11. THE MOLECULAR PATHWAYS INDUCED BY RADIATION AND LEADING TO THYROID CARCINOGENESIS

INTRODUCTION

The association between ionizing radiation and thyroid cancer is well established. It

was first proposed in 1950 in children who received X-ray therapy in infancy for

an enlarged thymus (1). During the following decades, numerous reports have docu-

mented an increased incidence of thyroid neoplasms in children after external radiation

for different benign conditions of the head, neck and thorax (2). Since the early 1960s,

when the use of radiotherapy for benign conditions was abandoned, the incidence

of radiation-associated thyroid malignancy in children gradually decreased (3). Cur-

rently, radiation therapy for malignancy continues to be a source of radiation-associated

thyroid cancer (4). An increased risk of thyroid cancer has also been linked to envi-

ronmental irradiation. This was documented in survivors of atomic bomb explosions

in Japan in 1945(5), and in residents of the Marshall Islands exposed to fallout after

detonation of a thermonuclear device on the Bikini atoll in 1954 (6). In the U.S.,

exposure to radioiodines from atmospheric nuclear tests in Nevada in the 1950s has

been suggested to lead to an excess of thyroid neoplasms (7, 8). In April 1986, an

accident at the Chernobyl Nuclear Power Station in the former USSR produced the

most serious environmental disasters ever recorded and led to a dramatic increase in

the frequency of childhood thyroid cancer in contaminated areas of Belarus, Ukraine,

and western Russia (9, 10). This tragic disaster has created one of the most striking

paradigms of radiation-induced thyroid tumors and allowed significant progress in the

understanding of the molecular pathways induced by radiation. In this chapter, I

review the genetic events and molecular mechanisms underlying radiation carcino- genesis in the thyroid gland.

RET/PTC REARRANGEMENTS

Over the last decade, rearrangements of the RET proto-oncogene have been identi- fied as the most common genetic event in thyroid tumors associated with radiation exposure.

The RET proto-oncogene is located on chromosome 10q11.2 and encodes a cell membrane receptor tyrosine kinase (11, 12). The receptor consists of three functional domains: an extracellular domain containing a ligand-binding site, a transmembrane domain, and an intracellular domain that includes a region with protein tyrosine kinase activity. The ligands for RET receptor are neurotrophic factors of the glial cell-line derived neurotrophic factor (GDNF) family, including GDNF, neurtulin, artemin, and persephin (13). Binding of a ligand causes the receptors to dimerize, leading to autophosphorylation of the protein on tyrosine residues and initiation of intracellular signaling cascade. Wild-type RET is expressed in neuronal and neural-crest derived tissues including thyroid parafollicular C-cells, but not in thyroid follicular cells. In thy- roid follicular cells, RET can be activated by fusion to different constitutively expressed genes. The product of this rearrangement is a chimeric oncogene named RET/PTC (PTC for papillary thyroid carcinoma).

Structure of RET/PTC oncogenes

Since the original report on RET activation by rearrangement in papillary thy- roid carcinomas (14), three major types of the rearrangement have been identified:

RET/PTC1, RET/PTC2, and RET/PTC3 (Figure 1). All of them are formed by

Figure 1. Schematic representation of the wild type RET gene and three major types of RET/PTC rearrangement. The portion of RET participating in the fusion encodes the tyrosine kinase domain (black box) but lacks the transmembrane and extracellular domains. The genes fused with RET encode dimerization domains, either coiled-coil domain (CC) or cysteine residues forming disulfide bonds during dimerization (C18, C39), allowing ligand-independent dimerization and activation of the truncated RET receptor. Block arrows indicate breakpoints.

ment are located on chromosome 10q (17, 18). In contrast, RET/PTC2 is formed by reciprocal translocation between chromosomes 10 and 17, resulting in RET fusion to the terminal sequence of the regulatory subunit of the cyclic AMP-dependent protein kinase A (Figure 1).

Recently, several novel types of RET/PTC have been described, most of them in papillary carcinomas from patients with the history of radiation exposure. Five novel types (RET/PTC5, RET/PTC6, RET/PTC7, RET/KTN1, RET/RFG8) were found in post-Chernobyl tumors (20–23), and two other in tumors from patients subjected to therapeutic external radiation (RET/PCM-1, RET/ (24, 25).

