Gain-of-function mutations in the gene encoding the receptor tyrosine kinase RET have been identified as the aetiological factor for multiple endocrine neoplasia type 2A (MEN2A). MEN2A is a dominantly-inherited cancer predisposition syndrome characterized by medullary thyroid carcinoma, a tumour of the calcitonin-producing thyroid C-cells. There are three isoforms of RET: RET9, RET43 and RET51, and although in vitro evidence suggests they vary in cellular transformation activities, little is known about their function in tumorigenesis in vivo. To address this, we used RET51 cDNA to construct mice in which the most frequent MEN2A mutation, Cys-634-Arg, was expressed under the control of the human calcitonin promoter (CT-2A mice). These mice developed C-cell tumours resembling human MTC and follicular tumours resembling human papillary thyroid carcinoma (PTC) depending on the founder line examined. One founder line developed compound MTC/PTC at low frequency (8%) and pancreatic cystadenocarcinoma. CT-2A mice also displayed a developmental defect in thyroid follicular structure, in which much of the thyroid was occupied by large irregular cystic follicles thought to be derived from the ultimobranchial body, a developmental precursor of the thyroid gland. The CT-2A mice will provide a suitable model to further study the effects of the MEN 2A RET mutation in vivo.
The RET proto-oncogene encodes a receptor tyrosine kinase which is expressed in a variety of tissues of neural crest origin during development, including the peripheral and central nervous system and the kidney (Pachnis et al., 1993). RET, in conjunction with the GFR-alpha co-receptors, binds the GDNF family of neurotrophic factors (reviewed by Airaksinen et al., 1999; Baloh et al., 2000), resulting in dimerization and auto-phosphorylation of RET, and the initiation of signal transduction cascades (Sanchez et al., 1996; Durbec et al., 1996; Trupp et al., 1996). Targetted disruption of the ret gene in mice results in the developmental failure of the superior cervical ganglia, renal agenesis and intestinal agangliosis (Schuchardt et al., 1994), the latter bearing a strong resemblance to the human condition Hirschprung's disease, which can be caused by inactivating mutations in the RET gene (Edery et al., 1994; Romero et al., 1994).
Gain-of-function mutations in RET have been identified as the aetiological factor for the dominantly-inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2) (Mulligan et al., 1993; Hofstra et al., 1994). Three clinical subtypes of MEN2 have been defined: MEN2A, MEN2B and familial medullary thyroid carcinoma (FMTC) (Farndon et al., 1986; Schimke, 1984). Medullary thyroid carcinoma (MTC), a common feature of all subtypes of MEN2, is a tumour of the thyroid parafollicular C-cells, which are thought to be at least in part derived from the embryonic neural crest (Saad et al., 1984). In addition, MEN2A patients may also develop pheochromocytoma, a tumour of the adrenal chromaffin cells, and parathyroid hyperplasia, whilst MEN2B is characterized by mucosal neuromas and developmental abnormalities (reviewed by Ponder and Smith, 1996). Tumorigenic rearrangements of RET have been identified in papillary thyroid carcinoma (PTC), a tumour of the thyroid follicular epithelial cells, which is histologically distinct from MTC observed in MEN2 (Grieco et al., 1990; Bongarzone et al., 1993; Santoro et al., 1994). Chromosomal translocations juxtapose the carboxy-terminal kinase domain of RET to a transcriptionally active gene which provides an N-terminal dimerisation domain, resulting in a fusion protein with inappropriately regulated kinase activity (reviewed by Santoro et al., 1999).
The vast majority of mutations causing MEN2A result in the substitution of one of the six conserved cysteine residues of the RET extracellular domain. This may prevent the formation of an intramolecular Cys–Cys disulphide bond, leaving a free cysteine residue which forms an intermolecular disulphide bond with an adjacent RET molecule, leading to ligand-independent dimerization and inappropriate activation of RET (Santoro et al., 1995; Asai et al., 1995). The most common of these mutations results in the substitution of arginine for cysteine at codon 634 (Cys-634-Arg) (Eng et al., 1996). Most cases of MEN2B result from a single amino acid substitution, threonine to methionine, at codon 918 in the catalytic domain of RET (Eng et al., 1994; Rossel et al., 1995). This mutation is thought to cause the partial ligand-independent activation of the kinase and to change the substrate preference of RET from that of receptor-type to Src-related, non-receptor type kinases (Songyang et al., 1995).
