An Overview of Molecular Abnormalities. A 1993 Perspective. Leading to Thyroid Carcinogenesis: ROBERT F. GAGEL ABSTRACT

Transcription

1 An Overview of Molecular Abnormalities Leading to Thyroid Carcinogenesis: A 1993 Perspective ROBERT F. GAGEL University of Texas, M.D. Anderson Cancer Center, Houston, Texas, USA Key Words. Medullary thyroid carcinoma Papillaty follicular thyroid carcinoma. Oncogene. Mutation ABSTRACT This discussion reviews molecular abnormalities in thyroid carcinoma and points out areas where these molecular defects might be applicable to thyroid carcinogenesis induced by radiation exposure. Both medullary and papillary follicular thyroid carcinoma are discussed. The multiple endocrine neoplasia type 2 gene on chromosome 10 is one of the genes responsible for medullary thyroid carcinoma. Genes thought to be involved in papillary and follicular thyroid carcinoma include the gsp, ret, trk, ras, met, and p53 oncogenes. Research is continuing to: A) find new genes whose regulation and/or expression may be responsible for these disorders; B) determine the mechanisms by which gene mutations can lead to thyroid carcinogenesis, and C) devise methods to prevent or counter the effects of these mutational events. Stern Cells 1997;15(suppl2):7-13 INTRODUCTION Our understanding of molecular abnormalities in both medullary and differentiated thyroid carcinoma is limited, as the field began to develop in This review of thyroid carcinogenesis will focus on reports implicating the role of gene rearrangements in the proto-oncogenes in the development of benign and malignant thyroid tumors. Our attention has been focused on the potential effects of the nuclear accident at Chernobyl, and most recently by reports that in regions within Belarus surrounding or downstream from the Chernobyl site, there has been a marked increase in the incidence of thyroid carcinoma [ 1, 21. While the report of Kuzakov et al. [I] is not well controlled and while it is unclear at this time whether the actual incidence (which was high in 1991) is a true incidence of thyroid cancer or whether it may reflect ascertainment bias; it points out clearly that an increased incidence of differentiated thyroid carcinoma (predominately papillary thyroid carcinoma) is evident in children who were in the region of Chernobyl. An interesting and inexplicable observation is the finding of a very high incidence of thyroid tumors in the Gomel region of the Republic of Belarus. Thyroid carcinoma can be subcategorized into two major types. The first type results from a transformation process of the calcitonin-producing cell (C-cell), [ I] or the perifollicular cell, which causes medullary thyroid carcinoma. The second type is derived from the follicular epithelium and encompasses predominately the follicular, papillary and anaplastic carcinomas, and a variant of the latter known as Radiation Injury and the Chernobyl Catastrophe. STEM CELLS 1997;15(suppl2): AlphaMed Press. All rights reserved.