All novel types of RET/PTC are translocations and resulted from the fusion of the intracellular domain of RET to heterologous genes located on different chromosomes (Figure 2).

Figure 2. Schematic representation of the novel RET/PTC types found in papillary carcinomas associated with radiation exposure. Each fusion involves the tyrosine kinase (TK) domain of RET and the

portion of different genes that encode one or more putative coiled-coil domains (CC) essential for dimerization and RET TK activation. Chromosomal location of the RET fusion partners is shown in brackets. Arrows indicate breakpoints.

The genes fused with RET are constitutively expressed in thyroid follicular cells and drive the expression of the chimeric RET/PTC oncogene. In addition, these partners provide a dimerization domain essential for ligand-independent activation of the RET tyrosine kinase (26, 27). It allows ligand-independent dimerization of the chimeric protein and autophosphorylation of the truncated RET receptor. Indeed, it has been demonstrated that tyrosines 1015 and 1062, which are autophosphorylated in the wild-type RET only upon ligand binding, are constitutively phosphorylated in the RET/PTC chimeric protein (28). The ligand-independent activation of the RET tyrosine kinase is considered essential for the transformation of thyroid cells (29).

Another important role of the genes fused with RET is in determining a subcel- lular localization of the chimeric protein which lacks the transmembrane domain and cannot be anchored to the cell membrane. In RET/PTC3 protein, for instance, the N-terminal coiled-coil domain of ELE1 (RFG) not only mediates oligomerization of the receptor and chronic kinase activation, but is also responsible for the compartmen- talization of the chimeric protein at plasma membrane level, where most of the normal ELE1 (RFG) protein is distributed (30). Thus, different types of RET/PTC chimeric proteins, which have a similar RET tyrosine kinase portion but different N-terminal domains, may be distributed in varies cytoplasmic compartments, allowing them to interact with distinct sets of signaling proteins. This may provide an explanation for some variations in biological properties recently found between different RET/PTC types (reviewed in (31).

RET/PTC prevalence in sporadic and radiation-associated tumors

The prevalence of RET/PTC in papillary carcinomas varies significantly in different studies and geographic regions. In the U.S., the five largest series of adult papillary carcinomas showed the frequency ranging from 11% to 43% (32–36), with the cumu- lative incidence of 35%. A comparable rate of 30–40% have been found in series from Canada (37)) and Italy (34, 38, 39). In other regions, a wide variation in frequency of RET/PTC has been reported, ranging from 3% in Saudi Arabia (40) to 85% in Australia (41). A higher overall prevalence of RET/PTC has been noted in papillary carcinomas from children and young adults (38, 42–44). Among sporadic tumors of all age groups, RET/PTC1 is the most common type, comprising up to 60–70% of all rearrangements, whereas RET/PTC3 accounts for 20–30% of positive cases and RET/PTC2 for less than 10% (32, 36, 39).

An unusually high incidence of RET/PTC rearrangements has been documented in papillary carcinomas from patients with the history of radiation exposure, including those subjected to either accidental or therapeutic irradiation (Table 1).

In children affected by the Chernobyl nuclear accident, 67–87% of papillary carci- nomas removed 5–8 years after exposure and 49–65% of tumors removed 7–11 years after the accident harbored RET/PTC (42, 45–48). Remarkably, in tumors developed less than 10 years after the accident, RET/PTC3 was the most common type, whereas those developed after the longer latency had predominantly RET/PTC 1 (Table 2).

In patients subjected to therapeutic X-ray irradiation for benign or malignant condi-

tions, the prevalence of RET/PTC was also significantly higher than in the general

population, being reported at 52–87% (49–51). Exposure to ionizing radiation not only results in a higher prevalence in RET/PTC, but also promotes the fusion of RET to the unusual rearrangement partners, since seven out of eight novel RET/PTC types have been detected in tumors associated with radiation exposure. The novel types comprised up to 13% of all rearrangements found in post-Chernobyl tumors (48) (Table 2).