Alternative splicing generates three isoforms of the RET protein with distinct Carboxy-termini termed RET9, RET43 and RET51 according to the number of amino acids after the C-terminal splice site (Myers et al., 1995). Although biochemical comparisons between the RET9 and RET51 isoforms suggest differences in intracellular substrate binding capabilities (Lorenzo et al., 1997), and cellular transformation and differentiation activities (Rossel et al., 1997; Marshall et al., 1997), the functional significance of these in vitro observations to development and tumorigenesis in vivo remains to be determined. Transgenic mice expressing activated RET kinase in the thyroid have been generated to study RET function in vivo. Most studies have concentrated on the generation of mouse models of PTC by expressing rearranged RET in the thyroid follicular cells (Santoro et al., 1996; Jhiang et al., 1996; Portella et al., 1996; Powell et al., 1998). Transgenic models of MEN2A and MEN2B have also been made. Michiels et al. (1997) generated a mouse expressing the RET9 isoform with the MEN2A mutation Cys-634-Arg under the control of the C-cell specific rat calcitonin promoter. These mice developed C-cell tumours biologically and morphologically similar to human MTC, demonstrating that the shorter isoform of RET is capable of tumorigenesis in vivo. Acton et al. (2000) generated mice expressing both the wild-type and the MEN2B RET9 isoform under the control of the human calcitonin promoter. Expression of MEN2B RET9, but not wild-type RET9 resulted in the formation of MTC. The consequences of RET51 expression in vivo in transgenic mice has not been investigated. To address this issue, we have targetted expression of both wild-type (RET-WT) and Cys-634-Arg (RET-2A) human RET51 to the mouse thyroid using the C-cell specific human calcitonin promoter (Peleg et al., 1989). We report here that whilst thyroids of the RET-WT mice appeared normal, mice expressing RET-2A develop not only MTC, but also follicular tumours similar to mouse and human PTC. In addition, a developmental defect in thyroid follicular structure was observed.
Generation of transgenic lines and expression pattern of transgene
In all, five founder lines of RET mice were generated, three carrying the Cys-634-Arg mutation, and two with the wild-type RET cDNA. All lines transmitted the transgene in a Mendelian fashion, and it is therefore assumed that the transgene integrated into the mouse genome at one site only. Transgene copy number, as estimated by comparative Southern blot, was low: 1, 3 and 10 for the RET-2A lines and 3 and 10 for the RET-WT lines. These results are summarized in Figure 1b. Expression of the transgene was determined by RT–PCR, using a forward primer anchored in human RET cDNA and a reverse primer anchored in SV40pA, which should therefore be transgene-specific. The reaction was normalized using RT–PCR for the housekeeping HPRT gene. The results show strong transgene expression in the thyroid and pancreas, and weak expression in the lung. All other tissues examined were negative, including the adrenal gland and kidney (Figure 1c). It is important to note that for technical reasons the founder designated 2A-1 was made in the FVB strain, whereas all other founders were on the B6/CBA (F1) mixed strain.
Thyroid tumours in the RET transgenic mice
RET-WT mice showed no phenotype, even up to 2 years of age. RET-2A mice developed thyroid tumours, and the tumour profile varied markedly between transgenic founder lines (Table 1). Of the three transgenic lines generated, only one (2A-3) displayed the expected tumour phenotype, with mice developing MTC at a frequency of ca. 35% at 3–4 months old, and 70% at 12 months or older. The tumours were strongly positive by immunohistochemistry for calcitonin (Figure 2a) and RET (Figure 2b), but largely negative for the follicular cell markers thyroglobulin and TTF-1, and they were similar in morphology to human MTC and to the tumours observed in mice transgenic for the RET9-2A isoform (Michiels et al., 1997). This demonstrates that the RET51 isoform is capable of driving tumour formation in vivo. There was no evidence of invasion or metastasis, nor did the tumours have any effect on mortality, the mice living to 2 years and beyond. There were no gross or histological abnormalities in the adrenal glands or the kidneys, which is not surprising given the lack of transgene expression in these tissues; nor was there any pancreatic phenotype.
