ANGELA GRECO, EMANUELA ROCCATO AND MARCO A. PIEROTTI Istituto Nazionale Tumori, Department of Experimental Oncology Operative Unit #3 Via G. Venezian, 1 20133 Milan Italy
INTRODUCTION
The NTRK1 gene encodes the high affinity receptor for Nerve Growth Factor, and its action regulates neural development and differentiation. Deregulation of NTRK1 activity is associated with several human disorders. Loss of function mutations cause the genetic disease Congenital Insensitivity to Pain with Anhidrosis (CIPA). Constitutive activation of NTRK1 has been detected in several tumor types. An autocrine loop involving NTRK1 and NGF is responsible for tumor progression in prostate carci- noma and in breast cancer. Somatic rearrangements of NTRK1, producing chimeric oncogenes with constitutive tyrosine kinase activity, have been detected in a consistent fraction of papillary thyroid tumors.
The topic of this review is the thyroid TRK oncogenes; the modalities of activa- tion, the mechanism of action, and the contribution of activating sequences will be discussed.
NTRK1 proto-oncogene
NTRK1 (also known as TRKA) is the prototype of a family of genes which also includes NTRK2 (TRKB) and NTRK3 (TRKC), encoding tyrosine kinase receptors for the neurotrophins of the Nerve Growth Factor (NGF) family. NGF is the preferred ligand for NTRK1, brain-derived neurotrophic factor and NT4/5 are ligands for NTRK2, and NT3 is the ligand for NTRK3. Interestingly, NT3 is also capable of binding to NTRK1 and NTRK2, although with low affinity (Barbacid, M., 1995).
All the neurotrophins bind also the p75 low affinity receptor, which belongs to the TNF receptor family, and is devoid of kinase activity (Kaplan, D. R. et al., 2000).
Neurotrophins are responsible for the survival, differentiation and maintenance of specific population of neurons in the developing and adult nervous system (Davies, A. M., 1994). In particular, the NGF/NTRK1 signaling supports survival and differ- entiation of sympathetic and sensory neurons responsive to temperature and pain. In addition to its neurotrophic functions, NGF also stimulates proliferation of a number of cell types such as lymphocytes, keratinocytes and prostate cells (Otten, U., et al., 1989; Di Marco, E. et al., 1993; Djakiew, D. et al., 1991).
NTRK1 was originally isolated from a human colon carcinoma as a transforming oncogene activated by a somatic rearrangement that fused a non-muscle tropomyosin gene to a novel tyrosine kinase receptor (Martin-Zanca, D. et al., 1986). Cloning of the full length gene (Martin-Zanca, D. et al., 1989) and identification of the NGF as a ligand occurred few years later (Kaplan, D. R. et al., 1991).
The NTRK1 gene is located on chromosome 1q21–22 (Weier, H.-U. G. et al., 1995) and consists of 17 exons distributed within a 25 kb region (Greco, A. et al., 1996). The NTRK1 receptor is a glycosylated protein of 140 kDa, comprising an extracellular portion, including Ig-like and Leucine rich domains for ligand binding, a single transmembrane region, a juxta-membrane domain, a tyrosine kinase domain and a C-terminal tail. Following NGF binding, NTRK1 undergoes dimerization and autophosphorylation of five tyrosine residues (Y490, Y670, Y674, Y675, and Y785).
Activated receptor initiates several signal transduction cascades, including the Mitogen Activated Protein Kinase (MAPK), the phosphatidylinositol 3-kinase (PI3K) and the pathways. These signaling cascades culminate in the activation of transcription factors that alter gene expression (Kaplan, D. R. et al., 2000).
NTRK1 in human diseases
Deregulation of NTRK1 activity is associated with several human diseases. Mutations affecting different NTRK1 domains are associated with CIPA (Congenital Insensitiv- ity to Pain with Anhidrosis), a rare recessive genetic disease characterized by loss of pain and temperature sensation, defects in thermal regulation and occasionally mental retardation (Indo, Y. et al., 1996). CIPA is the consequence of a genetic defect in the differentiation and migration of neural crest elements. By studying the effects of differ- ent CIPA mutations on NTRK1 biochemical and biological properties, the molecular mechanisms responsible for the disease have been unveiled. CIPA mutations cause inactivation of the NTRK1 receptor by at least three different mechanisms, such as complete inactivation, protein processing alteration, and reduction of receptor activity (Greco, A. et al., 1999; Greco, A. et al., 2000; Miranda, C. et al., 2002a; Miranda, C. et al., 2002b).