2 8 Molecular Abnormalities in Thyroid Carcinoma Hurthle cell carcinoma. The close histologic relationship between the C-cell and the follicular cell has been a point of interest to both investigators interested in differentiated or papillary follicular thyroid carcinoma, as well as to those individuals who are interested in neoplasms of the C-cell. MEDULLARY THYROID CARCINOMA Much progress has been made towards mapping the multiple endocrine neoplasia type 2 (MEN2), or familial medullary thyroid carcinoma gene. In 1987, two groups reported linkage to a centromeric chromosome 10 locus [3, 41. There has been a considerable amount of work since that time in mapping the genetic locus with the acquisition of a large number of polymorphic DNA sequences within the region. Efforts now continue to develop yeast artificial chromosome libraries that contain DNA from this region. To date, the predisposing gene has not been cloned, although additional mutations have been identified. As the molecular basis is determined for both medullary thyroid carcinoma and differentiated carcinoma, a pattern is emerging suggesting that it is not a single molecular defect that leads to transformation of either cell type. There is now strong evidence that mutations in several tumor suppressor genes located on chromosomes lp and 22q are involved in the genesis of medullary thyroid carcinoma. PAPILLARY FOLLICULAR THYROID CARCINOMA At present there are six candidate genes (outlined below), for which a reasonable amount of evidence has accumulated to indicate involvement in the molecular defects of differentiated thyroid carcinoma [5]. Figure 1 shows a schematic diagram demonstrating the follicular cell adjacent to a follicle. The thyroid follicular cell normally synthesizes thyroglobulin and extrudes this protein into the follicle where it is stored; thyroglobulin is then taken back into the thyroid follicular cell and degraded when thyroid hormone is needed. Regulation of the thyroid follicular cell is, to a large extent, dependent upon a pituitary hormone called thyroid-stimulating hormone (TSH). The receptor for TSH, which has been cloned, is a transmembrane protein that is linked to at least two different transduction systems. The first pathway, considered to be the most important, is linked through the stimulatory subunit of a G protein that facilitates the production of cyclic AMP and the activation of protein kinase A, and sets in motion a series of phosphorylation events. There is a second pathway that in recent years has taken on greater importance, and that is the linkage of the same TSH receptor to a phospholipase C, which leads to the activation of protein kinase C and the production of inositol triphosphates. THYROID FOLLICULAR CELL Figure 1. Diagram of a thyroid follicular cell showing the several pathways implicated in the development of thyroid follicular neoplasms. The primary molecular defect in all examples except the p53 tumor suppressor gene is a membrane associated receptor or linker protein. The abbreviations,for these oncogenes ure discussed in the text.

3 Gagel 9 gsp A hot thyroid nodule is diagnosed by thyroidologists when the radioactive iodine scan of the thyroid gland shows an area of increased uptake in one region with relative suppression of uptake in the surrounding area and in the contralateral lobe. Based on studies performed by Bourne et al. showing abnormalities of the GS subunit in pituitary tumors, the possibility arises that abnormalities in the G-protein might be involved in the development of toxic adenomas, or hot nodules. In fact, such abnormalities have been found in a significant percentage of tumors in which hot nodules have been documented [6]. Table 1, adapted from a review by Wynford-Thomas [5], shows the characteristics of gsp (Ga subunit) mutations in thyroid tumors. Mutations in the alpha subunit of G protein have been found in both thyroid adenomas and carcinomas. Specific mutations have been identified in regions of the genes that are involved in activation of adenylate cyclase, and the mechanism of these mutations is believed to be an impairment of the GTPase activity of the protein. These mutations have been associated with increased adenylate cyclase activity in most tumors, again indicating that they are functional in a large percentage of tumors in which they have been found. ret and trk The first identification of molecular abnormalities in thyroid carcinoma came as a result of work performed by two groups of investigators [7-lo]. Takahashi and colleagues frst identified the c-ret proto-oncogene [9, lo]. An Italian collaborative group frst Table 1. Features of gsp (Ga subunit) mutations in thyroid tumorsa A Mutations are present in adenomas and carcinomas. A Mutations in codon? 