Genes crucial for cancer development can be mutated via various mechanisms, which may reflect the nature of the mutagen. In thyroid papillary carcinomas, mutations of genes coding for effectors along ...the MAPK pathway are central for transformation. BRAF point mutation is most common in sporadic tumors. By contrast, radiation-induced tumors are associated with paracentric inversions activating the receptor tyrosine kinases RET and NTRK1. We report here a rearrangement of BRAF via paracentric inversion of chromosome 7q resulting in an in-frame fusion between exons 1-8 of the AKAP9 gene and exons 9-18 of BRAF. The fusion protein contains the protein kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It has elevated kinase activity and transforms NIH3T3 cells, which provides evidence, for the first time to our knowledge, of in vivo activation of an intracellular effector along the MAPK pathway by recombination. The AKAP9-BRAF fusion was preferentially found in radiation-induced papillary carcinomas developing after a short latency, whereas BRAF point mutations were absent in this group. These data indicate that in thyroid cancer, radiation activates components of the MAPK pathway primarily through chromosomal paracentric inversions, whereas in sporadic forms of the disease, effectors along the same pathway are activated predominantly by point mutations.
Genes crucial for cancer development can be mutated via various mechanisms, which may reflect the nature of the mutagen. In thyroid papillary carcinomas, mutations of genes coding for effectors along ...the MAPK pathway are central for transformation.
BRAF
point mutation is most common in sporadic tumors. By contrast, radiation-induced tumors are associated with paracentric inversions activating the receptor tyrosine kinases RET and NTRK1. We report here a rearrangement of
BRAF
via paracentric inversion of chromosome 7q resulting in an in-frame fusion between exons 1–8 of the
AKAP9
gene and exons 9–18 of
BRAF
. The fusion protein contains the protein kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It has elevated kinase activity and transforms NIH3T3 cells, which provides evidence, for the first time to our knowledge, of in vivo activation of an intracellular effector along the MAPK pathway by recombination. The
AKAP9-BRAF
fusion was preferentially found in radiation-induced papillary carcinomas developing after a short latency, whereas
BRAF
point mutations were absent in this group. These data indicate that in thyroid cancer, radiation activates components of the MAPK pathway primarily through chromosomal paracentric inversions, whereas in sporadic forms of the disease, effectors along the same pathway are activated predominantly by point mutations.
Molecular
genetic aberrations and the related phenotypes were investigated in 191
papillary thyroid carcinomas (PTCs) from patients exposed at
young age to radioiodine released from the Chernobyl ...reactor. A high
prevalence of RET gene rearrangements (62.3%) with a
significant predominance of ELE1/RET (PTC3) over
H4/RET (PTC1) rearrangements was found in PTCs of the
first post-Chernobyl decade. NTRK1 rearrangements were
rare (3.3%). In 3.3%, we observed novel types of RET
rearrangements: GOLGA5/RET (PTC5),
HTIF/RET (PTC6), RFG7/RET (PTC7), and an
as yet undefined RFGX/RET . RET
rearrangements, preferentially ELE1/RET , are related to
rapid tumor development. At longer intervals after exposure to ionizing
radiation, the prevalence of RET rearrangements declines
with a shift from ELE1/RET to H4/RET ,
most significantly in female patients. The prevalence of specific types
of rearrangements is independent of age at irradiation. A significantly
higher prevalence of ELE1/RET was observed in the most
heavily contaminated Oblasts, Gomel and Brest, suggesting a
preferential formation of this type of rearrangement after high thyroid
doses. RET rearrangement is related to aggressive
growth: Rearrangement-positive PTCs were in a more advanced pT category
and more frequently in the pN 1 category at presentation
than rearrangement-negative PTCs. ELE1/RET is related to
the solid variant of PTC, H4/RET more frequently to
typical papillary structures. The genotype/phenotype evaluation of
post-Chernobyl PTCs reveals a characteristic spectrum of gene
rearrangements that lead to typical phenotypes with important
biological and clinical implications.
As part of ongoing studies on the RET rearrangement frequency in children with papillary thyroid carcinoma (PTC) after their exposure to radioactive iodine after the Chernobyl reactor accident, new ...methods for the detection of novel types of RET rearrangements are being developed. In this study, an improved reverse transcription-PCR strategy is used successfully to identify a new type of RET rearrangement. This rearrangement is designated PTC8 and the involved RET-fused gene (RFG) as RFG8. The identification of two reciprocal transcripts coding for the RFG8/RET and RET/RFG8 fusions suggests that the PTC8 rearrangement results from a balanced chromosomal translocation. With a view to clarify its role in tumor induction, we compared the fusion products with those of previously described RET rearrangements. We therefore sequenced and characterized the RFG8 cDNA, which showed no significant similarity to any functional protein described as yet. RFG8 is located on chromosome 18q21-22 and is expressed ubiquitously. Bioinformatic analysis predicts with a high probability that the corresponding rfg8 protein is located in the cytoplasm and is involved putatively in intracellular transport processes. Furthermore, we identified coiled-coil structures upstream of the breakpoint with one of the coiled-coils showing dimerization capability. Thus the rfg8/ret fusion protein exhibits structures for oncogenic activation that are similar to those observed in previously described RET fusions.
