Rend. Fis. Acc. Dncei s. 9, v. 4:367-375 (1993)

Onco log i a . - - Activation of the R E T oncogene in human thyroid carcinomas. Nota di

DOMENICO SALVATORE, MASSIMO SANTORO, MICHELE GRmco, GL~rRANCO FENZI,

VIERI GALI.I, ALl=REDO FUSCO e GIANCARLO VECCHIO, p resen ta ta (* ) dal Socio G.

Salvatore.

ABSTRACT. - - A novel oncogene has been recently found activated in human thyroid carcinomas. This oncogene was named RET/PTC and was shown to be a chimeric gene, product of the fusion of the tyrosine- kinase domain of the RET proto-oncogene to the 5'-terminal region of another gene named H4 or D 10S 170. A paracentric inversion (10) (q11.2-21) was demonstrated to be responsible for the H4/RET fusion. Thyroid tumors consist of a broad range of lesions ranging from the benign follicular adenomas to the differentiated papillary and follicular carcinomas and to the fatal anaplastic carcinomas. Papillary and follicular carcinomas have different clinical behaviours and are associated with different risk factors, thus they are probably related to different genetic events. In fact the activation of RET is restricted to the papillary subtype. Here we show the activation of the RET oncogene in 2 out of 10 new cases of papillary thyroid carcinomas.

KEY WORDS: Thyroid carcinoma; Oncogene; Gene rearrangement.

R~SSUNTO. - - Attivazione dell'oncogene RET nei tumori della tiroide. Recentemente abbiamo individuato la attivazione di un nuovo oncogene nei tumori urnani tiroidei. Tale gene ~ stato definito RET/PTC. Esso e un gene chimerico che risulta dalla fusione del dominio tirosino-chinasi del proto-oncogene RET con la porzione 5'-terminale di un altro gene detto H4 o D10S170. L'inversione (10) (q11.2-21) e responsabile della fusione H4/RET. I tumori tiroidei comprendono un vasto spettro di neoplasie che vanno dagli adenomi, tumori benigni, ai carcinomi papilliferi e follicolari sino agli anaplastici, tumori completamente indifferenziati. I diversi fattori di rischio e il differente comportamento clinico indicano che i carcinomi papilliferi probabilmente hanno una base genetica diversa dai carcinomi foUicotari. L'attivazione dell'oncogene RET ~ limitata ai carcinomi papilliferi. In questo lavoro riportiamo due esempi di attivazione detl'oncogene RET su 10 nuovi carcinomi tiroidei papilliferi analizzati.

INTRODUCTION

The thyroid provides an attractive mo d e l for studying the steps that are involved in

the neoplastic process. T u m o r s of the follicular epi thel ium of the h u m a n thyroid gland, in

fact, represent a multi-stage mode l of epithelial tumorigenesis (Williams, 1979, 1980).

Most of thyroid neoplasms originate f rom a single cell type, the thyroid follicular cell and

they comprise a broad spec t rum of tumors with different phenotypic characteristics and

variable biological and clinical behaviour (from the benign colloid a d e n o m a s through the

slowly progressive different ia ted papillary and follicular carcinomas to the invariably fatal

anaplastic carcinomas) (Hed inge r et al., 1989). The undif ferent ia ted or anaplast ic thyroid

carc inoma is a rare tumor, occurr ing p redominan t ly in the elderly, which p robab ly arises

as a progression from different ia ted carc inoma (Williams, 1980). Papillary and follicular

thyroid carcinomas, the mos t c o m m o n forms of differentiated thyroid cancer, have

distinct biological characteristics. Papillary carc inoma is mult ifocal and general ly metasta-

(*) Nella seduta del 1S giugno 1993.