201 or 227 result in continuous activation of adenylate cyclaye by iinpairing GTPase activity of the protein A Mutations are associated with increased adenylate cyclase activity in tumors *>Adapted from [5] demonstrated that there was activation of ret and trk oncogenes in papillary thyroid carcinoma [7,8]. These discoveries were first made by the use of a relatively simple but elegant assay for detecting transformation, depicted in Figure 2. High molecular weight DNA was extracted from either thyroid tumors or from normal blood cells from patients. DNA was then transfected into NIH 3T3 cells, a fibroblast-like cell line, using the calcium phosphate precipitation technique. After several weeks, subclones of rapidly proliferating transfectants were removed and then injected into nude mice that subsequently developed tumors. DNA derived from these tumors was used to develop a thyroid tumor library from which the c-ret and trk oncogenes were identified. (c. High molecular Transfection 1 0 weightdna prepared from tumor and normal blood cells \ \ n Figure 2. Transformation assay used to identify the c-ret and trk rearrangements found in papillary thyroid carcinoma. [ Identification of cloned DNA Analysis of tumor 4- DNA by Southern blotting

4 10 Molecular Abnormalities in Thyroid Carcinoma ret rearrangements, such as the chromosome 10 inversion shown in Figure 3, are found in 10%-50% of cases of papillary thyroid carcinoma. The low percentage of tumors which carries this rearrangement suggests that it evolves only in the later stages of transformation, or that it plays a role in only a small percentage of tumors. In fact, recent results suggest that ret is present in a high percentage of metastatic thyroid carcinomas. Thus, ret rearrangements may be more important in the evolution of carcinogenic states than in initiating cancer. The ret oncogene has also been linked to the predisposing locus for medullary thyroid carcinoma. What relationship there might be between papillary thyroid carcinoma and medullary thyroid carcinoma, and this abnormality, remains unknown. ras A second pathway within the thyroid follicular cell may mediate carcinogenesis via a cell surface growth factor receptor. It is well known that insulin growth factor 1, and perhaps also epidermal growth factor, are required for TSH action and normal growth and development. As indicated in Table 2, the rus 21 kda protein is found at the inner surface of the cell membrane and appears to perform a function similar to that of receptors for these growth factors (Le., degradation of GTP). This function is poorly understood but it is clear that mutations of either Harvey-, Kerstin- or N-rus are involved in the initiation of cell transformation [5]. Mutations have been found in all three rus genes in papillary and, to a lesser extent, follicular thyroid carcinoma (Table 3). Overall, the incidence of mutations of all three rus genes approximates 50% of thyroid carcinoma [5]. met Another tyrosine kinase, the H-met oncogene, is the receptor for a factor known as a hepatocyte growth factor. There is no evidence for rearrangement of the H-met gene, although it is overexpressed in papillary thyroid carcinoma. There has also I Papillary thyroid carcinoma oncogene s.'+x Chromosome 10 H4 PROMCYIER SEQUENCES INV(lO)(qll.zqZl) ret KlNASE SEQUENCES Figure 3. The most common rearrangement of the c-ret proto-oncogene involved in papillary thyroid carcinoma (PTC) is a chromosome 10 qll to 421 inversion in which the H4promoter sequence, ubiquitously expressed in the thyroid follicular cell, drives the expression of a truncated form of the c-ret proto-oncogene which contains the intracellular tyrosine kinase domain. I the inner surface of the cell membrane, and function to degrade A ras proteins alternate between GDP- and GTP-bound forms. A Mutation of codons 12/13 and 61 impair GTPase activity. A Effector sites are unknown. "Adapted from [5]. tions A Mutations occur in all three rus genes. A Mutations are found in about 50% of tumors, with the incidence of mutation lower in papillary than in follicular carcinoma. A The predominant mutation is substitution of glutamine for arginine at codon 61. A Mutations of K-ras may be more common in radiation-induced thyroid carcinoma. aadapted from [5].

