RESUMO
Papillary thyroid carcinomas (PTCs) account for 90% of human thyroid cancer cases, which represent 1% of all cancer cases. They are likely to develop from papillary thyroid microcarcinomas (PTMCs), found in up to 36% of healthy individuals, due to rare progression events (0.01%). Although the prognosis of PTCs is excellent, 5% to 10% of tumors display an unfavorable outcome. About 45% of PTCs exhibit activating BRAFV600E mutations. Rats of the inbred BD strains postnatally exposed to the carcinogen N-ethyl-N-nitrosourea developed PTMCs, which closely resembled their human counterparts judging from their histology, size, and marginal tendency to progress. DNA sequencing revealed mutations in exon 15 of the Braf gene identical to the human BRAFV600E mutation in 82% of the cases. Predominantly a 50:50 ratio of wild-type to mutant Braf alleles was seen regardless of tumor size or animal age, indicating that the Braf mutation is an early, if not the initial, event in rat PTMC development. Surprisingly, most PTMCs carrying a confirmed BrafV600E mutation did not display BrafV600E protein expression. As the BrafV600Egene is supposed to be the driver in PTC development, down-regulation of expression should contribute to the low risk for progression of PTMC. This model system will enable further insights into the molecular mechanisms of PTMC initiation and progression to PTC, further translating into targeted tumor prevention strategies/therapies.
Assuntos
Carcinoma Papilar/patologia , Etilnitrosoureia/efeitos adversos , Proteínas Proto-Oncogênicas B-raf/genética , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Alelos , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Éxons/genética , Feminino , Humanos , Imuno-Histoquímica , Masculino , Mutação , Prognóstico , Ratos , Análise de Sequência de DNA , Câncer Papilífero da Tireoide/induzido quimicamente , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/induzido quimicamente , Neoplasias da Glândula Tireoide/patologiaRESUMO
A single dose of N-ethyl-N-nitrosourea (ENU) during late prenatal or early postnatal development induces a high incidence of malignant schwannomas and gliomas in rats. Although T->A mutations in the transmembrane domain of the Neu (c-ErbB-2) gene are the driver mutations in ENU-induced malignant schwannomas, the molecular basis of ENU-induced gliomas remains enigmatic. We performed whole-genome sequencing of gliomas that developed in three BDIV and two BDIX rats exposed to a single dose of 80 mg ENU/kg body weight on postnatal day one. T:A->A:T and T:A->C:G mutations, which are typical for ENU-induced mutagenesis, were predominant (41% to 55% of all somatic single nucleotide mutations). T->A mutations were identified in all five rat gliomas at Braf codon 545 (V545E), which corresponds to the human BRAF V600E. Additional screening revealed that 33 gliomas in BDIV rats and 12 gliomas in BDIX rats all carried a Braf V545E mutation, whereas peritumoral brain tissue of either strain had the wild-type sequence. The gliomas were immunoreactive to BRAF V600E antibody. These results indicate that Braf mutation is a frequent early event in the development of rat gliomas caused by a single dose of ENU.