NTRK1 gain of function mutations have been described in some human tumors.
Activation through genomic rearrangements producing chimeric oncogenes has been detected in a consistent fraction of human papillary thyroid carcinoma, and it will be described later. A 75 amino acids deletion in the extracellular domain of the NTRK1 receptor, resulting in a mutated protein with in vitro transforming activity, has been
detected in a patient with acute myeloid leukaemia, indicating that constitutive acti- vation of the NTRK1 receptor may contribute to leukemogenesis (Reuther, G. W.
et al., 2000). In human neuroblastoma, expression of NTRK1 is a good prognos- tic marker, suggesting that lack of NTRK1 expression contributes to malignancy, presumably because it results in the loss of signaling pathways important for growth arrest and/or differentiation of the neural crest derived cells from which these tumors originate (Brodeur, G. M. et al., 1997). In prostatic carcinoma an autocrine loop involving NGF and NTRK1 is responsible for tumor progression (Djakiew, D. et al., 1991), and tumor growth can be blocked by NTRK1 kinase inhibitors (Weeraratna, A. T. et al., 2001). Recently, an autocrine NGF/NTRK1 loop has been demonstrated in breast cancer cells, suggesting that it could represent a potential therapeutic tar- get (Descamps, S. et al., 2001). It is interesting to outline that, at variance with other RTKs such as Met, FGFR, Kit and RET, no oncogenic activation of NTRK1 by point mutations have been found in human cancer. Moreover, the introduction of missense mutations releasing the oncogenic potential of different RTKs showed a distinct effect on NTRK1 receptor. This suggests that, despite the high degree of conservation of certain amino acid residues, the NTRK1 receptor diverges from other RTKs in terms of tridimensional structure and have a distinct auto-inhibitory mechanism (Miranda, C. et al., 2002c).
Thyroid TRK oncogenes
Papillary thyroid carcinoma (PTC) is the most frequent neoplasia originating from the thyroid epithelium, and accounts for about 80% of all thyroid cancers (Hedinger, C.
et al., 1988). A consistent fraction (50%) of PTC harbors chromosomal alterations causing somatic rearrangements, and consequent oncogenic activation, of two RTK genes, namely RET and NTRK1 (Pierotti, M. A. et al., 1996). For a long time RET and NTRK1 rearrangements represented the only known genetic alterations in PTC. Recently, mutations of BRAF, alternative to RTK rearrangements, have been reported by different groups, and they represent the most frequent alteration in PTC (Kimura, E. T. et al., 2003; Cohen, Y. et al., 2003; Soares, P. et al., 2003).
Both RET and TRK oncogenes have been discovered in our laboratory by DNA transfection/focus formation assay in NIH3T3 cells, starting from papillary thyroid tumor DNA. Transforming activity correlated with the presence of human RET and TRK sequences in the mouse transfectants DNA (Bongarzone, I. et al., 1989); this provided the basis for the isolation and characterization of the chimeric oncogenes, containing the receptor tyrosine kinase domain preceded by activating sequences from different genes.
Several TRK oncogenes have been isolated from thyroid tumors, differing in the activating genes (Figure 1). The TRK oncogene, identical to that first isolated from colon carcinoma, and containing sequences from the TPM3 gene on chromosome 1q22–23 (Wilton, S. D. et al, 1995), has been frequently found in thyroid tumors (Butti, M. G. et al., 1995). TRK-T1 and TRK-T2 derive both from rearrangement between NTRK1 and TPR gene on chromosome 1q25 (Miranda, C. et al, 1994);
however, they display different structure, especially for the different TPR portion
Figure 1. Schematic representation of NTRK1, TRKs and TPR proteins. In the oncoproteins the portions contributed by NTRK1 and activating sequences are indicated. The gray portions represent coiled-coil domains. On TPR the break sites of Met, TRK-T1 and TRK-T2 oncoproteins are indicated by arrows. TM: transmembrane domain, TK: tyrosine kinase domain, NPC: nuclear pore complex.