Activating point mutations of the BRAF gene have been recently described in a variety of human tumors. In a study published in the Journal of Clinical Investigation, we reported a novel mechanism of ...activation of this gene via paracentric inversion of chromosome 7q. The fusion protein, AKAP9-BRAF, contains the intact kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It exhibited constitutive activation of BRAF kinase and was transforming for NIH3T3 cells. This finding represents the first demonstration of RAF activation by chromosomal rearrangement in human tumors. AKAP9-BRAF was more common in radiation-induced thyroid tumors, whereas point mutations of BRAF predominated in sporadic tumors of the same type, demonstrating the association between environmental factors and specific mechanisms of BRAF activation.
Proteome analysis led to the identification and characterization of tumor‐associated protein variants by two‐dimensional electrophoresis and mass spectrometry. We focused on comparing the influence ...of genotoxic nitroso compounds N‐methyl‐N‐nitrosourea, diethylnitrosamine and N‐nitrosomorpholine and the nongenotoxic peroxisome proliferator Nafenopin as tumor‐inducing agents on the protein pattern of rat hepatomas. We found several tumor‐associated variants that represent members of the aldo‐keto reductase superfamily. Their induction and/or inhibition was specifically related to the carcinogen used for tumor induction. The most prominent tumor‐associated protein, rat aldose reductase‐like protein‐1 (rARLP‐1) (69% sequence identity to lens aldose reductase) and three additional types of rARLP‐1 were detected in nitroso compound‐induced rat hepatomas, while rat aldo‐keto reductase protein‐c (Rak‐c), a novel tumor‐associated variant (65% sequence identity with 3α‐hydroxysteroid dehydrogenase) was discovered in N‐methyl‐N‐nitrosourea‐induced hepatomas only. 3α‐Hydroxysteroid dehydrogenase and γ4‐3‐ketosteroid‐5β‐reductase, both liver‐specific enzymes, were reduced in amount in all hepatomas investigated, independent of their mode of induction. We conclude, that detoxification enzymes like 3α‐hydroxysteroid dehydrogenase (3α‐HSD) and γ4‐3‐ketosteroid‐5β‐reductase (5β‐Red) might be replaced in hepatomas by tumor‐associated proteins that are often present in the embryonal state, like the rARLPs or the Rak‐c protein. Their induction appears to reflect an altered constitutive pattern of detoxification enzymes, detoxifying toxic aldehydes being induced by nitroso compounds. In contrast, members of the aldo‐keto reductase superfamily have not been found in Nafenopin‐induced hepatomas. The pattern of tumor‐associated protein variants is apparently characteristic for a given group of initiating carcinogens. The hypothesis is proposed that carcinogens leave specific fingerprints at the proteome level of manifest liver tumors.
Material from paraffin sections of 109 human colorectal carcinomas, mostly obtained at autopsy, was analyzed for the presence of K-ras point mutations at codon 12, position 2. Mutations at this ...position were found in 23 cases (21.1%). Aneuploid colorectal carcinomas showed a significantly higher prevalence of K-ras point mutations than diploid tumors, suggesting an involvement of ras mutations in the development of aneuploidy. No differences in the prevalence of K-ras mutations were observed with respect to the patients' age, sex and tumor type. In metastases, the type of ras gene mutation was always identical to that of the respective primary tumor. Mutations were not found in metastases from primary tumors devoid of ras mutations. This renders a clonal selection of K-ras mutated cells from a wild-type primary tumor during the metastatic process unlikely. However, nearly twice as many ras gene mutations were seen in metastatic than in non-metastatic primary tumors.
A novel type of RET rearrangement, PTC5, was detected in papillary thyroid carcinomas of two patients exposed to radioactive fallout after Chernobyl. Reverse transcription-PCR and rapid amplification ...of 5'-cDNA ends revealed a fusion of the ret tyrosine kinase (TK) domain with a sequence identical to that described previously as ret-II. Ret-II is a transfection artifact in NIH3T3 cells and has not yet been detected in any human tumor. Overlapping sequences found in the expressed sequence tag databases enabled us to sequence the COOH terminus of the ret-fused gene 5 (RFG5). The combined data made it possible to assemble a full-length rfg5 protein sequence. Computer-assisted analysis of this sequence reveals four putative coiled-coil structures, possibly involved in dimerization, but no membrane-binding sequences. Northern blots show a ubiquitous RFG5 expression in various normal tissues, including the thyroid gland. In addition to the RFG5/RET, we also detected the reciprocal RET/RFG5 transcript in both tumor samples, suggesting that the rearrangement is based on a balanced reciprocal translocation. In agreement with other rearranged TKs, it is concluded that the transforming action of the new fusion protein rfg5/ret in thyroid tumors may be due to an activation of the ret TK by constitutive expression and dimerization potential of the 5'-fused rfg5 protein. Ret immunohistochemistry indicates that the fusion protein is expressed in all cells of PTC5 tumors, suggesting that RFG5/RET rearrangement is an early event in thyroid carcinogenesis.
PDF
