Endocrine-Related Cancer (2007) 14 445–452

HOOK3-RET: a novel type of RET/PTC rearrangement in papillary thyroid carcinoma

Raffaele Ciampi1, Thomas J Giordano2, Kathryn Wikenheiser-Brokamp1, Ronald J Koenig 3 and Yuri E Nikiforov 1,4

1Department of Pathology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA 2Department of Pathology, University of Michigan Medical School and Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan 48109-0054, USA 3Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA 4Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA (Requests for offprints should be addressed to Y E Nikiforov who is now at Department of Pathology, University of Pittsburgh, A713 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261, USA; Email: [email protected]) R Ciampi is now at Department of Endocrinology and Metabolism, University of Pisa, Pisa, Italy

Abstract Chromosomal rearrangements of the RET proto-oncogene (RET/PTC) are the common feature of papillary thyroid carcinoma (PTC). In this study, we report the identification, cloning, and functional characterization of a novel type of RET/PTC rearrangement that results from the fusion of the 30- portion of RET coding for the tyrosine kinase (TK) domain of the receptor to the 50-portion of the Homo sapiens hook homolog 3 (HOOK3) gene. The novel fusion was identified in a case of PTC that revealed a gene expression signature characteristic of RET/PTC on DNA microarray analysis, but was negative for the most common types of RET rearrangement. A fusion product between exon 11 of HOOK3 and exon 12 of RET gene was identified by 50RACE, and the presence of chimeric HOOK3-RET protein of 88 kDa was detected by western blot analysis with an anti-RET antibody. The protein is predicted to contain a portion of the coiled-coil domains of HOOK3 and the intact TK domain of RET. Expression of the HOOK3-RET cDNA in NIH3T3 cells resulted in the formation of transformed foci and in tumor formation after injection into nude mice, confirming the oncogenic nature of HOOK3-RET. Endocrine-Related Cancer (2007) 14 445–452

Introduction been shown to be activated in papillary carcinomas by Papillary thyroid carcinoma (PTC) is the most chromosomal rearrangement resulting in the AKAP9- common type of thyroid cancer and accounts for BRAF fusion (Ciampi et al. 2005). It is a rare event in w80% of all thyroid malignancies (Hundahl et al. sporadic papillary carcinomas and more common in 2000). Genetic alterations along the RET/RAS/BRAF/ radiation-induced tumors. Approximately, 10% of MAPK signaling pathway have been demonstrated to papillary carcinomas harbor point mutations in one be crucial in the pathogenesis of papillary carcinomas, of the RAS genes (N-RAS, H-RAS, K-RAS), with most as they are found in more than 70% of all tumors and of these tumors being the follicular variants of rarely overlap in the same tumor, suggesting that papillary carcinoma (De Micco 2003, Zhu et al. activation of a single effector of this pathway is 2003). Chromosomal rearrangements involving the sufficient for cell transformation (Kimura et al. 2003, RET proto-oncogene is another common alteration in Soares et al. 2003, Frattini et al. 2004). The most PTC (Fusco et al. 1987, Grieco et al. 1990). common genetic alteration of this pathway is the point The RET proto-oncogene resides on chromosome mutation V600E of the BRAF gene, which is found in 10q11.2 and codes for a membrane receptor tyrosine w40% of papillary carcinomas (reviewed in Ciampi & kinase (TK; Takahashi et al. 1985, Takahashi 1988). It Nikiforov 2005, Xing 2005). Recently, BRAF has also contains three functional domains: an extracellular