The predominant tumour phenotype of the 2A-1 line was morphologically similar to human PTC and the tumours in mice bearing RET/PTC rearrangements (e.g. Powell et al., 1998), in that the tumours consisted mainly of cuboidal thyroid follicular epithelium thrown into folds (Figure 2c,d), as described by Harach and Williams (1994). The tumours were positive for RET (Figure 2c,d) and for the transcription factor TTF-1 (Figure 2f), a specific marker of thyroid follicular cells, but surprisingly were negative for thyroglobulin, another follicular cell marker. MTC, defined by RET and calcitonin immunoreactivity, did occur in the 2A-1 line, albeit at low frequency (about 8%), and always in association with PTC (Figure 2e). Although the tumours in the 2A-1 and 2A-3 mice sometimes penetrated the thyroid capsule, there was no evidence of metastasis. As with 2A-3 mice, 2A-1 mice showed no gross or histological abnormalities in the adrenal glands or the kidneys, but some mice (22%) developed exocrine pancreatic tumours similar in appearance to cystadenoma or cystadenocarcinoma (Figure 2g). These tumours were always co-existent with the thyroid tumours and were presumably due to the high expression level of the transgene in the pancreas. In half the mice that developed pancreatic tumours, there was clear evidence of metastatic invasion of the spleen. Mice were sacrificed as soon as these tumours became apparent.
Mice from the 2A-2 line had no apparent thyroid or other phenotype.
Atypical thyroid development in RET-2A mice
In contrast to both non-transgenic and RET–WT mice, which displayed a normal uniform thyroid follicular phenotype, RET-2A-1 and RET2A-3 mice showed evidence of altered follicular development. Though normal follicles were present, much of the thyroid was occupied by cystic follicles, distinguishable by their large size, irregular shape and unusual colloid (Figure 3). The colloid of these follicles stains positively for Alcian blue, suggesting the presence of acid mucins (Figure 3d). Thyroglobulin immunostaining of these follicles displayed a denser, more granular appearance compared to non-transgenic or RET–WT mice (Figure 3e). Also present were numerous ciliated follicular epithelial cells that are absent in the wild-type thyroid (Figure 3f). These follicles bear a strong resemblance to the ‘second-type’ of follicle described by Wetzel and Wollman (1969) in the C3H mouse. These follicles occupy 5–10% of the C3H thyroid lobe, compared to 40–90% in the RET-2A lines. The C3H follicular type is believed to be derived from the ultimobranchial body, a developmental precursor of the thyroid gland (Wollman and Hilfer, 1978).
The RET9 and RET51 splice isoforms activated by MEN 2 mutations both act as transforming genes in transfected fibroblasts, but may differ in their biological properties. For example, RET51 activated by a 2B mutation is seven times more effective than RET9 at inducing neurite outgrowth in PC-12 cells (Rossel et al., 1997). In vitro biochemical differences between RET9 and RET51 have also been described (Segouffin-Cariou and Billaud, 2000, and references therein). For example the adaptor protein Shc binds to the pTyr1062 autophosphorylation site in both RET9 and RET51. Both the PTB and SH2 domains of Shc bind pTyr1062 in RET9, whereas pTyr1062 is a stronger docking site for the PTB domain in RET51 (Lorenzo et al., 1997). In addition the autophosphosphorylation site pTyr1096 is only present in RET51 and is a docking site for the adaptor protein Grb2. It was therefore of interest to examine the in vivo effects of RET51 with the MEN 2A Cys-634-Arg mutation when expressed in the mouse thyroid in comparison to the in vivo effects of RET9 (Michiels et al., 1997).