Assuntos
Etilnitrosoureia/efeitos adversos , Glioma/genética , Neurilemoma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Substituição de Aminoácidos , Animais , Genótipo , Glioma/induzido quimicamente , Mutagênese , Neurilemoma/induzido quimicamente , Mutação Puntual , Proteínas Proto-Oncogênicas B-raf/metabolismo , Ratos , Análise de Sequência de DNARESUMO
The incidence of neural tumors is sexually dimorphic in both, humans and rodents. The identification of genetic determinants contributing to sex-biased tumor development is an essential prerequisite for differential tumor prevention in males and females. F2 hybrids of inbred BDIV and BDIX rats, resistant and susceptible, respectively, to ethylnitrosourea-induced malignant peripheral nerve sheath tumors (MPNST) display a marked sex bias regarding tumor risk. Homozygous BDIV alleles at the Mss4 locus (90.9-111.2 Mb, chromosome 6) mediate MPNST resistance exclusively in female F2 rats according to a genome wide association analysis. This locus was functionally confirmed and fine mapped through MPNST induction in males and females of three congenic rat strains (BDIX.BDIV-Mss4a, b, d). As a consequence, it could be subdivided in Mss4.1 (98.8-99.7 Mb) mediating cancer resistance, and Mss4.2 (99.7-111.2 Mb) enhancing sex specificity. Positional candidate genes were selected through DNA sequencing and expression profiling using RNAs from trigeminal nerve tissue of parental and congenic male and female animals. The transregulatory fingerprint of BDIV or BDIX alleles at Mss4.1 and/or Mss4.2, respectively, provided insight into the processes influencing cancer risk in a sex-biased way. A group of genes, a fraction of which involved in Schwann cell differentiation, showed low, male-biased expression in nerve tissues under the control of BDIX susceptibility alleles, but high, female-biased transcript levels when controlled by BDIV resistance alleles at Mss4. The Esr2 gene located in Mss4.1 constitutes an interesting functional candidate together with a yet unidentified gene/enhancer in Mss4.2.
Assuntos
Predisposição Genética para Doença/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Nervo Trigêmeo/patologia , Alelos , Animais , Diferenciação Celular/genética , Feminino , Masculino , Ratos , Risco , Células de Schwann/patologia , Transcriptoma/genéticaRESUMO
We previously mapped several gene loci influencing cancer risk of inbred BDIV and BDIX rats, resistant and susceptible, respectively, to N-ethyl-N-nitrosourea (ENU)-induced malignant peripheral nerve sheath tumors (MPNSTs). On the basis of a genomewide association analysis using a (BDIV × BDIX) F(2) generation the Mss4 locus on rat chromosome 6 was predicted to mediate resistance to MPNST development in the trigeminal nerves, preferentially in females. F(2) females homozygous for D6Mit1 proved almost exclusively resistant to peripheral neurooncogenesis, with no effect detectable in males. To functionally verify Mss4, a congenic BDIX rat strain was generated carrying a corresponding BDIV genomic fragment. On treatment with ENU, congenic BDIX.BDIV-Mss4a rats showed a 2.4-fold lower MPNST rate and a 55-day-longer survival time compared with BDIX animals. The sex-specific effect observed in F(2) rats was less pronounced in BDIX.BDIV-Mss4a congenics, with males, too, being protected against MPNST but to a lesser extent than females. Transcription profiling using trigeminal nerve tissue of BDIX, BDIV, and BDIX.BDIV-Mss4a congenics of both sexes revealed 61 genes located in the Mss4a fragment differentially expressed between BDIV and BDIX rats. In congenic rats each gene either displayed trans-regulated BDIX-like expression strength or cis-regulated BDIV-like transcript levels or intermediate expression without marked sex differences. Genomewide a number of genes exhibiting male-biased expression in the BDIX rat strain displayed a reversal of the sexual dimorphism in congenic rats similar to the BDIV expression pattern, which might be the basis of preferential protection of females against MPNST development.