Endocrine-Related Cancer (2007) 14 445–452 Downloaded from Bioscientifica.comDOI:10.1677/ERC-07-0039 at 10/01/2021 01:14:55AM 1351–0088/07/014–445 q 2007 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.orgvia free access R Ciampi et al.: HOOK3-RET rearrangement in thyroid cancer ligand-binding domain with four cadherin-like repeats analysis of this case which resulted in the identifi- and one cysteine-rich region, a hydrophobic trans- cation, cloning, and functional characterization of a membrane domain, and an intracellular domain novel type of RET/PTC rearrangement. containing the TK domain. The ligands of the RET receptor belongs to the group of the glial cell line- derived neutrophic factor family and include neurturin Materials and methods (NRTN), persephin (PSPN), and artemin (ARTN; Tumor samples and cell lines Airaksinen et al. 1999). Binding of the ligand causes receptor dimerization, autophosphorylation of critical Snap frozen tissue from the index case was obtained tyrosine residues within the intracellular domain, and from the University of Michigan Health System activation of the signaling cascade. through the Tissue Procurement Service with Insti- In the thyroid gland, RET is expressed at high level tutional Review Board (IRB) approval. The index case ! ! in parafollicular C-cells, where point mutations of RET was a 14-year-old female with a 7 4 4 cm papillary are responsible for the development of medullary carcinoma of the right lobe. The tumor morphology thyroid carcinoma. In follicular thyroid cells, RET can was classical papillary type and there were four be activated by chromosomal rearrangement resulting positive central compartment lymph nodes. There in the fusion of the 30-portion of the RET gene to the 50- was no history of prior radiation exposure. Frozen portion of several unrelated genes, which is known as tissue samples from 129 PTC were obtained from the RET/PTC rearrangement (Fusco et al. 1987, Grieco Department of Pathology, University of Cincinnati et al. 1990). Three types of RET/PTC were originally following a protocol approved by the University of identified and remain by far the most common in Cincinnati IRB and through the Cooperative Human papillary carcinomas. Of them, RET/PTC1 is formed Tissue Network. by fusion with the H4 (also known as D10S170, CCDC6) gene (Grieco et al. 1990) and RET/PTC3 by Fluorescence in situ hybridization (FISH) fusion with the NCOA4 (RFG, ELE1) gene (Bongar- FISH was performed as previously described (Ciampi zone et al. 1994, Santoro et al. 1994). RET/PTC1 and et al. 2005). Briefly, a mix of the two bacterial artificial RET/PTC3 are intrachromosomal paracentric inver- chromosome (BAC) clones RP11-168L22 (GenBank sions since both genes participating in the rearrange- accession no. AC068707) and RP13-368N15 (GenBank ment are located on chromosome 10q (Pierotti et al. accession no. AL591169) were used to design a probe 1992, Minoletti et al. 1994). In contrast, RET/PTC2 is spanning the RET gene. The clone RP11-122A17 a translocation between chromosomes 10 and 17, (GenBank accession no. AC110275) was used as a resulting in the RET fusion with the regulatory subunit probe for the HOOK3 gene. The clones were purchased RIa of the cAMP-dependent protein kinase A from BAC/PAC Resources, Children’s Hospital, Oak- (Bongarzone et al. 1993). More recently, several land. The extracted DNA was labeled with Spectrum- novel types of RET/PTC have been described in single Green-dUTP and SpectrumRed-dUTP using a nick cases of PTC, all of which are interchromosomal translation kit (Vysis Inc., Downers Grove, IL, USA). translocations (Klugbauer et al. 1998, 2000, Klugbauer Touch-preparations from the tumor were prepared from & Rabes 1999, Nakata et al. 1999, Corvi et al. 2000, snap frozen tissue and fixed in 3:1 methanol:acetic acid, Salassidis et al. 2000, Saenko et al. 2003). pretreated with 10 mg collagenase H (Sigma), and In a previous study of PTC using DNA microarray co-denatured with 10 ng each labeled probe. Hybrid- analysis, we determined a distinct expression profile ization was carried at 37 8C overnight. Microscopy was for the RET/PTC rearrangement and established a performed with a Leica Microsystem TCS 4D confocal highly accurate mutational classifier based on the fluorescence microscope with digital image capture expression of several genes (Giordano et al. 2005). We (Leica, Wetzlar, Germany). identified a case of papillary carcinoma in which the classifier predicted the presence of RET/PTC 0 rearrangement, despite the negative testing of the 5 RACE tumor RNA for RET/PTC1 and RET/PTC3 rearrange- Total RNA from the index case was extracted using ments by RT-PCR. By fluorescence in situ hybrid- Trizol Reagent (Invitrogen) and subjected to 50RACE ization (FISH), we demonstrated that the tumor cells using the 50RACE System for Amplification of cDNA indeed harbor a RET rearrangement, but one that did Ends (Invitrogen). cDNA synthesis was performed using not correspond to the most common RET/PTC1 or a previously published primer corresponding to exon 13 RET/PTC3 types. In this study, we performed further of the RET gene, 50-CTGCTTCAGGACGTTGAA-30