The fact that post-Chernobyl tumors arising shortly after exposure had mostly RET/PTC3 and those after a longer latent period harbored predominantly RET/PTC1 suggests at least two possibilities. First, radiation may be more effec- tive in inducing RET/PTC3, whereas other factors (still unknown), responsible for the majority of thyroid cancers that are not associated with radiation exposure, lead mostly to RET/PTC1. Second, it is conceivable that radiation is equally efficient in generation of both types, but tumors initiated by RET/PTC3 have a higher growth rate and manifest several years earlier. The latter possibility is supported by the animal experiments demonstrating that RET/PTC3 transgenic mice develop more malignant phenotype and metastatic disease as compared to RET/PTC1 animals (52, 53), and by recent in-vitro functional studies showing that RET/PTC3 is a more potent activator of MAPK signaling pathway and more efficient in promoting proliferation of cultured thyroid cells than RET/PTC1 (54).

The occurrence of RET/PTC has been observed after high-dose irradiation of

human undifferentiated thyroid carcinoma cells (55) and of fetal human thyroid tis-

sues transplanted into SCID mice (56, 57). In both studies, the rearrangements were

detected by RT-PCR as soon as 2 days after exposure. In addition, fetal thyroid cells revealed both RET/PTC 1 and RET/PTC3 7 days after irradiation, while only RET/PTC 1 persisted and was detectable 2 months later (57). The effective dose of radiation in both studies was high (50 Gy) and lethal for dividing cells, and the cells irradiated in the study by Ito and colleagues (55) were already highly transformed and hence more susceptible to develop secondary genetic defects. Nevertheless, these observations suggest that radiation exposure may lead to the generation of RET/PTC rearrangements in thyroid cells.

Potential mechanisms of RET/PTC generation after radiation exposure

The high prevalence of RET/PTC in post-Chernobyl children and in populations affected by other types of environmental and therapeutic exposures as well as the

and RET/PTC rearrangement. The role of radiation appears particularly important for the RET/PTC3 type, since its high prevalence was uniquely associated with pap- illary carcinomas developed shortly after the Chernobyl accident. The mechanisms of RET/PTC generation in thyroid cells after radiation have been a subject of extensive study over the last years.

Analysis of the breakpoint sites revealed no long-sequence homology between the DNA regions involved in RET/PTC1 fusion in sporadic tumors (58) and in RET/PTC3 in post-Chernobyl carcinomas (59, 60), establishing that these rear- rangements result from illegitimate rather than homologous recombination. When the breakpoint sites within the RET and ELE1 genes were mapped and analyzed in 12 post-Chernobyl tumors with RET/PTC3, it appeared that in each tumor the relative position of a breakpoint in the RET gene corresponded to the location of a breakpoint in the ELE1 gene (60). Specifically, after aligning the genes in opposite orientations, the breakpoints were located just across from each other in 5 (42%) tumors (Figure 3, A), while in other tumors they could be aligned by sliding one gene with respect to another (Figure 3, B, C). Similar pattern could be deducted from another study where the breakpoints in 22 post-Chernobyl tumors with RET/PTC3 were characterized (61).

Such predilection for the breakpoint site in one gene to correspond to the breakpoint site located within the certain region of the other gene suggests the presence of a stable spatial relationship between these two chromosomal loci within the nucleus (Figure 3).

If the interaction between these loci exists, it should involve folding of chromosome 10 where both RET and ELE1 genes reside separated by a linear distance of ~18 Mb.

This is conceivable since one of the levels of DNA packaging involves chromatin arrangement into loops of different size attached to a chromosomal backbone (62).

Therefore, although two chromosomal loci are located at a considerable linear distance from each other, they may be closely spaced in the interphase nucleus because of their location at specific areas of chromosomal loop(s).

Indeed, it has been recently demonstrated that chromosomal regions containing the

RET and H4 genes are non-randomly positioned with respect to each other in the

interphase nuclei of normal thyroid follicular cells (63). Utilizing fluorescence in situ

hybridization, two-dimensional distances between RET and H4 were measured in

Figure 3. Alignment of the breakpoint sites involved in RET/PTC3 in post-Chernobyl tumors demonstrating three patterns of correspondence between the position of breakpoints in each tumor (A, B, C). Modified from Nikiforov et al. (1999) (60) with permission.

