Hormonal, genetic, and environmental factors play major roles in the complex etiology of breast cancer. When treated continuously with 17ß-estradiol (E2), the ACI rat exhibits a genetically conferred propensity to develop mammary cancer. The susceptibility of the ACI rat to E2-induced mammary cancer appears to segregate as an incompletely dominant trait in crosses to the resistant Copenhagen (COP) strain. In both (ACI x COP)F₂ and (COP x ACI)F₂ populations, we find strong evidence for a major genetic determinant of susceptibility to E2-induced mammary cancer on distal rat chromosome 5. Our data are most consistent with a model in which the ACI allele of this locus, termed Emca1 (estrogen-induced mammary cancer 1), acts in an incompletely dominant manner to increase both tumor incidence and tumor multiplicity as well as to reduce tumor latency in these populations. We also find evidence suggestive of a second locus, Emca2, on chromosome 18 in the (ACI x COP)F₂ population. The ACI allele of Emca2 acts in a dominant manner to increase incidence and decrease latency. Together, Emca1 and Emca2 act independently to modify susceptibility to E2-induced mammary cancer.

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BREAST cancer is one of the leading causes of cancer-related death in women in the United States. Risk of developing breast cancer is determined by both genetic and environmental factors, as well as by the interactions between genetic and environmental determinants. A number of genetic factors, such as a germline mutation of the BRCA1, BRCA2, or p53 genes, strongly predispose carriers to breast cancer (TONIN 2000; IAU et al. 2001; DITE et al. 2003). However, only a relatively small percentage of breast cancers, 5–10%, are due to inheritance of these highly penetrant mutations. Nonetheless, one of the most consistent and significant risk factors associated with breast cancer development in women not carrying a BRCA1, BRCA2, or p53 mutation is a family history of breast cancer (TONIN 2000; DITE et al. 2003). These observations, as well as twin studies, suggest that unidentified genetic factors with penetrance lower than that of BRCA1 or BRCA2 also play a significant role in determining breast cancer risk (LICHTENSTEIN et al. 2000; RISCH 2001; MACK et al. 2002).

Because estrogens stimulate proliferation of the mammary epithelium, the duration of exposure of the breast to estrogens is hypothesized to play a role in breast cancer development. For example, early age at menarche and late age at menopause each increase risk of developing breast cancer, presumably by extending the length of time the mammary epithelium is exposed to estrogens (KEY et al. 2001; BERNSTEIN 2002). It has also been suggested that specific polymorphisms in a number of genes involved in estrogen metabolism may be correlated with an increased risk of breast cancer (SHULL 2002); however, these correlations have been observed in some populations (YIM et al. 2001; KOCABAS et al. 2002; LEE et al. 2003) but not others (THOMPSON and AMBROSONE 2000; BERGMAN-JUNGESTROM and WINGREN 2001; MITRUNEN et al. 2001). Thus, genetic factors may play an important role in determining the capacity of estrogens to influence breast cancer risk. In addition to these epidemiological data, clinical trials have provided compelling evidence associating estrogen exposure to breast cancer risk in humans. For example, the selective estrogen receptor modulator, tamoxifen, reduces breast cancer risk in individuals with identified risk factors and reduces the incidence of contralateral breast cancer in patients previously treated for breast cancer (EARLY BREAST CANCER TRIALISTS' COLLABORATIVE GROUP 1998; FISHER et al. 1998). Furthermore, numerous epidemiological studies, including the Women's Health Initiative randomized controlled trial, demonstrated that combined estrogen and progestin hormone replacement therapy is associated with an increased risk of breast cancer (COLLABORATIVE GROUP ON HORMONAL FACTORS IN BREAST CANCER 1997; ROSSOUW et al. 2002).