Assuntos
Transformação Celular Neoplásica/induzido quimicamente , Etilnitrosoureia , Fatores de Troca do Nucleotídeo Guanina/genética , Neoplasias de Bainha Neural/induzido quimicamente , Animais , Animais Congênicos , Feminino , Loci Gênicos , Estudo de Associação Genômica Ampla , Incidência , Masculino , Neoplasias de Bainha Neural/epidemiologia , Neoplasias de Bainha Neural/mortalidade , Neoplasias de Bainha Neural/patologia , Ratos , Ratos Transgênicos , Caracteres Sexuais , Análise de SobrevidaRESUMO
Inbred rat strains BDIX and BDIV are constitutionally susceptible and resistant, respectively, to the development of malignant peripheral nerve sheath tumors (MPNST) induced by neonatal exposure to N-ethyl-N-nitrosourea (EtNU). They represent a model system for analysis of molecular and cellular processes underlying differential cancer susceptibility. A point mutation in the Neu/ErbB-2 gene is an early marker of Schwann precursor cells at high risk of malignant conversion and is diagnostic of the resulting MPNST predominantly developing in the trigeminal nerves. Initially considerable amounts of Neu/ErbB-2-mutant cells arise in nerve tissue of both rat strains subsequently disappearing in resistant BDIV rats, but persisting and giving rise to MPNST in susceptible BDIX animals. An almost identical cellular immune response-sequentially involving macrophages, T helper- and cytotoxic T lymphocytes-is mounted in the trigeminal nerves of EtNU-treated rats of both strains. In this study, T cell maturation was prevented by neonatal thymectomy following EtNU-exposure. While resistance against MPNST development significantly decreased in BDIV rats MPNST incidence and survival time remained unaltered in thymectomized BDIX rats. Contrary to euthymic animals a number of both thymectomized BDIV and BDIX rats developed MPNST lacking the Neu/ErbB-2-mutation. This suggests that Schwann cells initiated by other genetic alterations can progress to full malignancy in immune-compromised rats only. T cell-dependent resistance against tumorigenesis originating from non-Neu/ErbB-2-mutant Schwann precursors might thus be shared by both strains while BDIV T lymphocytes additionally prevent the development of Neu/ErbB-2-mutant MPNST. Rat strain-specific differences in the interaction of T lymphocytes with (pre)malignant Neu-mutant cells may thus critically contribute to susceptibility and resistance towards EtNU-induced MPNST development.
Assuntos
Neoplasias dos Nervos Cranianos/imunologia , Genes erbB-2/genética , Linfócitos T/imunologia , Doenças do Nervo Trigêmeo/imunologia , Alquilantes/farmacologia , Animais , Neoplasias dos Nervos Cranianos/induzido quimicamente , Neoplasias dos Nervos Cranianos/genética , Neoplasias dos Nervos Cranianos/patologia , Etilnitrosoureia/toxicidade , Estimativa de Kaplan-Meier , Depleção Linfocítica , Mutação Puntual , Ratos , Ratos Endogâmicos , Células de Schwann/efeitos dos fármacos , Células de Schwann/imunologia , Linfócitos T/efeitos dos fármacos , Nervo Trigêmeo/imunologia , Nervo Trigêmeo/patologia , Doenças do Nervo Trigêmeo/induzido quimicamente , Doenças do Nervo Trigêmeo/genética , Doenças do Nervo Trigêmeo/patologiaRESUMO
The identification of cancer susceptibility- and resistance-mediating genes is an essential prerequisite for prevention and early diagnosis of malignant tumors. Model organisms are helpful to identify variant alleles involved in pathways affecting individual cancer risk. BDIX and BDIV rats of both sexes are highly susceptible and resistant, respectively, to the development of N-ethyl-N-nitrosourea (ENU)-induced malignant peripheral nerve sheath tumors (MPNST), predominantly in the trigeminal nerves. Nevertheless, female (BDIV x BDIX) F(2) intercross rats have a lower MPNST incidence and a longer latency time than males. Six of seven autosomal gene loci (Mss1-Mss7) controlling genetic susceptibility and resistance in (BDIV x BDIX) F(2) hybrids exert allele- and sex-specific effects on tumor incidence and/or latency time of variable strength. Homozygous BDIV alleles at Mss4 or Mss7 located on rat chromosomes 6 and 10, respectively, are sufficient to cause almost complete resistance to ENU-induced MPNST development in female F(2) rats regardless of the genotype of the other locus. Both loci display only weak effects on male cancer risk. Survival curves of ENU-treated F(2) females depleted of animals with homozygous BDIV alleles at Mss4 and Mss7 are not significantly different from those of males, suggesting that these loci account mainly for the excess tumor resistance observed in female F(2) rats. By haplotype analysis Mss4 and Mss7 could be narrowed down to 20 and 12 Mb, respectively, providing a basis for the positional identification of candidate genes.
