Genetic control of estrogen action in the rat: mapping of QTLs that impact pituitary lactotroph hyperplasia in a BN × ACI intercross

Estrogens are important regulators of growth and development and contribute to the etiology of several types of cancer. Different inbred rat strains exhibit marked, cell-type-specific differences in responsiveness to estrogens as well as differences in susceptibility to estrogen-induced tumorigenesis. Regulation of pituitary lactotroph homeostasis is one estrogen-regulated response that differs dramatically between different inbred rat strains. In this article we demonstrate that the growth response of the anterior pituitary gland of female ACI rats to 17β-estradiol (E2) markedly exceeds that of identically treated female Brown Norway (BN) rats. We further demonstrate that pituitary mass, a surrogate indicator of absolute lactotroph number, behaves as a quantitative trait in E2-treated F₂ progeny generated in a genetic cross originating with BN females and ACI males. Composite interval mapping analyses of the (BN×ACI)F₂ population revealed quantitative trait loci (QTLs) that exert significant effects on E2-induced pituitary growth on rat chromosome 4 (RNO4) (Ept5) and RNO7 (Ept7). Continuous treatment with E2 rapidly induces mammary cancer in female ACI rats but not BN rats, and QTLs that impact susceptibility to E2-induced mammary cancer in the (BN×ACI)F₂ population described here have been mapped to RNO3 (Emca5), RNO4 (Emca6), RNO5 (Emca8), RNO6 (Emca7), and RNO7 (Emca4). Ept5 and Emca6 map to distinct regions of RNO4. However, Ept7 and Emca4 map to the same region of RNO7. No correlation between pituitary mass and mammary cancer number at necropsy was observed within the (BN×ACI)F₂ population. This observation, together with the QTL mapping data, indicate that with the exception of the Ept7/Emca4 locus on RNO7, the genetic determinants of E2-induced pituitary growth differ from the genetic determinants of susceptibility to E2-induced mammary cancer.     

Genetic mapping of Eutr1, a locus controlling E2-induced pyometritis in the Brown Norway rat, to RNO5

In certain rat strains, chronic estrogen administration can lead to pyometritis, an inflammation of the uterus accompanied by infection and the accumulation of intraluminal pus. In this article, we report that the Brown Norway (BN) rat is highly susceptible to pyometritis induced by 17β-estradiol (E2). The susceptibility of the BN rat to E2-induced pyometritis appears to segregate as a recessive trait in crosses to the resistant August × Copenhagen Irish (ACI) strain. In a (BN × ACI)F₂ population, we find strong evidence for a major genetic determinant of susceptibility to E2-induced pyometritis on rat chromosome 5 (RNO5). Our data are most consistent with a model in which the BN allele of this locus, designated Eutr1 (Estrogen-induced uterine response 1), acts in an incompletely dominant manner to control E2-induced pyometritis. Furthermore, we have confirmed the contribution of Eutr1 to E2-induced uterine pyometritis using an RNO5 congenic rat strain. In addition to Eutr1, we obtained evidence suggestive of linkage for five additional loci on RNO2, 4, 11, 17, and X that control susceptibility to E2-induced pyometritis in the (BN × ACI)F₂ population.     

Genetic identification of multiple loci that control breast cancer susceptibility in the rat.

We have used a rat model of induced mammary carcinomas in an effort to identify breast cancer susceptibility genes. Using genetic crosses between the carcinoma-resistant Copenhagen (COP) and carcinoma-sensitive Wistar-Furth rats, we have confirmed the identification of the Mcs1 locus that modulates tumor number. We have now also identified two additional loci, Mcs2 and Mcs3. These three loci map to chromosomes 2, 7, and 1, respectively, and interact additively to suppress mammary carcinoma development in the COP strain. They are responsible for a major portion of the tumor-resistant phenotype of the COP rat. No loss of heterozygosity was observed surrounding the three loci. A fourth COP locus, Mcs4, has also been identified on chromosome 8 and acts in contrast to increase the number of carcinomas. These results show that mammary carcinoma susceptibility in the COP rat is a polygenic trait. Interestingly, a polymorphism in the human genomic region homologous to the rat Mcs4 region is associated with an increased breast cancer risk in African-American women. The isolation of the Mcs genes may help elucidate novel mechanisms of carcinogenesis, provide information important for human breast cancer risk estimation, and also provide unique drug discovery targets for breast cancer prevention.     

Genetic bases of estrogen-induced pituitary tumorigenesis: identification of genetic loci determining estrogen-induced pituitary growth in reciprocal crosses between the ACI and Copenhagen rat strains

Estrogens stimulate proliferation and enhance survival of the prolactin (PRL)-producing lactotroph of the anterior pituitary gland and induce development of PRL-producing pituitary tumors in certain inbred rat strains but not others. The goal of this study was to elucidate the genetic bases of estrogen-induced pituitary tumorigenesis in reciprocal intercrosses between the genetically related ACI and Copenhagen (COP) rat strains. Following 12 weeks of treatment with the synthetic estrogen diethylstilbestrol (DES), pituitary mass, an accurate surrogate marker of absolute lactotroph number, was increased 10.6-fold in ACI rats and 4.5-fold in COP rats. Composite interval mapping analyses of the phenotypically defined F(2) progeny from the reciprocal crosses identified six quantitative trait loci (QTL) that determine the pituitary growth response to DES. These loci reside on chromosome 6 [Estrogen-induced pituitary tumor (Ept)1], chromosome 3 (Ept2 and Ept6), chromosome 10 (Ept9), and chromosome 1 (Ept10 and Ept13). Together, these six Ept loci and one additional suggestive locus on chromosome 4 account for an estimated 40% of the phenotypic variance exhibited by the combined F(2) population, while 34% of the phenotypic variance was estimated to result from environmental factors. These data indicate that DES-induced pituitary mass behaves as a quantitative trait and provide information that will facilitate identification of genes that determine the tumorigenic response of the pituitary gland to estrogens.     

