Emma Samuelson

Recurrent gene amplifications in human type I endometrial adenocarcinoma detected by fluorescence in situ hybridization

Determining what genes are actively involved in tumor development is important, because they may provide targets for directed therapy. Human tumors are greatly heterogeneous with respect to etiology and genetic background, which complicates the identification of common genetic aberrations. In contrast, genetic and environmental variation can be in part controlled in experimental animals, which facilitates identification of the important changes. In inbred BDII rats, which are genetically predisposed to endometrial adenocarcinomas (EAC), certain chromosome regions exhibit recurrent amplification in the tumors. Previous CGH analysis had shown that a subset of human EAC tumors exhibited increased copy numbers in the homologous chromosomal regions, located in human 2p21 approximately p25 and 7q21 approximately q31. Using fluorescence in situ hybridization analysis on imprints from 13 human EAC tumors, we determined the average copy numbers of each of 15 probes derived from cancer-related genes situated in these chromosome regions. Among the genes analyzed, those most often targeted by amplification were SDC1 and CYP1B1 in 2p21 approximately p25 and CDK6 and MET in 7q21 approximately q31, but all of the 15 genes tested were found to be amplified in at least two tumors.

Chromosome ideograms of the laboratory rat (Rattus norvegicus) based on high-resolution banding, and anchoring of the cytogenetic map to the DNA sequence by FISH in sample chromosomes

A detailed banded ideogram representation of the rat chromosomes was constructed based on actual G-banded prometaphase chromosomes. The approach yielded 535 individual bands, a significant increase compared to previously presented ideograms. The new ideogram was adapted to the existing band nomenclature. The gene locus positions in the rat draft DNA sequence were compared to the chromosomal positions as determined by dual-color FISH, using rat (RNO) chromosomes 6 and 15 and a segment of RNO4 as sample regions. It was found that there was generally an excellent correlation in the chromosome regions tested between the relative gene position in the DNA molecules and the sub-chromosomal localization by FISH and subsequent information transfer on ideograms from measurements of chromosomal images. However, in the metacentric chromosome (RNO15), the correlation was much better in the short arm than in the long arm, suggesting that the centromeric region may distort the linear relationship between the chromosomal image and the corresponding DNA molecule.

Molecular classification of spontaneous endometrial adenocarcinomas in BDII rats

Female rats of the BDII/Han inbred strain are prone to spontaneously develop endometrial carcinomas (EC) that in cell biology and pathogenesis are very similar to those of human. Human EC are classified into two major groups: Type I displays endometroid histology, is hormone-dependent, and characterized by frequent microsatellite instability and PTEN, K-RAS, and CTNNB1 (beta-Catenin) mutations; Type II shows non-endometrioid histology, is hormone-unrelated, displays recurrent TP53 mutation, CDKN2A (P16) inactivation, over-expression of ERBB2 (Her2/neu), and reduced CDH1 (Cadherin 1 or E-Cadherin) expression. However, many human EC have overlapping clinical, morphologic, immunohistochemical, and molecular features of types I and II. The EC developed in BDII rats can be related to type I tumors, since they are hormone-related and histologically from endometrioid type. Here, we combined gene sequencing (Pten, Ifr1, and Ctnnb1) and real-time gene expression analysis (Pten, Cdh1, P16, Erbb2, Ctnnb1, Tp53, and Irf1) to further characterize molecular alterations in this tumor model with respect to different subtypes of EC in humans. No mutation in Pten and Ctnnb1 was detected, whereas three tumors displayed sequence aberrations of the Irf1 gene. Significant down regulation of Pten, Cdh1, p16, Erbb2, and Ctnnb1 gene products was found in the tumors. In conclusion, our data suggest that molecular features of spontaneous EC in BDII rats can be related to higher-grade human type I tumors and thus, this model represents an excellent experimental tool for research on this malignancy in human.

Unraveling molecular signatures of immunostimulatory adjuvants in the female genital tract through systems biology

Sexually transmitted infections (STIs) unequivocally represent a major public health concern in both industrialized and developing countries. Previous efforts to develop vaccines for systemic immunization against a large number of STIs in humans have been unsuccessful. There is currently a drive to develop mucosal vaccines and adjuvants for delivery through the genital tract to confer protective immunity against STIs. Identification of molecular signatures that can be used as biomarkers for adjuvant potency can inform rational development of potent mucosal adjuvants. Here, we used systems biology to study global gene expression and signature molecules and pathways in the mouse vagina after treatment with two classes of experimental adjuvants. The Toll-like receptor 9 agonist CpG ODN and the invariant natural killer T cell agonist alpha-galactosylceramide, which we previously identified as equally potent vaginal adjuvants, were selected for this study. Our integrated analysis of genome-wide transcriptome data determined which signature pathways, processes and networks are shared by or otherwise exclusive to these 2 classes of experimental vaginal adjuvants in the mouse vagina. To our knowledge, this is the first integrated genome-wide transcriptome analysis of the effects of immunomodulatory adjuvants on the female genital tract of a mammal. These results could inform rational development of effective mucosal adjuvants for vaccination against STIs.

