RET Receptor Expression in Thyroid Follicular Epithelial Cell-Derived Tumors1

[CANCER RESEARCH 60, 2845–2849, June 1, 2000] Advances in Brief

RET Receptor Expression in Thyroid Follicular Epithelial Cell-derived Tumors1

Giuseppe Bunone, Mauro Uggeri, Piera Mondellini, Marco A. Pierotti, and Italia Bongarzone2 Division of Experimental Oncology, Istituto Nazionale Tumori, 20133 Milan, Italy

Abstract have been detected by molecular analyses. In particular, well-differentiated carcinomas of the papillary type are characterized by activa- The RET proto-oncogene encodes a receptor tyrosine kinase for trans- tion of the neurotrophin receptor tyrosine kinases, RET and NTRK1 forming growth factor-␤-related neurotrophic factors, which include proto-oncogenes (2). The other relevant oncogenic activation in dif- GDNF and neurturin. The expression of RET proto-oncogene was detected in several tissues, such as spleen, thymus, lymph nodes, salivary ferentiated thyroid carcinomas is that related to the presence of gland, and spinal cord, and in several neural crest-derived cell lines. RET mutated RAS oncogene in follicular carcinomas, whereas RAS activa- expression in the thyroid gland was reported to be restricted to neural tion has been described to be a rare event in papillary carcinomas (3). crest-derived C cells. The presence of RET mRNA or protein has not yet During follicular carcinogenesis, RAS mutations appear to occur early. been reported in thyroid follicular cells. We previously demonstrated the In fact, it is possible to detect RAS mutations in adenomas and even expression of oncogenic rearranged versions of RET in papillary thyroid in multinodular goiters (2–6). The last relevant genetic alteration carcinomas: tumors derived from thyroid follicular cells. To assess the detected in thyroid tumors is represented by abnormalities in the TP53 expression of the normal RET proto-oncogene in follicular cells, we ana- tumor-suppressor gene. Many reports have described TP53 mutations lyzed its expression in a panel of neoplasias originating from thyroid follicular epithelial cells: papillary carcinomas and both follicular adeno- in a fraction of poorly differentiated and in most undifferentiated or mas and carcinomas. We also demonstrated the presence of RET normal anaplastic thyroid carcinomas (7, 8). transcripts in two follicular thyroid carcinoma lymph node metastases. As far as RET alterations are concerned, germline and somatic point Moreover, we found the presence of the RET/ELE1 transcript, the recip- mutations, dominantly activating the receptor tyrosine kinase activity, rocal complementary form of the oncogenic fusion transcript ELE1/RET, have been associated with three variants of both inherited multiple in a papillary thyroid carcinoma specimen expressing the RET/PTC3 endocrine neoplasia type 2 (MEN 2A, MEN 2B, and FMTC) and oncogene, thus demonstrating that the RET promoter is active in those sporadic MTC (9). In contrast, in a high percentage (35%) of PTCs, cells after rearrangement. Finally, we show that in a papillary carcinoma- RET activation is due to oncogenic rearrangements of RET (2). These derived cell line expressing the proto-RET receptor and the related fusion proteins are generated after chromosome rearrangements in GFR␣2 co-receptor, GDNF treatment induced RET tyrosine phosphorylation and subsequent signal transduction pathway, indicating that RET which the RET tyrosine kinase domain is fused to the NH2 terminus could be active in thyroid follicular cells. of different gene products designated “activating genes” (2). The most frequently involved are H4/D10S170, RI␣, and ELE1, respectively Introduction generating the RET/PTC1, RET/PTC2, and RET/PTC3 oncogenes (10–12). The fusion products express an intrinsic and constitutive Thyroid neoplasias comprise a broad spectrum of lesions with tyrosine kinase activity. Therefore, RET represents a genetic element different phenotypes and variable clinical behaviors. Thyroid adeno- whose alterations (point mutations and structural rearrangements) are mas are benign neoplasms rarely capable of malignant progression. associated with the development of neoplasms originating from both PTC3 and FTC are the most common forms of thyroid cancer. Al- the neural crest-derived C cells (MTC) and the follicular epithelium though originating from the same follicular cell, PTCs and FTCs are cells (PTC). regarded as different biological entities (1). FTC, solitary and encap- The RET proto-oncogene is expressed during the development of sulated, is an aggressive tumor that often gives rise to distant hema- the lineage of neuroectodermal cells that give rise to thyroid C cells. togenous metastases. The PTC is multifocal and associated with However, the role of RET in the development of thyroid C cells is not previous radiation exposure, and it frequently invades cervical lymph clear. RET expression in thyroid follicular cells as well as its possible nodes. Anaplastic or undifferentiated thyroid carcinomas present a role in the differentiation or proliferation has not been yet reported. In dramatic invasive potential and are almost invariably fatal. All of the particular, its expression in thyroid follicular cells is a vexing ques- above-mentioned neoplasias originate from the malignant degenera- tion. However, it is important to mention that the presence of the tion of the thyroid follicular epithelium. Conversely, MTC develops reciprocal product of ELE1/RET rearrangement, RET/ELE1 transcript, from neural crest-derived C cells. These neoplasias usually present a poor outcome, spreading through both the lymphatic and hematic has been reported in thyroid tumors of children from Belarus after the endothelium. Chernobyl reactor accident (13) and is considered to be a consequence Specific gene alterations in the different types of thyroid tumors 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. Received 10/25/99; accepted 4/17/00. The costs of publication of this article were defrayed in part by the payment of page Here we report the expression of proto-RET in sporadic and non- charges. This article must therefore be hereby marked advertisement in accordance with radiation-related thyroid follicular cell neoplasias, PTC, adenomas, 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 and FTCs as well as in normal thyroid tissues. Moreover, we have (AIRC), Fondazione Italiana per la Ricerca sul Cancro (FIRC), CNR (Biotecnologie) No. found the RET/ELE1 transcript in a PTC specimen expressing the 97.01258.PF49, Project BIOMED2 No. BMH4-CT97-2157, and Project Biotechnology RET/PTC3 oncogene, demonstrating that in this case, the RET pro- No. BIO4-CT98-0556. 2 To whom requests for reprints should be addressed, at Division of Experimental moter is active after rearrangement. Finally, we show that in a pap- Oncology, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milan, Italy. Phone: illary carcinoma-derived cell line expressing normal RET protein, 39-02-2390746; Fax: 39-02-2390764; E-mail: [email protected]. 3 The abbreviations used are: PTC, papillary thyroid carcinoma; FTC, follicular thyroid GDNF treatment induced RET tyrosine phosphorylation and its sub- carcinoma; MTC, medullary thyroid carcinoma; RT-PCR, reverse transcription-PCR. sequent signal transduction pathway. These data indicate that RET can 2845

