Proteomics study of medullary thyroid carcinomas expressing RET germ-line mutations: identification of new signaling elements.

Proteomics may help to elucidate differential signaling networks underlying the effects of compounds and to identify new therapeutic targets. Using a proteomic-multiplexed analysis of the phosphotyrosine signaling together with antibody-based validation techniques, we identified several candidate molecules for RET (rearranged during transfection) tyrosine kinase receptor carrying mutations responsible for the multiple endocrine neoplasia type 2A and 2B (MEN2A and MEN2B) syndromes in two human medullary thyroid carcinoma (MTC) cell lines, TT and MZ-CRC-1, which express the RET-MEN2A and RET-MEN2B oncoproteins, respectively. Signaling elements downstream of these oncoproteins were identified after treating cells with the indolinone tyrosine kinase inhibitor RPI-1 to knock down RET phosphorylation activity. We detected 23 and 18 affinity-purified phosphotyrosine proteins in untreated TT and MZ-CRC-1 cells, respectively, most of which were shared and sensitive to RPI-1 treatment. However, our data clearly point to specific signaling features of the RET-MEN2A and RET-MEN2B oncogenic pathways. Moreover, the detection of high-level expression of minimally phosphorylated epidermal growth factor receptor (EGFR) in both TT and MZ-CRC-1 cells, together with our data on the effects of EGF stimulation on the proteomic profiles and the response to Gefitinib treatment, suggest the relevance of EGFR signaling in these cell lines, especially since analysis of 14 archival MTC specimens revealed EGFR mRNA expression in all samples. Together, our data suggest that RET/EGFR multi-target inhibitors might be beneficial for therapy of MTC.

RET oncoproteins induce tyrosine phosphorylation changes of proteins involved in RNA metabolism.

We report the identification of proteins induced in response to RET/PTC2, an oncogene implicated in thyroid cancers. Anti-phosphotyrosine antibody affinity resin was used to purify Tyr(P)-containing and interacting proteins from 293T and NIH3T3 cells which were transfected with kinase active or inactive RET/PTC and RETMEN2 oncogenes. Proteins were separated by one-dimensional SDS-PAGE, extracted by in-gel digestion, and identified by MALDI-TOF peptide mass fingerprinting. The expression and tyrosine phosphorylation of Sam68, a protein implicated in mRNA nucleocytoplasmic translocation and splicing, were further examined in RET-transfected cells and thyroid tumors. Of relevance, cells transfected with RETMEN2B examined for anti-phosphotyrosine bound proteins, showed other proteins implicated in splicing: DEAD-box p68 RNA helicase, SYNCRIP, and hnRNP K. Western blotting analysis suggested that these proteins are singularly tyrosine phosphorylated in RETMEN2B-transfected cells, and that they constitutively bind with Sam68. The study concludes that regulation of splicing factors is likely to be important in RET-mediated thyroid carcinogenesis.

RET receptor expression in thyroid follicular epithelial cell-derived tumors.

The RET proto-oncogene encodes a receptor tyrosine kinase for transforming growth factor-beta-related neurotrophic factors, which include GDNF and neurturin. The expression of RET proto-oncogene was detected in several tissues, such as spleen, thymus, lymph nodes, salivary gland, and spinal cord, and in several neural crest-derived cell lines. RET expression in the thyroid gland was reported to be restricted to neural crest-derived C cells. The presence of RET mRNA or protein has not yet been reported in thyroid follicular cells. We previously demonstrated the expression of oncogenic rearranged versions of RET in papillary thyroid carcinomas: tumors derived from thyroid follicular cells. To assess the expression of the normal RET proto-oncogene in follicular cells, we analyzed its expression in a panel of neoplasias originating from thyroid follicular epithelial cells: papillary carcinomas and both follicular adenomas and carcinomas. We also demonstrated the presence of RET normal transcripts in two follicular thyroid carcinoma lymph node metastases. Moreover, we found the presence of the RET/ELE1 transcript, the reciprocal complementary form of the oncogenic fusion transcript ELE1/RET, in a papillary thyroid carcinoma specimen expressing the RET/PTC3 oncogene, thus demonstrating that the RET promoter is active in those cells after rearrangement. Finally, we show that in a papillary carcinoma-derived cell line expressing the proto-RET receptor and the related GFRalpha2 co-receptor, GDNF treatment induced RET tyrosine phosphorylation and subsequent signal transduction pathway, indicating that RET could be active in thyroid follicular cells.