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(Santoro et al. 1994). Subsequent PCRs utilized primer from liver, medullary thyroid carcinoma, and TPC1 50-GCAGGTCTCCGCAGCTCACTC-30 for the first cell line harboring RET/PTC1 rearrangement (Ishizaka reaction and 50-CTTTCAGCATCTTCACGG-30 (San- et al. 1990) were used as controls. toro et al. 1994) for the second round of amplification. The RACE products were excised from the gel, purified Transformation assays and sequenced using an automated ABI 377 Sequencer (Perkin–Elmer, Waltham, MA, USA). NIH 3T3 cells were plated in six-well plates at a density of 3.4!105 and transfected 24 h later using Lipofecta- mine (Invitrogen) with 16 mg pcDNA3.1D HOOK3- Cloning and sequencing analysis RET plasmid. Cells transfected with the empty vector The open reading frame (ORF) of the chimeric cDNA were used as a negative control. After 1 day, cells were was amplified using primers spanning the start codon of split into three 100 mm3 dishes and cultured in DMEM HOOK3 (50-CACCATGTTCAGCGTAGAG-30)and with 10% fetal bovine serum (FBS). For the focus assay, the stop codon of RET (50-ACTATCAAACGTGTC- cells were cultured for 21 days, stained with 0.5% CATTAATTT-30). The PCR product was electro- crystal violet, and foci counted. For stable cell lines, phoresed on agarose gel, purified, and cloned into the cells were mass selected for 21 days in the media pcDNA3.1D/V5-His-TOPO expression vector using supplemented with 350 mg/ml G-418 Sulfate (Invi- the pcDNA3.1 Directional TOPO Expression kit trogen). A total of 5!106 pooled cells were suspended (Invitrogen). Several clones were fully sequenced and in 0.4 ml culture media and then injected subcu- the clone I-9 was chosen for further experiments. taneously into the flank of homozygous Nu/Nu female mice (Charles River Laboratories, Wilmington, MA, RT-PCR experiments USA). Cells expressing HOOK3-RET were injected into the left flank and cells containing empty vector were Total RNA was extracted using Trizol Reagent injected into the right flank of each of the six mice. (Invitrogen), reverse transcribed, and amplified by Tumors were removed 20–26 days later, measured, PCR using primers flanking the HOOK3-RET 0 0 weighed, and fixed in 10% formalin for histologic usion point: 5 -AGGCGGCAGGTTAAACTCTT-3 evaluation. located in the exon 11 of HOOK3 and 50- TCCAAATTCGCCTTCTCCTA-30 in the exon 12 of RET, with an expected product of 195 bp. Amplifi- Results cation was carried out for 35 cycles (94 8C for 40 s, 55 8C for 1 min, and 72 8C for 1 min 30 s). In order to We studied a case of papillary carcinoma that had study the expression of the wild-type HOOK3, semi- revealed an expression profile consistent with quantitative RT-PCR was performed for 30 cycles RET/PTC rearrangement in our previous study using primers 50-TCCAAATTCGCCTTCTCCTA-30 (Giordano et al. 2005). The presence of RET and 50-TGTTCTCAGCCTGTCCTTTTC-30, with the rearrangement in the majority of tumor cells was expected amplification product of 246 bp. The results confirmed with FISH using a probe spanning the RET were normalized using amplification of the house- gene. A split of one of the two RET signals was noted keeping gene phosphoglycerate kinase (PGK), as in the majority of tumor cells (Fig. 1A). described elsewhere (Argani et al. 1998). In order to identify the partner gene of RET in this rearrangement, we performed the 50RACE assay using total RNA from the index case and specific primers for Western blot analysis the RET gene designed along its 30-portion. Several Western blotting was performed as previously bands were present after the nested amplification and a described (Kunzli et al. 2002). Twenty microgram of prominent band was gel purified and sequenced. The proteins were loaded on a 7.5% polyacrylamide gel and sequencing revealed that exon 12 of RET was fused subjected to SDS–PAGE. After the electro-transfer, in-frame with exon 11 of the Homo sapiens hook membranes were incubated with anti-RET mouse homolog 3 (HOOK3; GenBank accession number: monoclonal antibody (clone RET-X-D210) generated BC056146; Fig. 2A). The breakpoint within the RET against the C-terminus of the RET protein in our gene was in the same position as in all the other known laboratory and previously validated (Knauf et al. 2003). types of RET/PTC rearrangement. The novel fusion The dilution of the antibody was 1:250. HRP-goat-anti- ORF resulted in a transcript with a predicted size of mouse secondary antibody (Zymed, San Francisco, 2331 bp (1122 bp from HOOK3 and 1209 bp from CA, USA) was used for the detection. Protein extracts RET) and a protein of 776 amino acids with a predicted