with affinity-purified antiphosphotyrosine polyclonal antiserum (Upstate Bio- technology). The antiphosphotyrosine immunoprecipitates were resolved by electrophoresis on 7.5% SDS polyacrylamide gels (PAGE). Proteins were transferred onto nitrocellulose filters and immunoblotted with the same antiphosphotyrosine antiserum, with anti-RET affinity-purified antibodies or the anti-Shc polyclonal antiserum (Upstate Biotechnology) essentially as described previously (15). Immunoreactive bands were visualized using horse- radish peroxidase-conjugated antirabbit or antimouse antisera and ECL detec- tion reagents (Amersham).

Results The RET proto-oncogene is expressed in a wide range of embryonal tissues and in parafollicular cells (C cells) of the thyroid gland, and usually is not detected in normal thyroid follicular cells. In this study Fig. 1. Proto-RET expression in PTCs. RT-PCR of total RNA isolated from human we detected the expression of RET proto-oncogene by a RT-PCR specimens. The numbers above the lanes indicate different patients. N, normal thyroid method (13) using two primer combinations, RETTM1-RETC2 and tissue; PTC3, sample of a papillary carcinoma expressing RET/PTC3 oncogene; ⌽, ⌽X174 HaeIII-digested marker; W, negative control. RETC1-RETC2, respectively able to amplify the region of normal RET encompassing the RET/PTC fusion point and the region of RET that is 3Ј-terminal to the fusion point present in all RET and RET/ be expressed in follicular thyroid cells and that it can display a role in PTCs genes. We analyzed 10 PTCs negative for the expression of RET the differentiation/proliferation of these cells. (PTC1, -2, and -3) or NTRK1-derived oncogenes. As controls, we used normal thyroid tissues, a PTC expressing the RET/PTC3 onco- Materials and Methods gene, and a MTC sample. Fig. 1 shows the expression of the RET Cell Line. The human thyroid carcinoma cell line NPA (14) was grown in proto-oncogene in the above-mentioned specimens. We detected the DMEM supplemented with 10% FCS. For GDNF or PDGF stimulation, the expression of RET mRNA at different levels in all of the samples cells were starved for 20 h in F12 medium and 0.5% FCS. Stimulation with 50 analyzed but not in the PTC expressing the RET/PTC3 oncogene. On ng/ml GDNF (Alomone Labs) or 50 ng/ml PDGF (Upstate Biotechnology) was the contrary, using primers specific for the RET tyrosine kinase performed for 10 min. domain, we detected a RT-PCR product in all of the analyzed spec- RNA Extraction and RT-PCR Analysis. Each frozen biopsy was me- imens. It was possible that the observed expression of RET was due to chanically disrupted in a Mikro-Dismembrator II (B. Braun) containing liquid the presence of parafollicular cells in the analyzed specimens. This nitrogen. Total RNA was extracted by the Ultraspec II (Biotecx Laboratories) was true for the normal thyroid tissues, but in the PTC samples the according to the manufacturer’s instructions. RT-PCR was performed as fol- presence of C cells should be irrelevant. lows: 5 ␮g of total RNA were reverse-transcribed at 42°C for 50 min in the To address the presence of RET mRNA in thyroid follicular tumors, presence of 500 ng of random hexamers and Superscript II reverse tran- scriptase (Life Technologies, Inc.) in a final