368 D. S A L V A T O R E E T AL.

sizes to regional lymph-nodes. Conversely, follicular carcinoma is solitary and encapsula- ted, it invades blood vessels and often spreads to the bones (Williams, 1980). Studies in humans have shown a causative relationship between thyroid cancer, mostly of the papillary subtype, and neck, head or upper chest irradiation in infancy and childhood. In 1950, Duffy and Fitzgerald suggested an association between thyroid carcinomas occurring in childhood and a previous history of irradiation to the thymus (Duffy and Fitzgerald, 1950). Prospective studies performed in Rochester, New York, on a thymus irradiated population and by the Japanese National Institute of Health-Atomic Bomb Casualty Commission Life Span Study on a defined sample drawn from the atomic bomb survivors, have supplied useful data concerning this risk (Sampson et al., 1969; De Groot and Paloyan, 1973; Greenspan, 1977). A relationship between radioiodine uptake and thyroid cancer was observed in a Marshall Islands population exposed to fall-out radiation (Conard et al., 1966). Moreover, the administration of radioiodine to rats produced thyroid carcinomas (Goldberg and Chaikoff, 1951, 1952). Also in iodide- deficient areas the rate of malignant thyroid transformation is higher. Some studies have also shown high number of thyroid carcinomas in endemic goiter regions and this incidence is mainly attributable to those with follicular histology (Wahner et al., 1966; Shaller and Stevenson, 1966).

There is growing evidence that tumor development and progression may result from a series of genetic alterations that affect the normal mechanisms controlling cell prolifera- tion. The mutations, so far identified, include inactivation of tumor-suppressor genes, as well as activating mutations of cellular proto-oncogenes (Bishop, 1991). The different biological characteristics of the papillary and follicular thyroid carcinomas could be due to the involvement of different genetic events in their pathogenesis.

The papillary thyroid carcinoma, the most frequent thyroid cancer in humans, is often characterized by the activation of a specific oncogene. In fact, an average of 20% of human thyroid carcinomas of the papillary subtype harbour an activated RET oncogene (Fusco et al., 1987; Bongarzone et al., 1989; Grieco et al., 1990; Santoro et al., 1992a ; Jhiang et al., 1992; Wajjwalku et al., 1992; Santoro et al., 1993). The RET proto-oncogene encodes a protein structurally related to transmembrane receptors with a cytoplasmatic tyrosine-kinase domain (Takahashi and Cooper, 1987; Takahashi et al., 1988). The RET oncogene was originally isolated by the transfection assay onto NIH/3T3 cells with a human T-cell lymphoma DNA (Takahashi et al., 1985); its activation was caused by recombination with the 5'-terminal sequence of another gene, structurally related with the zinc-finger family, named jr~ (Takahashi and Cooper, 1987). This rearrangement, like the two other subsequently reported, occurred in vitro during the transfection assay; no such rearrangements were in fact detected in the original tumor DNAs (Takahashi et al., 1985; Ishizaka et al., 1988; Koda, 1988). Conversely the activation of the RET oncogene in human thyroid carcinomas, that we have recently described, was found to have occurred in vivo, as a tumor-specific somatic event (Grieco et al., 1990). The activation of RET in thyroid carcinomas consists of the fusion of its tyrosine-kinase domain to the 5'- terminal region of another gene named H4 or D10S170. Thus a coding sequence 354 bp long, that belongs to the H4 gene, replaces the transmembrane and the extracellular