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molecular weight of 88.2 kDa (Fig. 2B). The presence of the fusion between these two genes was confirmed at a genomic level by two-color FISH using probes corresponding to the RET and HOOK3 genes (Fig. 1B). Since the pattern of the HOOK3 gene expression in human thyroid cells was not well studied, we analyzed it by semi-quantitative RT-PCR in normal thyroid (NT) tissue and in several other tissues, including liver, skeletal muscle, and kidney. Abundant HOOK3-RET mRNA was found in all NT tissue samples and in other tissues studied (Fig. 3). Figure 1 Fluorescence in situ hybridization (FISH) on interphase tumor cell nuclei from the index case. (A) Hybridization with the To confirm the presence of the fusion transcript in RET probe only shows split of one of the signals (arrows) the index case, we performed RT-PCR with primers indicating the presence of the RET gene rearrangement. (B) flanking the identified breakpoint of HOOK3-RET.The Two-color FISH with the RET probe (green) and the HOOK3 probe (red) confirming the HOOK-RET fusion (arrow). Full color expected 195 bp product was successfully amplified version available at http://erc.endocrinology-journals.org. (data not shown). Western blot analysis using an

Figure 2 (A) Sequence of the 50RACE product showing the fusion between HOOK3 and RET. (B) Genomic organization of the HOOK3 and RET genes and the HOOK3-RET fusion. The boxes indicate exons and the lines indicate introns. Black arrows indicate the location of breakpoint in the two genes. The chimeric cDNA is shown at the bottom of the panel with indication of its size (bp) and the number of corresponding amino acids.