The Glu632-Leu633 deletion in cysteine rich domain of Ret induces constitutive dimerization and alters the processing of the receptor protein.

Mutations of the RET gene, encoding a receptor tyrosine kinase, have been associated with the inherited cancer syndromes MEN 2A and MEN 2B. They have also further been associated with both familial and sporadic medullary thyroid carcinomas. Missense mutations affecting cysteine residues within the extracellular domain of the receptor causes constitutive tyrosine kinase activation through the formation of disulfide-bonded homodimers. We have recently reported that a somatic 6 bp in-frame deletion, originally coding for Glu632-Leu633, potently activates the RET gene. This activation is increased with respect to the frequent MEN 2A-associated missense mutation Cys634Arg. This finding specifically correlated to the clinic behavior of the corresponding tumor, which was characterized by an unusually aggressive progression with both multiple and recurrent metastases. By examining the possibility that this deletion acts in a manner similar to cysteine substitution, we have analysed the molecular mechanism by which this oncogenic activation occurs. Phosphorylated dimers of the deleted Ret receptor were detected in immunoprecipitates separated under non-reducing conditions. Like other Cys point mutations, this 6 bp deletion affecting two amino acid residues between two adjacent Cys, is capable of activating the transforming ability of Ret by promoting receptor dimerization. These results suggest that alteration to cysteine residue position or pairing is capable of inducing ligand independent dimerization. Furthermore, we present data demonstrating that the processing and sorting of the Ret membrane receptor to the cell surface is affected by mutation type.

Full activation of MEN2B mutant RET by an additional MEN2A mutation or by ligand GDNF stimulation.

Germline mutations of RET gene, encoding a receptor tyrosine kinase, have been associated with the MEN2A and MEN2B inherited cancer syndromes. In MEN2A mutations affecting cysteine residues in the extracellular domain of the receptor cause constitutive activation of the tyrosine kinase by the formation of disulfide-bonded homodimers. In MEN2B a single mutation in the tyrosine kinase domain (Met918Thr) has been identified. This mutation does not lead to dimer formation, but has been shown (both biologically and biochemically) to cause ligand-independent activation of the Ret protein, but to a lesser extent than MEN2A mutations. Intramolecular activation by cis-autophosphorylation of RetMEN2B monomers has been proposed as a model for activation, although alternative mechanisms can be envisaged. Here we show that the activity of RetMEN2B can be increased by stable dimerization of the receptor. Dimerization was achieved experimentally by constructing a double mutant receptor with a MEN2A mutation (Cys634Arg) in addition to the MEN2B mutation, and by chronic exposure of RetMEN2B-expressing cells to the Ret ligand GDNF. In both cases full activation of RetMEN2B, measured by 'in vitro' transfection assays and biochemical parameters, was seen. These results indicate that the MEN2B phenotype could be influenced by the tissue distribution or concentration of Ret ligand(s).

Somatic in frame deletions not involving juxtamembranous cysteine residues strongly activate the RET proto-oncogene.

Somatic RET mutations have been identified in a variable proportion (about 30-70%) of sporadic Medullary Thyroid Carcinoma (MTC) cases. They are represented by the Met918Thr substitution (exon 16) typical of Multiple Endocrine Neoplasia type 2B (MEN2B) and, to a lesser extent, by nucleotide changes occurring at one of five critical cysteine residues (exons 10 and 11) typical of MEN type 2A (MEN2A). An in vitro transforming activity has already been demonstrated for these mutations. A few different MTC somatic mutations have been reported so far whose biological activity has still to be tested. In this paper we report the identification, in two MTC tumor samples, of two interstitial deletions of 48 bp and 6 bp occurred in exons 10 and 11 respectively. Both were somatic heterozygous in frame mutations, not involving any cysteine residue. Moreover, the expression of a full length RET cDNA carrying one of the two deletions demonstrated a strong transforming capacity in NIH3T3 cells.

