Bernd Koelsch

Sex-biased suppression of chemically induced neural carcinogenesis in congenic BDIX.BDIV-Mss4a rats

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.

Allele-specific losses of heterozygosity on chromosomes 1 and 17 revealed by whole genome scan of ethylnitrosourea-induced BDIXBDIV hybrid rat gliomas

The induction of neural tumors by N‐ethyl‐N‐nitrosourea (EtNU) in inbred strains of rats has evolved as a valuable model system of developmental stage– and cell type–dependent oncogenesis. Tumor yield and latency times are strongly influenced by genetic background. Compared with BDIX rats, BDIV rats are relatively resistant to the induction of brain tumors by EtNU, with a lower tumor incidence and latency periods prolonged by a factor of 3. To characterize genetic abnormalities associated with impaired tumor suppressor gene function in neuro‐oncogenesis, losses of heterozygosity (LOHs) and microsatellite instability (MI) were investigated in brain tumors induced by EtNU in (BDIV×BDIX) F1 and F2 rats. The polymerase chain reaction was used to amplify 55 polymorphic microsatellite markers spanning the entire rat genome. The tumors displayed different histologies and grades of malignancy, corresponding to part of the spectrum of human gliomas. MI was not observed in any of the tumors. LOH of rat chromosome 1q was predominantly detected in oligodendrogliomas and mixed gliomas, with a 30% incidence in informative cases. 11p15.5, the human genome region syntenic to the consensus region of LOHs observed on rat chromosome 1, has been shown to be involved in the formation of gliomas in humans. Furthermore, rat brain tumors of different histologies often showed allelic imbalances on chromosome 17p. In both cases of LOH, there was a clear bias in favor of the parental BDIV allele, suggesting the involvement of tumor suppressor genes functionally polymorphic between the two rat strains. Mol. Carcinog. 26:163–171, 1999.

Braf Mutations Initiate the Development of Rat Gliomas Induced by Postnatal Exposure to N-Ethyl-N-Nitrosourea

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.

Chemically induced rat Schwann cell neoplasia as a model for early-stage human peripheral nerve sheath tumors: phenotypic characteristics and dysregulated gene expression.

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-β 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.

Genetic dissection of the Mss4 locus mediating sex-biased cancer resistance in the rat peripheral nervous system

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.

Chemically Induced Oncogenesis in the Peripheral Nervous System Is Suppressed in Congenic BDIX.BDIV-Mss1 and -Mss7 Rats

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.

Neuro-oncogenesis Induced by Nitroso Compounds in Rodents and Strain-Specific Genetic Modifiers of Predisposition

The alkylating substances N-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) are the most potent systemically acting neurocarcinogens in rodents. They induce tumors of the central (CNS) and peripheral nervous system (PNS), which are similar to their human counterparts. The neuro-oncogenic effect of both chemicals depends on the dose, the rodent species, the strain, and the developmental stage at exposure. Neuro-oncogenesis induced by MNU and ENU has been widely used as a model system including studies of molecular mechanisms of malignant transformation, screening of therapeutics, and lately, the genetics of predisposition toward the development of these tumors. The induction of malignant tumors in the PNS of differentially susceptible rat strains made it possible to identify gene loci influencing tumor risk in an allele- and sex-specific way and to gain insight into molecular and cellular mechanisms underlying tumor susceptibility and resistance.