A C T I V A ' H O N O F T H E R E T O N C O G E N E IN H U M A N T H Y R O I D C A R C I N O M A S 369

domains of the RET proto-oncogene (Grieco et al., 1990). The chimeric oncogene resulting from the H4/RET fusion has been denominated RET/PTC (Grieco et al.,. 1990). The same rearrangement was described in the human papillary thyroid carcinoma cell line TPC-1 (Ishizaka et al., 1990). Recently, cases in which the activation of RET was due to fusion to genes other than H4 have also been reported (Bongarzone et al. , 1993; Santoro et al., manuscript in preparation) and named RET/PTC2 and RET/PTC3, respectively. Figure 1 schematically shows the RET rearrangements so far described; in all the cases described the activation was due to a gene rearrangement leading to the truncation of the tyrosine-kinase domain. Only in the case of thyroid carcinomas did the activation occur in vivo and was caused by fusion of RET with H4, protein kinase A RI and a novel gene named RFG in the cases of RET/PTC 1, RET/PTC2 and RET/PTC3 respectively. The RET proto-oncogene expresses five major mRNA species which represent the products of alternative splicing and polyadenylation sites (Tahira et al., 1990). Two RET protein species of 140 and 160 kDa, possibly derived from a single polypeptide core of 120 kDa differently glycosilated, were identified as membrane proteins in neuroblastoma cells (Takahashi et al., 1991). As a consequence of the rearrangement, the RET/PTC 1 product is a 520 amino acid long protein that localizes in the soluble cytoplasmic fraction (Ishizaka et al., 1992; Lanzi et al., 1992). The chimeric RET/PTC1 oncogene is generated by an intrachromosomal rearrangement. Both H4 and RET genes are normally located on the long arm of chromosome 10, (Donghi et al., 1989; Ishizaka et al., 1989; Sozzi et al., 1991) and a chromosomal inversion (10) (q11.2-q21) is responsible for their fusion (Pierotti et al., 1992). The activation of RET is restricted in v ivo to carcinomas of the papillary histotype. An analysis of 286 cases of human thyroid tumors of different histological types collected at the Mayo Clinic, Rochester, Minnesota, at the Hospitals of Lyon, France, at

Fig. 1. - Schematic representation of the described rearrangements of the RET proto-oncogene. The tyrosine~ kinase domain (tk) of RET and the sequences replacing the normal RET NH2-terminal region are shown. Rearrangements indicated as RET-I and RET-II were shown to have occurred in vitro as artefacts of the transfection technique. Conversely RET/PTC-I, -II, and -III are three different versions of RET, activated by

gene rearrangements occurred in vivo in human thyroid carcinomas.

370 o. SALVATOREET AL.

the Istituto Nazionale Tumori of Milan, and at the Medical School of Naples, University Federico ]I, demonstrated the presence of activated form of the RET oncogene in 33 (19%) of 177 papillary carcinomas. In contrast, none of the other 109 thyroid tumors, which included 37 follicular, 15 anaplastic and 18 medullary carcinomas and 34 benign lesions, showed RET activation (Santoro et al., 1992a ). Non-thyroid neoplasias have been analyzed for the activation of the RET oncogene. None of 528 non-thyroid tumors showed rearrangement of the RET proto-oncogene (Santoro et al., 1993).

More systematic studies of the activation of RET in the different stages of thyroid tumorigenesis will clarify the prognostic and diagnostic relevance of the RET activation in human thyroid carcinomas. We herein report a study conducted on 10 thyroid carcinomas and show RET activation in two of them.

MATERIALS AND METHODS

D N A extraction and Southern blot analysis. The tumor samples were frozen in liquid nitrogen and stored frozen until DNA

extractions were performed. Thyroid tumors were obtained from the Istituto di Endocrinologia of the Medical School of Naples, University Federico ]1. High molecular weight DNA extractions from tumors and Southern blot analyses were performed according to standard procedures (Sambrook et al., 1989). Briefly, 10 micrograms of DNA were digested with restriction enzymes (Amersham Corp., Promega Biotec.), electrophoresed through 0.8% agarose, transferred to Nylon filters (Hybond-N, Amers- ham Corp.) and hybridized to 32p labeled probes by the random oligonucleotide primer kit (Amersham Corp.). Hybridizations and washings were carried out under stringent conditions, as previously described (Grieco et al., 1990). Autoradiography was performed by using Kodak XAR films at - 70 ~ for 1-7 days with intensifying screens.

RNA analysis by RToPCR. Total RNA was extracted by a reported procedure (Sambrook et al., 1989). To avoid

contamination pipette tips with filter plugs (USA/Scientific plastics, FL) were used throughout the experiments. Briefly, 1 ug total RNA was denatured for 10 minutes at 68 ~ then incubated with 200 units of reverse transcriptase (BRL) of Moloney Leukaemia Virus in a total of 20 microliters reaction mixture for 30 minutes at 37 ~ in the presence of 1 mM of all four deoxynucleotide triphosphates (Pharmacia) and 100 pmoles of random hexamers (Pharmacia). The cDNA was amplified by PCR as previously described (Santoro et al., 1992a ). The primers used for PCR amplification of the cDNAs of the RET/tYrc transcripts, were as follows: Forward primer: 5'-ATTGTCATCTCGCCGTTC-3'. Reverse primer: 5'-CTGCTrCAGGACGTTGAA-3'.