Although many studies have demonstrated that estrogens play a central role in breast cancer etiology (HARRIS et al. 1992; PIKE et al. 1993), the mechanisms through which estrogens contribute to breast cancer development are not well defined. Furthermore, human populations provide relatively limited power to identify genetic factors that modulate the action of estrogens in breast cancer development in women. Therefore, we have begun to explore these issues using the ACI rat, which is highly susceptible to mammary carcinogenesis when the circulating level of the naturally occurring estrogen, 17ß-estradiol (E2), is maintained in the upper physiologic range by exogenous administration (SHULL et al. 1997, 2001; HARVELL et al. 2000, 2001, 2002). This susceptibility to E2-induced mammary cancer exhibits an incompletely dominant pattern of inheritance in crosses between the ACI strain and the genetically related, but resistant, Copenhagen (COP) rat strain (SHULL et al. 2001). In the current study, two genetic determinants of susceptibility to E2-induced mammary cancer, Emca1 (estrogen-induced mammary cancer) and Emca2, were mapped to rat chromosomes 5 and 18, respectively, in crosses between the ACI and COP rat strains. Furthermore, we demonstrate that the genetic bases of susceptibility to E2-induced mammary carcinogenesis are distinct from those regulating sensitivity to estrogen-induced pituitary tumorigenesis.

Phenotypic characterization of F₂ progeny:

The phenotypically defined F₂ progeny utilized in these linkage studies were generated in crosses between the ACI rat strain and the resistant COP strain and have been described previously (SHULL et al. 2001). Briefly, implants containing E2 were placed subcutaneously when the rats were 63 ± 4 days of age. Ovary-intact females were used for these experiments because ovariectomy inhibits the rapid development of E2-induced mammary cancers in the ACI rat (SHULL et al. 1997). Beginning 5–7 weeks later, each animal was examined twice weekly for the presence of palpable mammary tumors. Unless necessitated by morbidity, most frequently due to E2-induced pituitary enlargement, rats were sacrificed when the largest palpable mammary tumor approximated 2 cm in diameter. The location and size of each macroscopic mammary tumor were noted at necropsy. All of the tumors were classified histologically as carcinoma.

Determination of genotype at polymorphic genetic markers:

DNA was isolated from the spleen of each F₂ rat using DNeasy spin columns (QIAGEN, Valencia, CA). Oligonucleotide primers specific to simple sequence repeats that are polymorphic in length between the ACI and COP rat strains were obtained from Invitrogen (Carlsbad, CA). Each DNA sample was amplified in a 10-µl reaction containing 30 ng of DNA, 1.25 units Taq polymerase (Invitrogen), 20 mM Tris (pH 8.4), 1.5 mM MgCl₂, 50 mM KCl, 198 nM of each of the forward and reverse primers, and 200 µM each of dATP, dGTP, dCTP, and dTTP. The reaction mixtures were incubated at 94° for 5 min and subjected to 30 cycles of PCR as follows: 94° for 30 sec, 55° for 30 sec, and 72° for 1.5 min, with the final cycle followed by incubation at 72° for 3 min. For a subset of markers, PCR products were resolved on 3 or 4% agarose gels and visualized with Cambrex GelStar nucleic acid gel stain (Fisher Scientific, Pittsburgh). Alternatively, PCR was performed with the addition of 1.0 µCi [-³²P]dATP (Amersham, Arlington Heights, IL). The DNA products were then denatured, resolved on 5 or 8% polyacrylamide gels, and visualized using a Molecular Dynamics PhosphorImager and ImageQuaNT 5.0 software (Sunnyvale, CA).

Analysis of linkage:

Genetic maps were constructed using MAPMAKER/EXP version 3.0 (LANDER and BOTSTEIN 1989). Genotypes were determined at 183 polymorphic markers in 129 (ACI x COP)F₂ rats. For analysis of the (COP x ACI)F₂ population, 137 markers were used. Markers were chosen to achieve a density of 1 marker every 20 cM. However, there are regions of the genome where this level of marker density was not achieved due to a lack of polymorphic markers between the genetically related ACI and COP strains. Nonetheless, there is no region of the genome where the marker density is such that a quantitative trait locus (QTL), were it to reside there, would be >20 cM from the nearest marker. Analyses of the (ACI x COP)F₂ and the (COP x ACI)F₂ females provide estimates of a minimum genome size of 2357 and 1827 cM, respectively. These values are consistent with the consensus size of the rat genetic map of 2250 cM (Rat Genome Database, http://rgd.mcw.edu/vcmap/).