Genetic identification of distinct loci controlling mammary tumor multiplicity, latency, and aggressiveness in the rat

The rat is considered an excellent model for studying human breast cancer. Therefore, understanding the genetic basis of susceptibility to mammary cancer in this species is of great interest. Previous studies based on crosses involving the susceptible strain WF (crossed with the resistant strains COP or WKY) and focusing on tumor multiplicity as the susceptibility phenotype led to the identification of several loci that control chemically induced mammary cancer. The present study was aimed to determine whether other loci can be identified by analyzing crosses derived from another susceptible strain on the one hand, and by including phenotypes other than tumor multiplicity on the other hand. A backcross was generated between the susceptible SPRD-Cu3 strain and the resistant WKY strain. Female progeny were genotyped with microsatellite markers covering all rat autosomes, treated with a single dose of DMBA, and phenotyped with respect to tumor latency, tumor multiplicity, and tumor aggressiveness. Seven loci controlling mammary tumor development were detected. Different loci control tumor multiplicity, latency, and aggressiveness. While some of these loci colocalize with loci identified in crosses involving the susceptible strain WF, new loci have been uncovered, indicating that the use of distinct susceptible and resistant strain pairs will help in establishing a comprehensive inventory of mammary cancer susceptibility loci.     

Multiple Susceptibility Loci for Radiation-Induced Mammary Tumorigenesis in F2[Dahl S x R]-Intercross Rats

Although two major breast cancer susceptibility genes, BRCA1 and BRCA2, have been identified accounting for 20% of breast cancer genetic risk, identification of other susceptibility genes accounting for 80% risk remains a challenge due to the complex, multi-factorial nature of breast cancer. Complexity derives from multiple genetic determinants, permutations of gene-environment interactions, along with presumptive low-penetrance of breast cancer predisposing genes, and genetic heterogeneity of human populations. As with other complex diseases, dissection of genetic determinants in animal models provides key insight since genetic heterogeneity and environmental factors can be experimentally controlled, thus facilitating the detection of quantitative trait loci (QTL). We therefore, performed the first genome-wide scan for loci contributing to radiation-induced mammary tumorigenesis in female F2-(Dahl S x R)-intercross rats. Tumorigenesis was measured as tumor burden index (TBI) after induction of rat mammary tumors at forty days of age via ¹²⁷Cs-radiation. We observed a spectrum of tumor latency, size-progression, and pathology from poorly differentiated ductal adenocarcinoma to fibroadenoma, indicating major effects of gene-environment interactions. We identified two mammary tumorigenesis susceptibility quantitative trait loci (Mts-QTLs) with significant linkage: Mts-1 on chromosome-9 (LOD-2.98) and Mts-2 on chromosome-1 (LOD-2.61), as well as two Mts-QTLs with suggestive linkage: Mts-3 on chromosome-5 (LOD-1.93) and Mts-4 on chromosome-18 (LOD-1.54). Interestingly, Chr9-Mts-1, Chr5-Mts-3 and Chr18-Mts-4 QTLs are unique to irradiation-induced mammary tumorigenesis, while Chr1-Mts-2 QTL overlaps with a mammary cancer susceptibility QTL (Mcs 3) reported for 7,12-dimethylbenz-[α]antracene (DMBA)-induced mammary tumorigenesis in F2[COP x Wistar-Furth]-intercross rats. Altogether, our results suggest at least three distinct susceptibility QTLs for irradiation-induced mammary tumorigenesis not detected in genetic studies of chemically-induced and hormone-induced mammary tumorigenesis. While more study is needed to identify the specific Mts-gene variants, elucidation of specific variant(s) could establish causal gene pathways involved in mammary tumorigenesis in humans, and hence novel pathways for therapy.     

Localization of Eutr2, a locus controlling susceptibility to DES-induced uterine inflammation and pyometritis, to RNO5 using a congenic rat strain

In some rat strains chronic administration of exogenous estrogens induces pyometritis, an inflammation of the uterus associated with infection, suggesting that there is genetic variation in susceptibility to estrogen-induced inflammation and pyometritis. In this article we report that following 10 weeks of treatment with the synthetic estrogen diethylstilbestrol (DES), Fisher 344 (F344) rats exhibit modest uterine inflammation and a 0% incidence of pyometritis. By contrast, under identical experimental conditions, Brown Norway (BN) rats exhibit significant inflammation and a 100% incidence of pyometritis. Similarly, we also observed profound uterine inflammation and a 100% incidence of pyometritis in a congenic rat strain in which a segment of RNO5 from the BN strain is carried on the F344 strain. These data suggest that a locus on RNO5 controls both the magnitude of DES-induced uterine inflammation and susceptibility to DES-induced pyometritis. This locus, designated Eutr2, resides within the same segment of RNO5 as the Eutr1 locus, which confers susceptibility to E2-induced pyometritis in an F2 population generated in a cross between the BN and August × Copenhagen 9935, Irish (ACI) strains. Jyotsna Pandey, Karen A. Gould contributed equally to this work.     