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Figure 3 Expression of the wild-type HOOK3 gene in thyroid and other tissues. Electrophoresis of RT-PCR products showing HOOK3 expression in the liver (L), skeletal muscle (SM), kidney (K), and three samples from normal thyroid (NT). PGK amplification was used as a control for RNA quantity. M, 123 bp DNA ladder; NC, negative control. antibody against the C-terminal portion of RET revealed a band of w88 kDa in the index case which corresponds to the predicted size of the chimeric HOOK3-RET protein (Fig. 4). Totesttheoncogeniccharacteristicsofthenovelfusion gene, the full-size coding sequence of HOOK3-RET cDNA was amplified by RT-PCR and cloned into the pcDNA3.1D/V5-His-TOPOmammalianexpressionvec- tor. Several clones were screened by restriction analysis and sequenced. The clone I-9 was chosen for the functionalstudies(GenBankaccessionno.NM_032410). The ability of HOOK3-RET protein to induce cell transformation was studied in an NIH3T3 cells focus assay. Cells transfected with the HOOK3-RET ORF Figure 5 (A) Formation of transformed foci in NIH3T3 cells transiently transfected with pcDNA3.1D/HOOK3-RET and the cDNA formed transfected foci at high efficiency when empty vector. The results represent the average number of foci compared with cells transformed with empty vector per plate in three separate plates. Similar results were obtained in two separate experiments. (B) s.c. injection of NIH3T3 cells (Fig. 5A). In addition, NIH3T3 cells stably transfected stably transfected with HOOK3-RET or empty vector in the flank with HOOK3-RET formed tumors in nude mice as of nude mice. Results are shown as average weight of tumors early as 10 days after injection. Tumors formed in all removed from six mice. mice injected and grew to 0.78–1.22 g (20–26 mm in size) 3 weeks after injection. In contrast, no tumors were detected after injection of cells transfected with empty vector (Fig. 5B). These results demonstrate that HOOK3-RET functions as an oncogene and is able to transform NIH3T3 cells both in vitro and in vivo. The prevalence of the HOOK3-RET fusion was studied by RT-PCR with primers flanking the fusion point in a series of 129 consecutive PTC cases. No additional tumors were positive for the fusion indicating that this novel RET/PTC rearrangement is not a common event in thyroid papillary carcinomas from the general population. Figure 4 Western blot analysis with the RET specific antibody of protein extracts from liver (A), medullary thyroid carcinoma (B), TPC1 cell line (C), and in the index case of papillary carcinoma (D). The index case shows a band corresponding to predicted Discussion molecular weight of 88.2 kDa of the HOOK3-RET protein (*). TPC1 cell line harboring RET/PTC1 rearrangement shows an In this study, we report the identification of a novel expected band corresponding to 60 kDa (**), while medullary RET/PTC rearrangement that results from fusion of the thyroid carcinoma shows two bands at 170 and 180 kDa RET and HOOK3 genes. Since the identification of the corresponding to the wild-type RET (***). Liver cell extract as expected does not reveal the RET protein. An additional band first RET partner in a chromosomal rearrangement (arrow) is likely to be due to non-specific antibody binding. reported in 1990 (Grieco et al. 1990), 12 different types