For linkage analysis, the likelihood-ratio statistic (LRS) values for genotype/phenotype associations were estimated using MAPMANAGER QTX version 0.29 (MANLY et al. 2001). Both qualitative and quantitative phenotypic indicators of susceptibility to E2-induced mammary cancer, including tumor positive/negative status at defined time points following initiation of E2 treatment and mammary tumor number at the time of sacrifice, were evaluated. For linkage analysis of sensitivity to pituitary tumorigenesis, pituitary masses were log₁₀ transformed to normalize the population distribution relative to the mean. For the analysis of the (ACI x COP)F₂ population, evidence for linkage was assessed using experimentwise threshold values that were obtained from analysis using 1000 permutations of the phenotypic data (CHURCHILL and DOERGE 1994). For this cross, linkage analysis was conducted using all 129 experimental animals. By contrast, for the (COP x ACI)F₂ population, linkage analysis was initially conducted using the 43 F₂ animals that exhibited the most highly susceptible (N = 21) and the most resistant (N = 22) phenotypes. When an LRS value suggestive of linkage, based on permutation testing (1000 permutations), was obtained, the remaining F₂ animals were genotyped at all markers assayed on that chromosome. Only markers on rat chromosomes (RNO) 4, 5, and 18 were used to genotype the entire 146 (COP x ACI)F₂ mapping population. Linkage was then assessed using permutation-derived thresholds obtained from tests performed using data from just these three chromosomes. The confidence interval for each QTL was estimated using the resampling technique referred to as bootstrap analysis (VISSCHER et al. 1996), which is part of the interval mapping function of MAPMANAGER QTX (MANLY et al. 2001).

A number of rats from each cross were excluded from the genetic analyses for a variety of reasons. Of the 136 (ACI x COP)F₂ rats produced and treated with E2, 7 could not be used for the genetic analyses, primarily because these animals were found dead in their cages or lost their estrogen implants during the course of the experiment. Of the 173 (COP x ACI)F₂ rats produced and treated with E2, 27 were excluded from the genetic analyses. The majority of these rats, 21 of the 27, were excluded because they were sacrificed following <28 weeks of E2 treatment primarily due to morbidity associated with E2-induced pituitary tumorigenesis prior to developing a mammary tumor. Most of the remaining rats were eliminated from the genetic analyses because they were found dead in their cages. Importantly, for both crosses, no rat with a mammary tumor for which tissue was available was excluded from the genetic analyses.

Statistical analysis:

Survival analysis methods were employed to assess the linkage of a given marker to a locus that controls tumor latency, which is defined as the number of days of E2 treatment prior to the appearance of the first palpable mammary tumor. Specifically, the Breslow test was used to compare tumor latency in the presence of censored observations, that is, animals that were either tumor free at the end of the study or sacrificed due to morbidity from pituitary tumor development prior to developing a mammary tumor. To eliminate spurious evidence for linkage when multiple comparisons are made, linkage was not assessed in this manner for every marker used in the genotypic analysis. Rather, the Breslow test was performed with only one or two markers residing within the intervals in which the LRS values exceeded the threshold for suggestive or significant evidence of linkage. The Cox proportional hazards model was used to examine pairwise interactions between loci with respect to tumor latency. The effect of genotype at specific markers on mammary tumor incidence was assessed using a chi-square test or Fisher's exact test, where appropriate. Linkage of markers on RNOX to a locus modulating mammary tumor development was assessed using the chi-square test. Differences in average mammary tumor multiplicity and pituitary mass among genotypic classes were evaluated using the Wilcoxon rank-sum/Mann-Whitney U-test. Allelic imbalance data were evaluated as described previously using likelihood-based parameter estimation and Bayesian methods (NEWTON and LEE 2000). P-values 0.05 were considered to be significant.

Mapping Emca1 to RNO5 in the (ACI x COP)F₂ population:

Genetic analysis using 129 (ACI x COP)F₂ progeny yielded significant evidence for linkage of a locus that modulates susceptibility to E2-induced mammary cancer to multiple markers on distal RNO5. An LRS value of 20.2 was obtained at D5Rat30 when the (ACI x COP)F₂ population was evaluated following 175 days of treatment (Figure 1A). This LRS value exceeded the permutation-derived thresholds of 10.5 and 17.8 for suggestive and significant evidence of linkage, respectively, with the tumor status following 175 days of treatment phenotype. Bootstrap analysis was performed as part of the interval mapping function of MAPMANAGER QTX to provide an estimate of the confidence interval for the location of this QTL. These data indicate that one or more genetic determinants of susceptibility to E2-induced mammary cancer resides within an interval of 38 cM on RNO5 defined by markers D5Rat53 and D5Rat57. This locus has been designated Emca1. The genetic models most consistent with the linkage data indicated that the ACI allele of Emca1 (Emca1^ACI) likely acts in a recessive or incompletely dominant manner. Consistent with a recessive mode of action for Emca1^ACI, mammary tumor incidence following 175 days of E2 treatment was significantly greater in rats homozygous for the ACI allele at D5Rat30 (61%) than in rats heterozygous (22%) or homozygous for the COP allele (17%; P < 0.01; Figure 1B). The incidence of mammary tumors at this time point did not differ between rats heterozygous or homozygous for the COP allele at D5Rat30 (P = 0.34; Figure 1B).