volume of 20 ␮l. Two ␮lofthe we analyzed the expression of RET proto-oncogene in a panel of cDNA reaction were then subjected to 30 PCR cycles (30 s at 95°C, 30 s at thyroid neoplasias and relative lymph node metastases originating 56°C, and 1 min at 72°C) using the AmpliTaq Kit (Perkin-Elmer) and 0.4 from these cells. Using primers specific for RET only (RETTM1- ␮mol/liter specific primers. The following primers were used in RT-PCR RETC2), we analyzed the expression of RET mRNA in three adeno- experiments: RETTM1 (5Ј-CTGTCCTCTTTCCCCTCATC-3Ј) and RETC2 mas (benign neoplasias of follicular cells) and in four FTCs (Fig. 2). (5Ј-TGCAGGCCCCATACAATTTG-3Ј) for the amplification of proto-RET These results indicated that, although at low levels, RET proto- only; RETC1 (5Ј-TGGGAATCCCTCGGAAGAA-3Ј) and RETC2 for the oncogene was expressed in these follicular thyroid cells. RET expres- amplification of both the proto- or oncogenic version of RET being designed sion was also detected in two FTC lymph node metastases (Fig. 2, Ј on the tyrosine kinase domain of RET; Aldo/F (5 -CGCAGAAGGGGTCCT- 248m and 266m). GGTGA-3Ј) and Aldo/R (5Ј-CAGCTCCTTCTTCTGCTGCGGGGTC-3Ј) for To confirm the possibility that the RET promoter was active in the amplification of the aldolase A housekeeping gene; GDNFR1 (5Ј-AGCAT- GTACCAGAGCCTGCAG-3Ј) and GDNFR2 (5Ј-TCGTTCTTCATAGGAG- thyroid follicular cells, we looked for expression of the reciprocal CACAC-3Ј) for the amplification of GFR␣1; TRNR2r (5Ј-CCAGTGTCAT- product of RET rearrangement in a PTC sample expressing the RET/ CACCACCTGCACG-3Ј) and TRNR2H3 (5Ј-AGCCGACGGTCTGGCTC- PTC3 oncogene. Genomic DNA of tumor samples expressing the TGCTGG-3Ј) for the amplification of GFR␣2; EST10 (5Ј-ACTGTCCT- RET/PTC3 oncogene was further investigated by PCR to find the GCTCTTTGAACC-3Ј) and RET39 (5Ј-TGGACTCAGTACTTCGACC-3Ј) breakpoint region. We previously have described the chromosomal for the amplification of ELE1/RET; and RET56 (5Ј-TGCCCCTTCAGTGT- mechanism generating the oncogenic version of ELE1/RET as an TCCTACT-3Ј) and EST4 (5Ј-CTTGATAACACTGGCAGGTT-3Ј) for the amplification of RET/ELE1. PCR products were electrophoresed on a 3% agarose gel containing ethidium bromide (0.5 ␮g/ml) and visualized under UV light. Genomic PCR Analysis. High-molecular weight DNA was extracted following standard procedures. ELE1/RET and RET/ELE1 fragments containing the breakpoints were amplified using the following primers: EST10 (5Ј- ACTGTCCTGCTCTTTGAACC-3Ј) and RET39 (5Ј-TGGACTCAGTACT- TCGACC-3Ј) for ELE1/RET; and RET56 (5Ј-TGCCCCTTCAGTGTTC- CTACT-3Ј) and EST4 (5Ј-CTTGATAACACTGGCAGGTT-3Ј) for RET/ ELE1. Sequencing. For sequencing reactions, a dye terminator cycle sequencing ready reaction kit (ABI Prism) was used. Reaction products were then analyzed using the ABI Prism 377 fluorescent DNA sequencer (Perkin-Elmer). Fig. 2. Proto-RET expression in follicular thyroid tumors. RT-PCR of total RNA from human adenomas (Ade) and FTCs. The numbers above the lanes indicate different Western Blot Analysis, Immunoprecipitation, and Antibodies. Protein patients. N, normal thyroid tissue; NPA, papillary carcinoma-derived cell line; ⌽, ⌽X174 samples were prepared as described previously (15) and immunoprecipitated HaeIII-digested marker; W, negative control. 2846