the interphase cells and compared with a theoretical Rayleigh model that describes a distribution of distances between two points of linear polymers that fold in a random manner. Previous studies have shown that interphase distances between random loci on the same chromosome conform to the Rayleigh distribution (62, 64). Indeed, in the control experiment, distances between the RET and D10S539 loci, the latter located on chromosome 10q between RET and H4 and is not known to participate in the rearrangement, were found to follow the Rayleigh distribution (Figure 4) (63). As for the RET and H4 distances, they showed a strong deviation from the Rayleigh model, primarily due to the loci either juxtaposed or closer than expected, indicating a non- random manner of RET and H4 interaction. In addition, as many as 35% of primary cultured thyroid cells had at least one pair of RET and H4 genes juxtaposed. These data suggest that generation of RET/PTC rearrangements in thyroid cells may be in part due to the structural organization of chromosome 10, resulting in non-random interaction and spatial approximation of these potentially recombinogenic DNA loci (Figure 4).

It remains unclear, however, whether RET/PTC is a direct consequence of DNA

breaks induced by ionizing radiation, or it forms indirectly, after DNA damage has

Figure 4. Distribution of interphase distances between RET and D10S539 (A) and RET and H4 (B) in cultured normal thyroid cells as compared with the theoretical Rayleigh distribution (solid line). Dark bars indicate the RET – H4 distances that were in excess over the number expected based on the Rayleigh distribution. From Nikiforova et al. (2000) (63).

been repaired. Important information in this respect can be obtained by mapping and characterization the breakpoint sites involved in the fusion. Thus, if the rearrange- ment occurred indirectly (as a result of activation or disruption of the recombina- tion machinery), the breakpoints are expected be located within recombinase signal sequences at both participating loci, have similarity in nucleotide composition, or cluster at certain specific hypersensitive DNA regions. However, as it had been con- vincingly demonstrated in post-Chernobyl tumors with RET/PTC3, the breakpoints within and surrounding ELE1 intron 5 and RET intron 11 were distributed randomly, with no breakpoints occurring at exactly the same base or within an identical sequence in any of 41 tumors reported in three different series (59–61). The breakpoints exhib- ited no particular nucleotide sequence or composition. In one study, no evidence of AT-rich regions, fragile sites, recombination-specific signal elements, or other target DNA sites (i.e. chi-like motifs, heptamer/nonamer signal sequences) implicated in illegitimate recombination in mammalian cells was found (60). These results favor the direct induction of RET/PTC as a result of random double-strand DNA breaks, rather than disruption of the recombination machinery. However, another study proposed an alternative mechanisms and found at least one topoisomerase I site exactly at or in close proximity to all breakpoints, suggesting the role of DNA breaks induced by this enzyme in the generation of RET/PTC3 (61).

PAX8-PPARfl REARRANGEMENT

Recently, a fusion has been identified in follicular thyroid carcinomas

with cytogenetically detectable translocation t(2;3)(q13;p25) (65). This rearrangement

RAS MUTATIONS

Point mutations of the three RAS genes, N-RAS, H-RAS, and K-RAS, have been found with various prevalence in thyroid follicular and papillary tumors. The initial reports have suggested that K-RAS mutations in follicular carcinomas may be associated with radiation exposure, since they constituted 3 out of 4 (75%) RAS mutations in patients subjected to radiation, in contrast to 6 out of 25 (25%) RAS mutations in sporadic tumors (70). However, the subsequent studies have not confirmed such an association (Table 3). Indeed, RAS mutations were found in 30% of thyroid tumors from patients with a history of external irradiation and in 42% of sporadic tumors, and similar prevalence of the K-RAS gene mutations was observed in the two groups (71).

In a series of post-Chernobyl tumors, no RAS mutations was detected in 33 papillary carcinomas; whereas 3 follicular adenomas and 1 follicular carcinoma harbored N- RAS codon 61 mutation (72). Similar findings were observed in another series of 31 post-Chernobyl papillary carcinomas (73). Two RAS mutations detected in 44 cinomas (65), whereas in the larger follow-up series, the prevalence was 26–35% (66, 67). However, when this alteration was studied separately in follicular carcinomas from patients without a history of radiation and in those exposed to radiation,

fusion was found in 5 out of 12 (42%) sporadic tumors and in all three (100%) tumors

associated with radiation (68). Despite the small number of follicular carcinomas in

the latter group, this finding points towards the possible association between radiation

exposure and rearrangement. Although papillary carcinoma is by far

the most common type of thyroid tumors associated with radiation, an increased risk

of follicular carcinoma development has also been documented in these populations

