[CANCER RESEARCH 60, 2845–2849, June 1, 2000] Advances in Brief RET Receptor Expression in Thyroid Follicular Epithelial Cell-derived Tumors 1 Giuseppe Bunone, Mauro Uggeri, Piera Mondellini, Marco A. Pierotti, and Italia Bongarzone 2 Division of Experimental Oncology, Istituto Nazionale Tumori, 20133 Milan, Italy Abstract The RET proto-oncogene encodes a receptor tyrosine kinase for trans- forming growth factor--related neurotrophic factors, which include GDNF and neurturin. The expression of RET proto-oncogene was de- tected in several tissues, such as spleen, thymus, lymph nodes, salivary gland, and spinal cord, and in several neural crest-derived cell lines. RET expression in the thyroid gland was reported to be restricted to neural crest-derived C cells. The presence of RET mRNA or protein has not yet been reported in thyroid follicular cells. We previously demonstrated the expression of oncogenic rearranged versions of RET in papillary thyroid carcinomas: tumors derived from thyroid follicular cells. To assess the expression of the normal RET proto-oncogene in follicular cells, we ana- lyzed its expression in a panel of neoplasias originating from thyroid follicular epithelial cells: papillary carcinomas and both follicular adeno- mas and carcinomas. We also demonstrated the presence of RET normal transcripts in two follicular thyroid carcinoma lymph node metastases. Moreover, we found the presence of the RET/ELE1 transcript, the recip- rocal complementary form of the oncogenic fusion transcript ELE1/RET, in a papillary thyroid carcinoma specimen expressing the RET/PTC3 oncogene, thus demonstrating that the RET promoter is active in those cells after rearrangement. Finally, we show that in a papillary carcinoma- derived cell line expressing the proto-RET receptor and the related GFR2 co-receptor, GDNF treatment induced RET tyrosine phosphoryl- ation and subsequent signal transduction pathway, indicating that RET could be active in thyroid follicular cells. Introduction Thyroid neoplasias comprise a broad spectrum of lesions with different phenotypes and variable clinical behaviors. Thyroid adeno- mas are benign neoplasms rarely capable of malignant progression. PTC 3 and FTC are the most common forms of thyroid cancer. Al- though originating from the same follicular cell, PTCs and FTCs are regarded as different biological entities (1). FTC, solitary and encap- sulated, is an aggressive tumor that often gives rise to distant hema- togenous metastases. The PTC is multifocal and associated with previous radiation exposure, and it frequently invades cervical lymph nodes. Anaplastic or undifferentiated thyroid carcinomas present a dramatic invasive potential and are almost invariably fatal. All of the above-mentioned neoplasias originate from the malignant degenera- tion of the thyroid follicular epithelium. Conversely, MTC develops from neural crest-derived C cells. These neoplasias usually present a poor outcome, spreading through both the lymphatic and hematic endothelium. Specific gene alterations in the different types of thyroid tumors have been detected by molecular analyses. In particular, well-differ- entiated carcinomas of the papillary type are characterized by activa- tion of the neurotrophin receptor tyrosine kinases, RET and NTRK1 proto-oncogenes (2). The other relevant oncogenic activation in dif- ferentiated thyroid carcinomas is that related to the presence of mutated RAS oncogene in follicular carcinomas, whereas RAS activa- tion has been described to be a rare event in papillary carcinomas (3). During follicular carcinogenesis, RAS mutations appear to occur early. In fact, it is possible to detect RAS mutations in adenomas and even in multinodular goiters (2– 6). The last relevant genetic alteration detected in thyroid tumors is represented by abnormalities in the TP53 tumor-suppressor gene. Many reports have described TP53 mutations in a fraction of poorly differentiated and in most undifferentiated or anaplastic thyroid carcinomas (7, 8). As far as RET alterations are concerned, germline and somatic point mutations, dominantly activating the receptor tyrosine kinase activity, have been associated with three variants of both inherited multiple endocrine neoplasia type 2 (MEN 2A, MEN 2B, and FMTC) and sporadic MTC (9). In contrast, in a high percentage (35%) of PTCs, RET activation is due to oncogenic rearrangements of RET (2). These fusion proteins are generated after chromosome rearrangements in which the RET tyrosine kinase domain is fused to the NH 2 terminus of different gene products designated “activating genes” (2). The most frequently involved are H4/D10S170, RI, and ELE1, respectively generating the RET/PTC1, RET/PTC2, and RET/PTC3 oncogenes (10 –12). The fusion products express an intrinsic and constitutive tyrosine kinase activity. Therefore, RET represents a genetic element whose alterations (point mutations and structural rearrangements) are associated with the development of neoplasms originating from both the neural crest-derived C cells (MTC) and the follicular epithelium cells (PTC). The RET proto-oncogene is expressed during the development of the lineage of neuroectodermal cells that give rise to thyroid C cells. However, the role of RET in the development of thyroid C cells is not clear. RET expression in thyroid follicular cells as well as its possible role in the differentiation or proliferation has not been yet reported. In particular, its expression in thyroid follicular cells is a vexing ques- tion. However, it is important to mention that the presence of the reciprocal product of ELE1/RET rearrangement, RET/ELE1 transcript, has been reported in thyroid tumors of children from Belarus after the Chernobyl reactor accident (13) and is considered to be a consequence of radiation exposure, which also transcriptionally activated the RET promoter. However, an alternative explanation implies that the RET promoter is active in a number of thyroid follicular cells. Here we report the expression of proto-RET in sporadic and non- radiation-related thyroid follicular cell neoplasias, PTC, adenomas, and FTCs as well as in normal thyroid tissues. Moreover, we have found the RET/ELE1 transcript in a PTC specimen expressing the RET/PTC3 oncogene, demonstrating that in this case, the RET pro- moter is active after rearrangement. Finally, we show that in a pap- illary carcinoma-derived cell line expressing normal RET protein, GDNF treatment induced RET tyrosine phosphorylation and its sub- sequent signal transduction pathway. These data indicate that RET can Received 10/25/99; accepted 4/17/00. 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 indicate this fact. 1 This work was partially supported by Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Italiana per la Ricerca sul Cancro (FIRC), CNR (Biotecnologie) No. 97.01258.PF49, Project BIOMED2 No. BMH4-CT97-2157, and Project Biotechnology No. BIO4-CT98-0556. 2 To whom requests for reprints should be addressed, at Division of Experimental Oncology, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milan, Italy. Phone: 39-02-2390746; Fax: 39-02-2390764; E-mail: bongarzone@istitutotumori.mi.it. 3 The abbreviations used are: PTC, papillary thyroid carcinoma; FTC, follicular thyroid carcinoma; MTC, medullary thyroid carcinoma; RT-PCR, reverse transcription-PCR. 2845 on July 17, 2015. © 2000 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from