Assuntos
Transformação Celular Neoplásica/genética , Neoplasias do Sistema Nervoso Periférico/genética , Animais , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/patologia , Cruzamentos Genéticos , Etilnitrosoureia , Feminino , Predisposição Genética para Doença , Fatores de Troca do Nucleotídeo Guanina/genética , Haplótipos , Estimativa de Kaplan-Meier , Masculino , Neoplasias do Sistema Nervoso Periférico/induzido quimicamente , Neoplasias do Sistema Nervoso Periférico/patologia , Ratos , Ratos Endogâmicos , Fatores Sexuais , Organismos Livres de Patógenos Específicos , Nervo Trigêmeo/patologiaRESUMO
Human malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft-tissue sarcomas with a poor prognosis that arise either in the context of neurofibromatosis 1 or sporadically. Inbred BDIX and BDIV rat strains highly susceptible and resistant, respectively, to the development of ethylnitrosourea-induced MPNST enable us to identify, by using methods not applicable in humans, variant alleles involved in the pathways underlying individual MPNST risk. On the basis of a genome-wide association analysis using reciprocal intercrosses of BDIX and BDIV, BDIV alleles of two loci on chromosome 10, Mss1 and Mss7, were predicted to lower the risk of MPNST, the latter locus with a female bias. In this study we confirm the two nonoverlapping loci by exposing two congenic strains, BDIX.BDIV-Mss1 (Mss1) and BDIX.BDIV-Mss7 (Mss7), each carrying a BDIV genomic segment spanning the respective locus, to ethylnitrosourea. Compared with BDIX rats, the rate of MPNST is reduced 6.2-fold and 2.0-fold for Mss1 and Mss7 rats of both sexes, respectively. Although a moderate gain of survival time (30-50 days) is seen in Mss1 rats of both sexes and Mss7 males, Mss7 females survive 134 days longer than BDIX females. BDIV alleles at Mss7 obviously cause a markedly increased intrastrain sex difference regarding survival time in Mss7 compared with BDIX rats. Fine mapping will lead to the identification of allelic variants modulating rat MPNST risk and subsequently to their human counterparts. This is of particular relevance, because so far neither gene nor anonymous sequence variants have been identified that influence the risk of human sporadic Schwann cell malignancy.
Assuntos
Carcinogênese/induzido quimicamente , Predisposição Genética para Doença , Neoplasias do Sistema Nervoso Periférico/etiologia , Locos de Características Quantitativas , Alelos , Animais , Cruzamento , Mapeamento Cromossômico , Etilnitrosoureia/efeitos adversos , Feminino , Estudos de Associação Genética , Genoma , Incidência , Masculino , Neoplasias do Sistema Nervoso Periférico/mortalidade , Polimorfismo de Nucleotídeo Único , Ratos , Ratos EndogâmicosAssuntos
Cisto Epidérmico/genética , Regulação da Expressão Gênica , Locos de Características Quantitativas/genética , Animais , Biópsia por Agulha , Modelos Animais de Doenças , Cisto Epidérmico/patologia , Feminino , Humanos , Imuno-Histoquímica , Região de Controle de Locus Gênico/genética , Masculino , Distribuição Aleatória , Ratos , Ratos Endogâmicos BN , Sensibilidade e EspecificidadeRESUMO
Most malignant human tumors display a high degree of intratumoral heterogeneity at the time of diagnosis that contributes to treatment failure. This also applies to malignant peripheral nerve sheath tumors (MPNSTs) and aggressive soft tissue sarcomas that arise sporadically or in the context of neurofibromatosis type 1. On average, MPNSTs measure 10 cm in diameter at diagnosis. To explore molecular changes associated with early malignant progression and that may be present in most, if not all, tumor cells, we generated expression profiles of ethylnitrosourea-induced trigeminal MPNSTs in rats. Because these tumors cause increased intracranial pressure, they become detectable when they are comparatively minuscule. Histologic analyses revealed close resemblance to human MPNSTs. Compared with normal trigeminal nerve tissue, 365 genes were markedly upregulated and 310 genes were consistently downregulated in all MPNST samples. The molecular signature characteristic of early-stage MPNSTs included upregulation of proliferation and tissue remodeling-associated genes, downregulation of genes involved in Schwann cell differentiation, and the absence of transcripts associated with neuronal components. The transforming growth factor-ß pathway was consistently upregulated in all tumor samples. These data suggest that the signaling pathways underlying early malignant progression of Schwann cells might be targeted to prevent tumor growth and/or to treat more advanced lesions.