Estrogen-Induced Rat Breast Carcinogenesis is Characterized by Alterations in DNA Methylation,Histone Modifications,and Aberrant microRNA Expression

Breast cancer is the most common malignancy in women continuing to rise worldwide. Breast cancer emerges through a multi-step process, encompassing progressive changes from a normal cell to hyperplasia (with and without atypia), carcinoma in situ, invasive carcinoma, and metastasis. In the current study, we analyzed the morphological changes and alterations of DNA methylation, histone methylation and microRNA expression during estradiol-17β (E2)-induced mammary carcinogenesis in female August Copenhagen Irish (ACI) rats. E2-induced breast carcinogenesis in ACI rats provides a physiologically relevant and genetically defined animal model for studying human sporadic breast cancer. The pattern of morphological changes in mammary glands during E2-induced carcinogenesis was characterized by transition from normal appearing alveolar and ductular hyperplasia to focal hyperplastic areas of atypical glands and ducts accompanied by a rapid and sustained loss of global DNA methylation, LINE-1 hypomethylation, loss of histone H3 lysine 9 and histone H4 lysine 20 trimethylation, and altered microRNAs expression. More importantly, these alterations in the mammary tissue occurred after 6 weeks of E2-treatment, whereas the atypical hyperplasia, which represents a putative precursor lesion to mammary carcinoma in this model, was detected only after 12 weeks of exposure, demonstrating clearly that these events are directly associated with the effects of E2 and are not a consequence of the preexisting preneoplastic lesions. The results of this study show that deregulation of cellular epigenetic processes plays a crucial role in the mechanism of E2-induced mammary carcinogenesis in ACI rats, especially in the tumor initiation process.     

Physical confirmation and mapping of overlapping rat mammary carcinoma susceptibility QTLs, Mcs2 and Mcs6

Only a portion of the estimated heritability of breast cancer susceptibility has been explained by individual loci. Comparative genetic approaches that first use an experimental organism to map susceptibility QTLs are unbiased methods to identify human orthologs to target in human population-based genetic association studies. Here, overlapping rat mammary carcinoma susceptibility (Mcs) predicted QTLs, Mcs6 and Mcs2, were physically confirmed and mapped to identify the human orthologous region. To physically confirm Mcs6 and Mcs2, congenic lines were established using the Wistar-Furth (WF) rat strain, which is susceptible to developing mammary carcinomas, as the recipient (genetic background) and either Wistar-Kyoto (WKy, Mcs6) or Copenhagen (COP, Mcs2), which are resistant, as donor strains. By comparing Mcs phenotypes of WF.WKy congenic lines with distinct segments of WKy chromosome 7 we physically confirmed and mapped Mcs6 to ~33 Mb between markers D7Rat171 and gUwm64-3. The predicted Mcs2 QTL was also physically confirmed using segments of COP chromosome 7 introgressed into a susceptible WF background. The Mcs6 and Mcs2 overlapping genomic regions contain multiple annotated genes, but none have a clear or well established link to breast cancer susceptibility. Igf1 and Socs2 are two of multiple potential candidate genes in Mcs6. The human genomic region orthologous to rat Mcs6 is on chromosome 12 from base positions 71,270,266 to 105,502,699. This region has not shown a genome-wide significant association to breast cancer risk in pun studies of breast cancer susceptibility.     

Partial inhibition of estrogen-induced mammary carcinogenesis in rats by tamoxifen: balance between oxidant stress and estrogen responsiveness

Epidemiological and experimental evidences strongly support the role of estrogens in breast tumor development. Both estrogen receptor (ER)-dependent and ER-independent mechanisms are implicated in estrogen-induced breast carcinogenesis. Tamoxifen, a selective estrogen receptor modulator is widely used as chemoprotectant in human breast cancer. It binds to ERs and interferes with normal binding of estrogen to ERs. In the present study, we examined the effect of long-term tamoxifen treatment in the prevention of estrogen-induced breast cancer. Female ACI rats were treated with 17β-estradiol (E2), tamoxifen or with a combination of E2 and tamoxifen for eight months. Tissue levels of oxidative stress markers 8-iso-Prostane F(2α) (8-isoPGF(2α)), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, and oxidative DNA damage marker 8-hydroxydeoxyguanosine (8-OHdG) were quantified in the mammary tissues of all the treatment groups and compared with age-matched controls. Levels of tamoxifen metabolizing enzymes cytochrome P450s as well as estrogen responsive genes were also quantified. At necropsy, breast tumors were detected in 44% of rats co-treated with tamoxifen+E2. No tumors were detected in the sham or tamoxifen only treatment groups whereas in the E2 only treatment group, the tumor incidence was 82%. Co-treatment with tamoxifen decreased GPx and catalase levels; did not completely inhibit E2-mediated oxidative DNA damage and estrogen-responsive genes monoamine oxygenase B1 (MaoB1) and cell death inducing DFF45 like effector C (Cidec) but differentially affected the levels of tamoxifen metabolizing enzymes. In summary, our studies suggest that although tamoxifen treatment inhibits estrogen-induced breast tumor development and increases the latency of tumor development, it does not completely abrogate breast tumor development in a rat model of estrogen-induced breast cancer. The inability of tamoxifen to completely inhibit E2-induced breast carcinogenesis may be because of increased estrogen-mediated oxidant burden.     

Generation of a polymorphic marker linked to thymoma susceptibility gene of rat 1 by genetically-directed representational difference analysis.

