The Natural Thyroid Diet

The Natural Thyroid Diet

Get Instant Access

MEN2A Hoppner W, 1998

Figure 6. Mechanism of disulfide bond-mediated RET dimerization.

Intramolecular and intermolecular disulfide bonds (SS), unphosphorylated (Y) and phosphorylated (Y*) tyrosine residues are indicated.

Figure 6. Mechanism of disulfide bond-mediated RET dimerization.

Intramolecular and intermolecular disulfide bonds (SS), unphosphorylated (Y) and phosphorylated (Y*) tyrosine residues are indicated.

RET molecules thus leading to constitutive receptor dimerization and hence activation (Figure 6) (Asai, N. et al., 1995; Santoro, M. et al., 1995; Borrello, M. G. et al., 1995).

Accordingly, transgenic mice in which the RET9 gene carrying the MEN2A-C634R mutation was expressed under the control of the human calcitonin promoter or under the MoMuLv LTR, developed C-cell tumors resembling human MTC (Michiels, F. M. et al., 1997; Kawai, K. et al., 2000).

RET mutations at codons 609, 618, and 620 lead to constitutive RET activity by causing ligand-independent disulfide-bridged homodimerization as "classical" RET-C634R mutation but, in addition, they markedly decrease the cell surface expression of RET (Carlomagno, F. et al., 1997; Chappuis-Flament, S. et al., 1998; Ito, S. et al., 1997).

Some tumors in MEN2 display a second hit, a somatic mutation involving the RET gene in the tumor clone precursor cell; the activated RET allele is amplified by chromosome 10 duplication in some tumors, or the normal RET allele is deleted in some others (Huang, S. C. et al., 2000).

RET in multiple endocrine neoplasia type 2B (MEN2B)

Most MEN2B cases (95%) are caused by the M918T mutation (exon 16) (Carlson, K. M. et al., 1994). Other rarer (5%) intracellular mutations involve codon 883 (exon 15) in the RET tyrosine kinase domain (Smith, D. P. et al., 1997).

The M918T substitution is also found in sporadic MTC (reviewed in Jhiang, S. M., 2000), with M918T mutation-positive tumors often displaying a more aggressive phenotype. Recently, a double mutation at codons 804 and 806 has been found in a Japanese patient that had clinical features characteristic of MEN2B (Miyauchi, A. et al., 1999). The M918T mutation does not cause constitutive dimerization but activates RET by an intramolecular mechanism (Santoro, M. et al., 1995). The methionine at codon 918 is highly conserved in receptor tyrosine kinases and it maps in the P+1 loop of the kinase domain that is predicted to interact with the protein substrate. A threonine is found at the equivalent position in cytosolic tyrosine kinases, and the two kinase classes (receptorial and cytosolic) have different signaling specificity (Marengere, L.E. et al., 1994). Accordingly, it was demonstrated that the MEN2B-M918T mutation changes the substrate specificity of RET kinase. Indeed, RET-MEN2B displays phosphorylation of intracellular proteins as well as autophos-phorylation sites different from RET-MEN2A (Santoro, M. et al., 1995). Thus, the shift of RET autophosphorylation sites and of RET intracellular substrates, rather than the modest rise of RET kinase activity (Borrello, M. G. et al., 1995), may be crucial for the oncogenic activity of RET-MEN2B and responsible for its specific neoplastic activity (Santoro, M. et al., 1995). Furthermore, GDNF stimulation seems to be necessary for the full activation of RET-MEN2B (Bongarzone, I. et al., 1998).

It is not known how the A883F affects RET function. However, residue 883 is located in a subdomain of RET that defines substrate preference (Smith, D.P. et al., 1997) thus suggesting that the alteration of substrate specificity may be the common etiologic thread that underlies the pathogenesis of MEN2B. The production of a mouse model of MEN2B by introduction of the corresponding mutation into the RET gene demonstrated that heterozygous mutant mice displayed several features of the human disease, including C-cell hyperplasia and pheochromocytoma, while homozygous displayed more severe thyroid adrenal disease as well as male infertility. Only homozygous mice did develop ganglioneuromas of the adrenal medulla and enlargement of the associated sympathetic ganglia (Smith-Hicks, C.L. et al., 2000).