Assuntos
Modelos Animais de Doenças , Etilnitrosoureia/toxicidade , Regulação Neoplásica da Expressão Gênica , Neoplasias de Bainha Neural/metabolismo , Fenótipo , Células de Schwann/metabolismo , Animais , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Estadiamento de Neoplasias/métodos , Neoplasias de Bainha Neural/induzido quimicamente , Neoplasias de Bainha Neural/genética , Ratos , Células de Schwann/efeitos dos fármacos , Células de Schwann/patologiaRESUMO
The inbred BD rat strains constitute a model system for analysis of the genetic basis of susceptibility or resistance to the development of neural tumors, as they exhibit distinct strain-specific differences regarding the sensitivity to tumor induction by the alkylating carcinogen N-ethyl-N-nitrosourea (EtNU). Among the different BD strains, BDIX and BDIV rats, respectively, are either highly susceptible or entirely resistant to the development of EtNU-induced malignant schwannomas of the peripheral nervous system (PNS), predominantly of the trigeminal nerves. We have previously mapped one locus associated with susceptibility/resistance to schwannoma induction to the telomeric third of chromosome 10 (Mss1) in segregating (BDIX x BDIV) crosses. We report on the genetic mapping of 6 further loci controlling tumor incidence or survival time on chromosomes 1 (Mss2), 3 (Mss3), 6 (Mss4), 13 (Mss5) and 15 (Mss6) as well as on chromosome 10 (Mss7) close to the centromere. Interestingly, most of these loci mediate gender-specific effects of variable strength ranging from minor influences on tumor development to complete tumor resistance. The gender specificity is reflected by the fact that male (BDIX x BDIV) F2 rats exhibit a 2-fold higher incidence of EtNU-induced schwannomas than females as well as a shorter survival time. A number of human nervous system tumors too arise with a marked gender bias. Genes mediating gender-specific predisposition of developing malignant schwannomas in the rat may be relevant for the human individual risk of developing nervous system tumors.
Assuntos
Alquilantes/toxicidade , Mapeamento Cromossômico , Etilnitrosoureia/toxicidade , Predisposição Genética para Doença , Herança Multifatorial , Neurilemoma/genética , Neoplasias do Sistema Nervoso Periférico/genética , Animais , Transformação Celular Neoplásica/induzido quimicamente , Feminino , Masculino , Neurilemoma/induzido quimicamente , Neoplasias do Sistema Nervoso Periférico/induzido quimicamente , Ratos , Ratos Endogâmicos , Fatores SexuaisRESUMO
Frequent genetic aberrations of malignant schwannomas induced by the alkylating agent N-ethyl-N-nitrosourea in hybrids from inbred BD rat strains include allelic imbalances of the telomeric 20 Mb of chromosome 5 (Dis-2) and of the telomeric 5 Mb of chromosome 10q32 (Dis-1) in 59 and 94% of the tumors, respectively. The Dis-1 minimal loss of heterozygosity consensus region extends from D10Rat4 to the telomere and harbors a putative tumor suppressor gene(s). We constructed a 6-Mb BAC/PAC contig containing more than 70 known genes, 18 mapped microsatellites, and further ESTs/reference RNAs. A continuous block of strongly conserved synteny with mouse chromosome 11E2 and human chromosome 17q25.3 was found. Combining the sequence information from the rat and closely related syntenic regions of different mammalian species produces nearly complete gene maps as a basis for a positional candidate approach and gives insight into mammalian genomic evolution.