Local cytokine and inflammatory responses to candidate vaginal adjuvants in mice

The current study was undertaken to explore the correlation of adjuvanticity and local inflammatory response elicited in the murine vagina and the draining lymph nodes following local administration of two candidate vaginal adjuvants, Toll like receptor (TLR) 9 agonist CpG ODN, and a non-TLR targeting molecule alpha-galactosylceramide (alpha-GalCer). Using real-time PCR array analysis, we could show that a group of 13 common cytokine genes are activated in the vagina within 24h after vaginal administration of these adjuvants, including Ccl2, Ccl7, Ccl12, Ccl19, Ccl20, Ccl22, Cxcl1, Cxcl5, Il10 and the Th1-inducing molecules Ifng, Cxcl9, Cxcl10 and Cxcl11. A high degree of inflammation in and damage to the epithelium was exclusively observed in the vagina of the CpG ODN treated mice, which was reversed within 48h. These results indicate that there is a group of common genes that correlate with the adjuvanticity of CpG ODN and alpha-GalCer in the vagina, and that alpha-GalCer induces less of local inflammatory reactions in the murine vagina compared to CpG ODN.

Amplification Studies of MET and Cdk6 in a Rat Endometrial Tumor Model and Their Correlation to Human Type I Endometrial Carcinoma Tumors

Cancer is known to be a genetic disease that is both polygenic and heterogeneous, in most cases involving changes in several genes in a stepwise fashion. The spectrum of individual genes involved in the initiation and progression of cancer is greatly influenced by genetic factors unique to each patient. A study of complex diseases such as cancer is complicated by the genetic heterogeneous background and environmental factors in the human population. Endometrial cancer (EC) is ranked fourth among invasive tumors in women. In Sweden, approximately 1300 women (27/100,000 women) are diagnosed annually. To be able to study the genetic alterations in cancer, the use of an animal model is very convenient. Females of the BDII strain are genetically predisposed to EC and 90% of female BDII rats develop EC during their lifetime. Thus, BDII rats have been used to model human EC with respect to the genetics of susceptibility and of tumor development. A set of rat EC tumors was analyzed using conventional cytogenetics and comparative genome hybridization (CGH). Chromosomal aberrations, i.e., gains, were found on rat chromosome 4 (RNO4). Using FISH analysis, we concluded that the Met oncogene and Cdk6 (cyclin-dependent kinase 6) were amplified in this set of EC tumors. The data from this investigation were used to analyze a set of human endometrial tumors for amplification of Cdk6 and Met. Our preliminary data are indicative for a good correlation between our findings in the BDII rat model for EAC and the situation in human EC. These data provide strong support for the use of animal model systems for better understanding and scrutinizing of human complex disease of cancer.

Recurrent allelic imbalance at the rat Pten locus in DMBA-induced fibrosarcomas.

The tumor‐suppressor gene PTEN (phosphatase and tensin homolog) is frequently inactivated in different types of human tumors. Less is known about the involvement of the homologous gene Pten in animal model systems of cancer. By sequencing one of the introns of rat Pten, we found an informative intragenic PCR marker suitable for genetic studies. Through use of this marker, the position of Pten in the genetic linkage map was localized to the distal part of rat chromosome 1 (RNO1) by analysis of F2 progeny from an intercross between inbred strains BN and LE. Subsequently, 22 markers from this region (including the intragenic Pten marker) were used to study the occurrence of allelic imbalance in distal RNO1 in fibrosarcomas that had been induced by DMBA in F1(BN×LE) rats. The analysis revealed that allelic imbalance was common in the vicinity of Pten, and there was loss or reduction of one of the Pten alleles in more than 60% of the fibrosarcomas. DNA sequencing was preformed to investigate whether the Pten allele remaining in the tumors was inactivated by mutation. However, no mutations were detected in the genomic sequence of Pten exons 5 to 9 in any of the fibrosarcomas, and normal mRNA transcripts were expressed in all tumors. Thus, based on the targeted selection for loss of Pten observed in some of these tumors and the absence of inactivation of the remaining allele, we suggest that haploinsufficiency of Pten may be an important factor in rat DMBA‐induced fibrosarcomas.