BUF/Mna (BUF) is a rat strain susceptible to spontaneous development of thymomas. We have previously shown that the thymoma susceptibility is controlled principally by a dominant susceptibility gene located on chromosome 7, thymoma susceptibility gene of rat 1 (Tsr1). To generate genetic markers tightly linked to Tsr1, we performed genetically directed representational difference analysis (GDRDA) with three combinations of the tester and driver DNAs. From 124 ?ACI/NMs x (BUF x ACI/NMs) F1? backcross rats, 12 rats with the ACI/BUF genotype in the Tsr1 region (A/B rats) and 13 rats with the ACI/ACI genotype in the region (A/A rats) were selected, and their DNAs were pooled, respectively. Three kinds of tester DNAs, i) inbred BUF, ii) (BUF x ACI)F1, and iii) the pool from the A/B rats, were subtracted by the driver DNA prepared from the pool of the A/A rats. The three combinations yielded one, two, and one polymorphic marker(s), respectively. One marker, D7Ncc28, was isolated commonly by the three combinations of subtraction, and another marker, D11Ncc12 was isolated only by the second combination. Linkage analysis demonstrated that D7Ncc28 was located in the 8.3 cM region where Tsr1 has been mapped. The three combinations of subtraction were shown to be almost equally capable of isolating polymorphic markers in a specific chromosomal region.     

Genetics of meprin in the rat

The locus encoding for meprin activity in kidney was found polymorphic in the rat. The BDIV and COP strains that carried theMep-1 ^a allele had high meprin concentration in the kidney, while the ACI, BN, LE and LEW strains that carried theMep-1 ^b allele had low meprin levels. TheMep-1 ^a allele that controlled the high activity enzyme was expressed in a dominant fashion in the (BN × COP) F1 progeny. The backcross studies showed that the segregation of alleles at theMep-1 and theGlo-1 loci was normal and that the genes were linked (x ² = 4.06,P < 0.05). The map distance between theMep-1 andGlo-1 genes was 41 ± 4 centimorgans.     

Genetic control of susceptibility to carcinogen-induced colorectal cancer in mice: The Ccs3 and Ccs5 loci regulate different aspects of tumorigenesis

Colorectal cancer (CRC) is a multistep disease that involves a two-way interaction between a complex genetic pre-disposition component, and a set of poorly understood extrinsic environmental factors. In mice, CRC can be induced by treatment with azoxymethane (AOM). Using a set of AcB/BcA recombinant congenic strains derived from CRC-susceptible A/J and CRC-resistant C57Bl/6J (B6) progenitors, we previously detected the Ccs3 locus (colon cancer susceptibility locus 3) as a major regulator of CRC susceptibility. Phenotyping of additional AcB/BcA strains for susceptibility to AOM-induced CRC has refined the Ccs3 interval to a 6.7 Mb segment on chromosome 3. In addition, the presence of intermediate susceptibility phenotypes in individual AcB/BcA strains suggested additional gene effects regulating CRC susceptibility in A/J and B6 strains. Those were investigated by linkage analysis and whole genome scanning in a set of 208 informative (B6 x A/J)F2 progeny, using tumor multiplicity as a quantitative measure of susceptibility. This analysis validated the important role of Ccs3 in regulating this trait, and additionally detected contribution from a second locus on the distal portion of chromosome 9 (LOD = 3.76), that was given the temporary designation of Ccs5. Ccs5 modulates tumor multiplicity in F2 animals bearing at least one A/J-derived susceptibility allele at Ccs3, with A/J-derived Ccs5 susceptibility alleles being inherited in a recessive manner. There is a strong additive effect of Ccs3 and Ccs5 on tumor multiplicity in F2 mice: mice doubly homozygotes for A/J or B6 alleles at Ccs3 and Ccs5 show tumor numbers similar to those of parental A/J and B6, respectively. Interestingly, the Ccs5 region overlaps several quantitative trait loci previously reported to regulate intestinal homeostasis and susceptibility to intestinal colitis in mice and humans. Our findings identify a novel two-locus system regulating CRC susceptibility in mice, of which the relevance to human CRC can now be tested experimentally.     

Analysis of candidate genes included in the mammary cancer susceptibility 1 (Mcs1) region

The rat strain COP is resistant to spontaneous and carcinogen-induced mammary cancer, whereas the strain WF is susceptible. Using genetic linkage analysis of (WF × COP) F₁× WF backcrosses, LC Hsu, LA Shepel and co-workers showed that a region at the centromeric end of Chromosome (Chr) 2 (2q1) segregates with the sensitivity to mammary cancer development. The responsible locus was named Mcs1 (for mammary cancer susceptibility 1). We have developed the chromosome map of the 2q1 region by localizing 18 genes, 4 ESTs, and several anonymous markers, using radiation hybrids and fluorescence in situ hybridization. The region containing Mcs1 was delineated to 2q12–q14. Five of the genes (Mef2c, Map1b, Ccnh, Rasa, Rasgrf2) were assigned to this region and were shown to be expressed in the rat mammary glands, while three possible functional candidate genes, Pi3kr1, Rad17, and Naip, were excluded from the critical region. Since cyclin H, encoded by Ccnh, plays an important role in the control of the cell cycle and since the proteins encoded by Rasa and Rasgrf2 control the activity of the RAS oncoprotein, the corresponding genes appeared as both functional and positional Mcs1 candidates. RT-PCR experiments on RNA extracted from mammary glands of the two rat strains (COP, WF) was done. No amino acid sequence difference was found between the two strains. These results argue against the hypothesis that any of these three genes is Mcs1. Received: 25 September 2000 / Accepted: 15 November 2000     

Rat mutations cvd and hob with cerebellar malformations map to chromosome 2.