(Greco, A. et al., 1997; Greco, A. et al., 1992). TRK-T3 is activated by TFG, a novel gene on chromosome 3q11–12 (Greco, A. et al, 1995). All TRK oncogenes but TRK- T1 retain the NTRK1 transmembrane domain. TRK oncogenes display constitutive, ligand-independent tyrosine kinase activity.
In experimental models TRK oncogenes recapitulate the biological effects of the NTRK1 receptor upon NGF stimulation. In fact, they induce morphological trans- formation of NIH3T3 mouse fibroblasts, and neuronal-like differentiation of PC12 cells (Greco, A. et al., 1993b). The mechanisms by which TRK oncogenes mediate their effects have been in part elucidated (Figure 2). TRK oncoproteins interact to and activate Shc, FRS2, FRS3, IRS1 and IRS2. All these molecules, except are recruited by the same tyrosine residue, corresponding to Tyr490 of NTRK1, most likely in a competitive fashion. Such interaction site is crucial for oncogenic activity, in so far its mutation to phenylalanine completely abrogate TRK oncogene biological activity. Moreover, by using a Shc dominant-negative mutant unable to recruit GRB2, we showed a crucial role of Shc adaptor in TRK-T3 biological activity. Conversely, mutation of the interaction site did not affect the oncogenic activity. It is worth noting that our studies on TRK oncogenes allowed the identification of novel proteins interacting with NTRK1 kinase, such as IRS1, IRS2 and FRS3 (Miranda, C. et al., 2001; Roccato, E. et al., 2002; Ranzi, V. et al., 2003).
The capability of TPM3, TPR and TFG to activate chimeric tyrosine kinase onco- genes is not restricted to NTRK1; in fact, they have been found fused to other kinase genes. TPM3 and TFG were reported to fuse to ALK in anaplastic large cell lymphoma
Figure 2. TRK oncogenes signaling pathways. Tyrosine residues are indicated with the number of the corresponding aminoacids of NTRK1.
(Hernandez, L. et al., 2002; Lamant, L. et al., 1999). TPR was first identified as part of the MET oncogene in HOS cells, fused to the TK domain of the hepatocyte growth factor receptor (Park, M. et al., 1986); subsequently it was detected fused to the raf oncogene during the transfection of a rat hepatocarcinoma (Ishikawa, F. et al., 1987).
Interestingly, TPR and TFG were first identified in rearranged, oncogenic versions.
TPM3 gene encodes a non-muscle tropomyosin isoform. TPR gene encodes a large protein of the nuclear pore complex; recent studies have shown that TPR is a phos- phorylated protein involved in mRNA export, through the formation of complexes with different interacting proteins (Shibata, S. et al., 2002; Green, D. M. et al., 2003).
TFG encodes a protein of still unknown function.
Role of activating sequences in TRK oncogenic activation
Despite the diversity in structure and function, all the NTRK1 activating proteins con- tain coiled-coil domains that promote protein dimerization/multimerization. Coiled- coil domains are characterized by heptad repeats with the occurrence of apolar residues
Figure 3. Prediction of coiled-coil domains in TPM3, TPR and TFG with the use of Paircoil program (Berger B., PNAS vol 92, 1995. pag. 8259–8263). The vertical scale represents relative coiled-coil probability; the horizontal scale represents amino acids number.
preferentially in the first (a) and fourth ( d ) positions (Lupas, A., 1996; Lupas, A. et al., 1991). This confers to the proteins the capability to fold into that are wound into a superhelix. In Figure 3 the coiled-coil domains detected in TRK activating sequences by sequence analysis with the COIL program are shown.
TPM3 contains numerous, overlapping coiled-coil domains. Several coiled-coil domains are present in TPR, and two of them fall in the region contained in MET and TRK-T1 oncogenes. It has been reported that mutations within the first coiled-coil domain drastically reduces MET transforming activity (Rodrigues, G. A. et al., 1993).