(69). Therefore, it is likely that radiation exposure may promote the development of

follicular tumors through the generation of rearrangement in a simi-

lar way it involves in the initiation of papillary thyroid carcinogenesis via RET/PTC

rearrangement.

post-Chernobyl papillary carcinomas in another study were not in critical codons 12, 13, or 61 of the gene (74). These data indicate that activating mutations of the RAS genes do not play a significant role in radiation-induced papillary carcinomas. Similar conclusion can be made for thyroid follicular tumors, the prevalence of RAS mutation in which shows no correlation with the history of radiation.

P53 MUTATIONS

Missense point mutations in exons 5–8 of the p53 tumor suppressor gene have been detected in 4 out of 22 (18%) papillary carcinomas developed in the patients with a history of childhood irradiation to the head and neck area (75). Several studies have reported the prevalence of p53 mutations in post-Chernobyl tumors. In one series, PCR-SSCP analysis revealed two (6%) somatic mutations, both in exon 5, in a series of 33 papillary carcinomas (72). One of those mutations was a missense mutation and another was a silent mutation, as detected by nucleotide sequencing.

Other studies reported a 0–23% prevalence of mutations in the critical exons of the p53 gene in pediatric post-Chernobyl papillary carcinomas (Table 4) (73, 76, 77).

Despite some variation in the results between these observations, the overall prevalence of p53 mutations in this post-Chernobyl population appears approximately 10%. This indicates that inactivation of the p53 tumor suppressor gene has only a limited role in radiation-induced thyroid carcinogenesis.

OTHER GENETIC EVENTS

A number of other genetic events have been explored as the possible mediators of

radiation-induced carcinogenesis in the thyroid gland. Mutations of the GSP and TSH

receptor genes were found to play either no role or rarely present in tumors asso-

ciated with external radiation (71) and in post-Chernobyl tumors (73, 78). It has

been recently suggested that mutations in mitochondrial DNA, particularly large-

scale deletions, were significantly more frequent in radiation-induced post-Chernobyl

tumors in comparison to sporadic neoplasms, and the frequency of these alterations

correlated with the levels of radioiodine contamination in the areas of patients’ resi-

dency (79). Recently, point mutation at nucleotide 1796 of the BRAF gene has been

Several types of genomic instability have also been studied in radiation-associated thyroid tumors. These studies were important in the light of the reports of a higher rate of germline mutations at minisatellite loci in children born from parents exposed to radiation after Chernobyl (81, 82). However, post-Chernobyl thyroid carcinomas revealed either no significant minisatellite or microsatellite instability (83), or low rate of microsatellite mutations (84).

CONCLUDING REMARKS

A large volume of information on the genetic alterations in thyroid tumors, gen- erated over the last decade, points towards a significant difference in the molecular pathways involved in the sporadic and radiation-induced carcinogenesis in the thy- roid gland. Thus, it appears that the molecular landscape of sporadic papillary and follicular carcinomas is dominated by point mutations, such as those of the BRAF and RAS genes, whereas large-scale chromosomal abnormalities, such as RET/PTC and rearrangements, are the most common abnormality in radiation- induced tumors. Since ionizing radiation is known to produce single strand and double strand DNA brakes, it is likely that radiation-induced DNA damage plays a direct role in promoting carcinogenesis through the generation of these chromosomal abnormal- ities.

Significant progress has been achieved in the understanding of why, despite a ran- dom distribution of radiation-induced DNA damage, some common cancer-associated chromosomal rearrangements are very specific. It is likely that spatial proximity of potentially recombinogenic chromosomal loci in the nuclei of normal human cells predisposes to these recurrent genetic alterations. It remains unclear, however, whether rearrangements occur directly by mis-rejoining of radiation-induced DNA breaks or they are stimulated by radiation exposure in an indirect way. Some data exist in sup- port of both mechanisms, and further studies are required to explore this important question in more detail. This is crucial for better understanding of the mechanisms of radiation-induced carcinogenesis in humans, and thyroid cancer represents a unique model to address it.

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