The forward primer was synthesized on the basis of the nucleotide sequence which replaces the 5' RET sequence, i.e. the H4 gene whereas the reverse primer was synthesized on the basis of the RET proto-oncogene sequence. The expected size of amplified DNAs was 365 base pairs (bp) for RET/TPC. Each microliter of the cDNA

ACTIVATION OF THE RET O N C O G E N E IN HUM.AN THYROID CARCINOMAS 371

reaction mixture was incubated with Taq polyrnerase (Cetus) in the presence of 100 pmoles of both forward and reverse primers. 35 cycles of PCR were performed with a thermal cycler (Perkin-Elmer-Cetus) at 94 ~ for 30 seconds, 55 ~ for 1 minute and 72 ~ for 2 minutes. After PCR, each reaction mix was loaded onto a 2% agarose gel.

R E S U L T S A N D D I S C U S S I O N

We have demonstrated that the RET/txFC oncogene originates from the fusion of the tyrosine-kinase domain of the RET proto-oncogene to a still uncharacterized gene, named H4 or D10S170 (Grieco et al., 1990). The breakpoint of the RET gene occurs in an intronic sequence that resides between its tyrosine-kinase and transmembrane encoding domains. This rearrangement can be detected by Southern blot analysis of the tumor DNA (Grieco et al., 1990).

We have analyzed 10 thyroid neoplastic samples belonging to the papillary histotype for RET activation by probing Southern blots with a 1 Kbp BgllI-BamHI RET-specific DNA fragment. This fragment (shown in fig. 2) is able to detect the region within the RET gene where the rearrangement can occur. In fact, this probe detects a 6.3 Kbp fragment

Fig. 2. - Activation of the RET oncogene in papillary thyroid carcinomas # 1 and # 3. Southern blot analysis of DNA extracted from seven different neoplastic specimens. 10 ug of DNA were digested with EcoRI or BamHI restriction enzymes (Amersham Corp.), as indicated under the blots, transferred to Nylon filters (Hybond N, Amersham Corp.) and hybridized to a 1.0 Kbp. BamHI-BgllI RET specific DNA probe (depicted in the map showed below). Lane 1: DNA from papillary carcinoma # 1; Lane 2: DNA from normal human thyroid; Lane 3: DNA from papillary carcinoma # 3; Lar, es 4-8: DNAs from papillary carcinomas 4-8. Arrows indicate the sizes of the normal fragments: respectively 6.3 Kb with EcoRI and 3.7 Kb with BamHI. The restriction map showed below represents the region of the RET oncogene where the rearrangement can occur: the striped area

indicates the probe used.

372 D. S A L V A T O R E E T AL.

after restriction of normal human DNA with EcoRI and a 3.7 Kbp BamHI fragment (Santoro et al., 1992a ). A schematic representation of the genomic restriction map of RET and of the probe that we have used is shown in fig. 2. Through this analysis we found that 2 (samples # 1 and #3) out of 10 thyroid papillary carcinomas were positive for RET rearrangement; in fact they showed additional rearranged bands and this result was demonstrable both with EcoRI and with BamHI restriction enzymes (fig. 2, lanes 1 and 3).