View larger version (16K): In this window In a new window Download PPT slide   FIGURE 1.— Emca1 in the (ACI x COP)F2 population. (A) LRS values across RNO5 were derived using tumor status following 175 days of E2 treatment as the phenotype. (B) The effect of genotype at D5Rat30 on mammary tumor incidence following 175 days of E2 treatment. A 1 above a bar indicates that the incidence is significantly different from that in the ACI homozygotes. (C) The effect of genotype at D5Rat30 on mammary tumor latency and incidence. (•) Rats homozygous for the ACI allele at D5Rat30; () rats heterozygous at D5Rat30; () rats homozygous for the COP allele at D5Rat30. (D) LRS values across RNO5 were derived using tumor multiplicity at sacrifice as the phenotype.

 
There was a highly significant association between mammary tumor latency and genotype at markers within Emca1 in the (ACI x COP)F₂ population (P < 0.01; Figure 1C). Here, Emca1^ACI appeared to act in an incompletely dominant manner; median mammary tumor latency was 165 days in rats homozygous for the ACI allele at D5Rat30 compared to 193 days in rats heterozygous at D5Rat30 and 231 days in rats homozygous for the COP allele at D5Rat30.

Genetic analysis also suggested that Emca1 is involved in determining mammary tumor multiplicity in the (ACI x COP)F₂ population. For analysis of tumor multiplicity data at the time of sacrifice, permutation-derived thresholds for suggestive and significant evidence of linkage were 10.5 and 17.8, respectively. A peak LRS value of 17.8 was obtained at D5Rat30 when the (ACI x COP)F₂ population was analyzed with respect to tumor multiplicity at the time of sacrifice (Figure 1D). For the tumor multiplicity phenotype, the confidence interval for Emca1 extended from D5Rat53 to D5Rat205. Here again, the action of Emca1^ACI seemed most consistent with an incompletely dominant model; tumor-bearing rats homozygous for the ACI allele at D5Rat30 in the Emca1 interval had an average of 3.5 [standard deviation (SD) 2.4] tumors, whereas tumor multiplicity averaged 2.2 (SD 1.6) and 1.6 (SD 1.3) in heterozygotes and COP homozygotes, respectively (P < 0.02; data not shown).

Emca1 modulates mammary carcinogenesis in the reciprocal (COP x ACI)F₂ population:

Significant evidence for linkage of a QTL with a major effect on mammary tumor development was also observed on RNO5 in a (COP x ACI)F₂ population. A total of 146 progeny from this cross were genotyped at markers spanning the Emca1 interval on RNO5, and linkage was evaluated as a function of whether each F₂ rat was tumor negative or positive at 14-day intervals following initiation of E2 treatment in a fashion analogous to the F₂ progeny from the reciprocal (ACI x COP) intercross. When the (COP x ACI)F₂ population was evaluated following 175 days of treatment, the time point giving the peak LRS value in the analysis of the (ACI x COP)F₂ population, an LRS score of 8.3 was obtained at D5Rat53. This LRS value was above the permutation-derived threshold of 5.2 for suggestive evidence of linkage but below the threshold of 12.2 for significant evidence of linkage for this phenotype. Interestingly, evaluation of the (COP x ACI)F₂ population at 189 days of treatment or later yielded significant evidence for linkage. The strongest evidence was obtained by evaluation of the (COP x ACI)F₂ population at 189 days of treatment, when linkage analysis yielded an LRS value of 13.8 in the interval between D5Rat53 and D5Rat95, which exceeded the permutation-derived thresholds for suggestive and significant evidence of linkage of 5.6 and 12.1, respectively (Figure 2A). In the (COP x ACI)F₂ population, the linkage data were most consistent with a recessive or incompletely dominant mode of action for Emca1. For this cross, the incidence of mammary tumors within 189 days of estrogen treatment was 76% in rats homozygous for the ACI allele at D5Rat53, significantly greater than either the 49% in heterozygotes (P < 0.01) or the 37% in rats homozygous for the COP allele at D5Rat53 (P < 0.01; Figure 2B). Tumor incidence did not differ significantly between rats heterozygous and homozygous for the COP allele at D5Rat53 at this time point (P = 0.37; Figure 2B).