5 Gagel 11 been an independent observation that parafollicular cells, or C-cells, may produce hepatocyte growth factors. This may provide one example in which there is interaction between two cell types within the thyroid gland milieu. P53 The p53 anti-oncogene, a tumor suppressor type of gene, has been implicated in the development of colon carcinoma and breast carcinoma. A mutation or loss of p53 has also been implicated in the transformation from a differentiated to an anaplastic thyroid carcinoma [I 11. The preceding sections describe the presently known molecular defects in papillary and follicular thyroid carcinoma. As one looks across the field of mutational events, it is evident that the incidence of mutations for any of these genes rarely exceeds 50%. Therefore, it is likely that other candidate genes remain undiscovered. STUDY OF THYROID CARCINOMA Table 4 outlines strategies for the identification of new molecular defects and attempts to integrate these into the subject of today s symposium: the increased incidence of thyroid carcinoma associated with radiation exposure. First, it will be important to study newly identified oncogenes. In fact, much of the progress that has already been made in this area has been merely a direct application of other Table 4. Strategies for identifieation work on other cancers. A second approach that we are planning to of new molecular defects pursue in our own cohort of patients with thyroid carcinoma is to A Study newly identified oncogenes attempt to identify the predisposing or causative genes in familial A Perform linkage analysis in farnilies with hereditary thyroid canpapillary thyroid carcinoma by using a linkage analysis and a can- cer didate gene approach. In addition, transgenic or knockout models A Make transgenic or gene knockout should be developed to assess the importance of a particular gene animal models. in carcinogenesis. Finally, groups of patients who have exposure to A Study populations known to be at environmental risks would also be interesting because one can greater risk for development of study those groups of patients from the beginning of exposure and thyroid carcinoma, wch as individuals exposed to irradiation. or try to understand the cascade of molecular events that evolves into living in iodine-deficient regions thyroid carcinoma. STRATEGIES FOR MOLECULAR ADDENDUM The period between 1993 and the publication of this review was a period of intense activity in the field of thyroid carcinogenesis. Studies in mid-1993 led to the identification of mutations of the c-ret rearrangement plays in the development of papillary thyroid carcinoma. The c-ret proto-oncogene is a member of the tyrosine kinase receptor family (Fig. 4) which is normally expressed in the thyroidal C cell, but not in the thyroid follicular cell [14]. Two classes of c-ret mutations have been identified in MEN 2 and FMTC. Mutation of a cysteine to another amino acid in the extracellular cysteine-rich domain results in dimerization, autophosphorylation, and activation of the receptor [ 15-17]. A second class of mutations affects the intracellular tyrosine kinase domain and causes receptor activation without dimerization [ 15-17]. More recent studies have identified a second component of the Ret tyrosine kinase complex, a receptor for glial cell-derived neurotrophic factor (GDNF) [ 181. Current information suggests the GDNF binds to the GDNF receptor, a membrane-associated receptor, which complexes with the Ret tyrosine kinase receptor [19,20]. The GDNF signal is transmitted to the intracellular compartment by the Ret tyrosine kinase receptor [19, 201. Whether mutations of GDNF or its receptor will be identified in MEN 2 or FMTC is unclear at this time, although approximately 5%-7% of patients with MEN 2 or FMTC have no identifiable c-ret proto-oncogene mutations, making this a possibility.

