that displayed by inherited RET918 tumors and includes tumors from patients with distant metastases. It is characterized by the overexpression of genes involved in proliferation and invasion (PTN, ESM1, and CEACAM6) or matrix remodeling (COL1A1, COL1A2, and FAP). Interestingly, RET918 tumors showed overexpression of the PTN gene, encoding pleiotrophin, a protein associated with metastasis. Using a MTC cell line, silencing of RET induced the inhibition of PTN gene expression. Overall, our results suggest that familial MTC and sporadic MTC could activate similar oncogenic pathways.
Françoise Galland, Ludovic Lacroix, Patrick Saulnier, Philippe Dessen, Geri Meduri, Michèle Bernier, Stéphane Gaillard, Jean Guibourdenche, Thierry Fournier, Danièle Evain-Brion, Jean Michel Bidart and Philippe Chanson
Non-functioning pituitary adenomas (NFPAs) may be locally invasive. Markers of invasiveness are needed to guide patient management and particularly the use of adjuvant radiotherapy. To examine whether invasive NFPAs display a specific gene expression profile relative to non-invasive tumors, we selected 40 NFPAs (38 of the gonadotroph type) and classified them as invasive (n=22) or non-invasive (n=18) on the basis of magnetic resonance imaging and surgical findings. We then performed pangenomic analysis with the 44k Agilent human whole genome expression oligonucleotide microarray in order to identify genes with differential expression between invasive and non-invasive NFPAs. Candidate genes were then tested in qRT-PCR. Prediction class analysis showed that the expression of 346 genes differed between invasive and non-invasive NFPAs (P<0.001), of which 233 genes were up-regulated and 113 genes were down-regulated in invasive tumors. On the basis of Ingenuity networks and the degree of up- or down-regulation in invasive versus non-invasive tumors, 35 genes were selected for expression quantification by qRT-PCR. Overexpression of only four genes was confirmed, namely IGFBP5 (P=0.02), MYO5A (P=0.04), FLT3 (P=0.01), and NFE2L1 (P=0.02). At the protein level, only myosin 5A (MYO5A) immunostaining was stronger in invasive than in non-invasive NFPAs. Molecular signature allows to differentiate ‘grossly’ invasive from non-invasive NFPAs. The product of one of these genes, MYO5A, may be a useful marker of tumor invasiveness.
Urbain Weyemi, Bernard Caillou, Monique Talbot, Rabii Ameziane-El-Hassani, Ludovic Lacroix, Odile Lagent-Chevallier, Abir Al Ghuzlan, Dirk Roos, Jean-Michel Bidart, Alain Virion, Martin Schlumberger and Corinne Dupuy
NADPH oxidase 4 (NOX4) belongs to the NOX family that generates reactive oxygen species (ROS). Function and tissue distribution of NOX4 have not yet been entirely clarified. To date, in the thyroid gland, only DUOX1/2 NOX systems have been described. NOX4 mRNA expression, as shown by real-time PCR, was present in normal thyroid tissue, regulated by TSH and significantly increased in differentiated cancer tissues. TSH increased the protein level of NOX4 in human thyroid primary culture and NOX4-dependent ROS generation. NOX4 immunostaining was detected in normal and pathologic thyroid tissues. In normal thyroid tissue, staining was heterogeneous and mostly found in activated columnar thyrocytes but absent in quiescent flat cells. Papillary and follicular thyroid carcinomas displayed more homogeneous staining. The p22phox protein that forms a heterodimeric enzyme complex with NOX4 displayed an identical cellular expression pattern and was also positively regulated by TSH. ROS may have various biological effects, depending on the site of production. Intracellular NOX4–p22phox localization suggests a role in cytoplasmic redox signaling, in contrast to the DUOX localization at the apical membrane that corresponds to an extracellular H2O2 production. Increased NOX4–p22phox in cancer might be related to a higher proliferation rate and tumor progression but a role in the development of tumors has to be further studied and established in the future.