View larger version (22K): In this window In a new window Download PPT slide   FIGURE 2.— Emca1 in the (COP x ACI)F2 population. (A) LRS values across RNO5 were derived using tumor status following 189 days of E2 treatment as a phenotype. (B) The effect of genotype at D5Rat53 on mammary tumor incidence following 189 days of E2 treatment. A 1 indicates that the incidence is significantly different from that in the ACI homozygotes. (C) The effect of genotype at D5Rat53 on mammary tumor latency and incidence. (•) Rats homozygous for the ACI allele at D5Rat53; () rats heterozygous at D5Rat53; () rats homozygous for the COP allele at D5Rat53.

 
In the (COP x ACI)F₂ population, there was a significant association between mammary tumor latency and genotype at markers within Emca1 (P = 0.03; Figure 2C). Consistent with the hypothesis that Emca1 acts in an incompletely dominant manner, median mammary tumor latency was 155 days in rats homozygous for the ACI allele at D5Rat53, one of the peak markers in the Emca1 interval in this cross, compared to 190 days in rats heterozygous at D5Rat53 and 212 days in rats homozygous from the COP allele at D5Rat53.

In the (COP x ACI)F₂ population, which had a significantly lower average tumor number than the (ACI x COP)F₂ rats, analysis of tumor multiplicity at sacrifice yielded only suggestive evidence for linkage (LRS = 7.0) between D5Rat53 and D5Rat95 in the Emca1 genetic interval (data not shown; SHULL et al. 2001).

Emca2 maps to RNO18:

Modest evidence for a locus modulating susceptibility to E2-induced mammary carcinogenesis was observed on RNO18 in the (ACI x COP)F₂ population evaluated as a function of whether each rat was tumor negative or positive at 14-day intervals following initiation of E2 treatment. Analysis of the (ACI x COP)F₂ females following 189 days of treatment yielded a peak LRS value of 7.4 at the marker D18Rat21 (Figure 3A). Although this LRS value fell below the permutation-derived threshold of 10.6 for suggestive evidence of linkage, we continued to evaluate the impact of genotype in this region on mammary carcinogenesis because data obtained from a cross between the ACI and Brown Norway (BN) rat strains suggest that this genetic interval harbors a locus that modulates E2-induced mammary carcinogenesis in that cross (M. TOCHACEK, B. SCHAFFER and J. D. SHULL, unpublished data). Consistent with the hypothesis that there may be a locus on RNO18 that modulates E2-induced mammary carcinogenesis, the mammary tumor incidence following 189 days of E2 treatment was greater in rats homozygous for the ACI allele at D18Rat21 than in those homozygous for the COP allele; however, this difference fell short of statistical significance (P = 0.06; Figure 3B). By contrast, the difference in mammary tumor incidence following 189 days of E2 treatment between rats heterozygous for the ACI allele of D18Rat21 and those homozygous for the COP allele (55 vs. 21%) did achieve significance (P = 0.01). No significant difference in mammary tumor incidence was observed at this time point between rats homozygous and heterozygous for the ACI allele at D18Rat21 (44 vs. 55%; P = 0.67).

View larger version (19K): In this window In a new window Download PPT slide   FIGURE 3.— Emca2 in the (ACI x COP)F2 population. (A) LRS values across RNO18 were derived using tumor status following 189 days of E2 treatment as a phenotype. (B) The effect of genotype at D18Rat21 on mammary tumor incidence following 189 days of E2 treatment. The 1 indicates that the incidence is significantly different from that in the heterozygotes. (C) The effect of genotype at D18Rat21 on mammary tumor latency and incidence. (•) Rats homozygous for the ACI allele at D18Rat21; () rats heterozygous at D18Rat21; () rats homozygous for the COP allele at D18Rat21.