Our results show that the RET51 isoform, activated by the MEN 2A mutation, is capable of causing MTC in vivo. The MEN 2A-3 line of mice develop C-cell hyperplasia by 3 months in 35% of cases and bilateral MTC by 6–12 months in 52% of cases rising to 68% in mice over 1 year of age. The tumours were strongly positive for calcitonin and RET by immunohistochemistry, but generally negative for the follicular cell marker thyroglobulin. This phenotype is similar to that described by Michiels et al. (1997) in MEN 2A RET9 mice, although the penetrance of tumour formation in our mice is lower. This could imply lower activity of the RET51 isoform, although any comparison in penetrance between our mice and the RET9 mice of Michiels et al. (1997) must be made with caution as there were differences both in the promoter used (rat compared to human), and in genetic background (DBA in the case of the RET9 mice). In our study we also generated wild-type RET51 mice. These mice were indistinguishable from non-transgenics, demonstrating that the MEN 2A mutation and not RET overexpression is responsible for the tumorigenic and developmental phenotypes observed.
The RET9 mice of Michiels et al. (1997) developed uncomplicated MTC. In addition to MTC formation, our mice of both 2A-1 and 2A-3 lines had a developmental abnormality of the thyroid gland and in line 2A-1 the predominant carcinoma was PTC, with MTC occurring only rarely (approximately 8% of cases) and only in association with PTC. This result was surprising as in humans PTC is only rarely associated with MEN2A (Decker, 1993; Oishi et al., 1995), and although compound follicular-parafollicular cell carcinoma has been described in humans (Ljungberg et al., 1983), there is no association with MEN 2A. Recently, wild-type RET transcripts and functional RET protein were detected in both PTC and follicular tumour cell lines, suggesting that RET may be active and contribute to the transformation of both follicular and parafollicular cells of the thyroid (Bunone et al., 2000). Our observation, that Cys-634-Arg RET51, in addition to RET rearrangement, is capable of driving PTC formation in vivo, support this hypothesis. It is surprising that the PTC tumours were negative by immunohistochemistry for thyroglobulin, a marker of follicular thyroid epithelial cells, but positive for TTF-1, a transcription factor known to regulate thyroglobulin expression in the thyroid (Fabbro et al., 1994). The lack of thyroglobulin staining suggests that the tumours may consist of poorly differentiated follicular cells. The penetrance of the PTC phenotype was about 50% at ages 3–24 months, which is comparable to the RET/PTC mice generated by Powell et al. (1998). Twenty-two per cent of our 2A-1 mice also developed exocrine pancreatic tumours. Human pancreatic cancer cell lines are known to express RET (Okada et al., 1999); but MEN 2A is not associated with pancreatic tumours in man.
Different mouse strains were used to generate the 2A-1 and 2A-3 founder lines, however the marked difference in tumour profile between the lines (2A-1 mice giving rise to PTC and rarely MTC and pancreatic tumours, and 2A-3 mice giving rise to MTC only) are unlikely to be due to differences in genetic background. When the 2A-1 and 2A-3 transgenics were introduced into the opposite genetic background in the F1 crosses (e.g. 2A-1 from FVB to FVB/CBA/B6 F1) (Table 1) the tumour profile remained consistent. Consequently, expression level or pattern differences of the transgene due to copy number and/or integration site differences between the two lines may have modified the effect of the transgene.
