title: Role of Oncogene-Induced Senescence in Thyroid Carcinogenesis
Creators: name: Vizioli, Maria Grazia
Publisher: name: The Open University
Year: 2012

description: Oncogene-induced senescence (OIS) is an irreversible G1 cell cycle arrest triggered by aberrant expression of oncogenes. The arrest observed during senescence is implemented mainly through activation of p53 and the upregulation of the cyclin-dependent kinase (CDK) inhibitors, p16<sup>INK4a</sup> and p21<sup>CIP1</sup>. <i>In vivo</i> OIS may act as a barrier to neoplastic transformation, and bypass of OIS is a prerequisite for tumorigenesis. <br><br>In this work the role of OIS in thyroid carcinogenesis was investigated by in combination of <i>in vitro</i>and <i>in vivo</i> approaches. <br><br>We found that the expression of different thyroid tumour-associated oncogenes (BRAF, RAS, RET and TRK) in human primary thyrocytes triggers senescence, as demonstrated by the presence of OIS hallmarks. For instance, cells appeared flatted and enlarged; they displayed senescence-associated B-galactosidase activity (SA-β-Gal), senescence associated heterochromatic foci (SAHF), and high expression levels of the CDK inhibitors p16<sup>INK4a</sup> and p21<sup>CIP1</sup> and p53. Additionally, using RNA-interference strategy we directly assessed the individual contribution of p16<sup>INK4a</sup> in BRAF<sup>V600E</sup>-induced senescence. In our experimental setting the inactivation of p16<sup>INK4a</sup> did not have any impact on oncogene-induced senescence suggesting the possibility that p16<sup>INK4a</sup> may cooperate with other factors in triggering senescence. <br><br>A major consequence of oncogene activation is the secretion of a plethora of proteins including growth factors, cytokines, and chemokines. Some of these secreted proteins are known to be regulators of OIS. For example, IGFBP7 is recently proposed as a mediator of BRAF<sup>V600E</sup>-induced senescence in melanocytes. <br><br>We showed that IGFBP7 gene is frequently downregulated in Papillary thyroid tumour (PTC); restoration of its expression in a PTC-derived, IGFBP7-negative cell line affects growth properties, migration and invasion, and this is associated to the induction of apoptosis. Thus, our results implicate IGFBP7 as a tumour suppressor protein in thyroid tumours. Additionally, we investigated the possible involvement of IGFBP7 in our cellular senescence model. Our data indicate that IGFBP7 expression is not changed during OIS. <br><br>We also established and characterized OIS inducible system in our cellular model; we transduced human primary thyrocytes with a retroviral vector carrying an activated form of RAS oncogene coupled to the ligand binding domain of the estrogen receptor. Upon addition of 4-hydroxytamoxifen (4-OHT), we observed a robust induction of senescence, including significantly reduced incorporation of BrdU and high levels of p16<sup>INK4a</sup> p21<sup>CIP1</sup>, and p53. Moreover, we assessed the relative importance of tumour suppressor proteins, p16<sup>INK4a</sup> and p53 in mediating OIS in H-RAS<sup>G12V</sup> transfected thyrocytes. Surprisingly, silencing of p53 expression did not alter the growth arrest induced by H-RAS<sup>G12V</sup>, whereas p16<sup>INK4a</sup> seems to be the dominant effector of senescence in this experimental setting, as its specific inactivation delays the onset of senescence. <br><br>We also found that senescence induced by H-RAS<sup>G12V</sup> is associated with activation of inflammatory transcriptome including IL8 and its receptor CXCR2. We reported that knocking down the chemokine receptor CXCR2 either IL8 prevents H-RAS<sup>G12V</sup> cells from undergoing OIS. To translate information from <i>in vitro</i>experiments to a clinic setting, senescence markers identified in cultured cells were used to detect OIS in a panel of thyroid tumours characterized by different aggressiveness. Our immunohistochemical analysis showed that the expression of OIS markers is upregulated at early stages of the thyroid tumour and lost during tumour progression. These evidences support the notion that OIS may counteract oncogenic activity in thyroid tumours, and its escape allows thyroid tumour progression.