Endocrine-Related Cancer (2011) 18 743–757 Evidence of oncogene-induced senescence in thyroid carcinogenesis Maria Grazia Vizioli, Patricia A Possik1, Eva Tarantino2, Katrin Meissl1, Maria Grazia Borrello, Claudia Miranda, Maria Chiara Anania, Sonia Pagliardini, Ettore Seregni3, Marco A Pierotti4, Silvana Pilotti2, Daniel S Peeper 1 and Angela Greco Molecular Mechanisms Unit, Department of Experimental Oncology and Molecular Medicine, IRCCS Foundation - Istituto Nazionale dei Tumori, Via G. Amadeo, 42 20133 Milan, Italy 1Division of Molecular Genetics, The Netherlands Cancer Institute, Plesmanlaan, 121 1066CX Amsterdam, The Netherlands 2Department of Pathology, 3Nuclear Medicine Division, Department of Diagnostic Imaging and Radiotherapy and 4Scientific Directorate, IRCCS Foundation - Istituto Nazionale dei Tumori, Via G. Venezian, 1 20133 Milan, Italy (Correspondence should be addressed to A Greco; Email: [email protected]) Abstract Oncogene-induced senescence (OIS) is a growth arrest triggered by the enforced expression of cancer-promoting genes and acts as a barrier against malignant transformation in vivo. In this study, by a combination of in vitro and in vivo approaches, we investigate the role of OIS in tumours originating from the thyroid epithelium. We found that expression of different thyroid tumour-associated oncogenes in primary human thyrocytes triggers senescence, as demon- strated by the presence of OIS hallmarks: changes in cell morphology, accumulation of SA-b-Gal and senescence-associated heterochromatic foci, and upregulation of transcription of the cyclin- dependent kinase inhibitors p16INK4a and p21CIP1. Furthermore, immunohistochemical analysis of a panel of thyroid tumours characterised by different aggressiveness showed that the expression of OIS markers such as p16INK4a, p21CIP1 and IGFBP7 is upregulated at early stages, and lost during thyroid tumour progression. Taken together, our results suggest a role of OIS in thyroid carcinogenesis. Endocrine-Related Cancer (2011) 18 743–757 Introduction a tumour measuring %1 cm in diameter and is Thyroid tumours represent the most common endo- considered the early stage of PTC. It has an indolent crine malignancy; the majority originate from thyroid course and very favourable prognosis despite the follicular cells and include differentiated follicular presence of multifocality within the thyroid and the thyroid carcinoma (FTC), papillary thyroid carcinoma synchronous nodal loco-regional spread (Sakorafas (PTC), poorly differentiated thyroid carcinomas et al. 2005). The molecular mechanisms underlying (PDTC) and undifferentiated anaplastic thyroid carci- PTC pathogenesis have been partially elucidated. nomas (ATC) (Sheils 2005). PTCs are associated with genetic alterations leading PTC accounts for w80% of all thyroid cancers; to the activation of the ERK1/2 signal pathway, generally it is a slow-growing tumour type with a good including point mutations of the BRAF and RAS prognosis, although rare, aggressive forms with local genes, and rearrangements of RET and NTRK1 tyrosine invasion or distant metastasis can occur. In addition to kinase receptors, producing RET/PTC and TRK classical PTC, also known as not otherwise specified oncogenes respectively (Greco et al. 2009). Of note, (NOS), several histological variants, differing in BRAF mutations have been reported in a consistent morphological pattern as well as in prognosis, have fraction of PTMC (Lupi et al. 2007), thus supporting been described, including papillary thyroid micro- the notion that it represents an earlier stage of disease carcinomas (PTMC), solid variant (SV), PDTC and tall which eventually evolves into PTC. Nevertheless, the cell variant (TCV) (Sheils 2005). PTMC is defined as molecular mechanisms driving the evolution of PTMC Endocrine-Related Cancer (2011) 18 743–757 Downloaded from Bioscientifica.comDOI: 10.1530/ERC-11-0240 at 09/30/2021 03:39:20AM 1351–0088/11/018–743 q 2011 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.orgvia free access M G Vizioli et al.: Oncogene-induced senescence in thyroid tumours in overt carcinoma or, vice versa, keeping PTMC dermal neurofibromas, murine lung adenomas, human proliferation under control, as well as the mechanisms and murine prostatic adenomas, murine pancreatic responsible for the most aggressive forms of PTC, intraductal neoplasias and murine lymphomas (Braig remain to be elucidated. et al. 2005, Chen et al. 2005, Collado et al. 2005, When expressed in primary cells, activated onco- Courtois-Cox et al. 2006, Dankort et al. 2007, Ha et al. genes can block cellular proliferation by inducing 2007). The increasing evidence of OIS involvement in senescence or apoptosis (Campisi 2005, Mooi & human cancer suggests the