BAC CGH-array identified specific small-scale genomic imbalances in diploid DMBA-induced rat mammary tumors

BackgroundDevelopment of breast cancer is a multistage process influenced by hormonal and environmental factors as well as by genetic background. The search for genes underlying this malignancy has recently been highly productive, but the etiology behind this complex disease is still not understood. In studies using animal cancer models, heterogeneity of the genetic background and environmental factors is reduced and thus analysis and identification of genetic aberrations in tumors may become easier. To identify chromosomal regions potentially involved in the initiation and progression of mammary cancer, in the present work we subjected a subset of experimental mammary tumors to cytogenetic and molecular genetic analysis.MethodsMammary tumors were induced with DMBA (7,12-dimethylbenz[a]anthrazene) in female rats from the susceptible SPRD-Cu3 strain and from crosses and backcrosses between this strain and the resistant WKY strain. We first produced a general overview of chromosomal aberrations in the tumors using conventional kartyotyping (G-banding) and Comparative Genome Hybridization (CGH) analyses. Particular chromosomal changes were then analyzed in more details using an in-house developed BAC (bacterial artificial chromosome) CGH-array platform.ResultsTumors appeared to be diploid by conventional karyotyping, however several sub-microscopic chromosome gains or losses in the tumor material were identified by BAC CGH-array analysis. An oncogenetic tree analysis based on the BAC CGH-array data suggested gain of rat chromosome (RNO) band 12q11, loss of RNO5q32 or RNO6q21 as the earliest events in the development of these mammary tumors.ConclusionsSome of the identified changes appear to be more specific for DMBA-induced mammary tumors and some are similar to those previously reported in ACI rat model for estradiol-induced mammary tumors. The later group of changes is more interesting, since they may represent anomalies that involve genes with a critical role in mammary tumor development. Genetic changes identified in this work are at very small scales and thus may provide a more feasible basis for the identification of the target gene(s). Identification of the genes underlying these chromosome changes can provide new insights to the mechanisms of mammary carcinogenesis.

Analysis of chromosomal aberrations involving chromosome 1q31→q53 in a DMBA-induced rat fibrosarcoma cell line: amplification and overexpression of Jak2

In a study of DMBA-induced rat fibrosarcomas we repeatedly found deletions and/or amplifications in the long arm of rat chromosome 1 (RNO1). Comparative genome hybridization showed that there was amplification involving RNO1q31→q53 in one of the DMBA-induced rat fibrosarcoma tumors (LB31) and a cell culture derived from it. To identify the amplified genes we physically mapped rat genes implicated in cancer and analyzed them for signs of amplification. The genes were selected based on their locations in comparative maps between rat and man. The rat proto-oncogenes Ccnd1, Fgf4, and Fgf3 (HSA11q13.3), were mapped to RNO1q43 by fluorescence in situ hybridization (FISH). The Ems1 gene was mapped by radiation hybrid (RH) mapping to the same rat chromosome region and shown to be situated centromeric to Ccnd1 and Fgf4. In addition, the proto-oncogenes Hras (HSA11p15.5) and Igf1r (HSA15q25→q26) were mapped to RNO1q43 and RNO1q32 by FISH and Omp (HSA11q13.5) was assigned to RNO1q34. PCR probes for the above genes together with PCR probes for the previously mapped rat genes Bax (RNO1q31) and Jak2 (RNO1q51→q53) were analyzed for signs of amplification by Southern blot hybridization. Low copy number increases of the Omp and Jak2 genes were detected in the LB31 cell culture. Dual color FISH analysis of tumor cells confirmed that chromosome regions containing Omp and Jak2 were amplified and were situated in long marker chromosomes showing an aberrant banding pattern. The configuration of the signals in the marker chromosomes suggested that they had arisen by a break-fusion-bridge (BFB) mechanism.

Absence of Ras mutations in rat DMBA-induced mammary tumors.

Animal cancer models reduce genetic background heterogeneity and thus, may facilitate identification and analysis of specific genetic aberrations in tumor cells. Rat and human mammary glands have high similarity in physiology and show comparable hormone responsiveness. Thus, spontaneous and carcinogen (e.g., NMU and DMBA)‐induced rat mammary models are valuable tools for genetic studies of breast cancer. In NMU‐induced rat mammary tumors, activating mutations in Hras codon 12 have frequently been reported and are supposed to contribute to the mammary carcinogenic process. Involvement of Ras mutations in DMBA‐induced tumors is less clear. In the present study we investigated the mutation status of the three Ras genes, Hras, Kras, and Nras, in DMBA‐induced rat mammary tumors. We examined codons 12, 13, and 61 of all three genes for mutations in 71 tumors using direct sequencing method that in experimental conditions is sensitive enough to detect single nucleotide mutations even when present in only 25% of the test sample. No activating Ras gene mutation was found. Thus, in contrast to NMU‐induced rat mammary tumor, tumorigenesis in DMBA‐induced rat mammary tumors seems to be independent on activating mutations in the Ras genes. Our finding suggests that the genetic pathways selected in mammary tumor development are influenced by and perhaps dependent on the identity of the inducing agent, again emphasizing the importance of tumor etiology on the genetic changes in the tumor cells.