Fig. 3. Identification of the RET/ELE1 transcript in a tumor sample expressing RET/PTC3 oncogene. A, PCR on genomic DNA of sample 293 with specific primers for both transforming and reciprocal products of the rearrangement between RET and ELE1 genes. On the right and left are the direct sequence analyses of the genomic rearrangement of RET and ELE1 genes, respectively. B, RT-PCR analysis on total RNA of sample 293. PCR was carried out for transforming ELE1/RET and reciprocal RET/ELE1 fusion sequences. On the right and left are the direct sequence analyses of the two transcripts RET/ELE1 and ELE1/RET, respectively. ⌽, ⌽X174 HaeIII-digested marker. inversion within chromosome 10, band q11.2, where the genes ELE1 to activate the signal transduction pathway upon GDNF binding. In and RET are located (16). In Fig. 3A is shown the PCR products of Fig. 4C is shown Shc phosphorylation after GDNF treatment in these both genomic rearrangements between the ELE1 and RET genes. We cells. Similar results were obtained upon PDGF stimulation. Taken also analyzed the genomic sequence around the breakpoint region by together, these data demonstrated the presence of a functional RET direct sequencing. Using specific primers for the RET and ELE1 receptor in follicular thyroid cells. genes, we detected the expression of ELE1/RET (RET/PTC3) and of the reciprocal RET/ELE1 transcript by RT-PCR (Fig. 3B). We also Discussion sequenced the breakpoint regions of both the transforming and the reciprocal products of rearrangement. The RET-specific primer was The expression of the RET proto-oncogene was detected in several located at the 5Ј end of the fusion point, and the ELE1-specific primer tissues, including spleen, thymus, lymph node, salivary gland, and was on its 3Ј end. Fig. 3B shows the products of this RT-PCR, spinal cord, and in several neural crest-derived cell lines. On the other demonstrating the expression of both the transforming and reciprocal hand, the expression of this tyrosine kinase receptor in the thyroid products of ELE1/RET rearrangement. Moreover, direct sequencing of gland was reported to be restricted to parafollicular cells. In fact, the the fragment confirmed the specificity of the RT-PCR products. presence of RET mRNA or protein has yet to be fully documented in To study the biochemical activity of RET tyrosine kinase receptor thyroid follicular cells. in those thyroid follicular cells expressing the receptor, we first Here we have shown RET proto-oncogene expression in 10 PTC analyzed the expression of the RET gene, and GFR␣1 and GFR␣2 specimens selected for being negative for the expression of the main co-receptors in the PTC cell line NPA. We found the expression of oncogenic versions of RET reported to occur in these tumors (2). It is RET mRNA by RT-PCR together with that of GFR␣2, a glyco- possible that the RET expression was due to neural crest-derived C sylphosphatidyl inositol-anchored co-receptor for RET signaling. On cells contaminating the analyzed tissue specimens. Although C cells the other hand, we did not find expression of GFR␣1 (Fig. 4A). To should not be present in these PTC specimens, as documented by a demonstrate that the RET proto-oncogene product expressed on thy- careful pathological examination, to ensure that their presence did not roid follicular cells was a functional receptor, we further investigated interfere with our study, we analyzed RET expression on an additional the expression of RET protein and its activation by GDNF. We panel of neoplasias originating from thyroid follicular cell carcino- immunoprecipitated RET protein from NPA cells before and after mas, follicular adenomas, and two related lymph node metastases. We treatment with GDNF. We detected the expression of RET receptor in found RET proto-oncogene expressed in both adenomas and FTCs and NPA cells and GDNF-induced RET tyrosine phosphorylation (Fig. in two FTC lymph node metastases. Moreover, we have shown that 4B). We also investigated the ability of RET tyrosine kinase receptor GDNF treatment induced RET tyrosine phosphorylation and activated 2847