In this paper, we executed genome mapping and comparative mapping analyses for cvd and hob, autosomal recessive mutations with cerebellar vermis defect and cerebellar dysplasia in the rat. For the linkage analysis, we produced three sets of backcross progeny, (ACI x CVD)F(1) and (F344 x CVD)F(1) females crossed to a cvd homozygous male rat, and (HOB x WKY)F(1) males crossed to hob homozygous female rats. Analysis of the segregation patterns of simple sequence length polymorphism (SSLP) markers scanning the whole rat genome allowed the mapping of these autosomal recessive mutations to rat Chromosome (Chr) 2. The most likely gene order is D2Mgh12 - D2Rat86 - D2Mit15 - D2Rat185 - cvd - D2Rat66 - D2Mgh13, and D2Mit18 - Fga -D2Mit14 - D2Rat16 - hob - D2Mgh13. Crossing test between a proven cvd heterozygous and a hob heterozygous rats demonstrated their allelism. Furthermore, comparative mapping indicated the cvd locus corresponds to mouse chromosome 3 and a strong candidate gene Unc5h3, a causative gene for the rostral cerebellar malformation mouse, was implicated.     

Genetic mapping of a Ptch1-associated rhabdomyosarcoma susceptibility locus on mouse chromosome 2

Mutations in the Patched (Ptch1) gene are responsible for various familial and sporadic cancers. Ptch1(neo67/+) mice, in which exons 6 and 7 are deleted, show genetic background-dependent susceptibility to the development of muscle tumors resembling human rhabdomyosarcoma (RMS); BALB/c (BALB) is a susceptible strain whereas C57BL/6 (B6) shows resistance. A genome-wide linkage analysis was carried out using Ptch1(neo67/+)mice produced from B6 x (BALB x B6) backcrosses to identify loci involved in the control of RMS susceptibility. Quantitative trait locus mapping with the censored tumor latency time as the quantitative parameter was used to detect a significant RMS susceptibility modifier locus, Parms1 (Patched-Associated RMS 1), on chromosome 2 between D2Mit37 and D2Mit102 (LRS = 10). A Kaplan-Meier survival curve revealed that mice with the B6/BALB genotype develop tumors more frequently and much faster as compared to mice homozygous for the B6 allele (P = 0.02). Additional loci not reaching linkage significance were also detected for medulloblastoma resistance.     

Genetic bases of renal agenesis in the ACI rat: mapping of Renag1 to chromosome 14

Unilateral renal agenesis (URA) is a common developmental defect in humans, occurring at a frequency of approximately 1 in 500–1000 births. Several genetic syndromes include bilateral or unilateral renal agenesis as an associated phenotype. However, URA frequently occurs in individuals not afflicted by these syndromes and is often asymptomatic. Although it is clear that genetic factors contribute to the etiology of URA, the genetic bases of URA are poorly defined at this time. ACI rats, both males and females, exhibit URA at an incidence of 5%–15%. In this article we characterize the incidence of URA in female and male F₁, F₂, and backcross (BC) progeny from reciprocal genetic crosses between the ACI strain and the unaffected Brown Norway (BN) strain. Through interval mapping analyses of 353 phenotypically defined female F₂ progeny, we mapped to rat Chromosome 14 (RNO14) a genetic locus, designated Renag1 (Renal agenesis 1), that serves as the major determinant of URA in these crosses. Further genotypic analyses of URA-affected female and male F₂ and BC progeny localized Renag1 to a 14.4-Mb interval on RNO14 bounded by markers D14Rat50 and D14Rat12. The data from these genetic studies suggest that the ACI allele of Renag1 acts in an incompletely dominant and incompletely penetrant manner to confer URA. James D. Shull and Cynthia M. Lachel authors contributed equally to this work.     

Role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated growth by estradiol, prolactin and progesterone in human and rat mammary tumor cells: studies using TGF-alpha and EGF receptor antibodies

The biological role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated proliferation of primary human and rat mammary tumor cells was studied using antibodies against TGF-alpha and its receptor. A monoclonal antibody, MAb-425 against human EGF receptor was added to in vitro soft agar, clonogenic cultures of human breast carcinoma cells under basal and estradiol(E2)-stimulated conditions. The antibody had an antagonist effect on colony growth in 4 of 10 tumors and an agonist effect in 4 (72 and 153% of control). E2-stimulated colony growth in 5 tumors (167% of control) and the antibody blocked E2-stimulation in 3 of the 5. Inhibition of E2-stimulated growth in 3 and basal growth in 4 other tumors by the EGF receptor antibody suggest that endogenously secreted TGF-alpha has a role as an autocrine/paracrine growth factor in constitutive and E2-stimulated tumor cell proliferation in a majority of human tumors. A polyclonal antibody against TGF-alpha was used to study the role of TGF-alpha in E2-, prolactin(Prl)- and progesterone(Prog)-stimulated proliferation of NMU(nitrosomethylurea)-induced rat mammary tumor cells under similar culture conditions. TGF-alpha, E2, Prl and Prog stimulated colony growth equally to 176, 187, 168 and 181% of control. The antibody produced significant and similar inhibition of TGF-alpha and E2-stimulated growth (95 and 83%). In contrast, inhibition of Prl- and Prog-stimulated growth by the antibody was only 24 and 37%. The TGF-alpha ligand antibody did not have an agonist or antagonist effect when added alone. Thus, TGF-alpha seems to be a major stimulatory growth factor mediating E2-induced tumor cell proliferation in rat mammary tumors. It is less important in Prl- and Prog-induced tumor growth and not essential for basal growth in these tumors. We conclude that TGF-alpha is a biologically important autocrine/paracrine growth factor in primary human breast cancer cell proliferation and in E2-induced rat mammary tumor growth.     