The full oncogenic activity of Ret/ptc2 depends on tyrosine 539, a docking site for phospholipase Cgamma.

RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of proto-RET, a gene coding for a receptor-type tyrosine kinase (TK) whose ligand is still unknown. RET/PTCs encode fusion proteins in which proto-RET TK and C-terminal domains are fused to different donor genes. The respective Ret/ptc oncoproteins display constitutive TK activity and tyrosine phosphorylation. We found that Ret/ptcs associate with and phosphorylate the SH2-containing transducer phospholipase Cgamma (PLCgamma). Two putative PLCgamma docking sites, Tyr-505 and Tyr-539, have been identified on Ret/ptc2 by competition experiments using phosphorylated peptides modelled on Ret sequence. Transfection experiments and biochemical analysis using Tyr-->Phe mutants of Ret/ptc2 allowed us to rule out Tyr-505 and to identify Tyr-539 as a functional PLCgamma docking site in vivo. Moreover, kinetic measurements showed that Tyr-539 is able to mediate high-affinity interaction with PLCgamma. Mutation of Tyr-539 resulted in a drastically reduced oncogenic activity of Ret/ptc2 on NIH 3T3 cells (75 to 90% reduction) both in vitro and in vivo, which correlates with impaired ability of Ret/ptc2 to activate PLCgamma. In conclusion, this paper demonstrates that Tyr-539 of Ret/ptc2 (Tyr-761 on the proto-RET product) is an essential docking site for the full transforming potential of the oncogene. In addition, the present data identify PLCgamma as a downstream effector of Ret/ptcs and suggest that this transducing molecule could play a crucial role in neoplastic signalling triggered by Ret/ptc oncoproteins.

Age-related activation of the tyrosine kinase receptor protooncogenes RET and NTRK1 in papillary thyroid carcinoma.

Oncogenic rearrangements of RET and NTRK1 proto-oncogenes are frequently detected in papillary thyroid carcinomas. Several studies have shown an association between ionizing radiation and development of this tumor type. In addition in vitro irradiation of tumor cell lines induced rearrangements of RET similar to those observed in human papillary thyroid carcinomas. These two observations could be related to the reported increased incidence of papillary thyroid carcinomas in children living in contaminated areas around Chernobyl, given that it has been demonstrated that about 60% of them presents a RET oncogenic activation. However, this high frequency of RET positivity in radiation exposed children does not rule out the possibility that age could also play a role in the development of RET positive tumors. To assess this possibility we looked for a relationship between the presence of RET and NTRK1 oncogenic rearrangements and age at surgery in a sample of 92 consecutive patients. Our results show that, in papillary thyroid carcinoma, the frequency of RET and NTRK1 activation is significantly higher in the group of patients aged 4-30 years, thas supporting the concept that age could be contributing to this thyroid specific carcinogenic process.

Oncogenic rearrangements of the RET proto-oncogene in papillary thyroid carcinomas from children exposed to the Chernobyl nuclear accident.

Since the Chernobyl nuclear reactor accident, a striking increase of thyroid carcinoma has been reported in children exposed to radiation in Belarus. Because of its unprecedented scale and its emotional implications, this finding has raised concern and called the attention of the scientific community to this major health problem. Although epidemiologically documented, a direct correlation between thyroid cancer and radiation exposure has not been definitely proven at the molecular level. On the assumption that ionizing radiation could cause specific and common cancer-associated genetic lesions, an analysis of oncogene activation and/or tumor suppressor gene inactivation would help to define radiation-induced thyroid carcinomas. Therefore, we have analyzed by different molecular approaches, including Southern blotting, DNA transfection assay on NIH-3T3 cells, and reverse transcription-PCR analysis, six papillary carcinomas from children living in the region of Belarus at the time of the Chernobyl nuclear accident to identify tumor-specific gene rearrangements of the proto-oncogenes RET and TRK, previously found activated in a tumor type-specific manner in papillary thyroid carcinoma. Using Southern blot analysis in four cases, we could detect specific rearranged bands indicating an oncogenic activation of RET that in three cases resulted in rearranged sequences provided by the same activating gene. Moreover, the DNA of the last three cases showed a biological activity in transforming NIH-3T3 cells after the DNA-mediated transfection assay, and the respective NIH-3T3 transfectants were found to express the oncogenic fusion transcripts. These results support the possibility that RET oncogenic activation could represent a major genetic lesion associated with thyroid carcinoma in children exposed to the Chernobyl nuclear accident.