 
The Breslow test was employed to assess the effect of genotype on mammary tumor latency. These analyses revealed a significant association between mammary tumor latency and genotype at D18Rat21 in the (ACI x COP)F₂ population (P = 0.04; Figure 3C). The median latency was 191 days in rats homozygous for the ACI allele at D18Rat21 and 188 days in rats heterozygous at this marker, in contrast to 219 days in the rats homozygous for the COP allele at D18Rat21. These observations suggest that the ACI allele of a locus on RNO18 acts in a dominant manner to reduce tumor latency and increase incidence in the (ACI x COP)F₂ population. This locus has been designated Emca2. There was no clear evidence for any interaction between Emca1 and Emca2 with regard to modulation of tumor latency (data not shown). No association between tumor multiplicity and genotype at any marker on RNO18 was observed (data not shown).

By contrast, there was no evidence for a locus on RNO18 modulating E2-induced mammary cancer development in the (COP x ACI)F₂ population. Regardless of the phenotype used to analyze this cross, LRS values on RNO18 fell below the permutation-derived threshold for evidence suggestive of linkage (data not shown). The highest LRS value, 4.8 at D18Rat43, was obtained when the (COP x ACI)F₂ population was evaluated following 189 days of treatment (data not shown). This value fell below the threshold of 5.6 for suggestive evidence of linkage with this phenotype. Likewise, there was no observed association between tumor latency and genotype at D18Rat21 (P = 0.67). Thus, Emca2 did not have any perceptible effect on E2-induced mammary carcinogenesis in the (COP x ACI)F₂ population.

Allelic imbalances in Emca1 and Emca2 intervals:

Because the Emca1 and Emca2 intervals in the rat are syntenic with regions of the human genome that frequently show somatic deletions in human breast cancer, we hypothesized that Emca1 and Emca2 may be inactivated by somatic genetic events, such as loss of heterozygosity, during E2-induced mammary carcinogenesis (CALLAHAN et al. 1992; BIECHE and LIDEREAU 1995; EIRIKSDOTTIR et al. 1995; WEITH et al. 1996; TIRKKONEN et al. 1998; AN et al. 1999; LOVEDAY et al. 2000; RICHARD et al. 2000). Frequent somatic loss would suggest that these Emca loci act as classical tumor suppressor genes and would allow fine-structure genetic mapping by deletion analysis. Therefore, we screened a panel of 21 E2-induced mammary tumors from (ACI x COP)F₁ females for allelic imbalances at markers on RNO5 and 18. For these analyses, the relative abundance of the PCR products amplified from the ACI and COP alleles at markers on RNO5 and 18 were quantitated from DNA isolated from E2-induced mammary tumors from (ACI x COP)F₁ rats. These data were normalized to the relative abundance of the PCR products amplified for each allele from DNA isolated from the spleen of each animal. The frequency of allelic imbalance events on these two chromosomes was then compared to that observed at five unlinked background markers on five chromosomes not containing a locus that modulates mammary carcinogenesis. At these control markers, 12 allelic imbalances were detected and the number of allelic imbalances varied from 0 to 5 (0 to 24%) per marker (data not shown). Four of these 12 allelic imbalances (33%) represented either loss of the COP resistance allele or gain of the ACI susceptibility allele, a frequency that did not differ significantly (P > 0.05) from that expected in the absence of allelic bias.

In the 21 tumors analyzed, the number of allelic imbalance events detected for each of the markers assayed on RNO5 varied from 4 to 9 (19 to 43%; Figure 4). Furthermore, 38 of the 51 (75%) allelic imbalance events detected revealed an overabundance of the ACI allele relative to the COP allele, suggesting that allelic imbalance events involving either loss of the COP allele or gain of the ACI allele occur more frequently than would be expected if there were no allelic bias in these somatic events (P < 0.02). Fourteen of the 21 tumors (67%) displayed allelic imbalance events at one or more markers on RNO5. Significantly, 7 of 21 tumors (33%), displayed regions of significant allelic imbalance—either loss of the COP resistance allele or gain of the ACI susceptibility allele—that spanned two or more adjacent markers in the Emca1 interval. These data were evaluated for statistical significance using previously described methods (NEWTON and LEE 2000). When a conditional hypothesis testing method was used to evaluate the data generated for RNO5, evidence of excessive allelic imbalance fell short of the threshold of statistical significance (P = 0.14). However, likelihood analysis did suggest that there was excessive allelic imbalance in the Emca1 interval. Whereas the neutral background loci were imbalanced 20% of the time, the apparent hot spot for allelic imbalance on RNO5 was imbalanced 50% of the time. This likelihood analysis yielded a Bayes factor of 1.4, providing evidence for excessive allelic imbalances in the Emca1 interval on RNO5.