Downloaded from Bioscientifica.com at 10/01/2021 01:14:55AM via free access www.endocrinology-journals.org 449 R Ciampi et al.: HOOK3-RET rearrangement in thyroid cancer of RET/PTC have been identified resulting from the this tumor, HOOK3-RET. To our knowledge, this is only fusion of RET to different partner genes (Table 1; the second example of a novel chromosomal rearrange- Bongarzone et al. 1993, 1994, Santoro et al. 1994, mentbeing detectedbased ontheinformation obtainedby Klugbauer et al. 1998, 2000, Klugbauer & Rabes 1999, gene expression profile analysis. Previously, the analysis Nakata et al. 1999, Corvi et al. 2000, Salassidis et al. of gene expression profiles allowed identification of the 2000, Saenko et al. 2003). RET/PTC1 and RET/PTC3 fusion between the TMPRSS2 and either the ETV1 or result from intrachromosomal inversions, and consti- the ERG genes in prostate cancer (Tomlins et al. 2005). tute the two most common RET/PTC rearrangement The identification of HOOK3-RET provides further types found in sporadic and radiation-associated confirmationoftheaccuracyoftheidentifiedtranscription papillary carcinomas. RET/PTC2 and the more signaturesassociatedwithspecificmutations.Italsooffers recently identified rare types of RET/PTC are all evidence that different types of RET/PTC rearrangement interchromosomal translocations. Most of these rare lead to similar effects with respect to alteration of the RET/PTC types have been found in papillary carci- expression of many genes. In addition, it validates the use nomas from patients with a history of either environ- ofsuchanapproachtosearchfor novel genetic alterations mental (after Chernobyl) or therapeutic exposure to inpapillarycarcinomasthatshowtranscriptionsignatures ionizing radiation (Klugbauer et al. 1998, 2000, ofothermutations,suchasthoseofRASgenes,butlacking Klugbauer & Rabes 1999, Corvi et al. 2000, Salassidis mutations affecting the known hot spots within these et al. 2000, Saenko et al. 2003). The one exception is genes (Giordano et al. 2005). the ELKS-RET fusion identified in a case of papillary It has been established that different RET partner carcinoma with no apparent history of radiation genes involved in RET/PTC rearrangement share exposure (Nakata et al. 1999). In the present study, several common characteristics important for the we identify yet another novel RET/PTC rearrangement oncogenic function of the chimeric gene. They are all in a patient with no radiation exposure history. expressed in thyroid follicular cells, providing an The novel rearrangement type reported herein resulted active promoter for the fusion gene. In addition, they from the fusion of RET to yet another gene, HOOK3, all encode putative dimerization domains, either a located on chromosome 8. The presence ofRET/PTC was coiled–coil or leucine zipper, essential for dimerization predicted in this tumor based on the analysis of gene and ligand-independent activation of the RET (TK; expression profiles obtained by DNA microarray analysis Tong et al. 1997, Jhiang 2000). As for the RET gene, (Giordano et al. 2005). The analysis defined distinct gene virtually all breakpoints occur within its 1.8 kb intron expression signatures for papillary carcinomas carrying 11, leaving intact the TK domain of the receptor and specific types of mutations, i.e., BRAF, RAS,andRET/ therefore enabling the RET/PTC oncoprotein to bind PTC. One papillary carcinoma showed an expression SHC via Y1062 and activate the RAS–RAF–MAPK profile highly consistent with the presence of RET/PTC, pathway in thyroid cells (Knauf et al. 2003). but revealed no RET/PTC1 or RET/PTC3 rearrangement, All of these structural features can be found in the suggesting that it may harbor either a known rare type of HOOK3-RET chimeric gene. HOOK3 encodes a rearrangement or a novel type of RET/PTC. Further protein belonging to a recently identified family of analysis now reveals a novel RET/PTC rearrangement in human cytosolic coiled-coil proteins that link

Table 1 RET/PTC rearrangement types in papillary thyroid carcinoma (PTC)

No. RET/PTC type Partner gene Chromosomal positions Reference

1 RET/PTC1 H4 (CCDC6, D10S170) inv10(q11.2;q21) Grieco et al. (1990) 2 RET/PTC2 PRKAR1A t(10;17)(q11.2;q23) Bongarzone et al. (1993) 3 RET/PTC3 RET/PTC4 NCOA4 (RFG, ELE1) inv10(q11.2;q10) Bongarzone et al. (1994), Santoro et al. (1994) and Fugazzola et al. (1996) 4 RET/PTC5 GOLGA5 (RFG5) t(10;14)(q11.2;q32) Klugbauer et al. (1998) 5 RET/PTC6 HTIF1 (TRIM24) t(7;10)(q32–34;q11.2) Klugbauer & Rabes (1999) 6 RET/PTC7 TIF1G (RFG7, TRIM33) t(1;10)(p13;q11.2) Klugbauer & Rabes (1999) 7 ELKS-RET ELKS (RAB6IP2) t(10;12)(q11.2;p13.3) Nakata et al. (1999) 8 RET/PTC8 KTN1 t(10;14)(q11.2;q22.1) Salassidis et al. (2000) 9 RET/PTC9 RFG9 t(10;18)(q11.2;q21–22) Klugbauer et al. (2000) 10 PCM1-RET PCM1 t(8;10)(p21-22;q11.2) Corvi et al. (2000) 11 RFP-RET RFP (TRIM27) t(6;10)(p21;q11.2) Saenko et al. (2003) 12 HOOK3-RET HOOK3 t(8;10)(p11.21;q11.2) Current report