A sequence analysis of the genomic regions involved in the rearrangements between TPM3 and NTRK1 genes producing TRK oncogenes in papillary thyroid carcinomas.

Papillary thyroid carcinomas have frequently been found to display oncogenic rearrangements of the NTRK1 gene, which encodes the high-affinity nerve growth factor receptor. Replacement of its extracellular domain by sequences coding for the 221 amino-terminal residues of the TPM3 gene was responsible for the oncogenic NTRK1 activation in three of eight of these tumors. In all of them, the illegitimate recombination involved the 611-bp NTRK1 intron placed upstream of the transmembrane domain and the TPM3 intron located between exons 7 and 8. Therefore, due to the splicing mechanism, all of the TPM3/NTRK1 gene fusions encoded an invariable transcript and the same chimeric protein of 70 kDa, which was constitutively phosphorylated on tyrosine. In two of the three tumors the simultaneous presence of the reciprocal products of the TPM3/NTRK1 recombination, 5'TPM3-3'NTRK1 and 5'NTRK1-3'TPM3 sequences, respectively, and the previously demonstrated localization of both genes on the long arm of chromosome 1 lead us to suggest that an intrachromosomal inversion could be responsible for their recombination. In an attempt to understand the molecular basis that predisposes NTRK1 and TPM3 genes to be a recurrent target of illegitimate recombination, we have determined the nucleotide sequence around the breakpoints of the recombination products in all three patients as well as those of the corresponding regions from the normal TPM3 and NTRK1 genes. In these regions, a search for common features usually involved in illegitimate recombination in mammalian cells revealed the presence of some recombinogenic elements as well as pal-indromes, direct and inverted repeats, and Alu family sequences.

Loss of function effect of RET mutations causing Hirschsprung disease.

We have introduced three Hirschsprung (HSCR) mutations localized in the tyrosine kinase domain of RET into the RET/PTC2 chimaeric oncogene which is capable of transforming NIH3T3 mouse fibroblasts and of differentiating pC12 rat pheochromocytoma cells. The three HSCR mutations abolished the biological activity of RET/PTC2 in both cell types and significantly decreased its tyrosine phosphorylation. By contrast, a rare polymorphism in exon 18 does not alter the transforming capability of RET/PTC2 or its tyrosine phosphorylation. These data suggest a loss of function effect of HSCR mutations which might act through a dominant negative mechanism. Our model system is therefore capable of discriminating between causative HSCR mutations and rare polymorphisms in the tyrosine kinase domain of RET.

Frequent activation of ret protooncogene by fusion with a new activating gene in papillary thyroid carcinomas.

Tumor specific rearrangements of ret gene are frequently detected in papillary thyroid carcinomas. These rearrangements result in the formation of chimeric genes showing the tyrosine kinase domain of ret fused with the 5' end sequences of different genes. We examined a series of 52 patients and identified 10 cases of ret fusion with D10S170 locus resulting in the generation of ret/PTC1 oncogene, 2 cases with the gene encoding the regulatory subunit RI alpha of PKA (ret/PTC2), and finally 6 cases, here described, with a newly discovered gene called ele1 localized on chromosome 10 and leading to the formation of ret/PTC3 oncogene. Our results show the expression of the ret/PTC3 hybrid gene in all the 6 cases and demonstrated its association with the synthesis of 2 constitutively phosphorylated isoforms of the oncoprotein (p75 and p80). The chromosome 10 localization of both ret and ele1 and the detection, in all cases, of a sequence reciprocal to that generating the oncogenic rearrangements, strongly suggest that ret/PTC3 formation is a consequence of an intrachromosomal inversion of chromosome 10.

trk and ret proto-oncogene expression in human neuroblastoma specimens: high frequency of trk expression in non-advanced stages.