TFG contains a single coiled-coil domain, of approximately three heptads, shorter than typical coiled-coil domains (Figures 3 and 4). However, the presence of a hydropho- bic residue in position a, would increase the strength of association, despite the short length (Greco, A. et al., 1995). The contribution of TFG coiled-coil domain to TRK- T3 oncogenic activation has been elucidated by studies employing mutants where the domain was either deleted or mutated at leucine residues in position d of each hep- tad. We have demonstrated that coiled-coil domain plays a crucial role in TRK-T3 oncogenic activation by mediating oncoprotein complexes formation, an essential step for tyrosine kinase activation. Our studies support the model by which coiled-coil
domains mediate protein oligomerization of RTK oncogenes leading to constitutive, ligand-independent tyrosine kinase activity (Greco, A. et al., 1998). The TFG coiled- coil domain is predicted to fold into trimers. By size-exclusion chromatography we have demonstrated that the wild type TRK-T3 protein is part of high molecular weight complexes, compatible with the assembly of six oncoproteins molecules, or including other proteins. However, the precise composition of TRK-T3 complexes remains to be determined (Roccato, E. et al., 2003).
Studies on receptor and non receptor tyrosine kinase chimeric oncogenes have demonstrated that the importance of activating genes is related to the coiled-coil domains which mediate the activation above reported. However, an attractive hypoth- esis is the possibility that activating sequences may contribute with other functions, apart from dimerization. In addition, since the oncogenic rearrangements disabled one allele of the activating gene, the reduced activity of the corresponding protein could play a role in thyroid carcinogenesis. In this respect the thyroid TRK-T3 oncogene represents an attractive model for at least three reasons: 1) the activating portion is provided by TFG, a gene coding for a novel protein whose function remains to be unveiled; 2) the TFG portion contained in TRK-T3 display a single, short coiled-coil domain, corresponding to 14% of the aminoacid residues; 3) TFG might interact with other proteins (see later).
After our initial isolation as part of the TRK-T3 oncogene, the normal TFG coun- terpart was cloned and characterized (Figure 4).
Figure 4. Schematic representation of the TRK-T3 oncogene. The portions contributed by TFG and NTRK1 are indicated. In the box the TFG aminoacidic sequence is reported; specific domains and consensus sites are indicated. CC: coiled-coil domain; TM: transmembrane domain; CK2: putative phosphorylation site for CK2; PKC: putative phosphorylation site for PKC.
TFG gene is ubiquitously expressed in human adult tissues and it is conserved among several species, including C. elegans. In addition to the coiled-coil domain, the TFG protein also contains putative phosphorylation sites for PKC and CK2, glycosilation sites, as well SH2- and SH3-binding sites (Mencinger, M. et al., 1997). Several of these sites are identical in TFG proteins from different species, indicating that the pro- tein might be involved in basic cell processes (Mencinger, M. et al., 1999). We have recently identified a PB1 domain, which encompasses almost entirely the TFG N- terminal portion (Roccato, E. et al., 2003). PB1 is a novel protein module mediating protein oligomerization: in fact it is capable of binding to target proteins containing PC motifs and, as recently discovered, other PB1 domains (Terasawa, H. et al., 2001;
Nakamura, K. et al., 2003). Based on the peculiarity of TFG, we have recently focused our interest on the role of sequences outside the coiled-coil domain in TRK-T3 onco- genic activation. On the whole our studies demonstrate that the regions outside the coiled-coil domain give a great contribution to TRK-T3 activation. When deleted, complexes formation is unaffected; however transforming activity is reduced to dif- ferent extent. More detailed information was provided by studies employing point mutants: transforming activity was significantly reduced by mutating a putative SH2- binding motif, whereas it was abrogated by the mutation of the conserved Lys residue within the PB1 domain. These evidences strongly support the notion that proteins interacting with TFG might play a role in TRK-T3 oncogenic activity (Roccato, E.
et al., 2003). The identification of such proteins will give an important contribution to the definition of the modalities by which TRK-T3 triggers thyroid carcinogenesis, as well as to the discovery of TFG normal function.