Downloaded from Bioscientifica.com at 10/01/2021 01:14:55AM via free access 450 www.endocrinology-journals.org Endocrine-Related Cancer (2007) 14 445–452 cytoplasmic organelles to microtubules (Walenta et al. tyrosine kinase and the regulatory subunit RI alpha of 2001). The three known human HOOK proteins, cyclic AMP-dependent protein kinase A. Molecular and HOOK1, HOOK2, and HOOK3, contain conserved Cellular Biology 13 358–366. N-terminal domains which attach to microtubules, and Bongarzone I, Butti MG, Coronelli S, Borrello MG, Santoro M, more variable C-terminal domains which mediate Mondellini P, Pilotti S, Fusco A, Della Porta G & Pierotti binding to different organelles. The C-terminal portion MA 1994 Frequent activation of ret proto-oncogene by fusion with a new activating gene in papillary thyroid of HOOK3 binds to Golgi membranes in vitro, carcinomas. Cancer Research 54 2979–2985. suggesting that it may participate in defining the Ciampi R & Nikiforov YE 2005 Alterations of the BRAF organization and localization of the mammalian Golgi gene in thyroid tumors. Endocrine Pathology 16 163–172. complex within the cell (Walenta et al. 2001). Similar Ciampi R, Knauf JA, Kerler R, Gandhi M, Zhu Z, Nikiforova to all other family members, the HOOK3 protein MN, Rabes HM, Fagin JA & Nikiforov YE 2005 contains an extended central coiled-coil motif, which Oncogenic AKAP9-BRAF fusion is a novel mechanism was shown to mediate homodimerization in the of MAPK pathway activation in thyroid cancer. Drosophila Hook protein (Kramer & Phistry 1996, Journal of Clinical Investigation 115 94–101. Sevrioukov et al. 1999). In the HOOK3-RET fusion, Corvi R, Berger N, Balczon R & Romeo G 2000 RET/PCM- the first 11 exons of the HOOK3 gene are fused to 1: a novel fusion gene in papillary thyroid carcinoma. exons 12–20 of the RET gene. Two large coiled-coil Oncogene 19 4236–4242. domains of HOOK3 encoded by exons 7–11 (aa. 171– De Micco C 2003 Ras mutations in follicular variant of 226 and 244–359) are preserved in the HOOK3-RET papillary thyroid carcinoma. American Journal of Clinical Pathology 120 803–804. protein, indicating that it possesses a dimerization Frattini M, Ferrario C, Bressan P, Balestra D, De Cecco L, domain found in all known RET fusion partners. In the Mondellini P, Bongarzone I, Collini P, Gariboldi M, Pilotti RET gene, the breakpoint is located within intron 11, S et al. 2004 Alternative mutations of BRAF, RET and similar to most other rearrangement types. Finally, we NTRK1 are associated with similar but distinct confirmed that the wild-type HOOK3 gene is expressed gene expression patterns in papillary thyroid cancer. in thyroid follicular cells, providing an active promoter Oncogene 23 7436–7440. to drive the expression of the TK domain of RET. Fugazzola L, Pierotti MA, Vigano E, Pacini F, These qualities are expected to enable the HOOK3- Vorontsova TV & Bongarzone I 1996 Molecular and RET with oncogenic properties, which indeed were biochemical analysis of RET/PTC4, a novel oncogenic identified in both in vitro and in vivo experiments. rearrangement between RET and ELE1 genes, in a In summary, we report a novel RET/PTC rearrange- post-Chernobyl papillary thyroid cancer. Oncogene 13 ment resulting from RET fusion to the HOOK3 gene 1093–1097. which leads to the formation of an oncogene. Fusco A, Grieco M, Santoro M, Berlingieri MT, Pilotti S, Pierotti MA, Della Porta G & Vecchio G 1987 A new oncogene in human thyroid papillary carcinomas and their Acknowledgements lymph-nodal metastases. Nature 328 170–172. Giordano TJ, Kuick R, Thomas DG, Misek DE, Vinco M, This study was supported by NIH grant R01 CA88041. Sanders D, Zhu Z, Ciampi R, Roh M, Shedden K et al. 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