the reciprocal RET/ELE1 transcript. These authors proposed that the RET promoter might be activated by radiation exposure, thus trigger- ing the expression of RET/ELE1 transcripts. We have now found the reciprocal product of the ELE1/RET rearrangement expressed in a tumor sample from a patient with a non-radiation-related cancer, thus implying that the RET promoter region is active independent of radiation exposure. In this case, both the structural features of this tumor and the location of the two metastatic specimens should rule out a significant contribution of type C cells. The combination of our in vivo and in vitro results strongly supports the concept that the thyroid follicular component can express a functional RET receptor, which may be activated in the presence of specific ligands in the thyroid microenvironment. Because C cells express the RET receptor, the concept that RET ligands are present in this microenvironment is highly plausible. In recent years, it has been shown that there are some interconnec- tions between follicular- and parafollicular-type C cells. The microenvironment provided by MTC cells has the capacity to stimulate the proliferation of follicular cells, resulting in hyperplastic and adenom- atous follicles, and as suggested recently, the latter can ultimately acquire a fully developed neoplastic phenotype (either follicular or papillary; Refs. 17, 18). The opposite situation has also been described: C-cell hyperplasia was recognized in some patients with Hashimoto thyroiditis as well as in thyroid adjacent to follicular and papillary neoplasms (17, 18). A large amount of evidence supports the concept that RET oncogenic activation is important for both follicular and parafollicular cell components. In fact, RET is involved in the tumorigenesis of almost all of the hereditary MTCs and in a propor- tion (ϳ50%) of sporadic MTCs. The importance of RET oncogenic rearrangements in sporadic and radiation-induced papillary thyroid tumorigenesis has been fully demonstrated. Approximately 35% of sporadic and Ͼ60% of radiation-induced tumors carry an oncogenic version of the RET gene. Biochemical studies of the RET/PTC onco- protein signal transduction pathways have demonstrated that they recruit cytoplasmic proteins containing SH2 domains such as phospholipase C␥ (19) phosphatidylinositol 3-kinase, the GTPase-activating protein Ras, Src kinase, and the adapter proteins Shc and Grb2 (20–22). It has been also demonstrated that activated RET constructs stimulate JNK activation in different cell lines (23). Additional results also support the concept that RET oncogenic activation is an early event in thyroid carcinogenesis and that further or concomitant molecular events could determine neoplastic progression. We propose that RET stimulation can constitute a factor contrib- Fig. 4. Expression of proto-RET in papillary carcinoma-derived cell line NPA. A, uting to the transformation of both follicular and parafollicular cells. RT-PCR analysis of RET and GFR␣1 and GFR␣2 in NPA cells. Aldo, aldolase A Further efforts must be aimed to better define the role of RET in these ⌽ ⌽ housekeeping gene; , X174 HaeIII-digested marker. B, RET tyrosine phosphorylation cells and to clarify the question of their histogenetic origin (17). induced by GDNF. RET protein was immunoprecipitated from NPA cells before (Ϫ) and after (ϩ) treatment with GDNF. Proteins were immunoprecipitated (IP) and immuno- Finally, an interesting question still to be answered is why the same blotted (IB) with the monoclonal antiserum antiphosphotyrosine (␣Ptyr) or with the gene with different mechanisms is involved in tumorigenesis of both anti-RET antiserum (␣RET). C, Shc phosphorylation after RET activation. Shc proteins were immunoprecipitated (IP) from NPA cells before (Ϫ) and after (ϩ) treatments with follicular and parafollicular thyroid cell components. GDNF and PDGF. The immunoprecipitates were resolved as in B and immunoblotted (IB) ␣ with anti-Shc antiserum ( Shc). Acknowledgments

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Giuseppe Bunone, Mauro Uggeri, Piera Mondellini, et al.

Cancer Res 2000;60:2845-2849.

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