6 ~~ ~ 12 Molecular Abnormalities in Thyroid Carcinoma extracellular domains trk receptor kinase tyrosine kinase domain I extracellular domains trk fusion kinase tyrosine kinase domain CO-OH ICO-OH A 7 glycosylation site... disulfide bond B 7 exons of tropomycin, L7a ribosomal Drotein or TPR Figures 4A and 4B. Schematic representation of the normal trk receptor (4A) and the efsect of the chromosomal rearrangement which leads to activation of trk tyrosine kinase activity as discussed in [5]. The Ret tyrosine kinase is not normally expressed in the thyroid follicular cell. Three rearrangements of c-ret, designated PTCl, PTC2, and PTC3 [21], have been identified and result in expression of Ret tyre sine kinase activity within the thyroid follicular cell. Targeted expression of PTCl in a transgenic mouse results in histologic features of papillary thyroid carcinoma (PTC) [22]. Mice with a high copy number of the transgene also develop hypothyroidism, suggesting a relationship between Ret expression and regulation iodide transport or thyroid hormone syntheses [22]. Further exploration of this model system may result in an understanding of the defects in iodide transport seen in papillary thyroid carcinoma. Finally, evidence has accumulated that the initial increase in pediatric papillary thyroid carcinoma observed in children exposed to radiation from the Chernobyl disaster is real. Furthermore, the PTC rearrangement (Fig. 3) has been identified in approximately 60% of these children, suggesting that radiation-induced recombination is a potent stimulus for generation of this rearrangement. These findings also provide additional evidence for a causative role for this rearrangement in the genesis of PTC. The pace of discovery in this field has been rapid, suggesting the molecular defects in thyroid carcinoma may be elucidated over the next decade. REFERENCES 1 Kazakov VS, Demidchik EP, Astakhova LN. Thyroid cancer after Chemobyl. Nature 1992;359:21. 2 Baverstock K, Egloff B, Pinchera A et al. Thyroid cancer after Chernobyl. Nature 1992;359: Simpson NE, Kidd KK, Goodfellow PJ et al. Assignment of multiple endocrine neoplasia type 2A to chromosome 10 by linkage. Nature 1987;328: Mathew CG, Chin KS, Easton DF et al. A linked genetic marker for multiple endocrine neoplasia type 2A on chromosome 10. Nature 1987;328: Wynford-Thomas D. Molecular basis of epithelial tumorigenesis: the thyroid model. Clin Rev Oncogenesis 1993;4: Lyons J, Landis CA, Harsh G et a]. Two G protein oncogeneses in human endocrine tumors. Science 1990;249: Santoro M, Rosati R, Grieco M et al. The Pet proto-oncogene is consistently expressed in human pheochromocytomas and thyroid medullary carcinomas. Oncogene 199O;S:lS Lanzi C, Borrello MG, Bongarzone I et al. Identification of the product of two oncogenic rearranged forms of the RET proto-oncogene in papillary thyroid carcinomas. Oncogene 1992;7: Takahashi M, Buma Y, Iwamoto T et al. Cloning and expression of the ret proto-oncogene encoding a tyrosine kinase with two potential transmembrane domains. Oncogene I988;3: Takahashi M. Structure and expression of the ref transforming gene. IARC Sci Pub1 1988;92: Fagin JA, Matsuo K, Karmakar A et al. High prevalence of mutations of the ps3 gene in

7 Gagel poorly differentiated human thyroid carcinomas. J Clin Invest 1993;91: Mulligan LM, Kwok JBJ, Healey CS et al. Germline mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A (MEN 2A). Nature 1993;363: Donis-Keller H, Dou S, Chi D et al. Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC. Hum Mol Genet 1993;2: Takahashi M, Ritz J, Cooper GM. Activation of a novel human transforming gene, ret, by DNA rearrangement. Cell 1Y85;42: Xing S, Smanik PA, Oglesbee MJ et al. Characterization of ref oncogenic activation in MEN 2 inherited cancer syndromes. Endocrinology 1996; 137: Asai N, Iwashita T, Matsuyama M et al. Mechanisms of activation of the ret proto-oncogene by multiple endocrine neoplasia 2A mutations. Mol Cell Biol 1995;15: Santoro M, Carlornagno F, Romano A et al. Activation of RET as a dominant transforming gene by germline mutations of MEN 2A and MEN 2B. Science 1995;267: Jing S, Wen D, Yu Y et al. GDNF-induced activation of the Ret protein tyrosine kinase is mediated by GDMFR-a, a novel receptor for GDNF. Cell 1YY6;85: Durbec P, Marcos-Gutierrez CV, Kilkenny C et al. GDNF signalling through the ret receptor tyrosine kinase. Nature 1996;381: Trupp M, Arenas E, Falnzilber M et al. Functional receptor for GDNF encoded by the c-ref protooncogene. Nature 1996;381: Bongarzone I, Butti MG, Coronelli S et al. Frequent activation of RET protooncogene by fusion with a new activating gene in papillary thyroid carcinomas. Cancer Res 1994;54: Jhiang SM, Sag& JE, Tong Q et al. Targeted expression of the ret/ptcl oncogene induces papillary thyroid carcinomas. Endocrinology 1996; 137: