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Characterization of Mcs7, an Inherited Mammary Carcinoma Susceptibility Locus on Rat Chromosome 10
Background/Hypothesis: Inbred rat strains vary in their susceptibility to induction of mammary carcinomas with chemical carcinogens. The Wistar Kyoto (WKy) rat is resistant, while the Wistar Furth (WF) strain is sensitive. Analysis of genetic crosses between these strains indicated several WKy quantitative trait loci (QTLs) associated with both susceptibility and resistance. The mammary carcinoma susceptibility locus, Mcs7, located on chromosome 10, is associated with increased susceptibility. Mcs7 differs from other WKy QTLs in that it contains a region of allelic imbalance (AI) in mammary carcinomas from (WKy X WF) F1 rats. This region is homologous to the distal q arm of human chromosome 17, a common site of AI in breast cancer. Together, these observations support the hypothesis that the locus contains one or more genes controlling susceptibility, and that they may be contained within the region of allelic imbalance.
Aims: The goal of this research is to identify candidate genes in the Mcs7 region modulating mammary cancer susceptibility, so that they can be evaluated for their role in human breast cancer. The specific aims are: (1) to verify the association of the Mcs7 locus with increased susceptibility by testing congenic rats with the WKy Mcs7 locus on the genetic background of the WF strain for susceptibility to 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis; (2) to identify potential candidate genes in the Mcs7 locus associated with this trait by (a) collecting recombinant congenics with Mcs7 sub-loci for phenotyping, including those containing the region of greatest frequency of AI, (b) selecting the sub-locus with the strongest association to the trait to find potential candidate genes from comparative genetic maps, and (c) comparing the WF and WKy strains for sequence polymorphisms and differences in mRNA/protein expression of potential candidate genes; and (3) to use gene chips for comparison of mammary gland RNA expression in WF.WKy-Mcs7 congenics with WF controls, providing information as to the molecular pathways modulated by Mcs7 genes, and possibly finding candidates in the Mcs7 locus.
Study Design: Congenic lines were initiated by crosses between WKy and WF strains and selection of male carriers heterozygous for markers in the Mcs7 locus. Male carriers will be backcrossed continually to WF females, until the rats are fully congenic (N10 generation). Testing for the phenotype of increased susceptibility will be done using DMBA to induce mammary carcinomas in the N8 (semi-speed congenic) and the N10 (full congenic) generations. Other lines will be generated for phenotyping using male carriers heterozygous for sub-regions of the Mcs7 locus. If multiple sub-regions are associated with the trait, the sub-locus with the strongest association will be chosen for finding potential candidate genes from other species¡¯ comparative genetic maps. Gene chip analysis of mammary gland RNA will be done to identify differences in gene expression between WF.WKy-Mcs7 congenics and WF controls, in both DMBA-treated and untreated rats. Additional chips may be used to compare mammary gland RNA from rats with Mcs7 sub-loci to RNA of WF controls. Potential candidate genes identified using congenics and gene chips will be tested for polymorphism between the WKy and WF strains by sequence comparison. Additionally, strain differences in mRNA/protein expression of potential candidate genes will be assessed by real time quantitative PCR (RT-PCR) and western blotting.
Potential Outcomes/Benefits: Since the identification of low-penetrance, high-frequency genes controlling breast cancer risk directly in humans is difficult, the rat model provides an excellent alternative to locate potential human modifier genes using comparative genomics. Once genes controlling susceptibility are identified in the rat, their human homologues can be evaluated for their role in breast cancer risk. These genes can then be used to assess breast cancer risk in populations, to identify susceptible women, and to provide targets for chemopreventive agents.
Breast cancer incidence is controlled by both environmental and hereditary factors. Although research efforts have identified some genes that are responsible for hereditary breast cancer, many cases with strong family histories cannot be attributed to these genes. Discovery of these genes is important in identifying women with increased susceptibility to breast cancer, so that preventive measures may be taken. Characterization of the genes and their functions also may aid in the development of drugs that can be used for prevention. Since studies in humans to identify genes are difficult to carry out, animal models can be used to both define hereditary patterns of breast cancer susceptibility and to identify genes for evaluation in humans. The rat model is excellent for the study of breast cancer, as the rat mammary gland is structurally and developmentally similar to humans. Rat strains vary in their susceptibility to mammary cancer. The Wistar-Kyoto (WKy) strain is resistant, while the Wistar-Furth (WF) strain is susceptible. Our research involves using the genetic differences in rats to determine which genes confer the traits of susceptibility and resistance to mammary cancer. The goal of the proposed research is to identify these genes in the rat, which will lead to the finding of homologous (similar) genes in humans for testing in population studies.
My lab has previously identified loci, or regions of DNA, that are associated with susceptibility and resistance to chemically-induced mammary carcinogenesis in several rat strains. The WKy rat is resistant, but has individual loci associated with both resistance (Mcs5, -6, -8) and susceptibility (Mcsm1 and Mcs7). The proposed research will focus on the susceptibility locus, Mcs7. Interestingly, when our lab analyzed the Mcs loci using DNA from rat mammary carcinomas in offspring derived from a genetic cross between WKy and WF animals, a genetic aberration, allelic imbalance, was detected only in the Mcs7 region. The locus affected by allelic imbalance in the rat is homologous to an area of human chromosome 17, a common site of genetic aberrations in human breast cancer. We hypothesize that the Mcs7 locus contains one or more genes that control susceptibility.
We will study the independent effect of the Mcs7 locus on susceptibility without the other Mcs loci using congenic rats. These rats will have the background genetics of the susceptible strain (WF), and carry only a small region of WKy DNA (Mcs7 locus). We predict that congenics carrying the WKy Mcs7 locus will have an increased susceptibility to chemically-induced mammary cancer compared with the WF strain. Since the Mcs7 locus is large and contains many genes, we will narrow the region associated with susceptibility by generating congenics with sub-regions of the Mcs7 locus and testing for susceptibility. Once the locus associated with susceptibility is made smaller, we will use computer databases to find candidate genes (those which control the trait) in the region for testing. To test candidates, we will look for differences between the WF and WKy strains in: (1) base pair DNA sequence (polymorphisms), (2) expression level in the mammary gland, and (3) expression of the proteins encoded by these genes in the mammary gland.
As a complementary strategy, we will compare gene expression in the mammary glands of congenic rats with WF rats using gene chips. Chips allow for the simultaneous measurement of thousands of genes and will find differences in expression of genes throughout the genome. By analyzing expression differences, we have the potential to identify genes that are under the control of Mcs7 genes. This information will aid in determining how the biological processes occurring in the mammary gland are affected by Mcs7 genes.
Thus, the identification of Mcs7 susceptibility genes, their downstream effectors, and possible human homologues will enhance our understanding of breast cancer genetics. Such knowledge could help identify women at higher risk to developing breast cancer and also provide unique targets for customized molecular drugs to alleviate this increased genetic sensitivity.