RET in familial medullary thyroid carcinoma (FMTC)

FMTC mutations can be found either in the extracellular or in the tyrosine kinase domain of RET. The ones occurring in the extracellular RET domain are the same mutations also associated with MEN2A: substitution of cysteines 609, 611, 618, 620 (exon 10), 630 and 634 (exon 11). The mutations in the tyrosine kinase domain occur at residues 768, 790, 791 (exon 13), 804, 844 (exon 14) or 891 (exon 15) (see references in

Table 3). Rare mutations have been recently reported, such as a 9-base pair duplication in exon 8 in a FMTC family (Pigny, P. et al., 1999) or mutations at codons 778 and 804 on the same RET allele which are associated with both FMTC and prominent corneal nerves (Kasprzak, L. et al., 2001). FMTC mutations occurring in the intracellular RET domain were thought to be infrequent and only a few number of families bearing the RET mutation within exons 13, 14 and 15 have been described. However, in the past two years the frequency of detection of these mutations has increased (Niccoli-Sire, P. et al., 2001) due to more accurate analysis and screening. No data are yet available on the mechanisms of activation of FMTC mutations occurring in RET tyrosine kinase domain. Patients with RET mutations in exons 13, 14 and 15 exhibit a mild C-cell disease phenotype (Berndt, I. et al., 1998; Fattoruso, O. et al., 1998) confirmed by in vitro studies. In fact, mutant RET proteins carrying mutations at residues 768, 804 or 891 display lower transforming activity (Pasini, A. et al., 1997; Iwashita, T. et al., 1999) compared with RET substitutions at codons 634, 918 or 883 strongly associated with MEN2A and MEN2B, respectively. Computer modeling has suggested that the E768D substitution modifies the kinase activity ofthe receptor by altering the substrate specificity or the ATP-binding capacity (Pasini, A. et al., 1997). As for its location, also the substitution at position 804 may exert an activating effect by altering the kinetics of interactions with normal cellular substrates or by modifying the range of substrates that are phosphorylated (Bolino, A. et al., 1995; Eng, C. et al., 1995; Iwashita, T. et al., 1999; Pasini, A. et al., 1997).

Recent studies (Feldman, G. L. et al., 2000; Brauckhoff, M. et al., 2002) have reported cases of patients harboring RET germline mutations in exons 14 and 15 (at codons 790, 791, 804) resulting in papillary microcarcinoma. Rey and colleagues (Rey, J. M. et al., 2001) also described the case of a kindred in which a novel single point germline RET mutation (K603E in exon 10) cosegregates with medullary and papillary thyroid carcinomas (PTCs). Despite the low number, these observations suggest that there might be a correlation between the occurrence of PTC and RET germline mutations in exons 13 and 14 that may play a role in pathogenesis of PTC. Of note, PTC seems to be present just in patients with low penetrance RET germline mutations. It remains an open question whether the simultaneous occurrence of inherited MTC and PTC is coincidental or the result of partly common pathogenic pathways. Reynolds and coworkers (Reynolds, L. et al., 2001) found the co-existence ofMTC and PTC in transgenic mice expressing the long isoform of RET-MEN2A and suggested that this might be due to 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 (Kovacs, C. S et al., 1994).

MEN2/HSCR paradox: the same mutation of RET with both gain and loss of function

In 20 to 30% of families with a mutation at cysteine residues 609, 611, 618 or 620 and no additional mutations within the coding sequence of RET, MEN2A and FMTC associate with HSCR (Mulligan, L.M. et al., 1994; Attie, T. et al., 1995; Borst, M. J. et al., 1995; Nishikawa, M. et al., 2003). The occurrence of HSCR in MEN2A/FMTC