Innate resistance to lethal mousepox is genetically linked to the NK gene complex on chromosome 6 and correlates with early restriction of virus replication by cells with an NK phenotype.

Most inbred strains of mice, including DBA/2 (D2), are highly susceptible to the lethal effects of ectromelia virus, but C57BL/6 (B6) mice are innately resistant. Resistance is controlled by multiple, unlinked, autosomal dominant genes. Of 101 male (B6 x D2)F1 x D2 backcrossed (N2) mice, 18 died after ectromelia virus challenge and all were homozygous for the D2 allele at the proline-rich protein (Prp) locus on distal chromosome 6 (P < 0.001). This association was suggested by the patterns of susceptibility to lethal mousepox in recombinant inbred strains derived from B6 and D2 mice (D. G. Brownstein, P. N. Bhatt, L. Gras, and R. O. Jacoby, J. Virol. 65:1946-1951, 1991). The association between the Prp locus and susceptibility to lethal mousepox also held for N2 male mice that were castrated as neonates, which increased the percentage that were susceptible to 40. Spleen virus titers were significantly augmented in B6 (NK1.1+) mice depleted of asialo GM1+ or NK1.1+ cells, whereas spleen virus titers were unaffected in D2 (NK1.1-) mice depleted of asialo GM1+ cells. These results suggest that a gene or genes within the natural killer gene complex, adjacent to the Prp locus, determine strain variations in resistance to lethal ectromelia virus infection.     

Anecdotal,Historical and Critical Commentaries on Genetics: The Utility of Comparative Genetics to Inform Breast Cancer Prevention Strategies

My research seeks to aid in developing approaches to prevent breast cancer. This research evolved from our early empirical studies for discovering natural compounds with anticancer activities, coupled with clinical evaluation to a genetics-driven approach to prevention. This centers on the use of comparative genomics to discover risk-modifying alleles that could help define population and individual risk and also serve as potential prevention drugable targets to mitigate risk. Here, we initially fine map mammary cancer loci in a rat carcinogenesis model and then evaluate their human homologs in breast cancer case-control association studies. This approach has yielded promising results, including the finding that the compound rat QTL Mcs5a''s human homologous region was associated with breast cancer risk. These and related findings have the potential to yield advancements both in translation-prevention research and in basic molecular genetics.WRITING this Perspectives for Genetics allows me to examine how a cancer biologist focused on cancer prevention morphed into a practicing geneticist. In addition, it allows me to review a decade of our investigations into the complexity of the genetic risk to breast cancer development using comparative genomics.Our comparative genomic strategy consists of genetically identifying mammary cancer risk loci using fine mapping studies in a rat mammary carcinogenesis model. Human homologs of these loci are then evaluated in human breast cancer association studies for their potential to modify risk. This genetics approach provides an integrated discovery platform to identify and mechanistically characterize novel breast cancer risk alleles. We predict that this platform will serve as a foundation for a cancer prevention drug development pipeline.My early work focused on the etiology and prevention of breast cancer. It is work on these interrelated areas that led me to investigate breast cancer genetics. While studying the etiology of breast cancer after joining the faculty of the University of Wisconsin, my interest targeted early events in the etiology of cancer. These range from altering the metabolic activation of environmental xenobiotics to metabolites capable of adducting DNA to destroying clones premalignant cells. At the time we began work in this area, cancer chemoprevention was an emerging field that was assumed to be less complex than cancer therapy. This was, in part, based on the fact that normal and premalignant tissues were genetically more stable than cancer cells and thus less likely to develop resistance to anticancer drugs.Our chemoprevention studies focused on a novel class of nontoxic monoterpenes widely found in the essential oils of fruits. These compounds were found to have both preventive and therapeutic anticancer activities in being able to inhibit both premalignant and malignant cells. Our lead compound was limonene, found in orange peel oil, and the first monoterpene we entered into FDA-approved clinical trials was perillyl alcohol (POH), found originally in lavender oil. For expediency, our first trial was a therapeutic one. This therapeutic phase I trial showed limited promising results (Ripple et al. 2000). We later discovered that POH inhibited the antiapoptotic ability of cancers via a calcium channel interaction that led to the downregulation of NFκB (Berchtold et al. 2005). This mechanism of action could underlie the cytostatic and cytotoxic actions of POH toward both premalignant and malignant cells.The monoterpenes and POH were found through empirical screening. Like the monoterpenes, many chemopreventive and therapeutic agents are found to be of low overall efficacy. Many also have undesirable toxicity, in part due to the lack of target specificity. As such, we felt the need to develop nonempirical methods to develop prevention strategies and drugs.To develop chemopreventative agents for common diseases, we sought an approach that would identify both appropriate drug targets and high-risk populations. For example, we aimed to develop prevention strategies for the large number of individuals at risk for breast cancer but not those who specifically carried the rare but highly penetrant susceptibility alleles of the breast cancer genes such as Brca1 and -2; these and other highly penetrant breast cancer risk alleles collectively account for <25% of inherited breast cancer risk in humans (Pharoah et al. 2008).We thus sought to identify moderately penetrant breast cancer susceptibility alleles that were common (high population frequency). Ten years ago it was difficult to identify such loci directly in human populations. In fact, most association studies at that time were based on a “candidate gene” approach; these studies were rarely successful (Pharoah et al. 2007). We thus adapted a comparative genomics strategy in which such loci are identified in a model organism using a nonbiased linkage approach and then evaluated in humans. We chose what we believe is the in vivo breast cancer model most closely related to the human—the rat.The rat, in contrast to the mouse and like the human, develops a spectrum of hormonally responsive and nonresponsive breast cancers. Importantly, almost all rat and human cancers have a ductal cell origin (Gould 1995). At the time we began this research, however, the rat had far fewer genetic resources and tools than the mouse (Gould 1995). This can be illustrated by our need to use a M13 minisatellite marker to identify our first rat mammary susceptibility QTL (Hsu et al. 1994). Over the course of this research and subsequent studies, rat geneticists have substantially narrowed this technology gap (see Aitman et al. 2008). For example, in pursuing this project we developed a technology that produced the first gene inactivation (“knockout”) rat models (Zan et al. 2003).