Assuntos
Mapeamento de Sequências Contíguas , Genes Supressores de Tumor , Ligação Genética , Neurilemoma/genética , Animais , Sequência de Bases , Cromossomos Artificiais Bacterianos , Primers do DNA , Etiquetas de Sequências Expressas , Perda de Heterozigosidade , Dados de Sequência Molecular , RatosRESUMO
The LEW/Ztm-ci2 rat is an autosomal recessive mutant that displays circling behavior, deafness, progressive retinopathy, locomotor hyperactivity, ataxia, and opisthotonus. We performed a genome-wide scan of a (LEW/Ztm-ci2 x BN/Ztm) F1 x LEW/Ztm-ci2 backcross population with anonymous microsatellite markers to analyze the genetics of this mutant rat. This linkage analysis demonstrated a very strong association of RNO10 SSLP markers to the phenotype with a core region in the central part of the chromosome. The knowledge of genes mapping to this part of the rat genome and their linkage to SSLP markers is still poor. We developed SSLP markers closely linked to genes, which might be responsible for the mutant phenotype by using the growing amount of rat-specific DNA sequences available at World Wide Web databases. Application of this method facilitated the search for candidate genes for the phenotype of the LEW-ci2 rat. We were able to map Myo15 and its neighboring genes, Znf179 and Aldh3a1, to the region of interest and Myo1c to a more distal location on RNO10. Further rat BAC clones were used to create a physical map of the region of interest. This map revealed the position of further genes. Among those is Kcnj12. Owing to their localization on RNO10 and their involvement in a similar pathology in human and mouse, Myo15 and Kcnj12 can be regarded as candidate genes for the deafblind phenotype of the LEW-ci2 rat.
Assuntos
Cegueira/genética , Surdez/genética , Animais , Sequência de Bases , Comportamento Animal , Mapeamento Cromossômico , DNA Complementar/genética , Marcadores Genéticos , Humanos , Camundongos , Miosinas/genética , Fenótipo , Canais de Potássio/genética , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Ratos MutantesRESUMO
Rats of the inbred BD strains strongly differ in their susceptibility to the induction of tumors of the central (CNS) and peripheral nervous system (PNS) by N-ethyl-N-nitrosourea (EtNU). Malignant schwannomas induced in (BDIX x BDIV) and (BDIX x BDVI) rat hybrids were analyzed to identify genetic alterations associated with EtNU-induced tumorigenesis in the PNS. EtNU-induced schwannomas exclusively exhibit an A:T T:A transversion mutation of the neu/Erbb-2 gene located on chromosome 10, with subsequent loss of the wild-type neu/Erbb-2 allele at a post-initiation stage. Targeted allelic deletion mapping previously revealed losses of heterozygosity (LOH) at the distal end of chromosome 10 in a large majority of (BDIX x BDIV) schwannomas. The aims of the present study were (i) to scan the whole genome for further LOHs; (ii) to narrow down the consensus regions of frequently occurring allelic deletions using tumors from different crosses of BD rats; and (iii) to determine the sequence of genetic alterations during schwannoma development. A limited number of (BDIX x BDIV) F(1) tumors were initially screened for LOH and microsatellite instability (MI) by amplifying 58 microsatellite markers spanning the whole genome. LOHs on chromosome 5 were detected in 9/17 tumors, with random loss of the parental alleles. Ninety-two schwannomas from different BD rat-crosses were then analyzed to solidify these data and to determine the consensus region of frequent LOHs. The results indicate that LOHs on chromosomes 10 and 5 are required for the development of EtNU-induced malignant schwannomas from immature neu/Erbb-2 mutant glial cells, and that putative tumor suppressor genes are localized on chromosome 10q32.3, corresponding to human chromosome 17q25.3, and the telomeric region of mouse chromosome 11, and on the telomeric quarter of chromosome 5. MI was detected in <0.2% of cases.