Genomic features of NTRK1 oncogenic rearrangements
The NTRK1 genomic rearrangements present in the tumor DNA have been cloned and characterized (Figure 5) (Greco, A. et al., 1997; Greco, A. et al., 1995, Butti, M.G.
et al, 1995). All the breakpoints fall within a NTRK1 region of 2.9 Kb, showing a GC content of 58.8% (Greco, A. et al., 1993a). The NTRK1 rearrangements are balanced;
in fact, in addition to the oncogenic rearrangement containing the moiety of the receptor, the reciprocal product of the rearrangement, containing the portion of NTRK1 fused to the portion of the activating genes, was present in tumor DNA.
Figure 5. Genomic structure of the NTRK1 gene. The break sites of the different TRK oncogenes are indicated.
Sequencing of the breakpoint regions showed no homologies between the joined extremities. This suggests that the Non Homologous End Joining (NHEJ) mechanism, which requires little or not sequence homology, could be involved in the generation of TRK oncogenes. The NHEJ pathway is activated by ionizing radiations, and this is consistent with the association of PTC with therapeutic or accidental radiation exposure. Analysis of the breakpoint regions in different tumors demonstrated that all the rearrangements are conservative, involving deletion, insertion or duplication of only few nucleotides. In a tumor carrying the TRK-T2 oncogene a peculiar rearrangement was found, with the end of NTRK1 joined to sequences from chromosome 17;
however, such additional rearrangement does not contribute to oncogenic activation (Greco, A. et al., 1997).
No cytogenetic studies are available for tumors carrying NTRK1 rearrangements;
therefore the type of chromosomal rearrangement generating TRK oncogenes is not documented. A t(1 ;3) translocation is most likely responsible for the generation of TRK-T3 oncogene (TFG/NTRK1 rearrangement). For TRK, TRK-T1 and TRK- T2, produced by rearrangements with genes located on the q arm of chromosome 1, similarly to NTRK1, three mechanisms of rearrangement can be postulated: deletion, inversion, and reciprocal translocation between the two chromosome 1 homologues.
The presence in the tumor DNA of the reciprocal products of the rearrangement allowed us to exclude the deletion. Sequence data recently available in public databases unveiled that NTRK1 has transcriptional orientation opposite to that of TPM3 and TPR. Therefore, intrachromosomal inversion is the only mechanism capable to pro- duce TRK, TRK-T1 and TRK-T2 oncogenes (Figure 6).
The thyroid epithelium is very prone to chromosomal rearrangements. These include the RET and NTRK1 oncogenes in PTC, and the fusion gene associated with follicular thyroid tumors. This predisposition to gene rearrange- ments is a peculiarity of thyroid epithelium, at variance with other epithelia, and the understanding of the molecular basis underlying such predisposition represents a fasci- nating topic. Recently, Nikiforova et al (2000) have shown that, in thyroid interphase nuclei, RET and H4 loci display a distance reduced with respect to other cell type, and suggested that this spatial contiguity may provide the structural basis for the generation of the thyroid RET/H4 (PTC1) oncogene. It is very important to assess whether or not this attractive model also apply to NTRK1 and its partners, rearranging genes.
Another possibility is that the high frequency of chromosomal rearrangements in thy- roid tumors might reflect the thyrocyte intrinsic capability to repair DNA DSBs, either spontaneous or induced. Yang et al (1999) showed that human thyrocytes exposed in vitro to ionizing radiations failed to induce apoptosis; instead, they showed a significant increase of DNA end-joining activity. In this respect the analysis of the DNA repair kinetics and the status of the enzymes involved in DNA repair in human thyrocytes deserve to be investigated.
CONCLUSIONS
Rearrangements of RET and NTRK1 are frequently detected in human papillary thyroid carcinoma. TRK oncogenes involve different activating genes containing
Figure 6. Ideogram of chromosomes 1 and 3 showing the localization of NTRK1, TPM3, TPR and TFG genes. The arrows indicate the transcriptional orientation.
coiled-coil domains chat mediate protein dimerization and consequent tyrosine kinase activation. However, also regions outside the coiled-coil domain contribute to onco- genic activation, with modalities presently under investigation. The high proneness of thyroid epithelium to chromosomal rearrangements might reflect structural and enzymatic properties of thyrocytes with respect to DNA repair. The identification and study of such properties will elucidate the mechanisms responsible for the generation of thyroid oncogenes.
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