Comparative genetics studies:

The first major results of these genomewide comparative studies were published by Shepel et al. (1998) in Genetics. In this study we crossed two rat strains with large differences in their susceptibility to the induction of mammary carcinomas by the chemical carcinogen dimethylbenzanthracene (DMBA). The susceptible strain was the Wistar-Furth (WF) rat, while the resistance strain was the Copenhagen (COP) rat. F₁ hybrid rats were backcrossed (WF × COP) F₁ × WF or intercrossed (F₁ × F₁). Large groups of these rats were orally gavaged with DMBA, and the average number of mammary carcinomas per rat was quantified at necropsy. Rats were also genotyped using microsatellite markers, which had become available for the rat in the 1990s.The QTL genetically identified in this study accounted for most of the genetic variance controlling susceptibility to mammary cancer by identifying the Mammary carcinoma susceptibility (Mcs) loci—Mcs1, -2, -3, and -4. The COP allele of Mcs1, -2, and -3 conferred resistance while Mcs4 conferred an increased susceptibility to mammary cancer development. This study demonstrated the ability to use the rat model to identify the major COP vs. WF polymorphic loci controlling susceptibility. These loci interacted in an additive manner. Interestingly, the almost completely mammary cancer-resistant COP rat strain was shown to carry a polymorphic allele at the Mcs4 locus predicted to increase mammary cancer risk.In extending this study, we asked whether other mammary cancer-resistant strains varied at polymorphic mammary cancer susceptibility loci shared with those genetically identified in the WF × COP cross. A similar analysis was performed by conducting a QTL analysis of a cross between WF and a second resistant strain Wistar-Kyoto (WKy). In this backcross analysis we genetically identified four loci that accounted for most of the genetic variance associated with the susceptibility phenotype. As with the COP cross, the WKy cross identified three loci in which the WKy allele contributed to resistance and one locus at which the WKy allele contributed to increased susceptibility (Lan et al. 2001). Of these four WKy loci, only one broadly overlapped with those identified in the COP × WF cross, i.e., Mcs2 (COP) with Mcs6 (WKy). This study also used a novel statistical approach developed by our statistical collaborator, Christina Kendziorski, to identify alleles with no main effect that modify QTL with main effects. Mcs-modifier 1 (Mcsm1) was the most strongly supported locus of this class. The WKy allele of this locus fully negated the effects of the resistance conferred by the WKy allele of the Mcs8 QTL. Thus it appeared that there could be a large number of polymorphic loci in rats that could contribute to mammary cancer risk.It is important to keep in mind that genetically identified QTL are the product of statistical modeling and analysis of segregating populations from crosses. It is thus critical that their existence be confirmed in more homogeneous genetic material. An established method for QTL validation is to breed and phenotype congenic animals carrying only the region surrounding the QTL allele of interest on an alternative genetic background. So far we have generated and characterized six of the eight candidate WKy and COP QTL by genetically introgressing them onto the WF background. All six have the phenotype predicted by our quantitative models.Most congenic substitutions include tens of megabases encompassing the introgressed allele. The next step is to fine map this congenic interval to first determine whether this interval harbors more than one independent susceptibility locus. In addition, the fine mapping process allows for an increased genomic resolution of the locus and thus a more limited set of candidates. We have fine mapped two Mcs loci–Mcs1 (COP) and Mcs5 (WKy). Each was found to be complex, containing at least three separable subloci termed Mcs1a, -b, -c and Mcs5a, -b, -c. In the case of Mcs1, all three identified loci within it contributed to the cancer resistance phenotype of Mcs1. This led us to speculate that this apparent clustering might be biologically “random”; their strong-combined phenotype allowed us to readily identify Mcs1 over the experimental background. In contrast, the Mcs5 also had at least three subloci, Mcs5a, -b, and -c, but two of these, a and c, contribute to resistance while b confers an increased sensitivity. Each of the three had similar absolute relative risk (RR) contributions. If they interact in a purely additive manner, it might have been difficult to identify Mcs5. However, Mcs5 had the strongest of LOD scores of any identified locus in the WKy cross (Lan et al. 2001). When we explored the interaction of the alleles at the Mcs5 loci, we found complex epistatic interactions. The strongest was the complete neutralization of the effect of the sensitive WKy allele of Mcs5b by the resistant WKy allele of Mcs5a (Samuelson et al. 2005).It is interesting to explore an alternative hypothesis that suggests that the clustering of mammary cancer susceptibility alleles arise from evolutionary selection. Data supporting such a possibility in rodents has been published by Petkov et al. (2005). Their findings suggest that alleles controlling certain phenotypes cluster to assure joint inheritance, in that in concert with one another, they provide for an enhanced survival advantage. This could account for the clustering of risk-related genes at the Mcs1 and Mcs5 QTL.Many of the most comprehensive published mammalian fine-mapping studies achieve mapping resolutions in the order of several megabases. Such intervals, while carrying a limited number of genes, often require choosing one or more candidate genes