pedigrees is difficult to be explained with a gain-of-function mutation in RET, which is typical for the MEN 2 mutations. RET proteins containing these specific cysteine mutations have been shown to translocate to the cell surface with low efficiency (Ito, S. et al., 1997; Chappuis-Flament, S. et al., 1998). Moreover, a kinase activity under the threshold required for cell survival (Takahashi, M. et al., 1999), or an inability to respond to GDNF, and protect RET-expressing cells from apoptosis (Mograbi, B. et al., 2001) have also been proposed as explanations. The mutation of each cysteine involved in MEN2A/FMTC promotes the aberrant formation of disulfide-linked RET homodimers, causing a constitutive activation of RET. However, mutation of Cys634 is approximately fivefold more strongly activating than mutations of cysteine 609, 611, 618, or 620 (Chappuis-Flament, S. et al., 1998). As a result, the low level of the RET covalent dimers resulting from mutations of cysteine 609, 611, 618, or 620, might be sufficient to activate the RET signaling pathways in the thyroid C-cell and chromaffin cell, leading to hyperplasia or tumor formation. On the other hand, insufficient RET protein is available in the developing enteric nervous system, thereby leading to HSCR. However, the fact that HSCR and MEN2A/FMTC are associated in only a fraction of families with a mutation at codon 609, 611, 618, or 620, indicates that other genetic or environmental factors might influence the clinical expression of the enteric phenotype (Mulligan, L. M. et al., 1994; Decker, R. A. et al., 1998).

RET in sporadic MTC (sMTC)

The majority of MTC cases (75%) have no associated family history. However, it has been found recently that 3-7% of sporadic cases represent occult or de novo MEN2 cases as determined by finding germline RET mutations (Wohllk, N. et al., 1996).

Somatic RET mutations are found, however, in 30 to 70% of true sporadic medullary thyroid carcinomas and, rarely, in pheochromocytomas. Mutation at the codon 918 is largely predominant but also mutations at the cysteine codons 609, 611, 620, 630, 634 were found in sporadic MTC cases. Recently three new somatic missense mutations (at codons 639, 641 and 922) of the RET gene associated with sporadic MTC have been described (Kalinin, V. N. et al., 2001). The functional significance of RET mutations, however, is unclear. They have been shown to be present heterogeneously, suggesting that they have occurred during tumor evolution rather than being the initiating step (Eng, C. et al., 1996b). In addition, loss of heterozygosity (LOH) has been found in up to 30% of tumors on chromosomes 1p, 3p, 3q, 11p, 13q, 17p, 22q (Mulligan, L. M. et al., 1993a).

RET-MEN2A and RET-MEN2B signaling

The functions of RET in normal and tumorigenic cells are mediated by a complex series of downstream interactions. RET-activated pathways, such as AKT pathway, have been shown to be important for cell signaling mediated by both ligand-dependent and -independent activation of RET and to have a role in cell survival, proliferation and oncogenic transformation by all RET oncogenic forms (reviewed in Manie, S.

et al., 2001). However, RET-MEN2A is considered a constitutively activated kinase, whereas RET-MEN2B appears to be an activated kinase with in addition altered catalytic properties (Santoro, M. et al., 1995; Borrello, M. G. et al., 1995). Consistent with this fact, differences in cell signaling triggered by each of these oncogenic forms have been emerging. Phosphorylation of Tyr864 and Tyr952 is crucial for transformation triggered by RET-MEN2B while Tyr905 phosphorylation is essential for transformation promoted by RET-MEN2A (Iwashita, T. et al., 1996). Mutation of Tyr1062 multidocking site markedly impaired the transforming activity of all MEN2 mutants (Iwashita, T. et al., 1996). However it was reported that the level of phosphorylation of Tyr1062 is increased in cells expressing RET-MEN2B compared to those expressing RET-MEN2A, resulting in enhancement of activation of ERK and PI3K/AKT pathways (Salvatore, D. et al., 2001). In addition, DOK1, a Tyr1062-associated docking protein activating JNK pathways, more strongly binds RET-MEN2B than RET-MEN2A protein (Murakami, H. et al., 2002). Consequently, enhanced signaling via Tyr1062 has been suggested to be involved in MEN2B phenotype. Furthermore, recent studies obtained by differential display analysis of gene expression in cells expressing either RET-MEN2A or RET-MEN2B mutant proteins identified genes predominantly induced by either mutations (Watanabe, T. et al., 2002).

Was this article helpful?

0 0
10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook

Post a comment