A consistent feature of the 2A-1 and 2A-3 transgenic lines that was not seen in the RET9 transgenics was atypical thyroid architecture, characterized by large colloid-filled follicles and the presence of ciliated and squamous epithelial cells, usually absent in the normal thyroid, together with the presence of acid mucins as demonstrated by Alcian blue staining (Figure 3d). This is of interest because it relates to a continuing debate over the possible existence of an ultimobranchial stem cell of endodermal origin, which gives rise to a subset of both thyroid follicular cells and C-cells (Caillou, 1991; Kovacs et al., 1994). The atypical follicles closely resemble the ‘second type’ of follicle which constitutes 5–10% of the thyroid of C3H mice but is absent in all other strains (Wetzel and Wollman, 1969). These follicles are thought to be derived from the ultimobranchial bodies, embryological structures derived from the pharyngeal pouch endoderm and the neural crest endoderm (Wollman and Hilfer, 1978). In mammals, the ultimobranchial bodies fuse with the thyroid diverticulum to form the thyroid gland (Manley and Capecchi, 1995). The thyroid diverticulum gives rise to a majority of the follicular cells (85%), whilst the ultimobranchial bodies give rise to a minor component (15%). At least some of the C-cells are derived from the neural crest component of the ultimobranchial body (Le Douarin and Le Lievre, 1970). Fusion of the ultimobranchial body and the thyroid diverticulum fails to occur in double-mutant mice lacking both Hoxa and Hoxd, leading to the development of ectopic ultimobranchial bodies (Manley and Capecchi, 1998). Colloid-containing follicles and calcitonin-positive cells are present in these bodies supporting the view that both thyroid follicular epithelial cells and parafollicular C-cells may be derived from the ultimobranchial body. The presence of atypical colloid-containing follicles in the mice described here, both in the presence and absence of PTC, might possibly be a consequence of inappropriate expression of the transgene in a stem cell of ultimobranchial origin. The mice generated by Johnston et al. (1998) in which expression of the viral oncogene v-Ha-ras was driven by the human calcitonin promoter also provide evidence for an ultimobranchial stem cell giving rise to both thyroid follicular and parafollicular C-cells. Although they found a predominant phenotype of MTC, in mice derived from one founder there was also thyroglobulin positivity within the MTC and some evidence of papillary tumour outgrowths suggesting the possibility of partial thyroid epithelial differentiation. Both the atypical follicles and the co-existence of MTC and PTC in the mice we describe might be explained by transformation of such an ultimobranchial stem cell. Transformation occurring directly by activated RET expression in the ultimobranchial component of the pharyngeal endoderm, or possibly through a paracrine effect on the future follicular cells from RET expressing ultimobranchial C-cells, might explain both the atypical follicles and the co-existence of MTC and PTC in the mice we describe.
In conclusion, one of the transgenic lines described here, 2A-3, developed C-cell tumours typical of human MTC as described in the inherited cancer syndrome MEN2A. This line will provide a model to further study aspects of MEN 2A in vivo, for example the sequence of genetic events in tumour progression, and by crossing to other genetically modified mice the importance of RET-modulated signalling pathways identified in vitro to tumour progression in vivo. In addition, the formation of atypical thyroid glands in MEN 2A RET51 has added to the growing body of evidence for the dual neural crest and endodermal origin of thyroid C-cells.
Materials and methods
Construction of the transgene
The 1457 bp human calcitonin promoter was amplified by PCR from pCTGH-1 (a gift from Sara Peleg (Peleg et al., 1989)) and the SV40 early polyadenylation signal (SV40pA) were amplified by PCR from the plasmid pSV-β galactosidase (Promega). The resulting fragments were sub-cloned into pBluescript (Stratagene). Wild-type and Cys-634-Arg human RET51 cDNAs were cloned between the promoter and the SV40pA (see Figure 1a). Cloning of these cDNAs has been described previously (Borrello et al., 1995; Lorenzo et al., 1997). The transgene was linearized with ScaI, and the 8.6 Kb fragment isolated was used for pronuclear injection.
