Expression of the RET 1 PTCI Oncogene Impairs the Activity of TTF-1 and Pax-8 Thyroid Transcription Factors 1

The most frequent genetic alterations described thus far in human papillary thyroid carcinomas are somatic rearrangements of the RET proto-oncogene, which generate the chimeric RET/PTC oncogenes. We recently found that the expression of the RET/PTC1 oncogene blocked the expression of the thyroiddifferentiated phenotype in rat thyroid epithelial cell line PC CI 3 (PC). Here, we show that this block occurs at a transcriptional level; indeed, the thyroid-specific thyroglobulin and thyroperoxidase gene promoters were inactive in PC-FTC cells. Specific transcription factors, namely, TTF-1 and Pax-8, regulate the expression of differentiated functions in thyroid cells. Here, we show that Pax-8 is expressed at reduced levels in PC-PTC cells and that its adoptive overexpression is unable to restore the activity of target promoters. In contrast, TTF-1 expression is unaltered in PC-PTC cells; however, by using a synthetic promoter that contains its specific target sequence, we demonstrate that TTF-1 is inactive in PC-PTC cells. We conclude that the RET/PTCI oncogene alters the expression of the thyroiddifferentiated phenotype by at least two different mechanisms, i.e., down-regulation of Pax-8 protein and mRNA expression and impaired function of TTF-1 and Pax-8, which occurs at a posttranslational level. Received 5/14/97; revised 11/10/97; accepted 12/3/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdicate thistact. 1 This study was supported by the Associazione ltaliana perla Ricerca sul Cancro, by the Progetto Finalizzato Applicazioni Cliniche della Ricerca Oncologica of the Consiglio Nazionale delle Richerche, and by European Community Grants FI4C-CT96-0003, BMH4-CT96-0814, and BIO2CT93-0454. 2 To whom requests for reprints should be addressed, at Centro di Endocnnologia ed Oncologia Sperimentale del Consiglio Nazionale delle Richerche, Universita di Napoli “Federico II,” via S. Pansini 5, 80131 Naples, Italy. Phone: 39-81-7463056; Fax: 39-81-7463037; E-mail: masantor@unina.it. Introduction The RET gene encodes a receptor-type TK3 that has transforming activity (1). Oncogenic activation of RET is the genetic alteration most consistently observed in human thyroid papillary carcinomas (2). This activation consists of the fusion of the RET TK domain to the 5’-terminal region of heterologous genes, which leads to the generation of the chimeric RET/PTC oncogenes. Thus far, three RET/PTC variants have been isolated. In the case of RET/PTC1 , the most frequently described variant, the fusion occurs with the 5’-terminal region of the H4 gene (2); RET/PTC2 and RET/PTC3 are generated by the fusion of the RETTK-encoding domain with Ria, the gene encoding the regulatory subunit of the cAMPdependent protein kinase, and with the RFG gene, respectively (3, 4). RET is normally not expressed in thyroid follicular cells, but the heterologous promoters, provided by the fused genes, allow the chimeric oncogenes to be expressed in thyroid neoplastic cells (2). Moreover, the truncation of RET causes a cytosolic localization of the chimenc proteins and a constitutive activation of their TK function (5). The role played by RETactivation in thyroid papillary carcinomas is strengthened by the observation that its rearrangement is an early event in tumor progression because it is found in occult thyroid papillary carcinomas, which represent the early stages of papillary tumors (6). Finally, the formal demonstration that RET activation is able to initiate thyroid carcinogenesis was obtained in transgenic mice models; thyroid-targeted expression of the RET/PTC1 oncogene causes the generation of papillary tumors in vivo (7, 8). The thyroid-differentiated phenotype is characterized by the expression of a variety of proteins that are specifically synthesized by the thyroid follicular cells; the synthesis of these proteins is mainly regulated at transcriptional level. Four genes encoding thyroid-specific proteins have been cloned so far: Tg, TPO, TSH receptor (9), and, more recently, the thyroid iodide transporter (NIS; Ref. 10). The thyroidspecific expression of these genes depends on their promoters, which are activated by the binding of thyroid-specific transcription factors and of ubiquitous factors (9). TTF-1 and Pax-8 are the best characterized of the transcription factors that are able to bind and activate thyroid-specific gene promoters. TTF-1 is a homeodomain-containing protein that has a limited tissue distribution, being expressed in thyroid, lung, and diencephalon (1 1,12). Pax-8 is a member of the paired box-containing genes and is expressed in thyroid, kidney, and developing central nervous system (13). Tg and TPO promoters contain binding sites for both hF-i and Pax-8 3 The abbreviations used are: TK, tyrosine kmnase; Tg, thyroglobulmn; TPO, thyroperoxidase; TSH, thyrotropmn; NIS, Na /l symporter; PC, PC Cl 3; CAT. chloramphenicol acetyltransferase; LUC, luciferase; CMV, cytomegalovirus; PKA, protein kinase A.