We have recently demonstrated that a chromosomal inversion (10) (q11.2-q21) leads to the H4/RET fusion and that this inversion can be evidentiated by Southern blot analysis (Pierottiet al., 1992). In fact tumors bearing this chromosomal alteration show the presence of a rearranged restriction fragment also when tested with an NH2-terminal RET fragment. We have analyzed the two positive samples (# 1 and # 3) here described, also with the NH2- terminal proto-RET specific sequence and we found that they did not show any rearrangement (data not shown); this observation suggests that in these cases the mechanism of RET acfiieation could be different. Finally, in order to clarify whether in the two positive cases reported here, the activation of RET was due to its fusion to H4, samples # 1 and # 3 were analyzed by RT-PCR as ,already described (Santoro et al., 1992a). Only sample # 3 showed the amplification of a fragment of the expected size of 365 bp, confirming that in this case the activation of RET was due to an H4/RET fusion (fig. 3). In the other case (# 1 ) the activation of RET, demonstrated by Southern analysis, was due to its fusion to a gene different from H4 whose characterization will require further studies. In conclusion, we herein report two new examples of activation of the RET oncogene in human thyroid carcinomas.

The RET transforming gene has been found activated in vivo only in papillary thyroid carcinomas (Fusco et al., 1987; Grieco et al., 1990; Santoro et al., 1992; Jhiang et al.,

Fig. 3. - RT-PCR analysis of thyroid carcinomas # 1 and # 3. The primers, indicated in the Materials and methods section, were used for amplifying a fragment of the RET/PTC cDNA. After the RT-PCR 10% of the reaction products was loaded onto a 2% agarose get and stained with ethidium bromide. M: molecular weight marker; Lane 1: papillary carcinoma # 1; Lane 2: normal thyroid; Lane 3: papillary carcinoma # 3. Below the gel is depicted the map of the fusion point with the positions of the PET and H4 specific primers and the size of

the expected amplified product is also indicated.

ACTIVATION OF THE PET ONCOGENE IN HUMAN THYROID CARCINOMAS 373

1992; Wajjwalku et al., 1992), and in a papillary thyroid carcinoma cell line (Ishizaka et al.,

1990). No RET activation has been described in non-thyroid tumors (Santoro et al.,

1993) apart from some cases in which RET rearrangements occurred during the transfection procedure (Takahashi et al., 1985; Koda, 1988; Ishizaka et al., 1989).

The data reported here confirm the frequent activation of RET in thyroid carcinomas and support the hypothesis that the activation of PET is a genetic mutation characteristic of human thyroid carcinomas. In order to explain this observation it is possible to suppose that the rearrangement leading to RET activation occurs more frequently in the thyroid than in other tissues, or that the transforming potential of the activated RET is restricted to the thyroid follicular cell. In vivo studies in transgenic mice, have demonstrated that an activated RET under the transcriptional control of MMTV-LTR promoter is able to induce tumors in several tissues (Iwamoto et al., 1990) suggesting that the in vivo transforming potential of RET is not restricted to the thyroid. Clearly, the possibility of RET activation in some kinds of non-thyroid neoplasias not yet analyzed, cannot be excluded.

In consideration of the thyroid-specificity of PET activation, it is interesting to study the biological actMty of the RET/I:'TC product expressed in an established differentiated rat thyroid cell line (Santoro et al., 1992b ). These studies have in fact shown that the RET/I~C chimeric gene is able to de-differentiate the normal rat thyroid PC-C1-3 cell line.

In conclusion, we have herein shown the possibility of performing a systematic analysis of thyroid carcinomas by using the two complementary techniques of the Southern blot and the RT-PCR; we suggest that in future this kind of studies will help in elucidating the role played by the PET activation in the early stages of thyroid tumorigenesis (as in the (<occult~ thyroid carcinomas) or in thyroid tumors occurred in areas of high risk factors (such as for example the exposure to ionizing radiations). Finally, the systematic analysis of thyroid tumors for RET activation will clarify its prognostic significance with respect to thyroid tumors not carrying the R E T activation.

ACKNOWLEDGEMENTS

We are grateful to Prof. Gaetano Salvatore for his continuous and enthousiasthic support during the course of this work. We would also like to extend our thanks to Mr. Antonio Montoro for his technical assistance and to Mr. Franco D'Agnello, Fortunato Moscato and Mario Berardone for the art-work. This study was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), the Progetto Finalizzato CNR ACRO- Sottoprogetto 2, Biologia Molecotare and by funds obtained by the Regione Campania.

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