Forty-three fresh tumor specimens of human neuroblastoma belonging to different clinical stages were analyzed for the expression of 2 proto-oncogenes: trk, which encodes a tyrosine-kinase receptor for nerve growth factor (NGF) and ret, another receptor-type tyrosine kinase whose ligand is unknown. The mRNA expression of the trk gene was detected in 67.4% of cases, with increased frequency in I, II and IVs Evans' stages and in patients with favorable prognosis according to the Shimada classification. Moreover, trk expression inversely correlated with Nmyc-gene amplification. ret mRNA was found in 36.8% of cases and equally distributed in the different stages. In addition, ngfR (low-affinity NGF receptor)-gene expression was present in 9 out of 25 cases. The simultaneous presence of mRNA related to both forms of the NGF receptor, while not proving the presence of a functional receptor, indicates the existence of a sub-set of neuroblastoma cells potentially responsive to NGF.

Molecular characterization of a thyroid tumor-specific transforming sequence formed by the fusion of ret tyrosine kinase and the regulatory subunit RI alpha of cyclic AMP-dependent protein kinase A.

The ret oncogene frequently has been found activated in papillary thyroid carcinomas. A previous characterization of ret activation revealed recombination of its tyrosine kinase domain and sequences derived from an uncharacterized locus (D10S170). The mechanism leading to this recombination was identified as a paracentric inversion of the long arm of chromosome 10, inv(10)(q11.2q21), with the breakpoints occurring where ret and D10S170 were mapped. To further characterize the activation of ret in papillary thyroid carcinomas, we have now isolated and sequenced a second type of ret oncogenic rearrangement not involving the D10S170 locus. The nucleotide sequence indicated that the transforming activity was created by the fusion of the ret tyrosine kinase domain with part of the RI alpha regulatory subunit of protein kinase A (PKA). This is the first example of an oncogenic activity involving a PKA gene. PKA is the main intracellular cyclic AMP receptor, and its RI alpha subunit gene is located on chromosome 17q. RI alpha-ret transcripts encode two isoforms of the chimeric protein (p76 and p81), which display constitutive tyrosine phosphorylation as well as a tyrosine kinase enzymatic activity. Under nonreducing conditions, both isoforms are found in a dimeric configuration because of both homo- and heterodimer formation. Thus, the in vivo activation of ret in human papillary thyroid carcinomas is provided by the fusion of its tyrosine kinase domain with different genes and can be mediated by different mechanisms of gene rearrangement.

Cytogenetic and molecular genetic characterization of papillary thyroid carcinomas.

A combined cytogenetic and molecular analysis was performed on 11 cases of papillary thyroid carcinoma. A simple karyotypic abnormality was detected in five tumors, whereas six had no apparent chromosome change. In four of five rearranged cases the presence of a specific chromosomal abnormality involving chromosome 10 (cases 1 and 2) and chromosome 1 (cases 3 and 4) was associated with the rearrangement of two protooncogenes: RET and NTRKI (formerly trk), respectively, with different donor genes. Moreover, the chromosomal localization of the involved genes and the type of chromosomal change observed suggested that RET and NTRKI activation occurred by intrachromosomal rearrangements. The six cases with normal karyotype did not show RET or NTRKI activation. These findings suggest that a combined cytogenetic and molecular approach would be useful in understanding the pathogenesis of thyroid neoplasia.

Ultrastructural cytoskeleton alterations and modification of actin expression in the NIH/3T3 cell line after transformation with Ha-ras-activated oncogene.

Cytoskeleton alterations of NIH/3T3 fibroblast monolayers transfected with Ha-ras-activated oncogene were studied by immunofluorescence, immunoelectron microscopy, and immunoelectrophoretic analysis of actin isoforms. Transformation foci were found to consist of cells with a round shape and rare stress fibers that spread sparsely, forming rare focal contacts and fibronexuses. The loss of stress fibers in transformed cells was confirmed by staining with rhodamine-phalloidin and with a fluorescinated anti-non-muscle cell actin antibody. The transformed cells were anchored to the substrate prominently by filaments that contained fibronectin, as showed by immunoelectron microscopy. A down-regulation of alpha-actin isoform was observed by immunofluorescence and immunoblotting analysis using a specific monoclonal antibody. The diffuse distribution of alpha-actin, lacking a specific association with stress fibers, challenges the hypothesis of a connection between alpha-actin down-regulation and stress fiber loss.