Generation and screening of transgenic mice
The linearized DNA was injected into the pronuclei of fertilized oocytes from either FVB or B6/CBA mice. After overnight incubation (37°C/5% CO2) embryos surviving to two-cell stage were transferred into the oviducts of pseudopregnant (MF-1) females. Tail biopsies were taken from pups at weaning (3–4 weeks), DNA extracted and analysed by PCR using primers R1 (5′-GCGATGTTGTGGAGACCCAAG-3′) and R2 (5′-AGCACCGAGACGATGAAGGAG-3′) under the following conditions; denaturation at 95°C for 5 min, followed by 30 cycles of 94°C for 1 min, 57°C for 1 min and 72°C for 1 min, and a final extension step of 72°C for 10 min. This amplifies a 202 bp product from RET cDNA. Genotyping was confirmed by Southern blotting, using a radiolabelled 418 bp EcoRI–BglII fragment of RET51, which hybridizes to a 1286 bp EcoRI fragment of transgenic genomic DNA. An estimate of transgene copy number was made for each founder line based on a comparison of Southern blot signal strength with a range of plasmid DNA concentrations. Transgenic mice and their non-transgenic littermates were maintained for periods of up to 2 years under specific pathogen free conditions, and were routinely screened for the transgene by either Southern blot or PCR.
Expression pattern of the transgene by RT–PCR
Adult mice were sacrificed, tissues dissected and snap frozen in liquid nitrogen. Total RNA was extracted using the RNeasy miniprep kit (Qiagen) following the manufacturers instructions. RNAs were reverse transcribed using the Moloney murine leukaemia virus reverse transcriptase (Pharmacia) and the resulting cDNAs were analysed by PCR. The forward primer, F1 (5′-TATGACGACGGCCTCTC-3′) annealed to 3234 bp of human RET51, the reverse primer F2 (5′-AACTGGAAGAATCGCGG-3′) annealed to 405 bp of SV40pA. The PCR conditions were 40 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 1 min, followed by an extension step of 72°C for 10 min, yielding a product of 844 bp. A mock cDNA synthesis reaction (reverse transcriptase omitted) was included to ensure that the product generated did arise from RET cDNA and not from contaminating genomic DNA. As a control of cDNA quality, RT–PCR of the ubiquitously-expressed HPRT gene was performed using the primers H1 (5′-CCTGCTGGATTACATTAAAG-3′) and H2 (5′-GTCAAGGGCATATCCAAC-3′); the predicted product size is 352 bp.
Histology and immunohistochemistry
Thyroid glands removed from mice sacrificed at various ages were fixed in neutral buffered formalin (10% formaldehyde in phosphate buffer pH 7.5), processed through ethanol and xylene and embedded in paraffin. Serial sections (5 μM) were cut through the entire thyroid, mounted on poly-L-lysine microscope slides and processed for either histology by haemotoxylin/eosin staining (H&E) or for immunohistochemical analysis. Rabbit polyclonal antibodies to calcitonin and thyroglobulin were obtained from DAKO, and were used at dilutions of 1 : 3000 and 1 : 5000 respectively. Rabbit-anti RET antibody (Lorenzo et al., 1997) was used at 1 : 750 and rabbit-anti TTF-1, a gift from M Santoro (Lazzaro et al., 1991) was used at 1 : 500. For successful immunostaining of RET and TTF-1, antigen retrieval by heating slides in citrate buffer (Shi et al., 1991) was necessary. Immunostaining was visualized by using biotin-conjugated swine anti-rabbit antibody (1 : 200), followed by incubation with horseradish peroxidase (HRP) conjugated streptavidin (both DAKO). The HRP was developed using either diamino-benzidine (DAB), or cobalt-enhanced DAB (both Sigma).
Note added in proof
Since submission of this manuscript, Orlandi et al. (2001) have described a case of a patient with mixed medullary-follicular thyroid carcinoma. A somatic point mutation (Met-918-Thr) of the RET gene was detected, and the authors suggested that constitutive activation of RET may be involved in the development of the compound carcinoma (see References).
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We wish to thank Sara Peleg for kindly supplying the human calcitonin promoter DNA and Massimo Santoro for the anti-TTF-1 antibody. This work was supported by The Cancer Research Campaign (CRC).
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Reynolds, L., Jones, K., Winton, D. et al. C-cell and thyroid epithelial tumours and altered follicular development in transgenic mice expressing the long isoform of MEN 2A RET. Oncogene 20, 3986–3994 (2001). https://doi.org/10.1038/sj.onc.1204434
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