for intensive study. These are usually chosen on the basis of how they might functionally relate to the specific disease risk under investigation. This negates the potential of positional cloning to identify an unbiased candidate. As mentioned above, experience suggests that functional candidate selection rarely identifies disease-specific modifier genes. For example, in breast cancer, when 120 such published candidates (710 SNPs) were rigorously evaluated, none met minimal statistical significance in a study of a large population of women in a breast cancer case-control study (Pharoah et al. 2007).We explored the ability of ultrafine mapping to annotate the Mcs5a locus. We mapped this locus to >100-kb resolution by phenotyping congenic rats recombinant within this locus. We found it to contain two elements. The WKy allele of each element by itself failed to elicit a mammary cancer phenotype; however, when combined, the resistance phenotype was obvious. These elements, termed Mcs5a1 and Mcs5a2, synthetically interact, making Mcs5a one of the first-identified compound QTL in mammals. Because Mcs5a acts in a semidominant manner, we could use heterozygous congenic recombinants to ask whether both elements of Mcs5a needed to lie in cis on the same chromosome, or could they interact in trans from separate homologs. They interact only in cis (Samuelson et al. 2007). Another interesting observation arising from the fine mapping of Mcs5a is that it localizes to noncoding DNA. All four Mcs loci that we have fine mapped to high resolution are localized to noncoding DNA (in progress).The observations that the rat compound locus Mcs5a consists of two synthetically interacting elements separated by ∼50-60 kb (based on the human sequence), interact only when on the same chromosome, and are noncoding suggest the hypothesis that they may be localized in closer proximity than suggested by the linear genomic distance that separates them. Recent observations in our laboratory using chromosome confirmation capture suggest that most of the sequences between these elements form a CTCF-mediated loop bringing both elements in close physical proximity to each other. The ability of this compound locus to control local and interchromosomal gene expression is being studied (in progress).To determine whether our findings in our rat model could be extended to women, we next asked whether the human ortholog of Mcs5a (-a1 and -a2) could influence breast cancer risk. In contrast to the method of searching for modifier genes using genomewide association studies (GWAS), we restricted our search to an ∼100-kb region of the human genome. Focusing on this orthologous locus defined by comparative genomics vastly reduced the number of SNP-tagged alleles needed for testing for association, greatly reducing the statistical penalty for multiple testing. We tested several SNPs in the orthologous MCS5A1 and -5A2 regions of the human genome in a total of ∼12,000 women in a breast cancer case-control study. We found that a tagged SNP in both MCS5A1 and -5A2 was significantly associated with risk to breast cancer in this population of women. The minor allele of SNP rs56476643 (MCS5A1) acts in a recessive manner to increase risk. Its allele frequency is 25% and it increases risk in homozygous women by 19%. In contrast, the minor allele of MCS5A2 (rs2182317) has an allele frequency of 13% and acts in a dominant manner to reduce by 14% the risk of breast cancer in the 24% of women carrying one or two copies of this allele (Samuelson et al. 2007).Not only does this human study support the use of comparative genomics to identify human cancer risk modifier alleles, it also extends the resolution obtained in the rat in localizing the two genetic elements of the Mcs5a allele. The rat localizes Mcs5a1 and -a2 to 32 and 84 kb, respectively, while the human studies resolved these determinants to 5.7 kb and 16.8 kb (Samuelson et al. 2007). Thus, we have demonstrated a clear advantage in using comparative genomics to localize target regions within QTL.Both MCS5A1 and -5A2 have similar allele frequencies and genetic penetrance (relative risk) as do most breast cancer alleles identified by GWAS studies. However, unlike alleles identified by GWAS studies, those identified by comparative genomics also provide in vivo models to functionally characterize risk alleles. For example, it is often assumed that breast cancer modifiers are likely to act within breast tissue to modulate risk. Using the rat as a model we have been able to show that Mcs5a, a noncoding allele, acts to differentially regulate its neighboring FBXO10 gene in immune but not mammary tissues (Samuelson et al. 2007).It is also intriguing to consider the observation that breast cancer risk-associated alleles such as Mcs5a1 and -5a2 are either conserved over millions of evolutionary years or are highly mutable and functionally neutral, suggesting that these alleles do not significantly reduce fitness. If so, one then speculates that they would make good targets for chemoprevention drugs by possessing low toxicity and as such a good therapeutic index. In particular, converting sensitive to resistant allelic function with drug therapy would mimic the conserved resistance allele that persists in the human population and should therefore show a low side-effects profile.Our current research on these genetically identified Mcs loci focuses on molecular, cellular, and organismal mechanisms by which they modify risk. Not only will these investigations provide insight into the function of each noncoding Mcs locus, but collectively they will provide a mechanistic framework to facilitate integrative genetic studies of the plethora of polymorphic risk loci identified by GWAS in multiple diseases.