High frequency of activation of tyrosine kinase oncogenes in human papillary thyroid carcinoma.

We had previously detected a transforming oncogene, designated PTC, in 25% of 20 papillary thyroid carcinomas. In order to characterize further the transforming activity of this tumour histotype, a new panel of tumour specimens from 16 patients was analysed by using a modified calcium phosphate-DNA coprecipitation transfection protocol. Tumour DNA from 10 patients (62%) displayed a transforming activity due to activation of three different oncogenes identified in four cases as PTC, in four cases as TRK, and in two cases as N-RAS. The same structural alterations of PTC and TRK (gene rearrangements) as well as of N-RAS (point mutation) detected in the NIH3T3 transformants, were also found in the original tumour DNAs, thus indicating that their activation was not due to transfection procedures. Since both PTC, a novel rearranged form of RET, and TRK display a tyrosine protein kinase activity, it is proposed that the activation of this class of oncogenes is specifically involved in the pathogenesis of papillary thyroid cancer.

Modulation of the human Harvey-ras oncogene expression by DNA methylation.

A clone of the human transforming Ha-ras oncogene (pT24-C3) was methylated "in vitro" at its HpaII and HhaI sites and co-transfected with a selectable marker into NIH/3T3 cells. In the resulting colonies the normal or transformed morphology correlated respectively with a methylated or unmethylated status of the Ha-ras 1 promoter. A transfected, morphologically normal NIH/3T3 colony treated with 5'-azacytidine acquired a transformed phenotype growing in agar and in nude mice and showed demethylation of the promoter region of Ha-ras 1 gene. The cells of the reactivated colony produced human Ha-ras 1 mRNA and p21, and their DNA displayed transforming activity for NIH/3T3 recipient cells that was due to the original human Ha-ras 1 oncogene. We conclude that the methylation status of the promoter region of the human Ha-ras 1 modulates the expression and consequently the transforming activity of the oncogene.

DNA methylation affecting the transforming activity of the human Ha-ras oncogene.

A plasmid containing the transforming Ha-ras gene and designated pT24-C3 was methylated in vitro using the sequence-specific bacterial methyltransferases HpaII and HhaI. Aliquots of the plasmid were methylated by the single enzymes or by the two enzymes simultaneously (double methylation). The transforming activity of the treated plasmids was assayed in the standard transfection assay on NIH-3T3 cells. Double methylation reduced the transforming activity of pT24-C3 about 80%, whereas treatment with the single methylating enzymes did not significantly affect the oncogene activity. Southern blot analysis of the transformants obtained with the methylated or mock-methylated pT24-C3 plasmids indicated in all the examined DNAs the presence of human Ha-ras sequences with methylation degrees consistent with the treatment of the plasmids. The Mr 21,000 oncogene protein p21 was also detected in several examined transformants. The DNA-demethylating agent 5-azacytidine restored the transforming activity of the double-methylated pT24-C3 upon 24 h incubation of transfected NIH-3T3 cells. Southern blot analysis showed integration of human Ha-ras with a methylation profile intermediate between the double-methylated and mock-methylated plasmids. It is suggested that DNA methylation of specific CG-containing target sites can affect the transforming activity of a human oncogene.

Integration and expression of MCF-13 provirus in MCF-13-induced lymphomas.

Polytropic viruses were isolated from 11 MCF-13-induced lymphomas using multiple infectious cycles on the mink lung and SC1 cell lines. The viruses were characterized by host range infectious properties and by biochemical properties using Cleveland's polypeptide mapping of the p30 and gp 70 viral proteins demonstrating identity in their biological properties with the MCF-13 lymphoma-inducing virus. Restriction enzyme analysis of the high molecular weight tumoral DNAs confirmed the presence of proviral sequences homologous to the inducing provirus in the lymphoma genomes indicating that MCF-13 MuLV has integrated in the cellular genome of the induced lymphomas.