possibility of new thera- Peeper 2006). Both mechanisms are thought to play peutic approaches based on the functional restoration important roles in suppressing tumourigenesis in vivo, of OIS in tumour cells. as they prevent the proliferation of cells at risk of In this study, we investigate the occurrence of OIS neoplastic transformation (Michaloglou et al. 2005). in thyroid carcinogenesis with in vitro and in vivo The factors that direct the choice of cancer cells into studies. We found that PTC-associated oncogenes one or the other outcome have remained unclear trigger OIS in primary human thyrocytes, as demon- (D’Adda di Fagagna 2008); most likely they are related strated by changes in cell morphology and the to the oncogenic trigger and to the balance between presence of specific OIS markers. To assess whether proapoptotic and antiapoptotic factors. this mechanism represents a barrier to thyroid cancer Oncogene-induced senescence (OIS) was first in vivo, we performed immunohistochemical analysis identified in vitro and describes a stable cell-cycle in a panel of thyroid tumours characterised by different arrest that is triggered by the aberrant proliferative aggressiveness. OIS markers were found to be signals of oncogenes (Serrano et al. 1997). Senescent upregulated in PTMCs, whereas they were pro- cells are characterised by a flat and large morphology gressively lost in PTCs and ATC. Overall, our data with vacuoles (Denoyelle et al. 2006), an increase support a role for OIS in thyroid carcinogenesis. in senescence-associated b-galactosidase activity (SA-b-Gal; Dimri et al. 1995), and alterations in chromatin structure known as senescence-associated Materials and methods heterochromatic foci (SAHF), which may repress the Cell culture expression of proliferation-promoting genes (Narita Normal thyroid samples were obtained from patients et al. 2003, Adams 2007). Moreover, senescent cells undergoing surgery at IRCCS Foundation Istituto undergo dramatic changes in gene expression pattern, Nazionale dei Tumori (Milan, Italy). All patients including, activation of p53, increased levels of the gave their written informed consent, and the study cyclin-dependent kinase inhibitors, p16INK4a and was approved by the independent ethics committee p21CIP1 (Beausejour et al. 2003, Lowe et al. 2004) of IRCCS Foundation Istituto Nazionale dei Tumori. and secretion of multiple factors such as extracellular Primary thyrocyte cultures were established and proteases, matrix components, growth factors, pro- maintained in nutrient mixture Ham’s F12 medium inflammatory cytokines and chemokines (Kortlever (custom made by Invitrogen) containing 5% calf serum et al. 2006, Acosta et al. 2008, Kuilman et al. 2008, and bovine hypothalamus and pituitary extracts, as Wajapeyee et al. 2008). described previously (Curcio et al. 1994). Thyrocytes OIS is a mechanism of increasing importance in expressing RET/PTC1 oncogene, obtained from infec- tumours (Kuilman et al. 2010), as several reports have tion of primary thyrocytes with RET/PTC1 retroviral demonstrated that it occurs in several diverse pre- vector, have been previously described (Borrello et al. cancerous tissues from human and mice, and acts as 2005). HEK293T cells were maintained in DMEM a barrier to invasiveness and tumourigenesis. The first F9 medium supplemented with 10% FCS. direct evidence of cellular senescence in a growth arrested human neoplasm was reported in melanocytic nevi, premalignant lesions frequently carrying the Lentivirus production and transduction BRAFV600E mutation which rarely progress to mela- HEK293T cells (4!106) were transfected with 8 mgof noma (Michaloglou et al. 2005). The blunted pro- BRAFV600E lentiviral construct (HIV-CS-CG-Blast- liferative activity in nevi was associated with features BRAFV600E) and 3 mg of each of the helper plasmids of cellular senescence, including SA-b-Gal activity pMDLglpRRE, pHCMV-G and pRSVrev by calcium and elevated levels of p16INK4a. On the contrary, in phosphate in complete medium containing 25 mM melanoma, which is characterised by accelerated chloroquine. After 6–12 h of incubation cells were proliferation, cell senescence was extinguished. Later washed and refed with complete medium. Supernatant on, senescence markers were identified in human containing lentiviral particles was collected and frozen Downloaded from Bioscientifica.com at 09/30/2021 03:39:20AM via free access 744 www.endocrinology-journals.org Endocrine-Related Cancer (2011) 18 743–757 24 h after transfection, and subsequently added to SA-b-Gal assay thyrocytes for 6 h in the presence of 0.8 mg/ml Adherent cells were analysed for SA-b-Gal production polybrene (Sigma–Aldrich), followed by blasticidin using a Senescence-beta-Galactosidase