View larger version (30K): In this window In a new window Download PPT slide   FIGURE 4.— Allelic imbalance in the Emca1 interval in E2-induced mammary tumors. This figure illustrates the presence or absence of allelic imbalances at markers spanning the Emca1 interval in E2-induced mammary tumors from (ACI x COP)F1 rats. The markers assayed are listed at the top and the position of each marker along the chromosome in centimorgans, according to the Rat Genome Database (http://rgd.mcw.edu/vcmap/), is given in parentheses. (O) The centromere. () Indicates that no allelic imbalance was detected at that marker in the indicated number of tumors. () Indicates that an allelic imbalance event consistent with either a loss of the COP or a gain of the ACI allele was detected at that marker in the indicated number of tumors. () Indicates that an allelic imbalance event consistent with either a loss of the ACI or a gain of the COP allele was detected at that marker in the indicated number of tumors. "ND" indicates that a genotype at the indicated marker could not be determined. Map distances are not drawn to scale.

 
The number of allelic imbalance events detected for each of the markers assayed on RNO18 varied from 4 to 11 (19 to 52%; Figure 5). Twenty-two of the 30 (73%) allelic imbalance events detected represented either loss of the COP resistance allele or gain of the susceptible ACI allele. This frequency was not significantly different from what would be expected in the absence of allelic bias for somatic losses (P > 0.05). The majority of tumors, 20 of 21 (95%), had allelic imbalance events involving one or more markers on RNO18. Furthermore, 4 of 21 tumors (19%) displayed regions of significant allelic imbalance—either loss of the COP resistance allele or gain of the ACI susceptibility allele—spanning two or more adjacent markers in the Emca2 interval. Analysis of these data by conditional hypothesis testing yielded evidence for excessive allelic imbalance that fell short of statistical significance (P = 0.10). However, likelihood analysis of the observation that the neutral background loci were imbalanced just 20% of the time, whereas the apparent hot spot for allelic imbalance on RNO18 was imbalanced 70% of the time, yielded a Bayes factor of 4.3, providing evidence of excessive allelic imbalance in the Emca2 interval on RNO18.

View larger version (16K): In this window In a new window Download PPT slide   FIGURE 5.— Allelic imbalance in the Emca2 interval in E2-induced mammary tumors. This figure illustrates the presence or absence of allelic imbalances at markers spanning the Emca2 interval in E2-induced mammary tumors from (ACI x COP)F1 rats. The markers assayed are listed at the top and the position of each marker along the chromosome in centimorgans, according to the Rat Genome Database (http://rgd.mcw.edu/vcmap/), is given in parentheses. (O) The centromere. () Indicates that no allelic imbalance was detected at that marker in the indicated number of tumors. () Indicates that an allelic imbalance event consistent with either a loss of the COP or a gain of the ACI allele was detected at that marker in the indicated number of tumors. () Indicates that an allelic imbalance event consistent with either a loss of the ACI or a gain of the COP allele was detected at that marker in the indicated number of tumors. Map distances are not drawn to scale.

 

Analysis of linkage of mammary tumor phenotypes with markers outside of RNO5 and 18:

Linkage analysis revealed no significant evidence for linkage of a locus modulating the incidence of E2-induced mammary tumor development or mammary tumor multiplicity to any other marker examined in either the (ACI x COP)F₂ or the (COP x ACI)F₂ populations. However, suggestive evidence for a QTL on RNO3 (D3Rat41; LRS = 11.9; phenotype was the tumor incidence after 161 days of treatment), RNO6 (D6Mit10; LRS = 15.0; phenotype was the tumor number at sacrifice), and RNO13 (D13Rat33; LRS = 11.1; phenotype was the tumor incidence after 203 days of treatment) was obtained following analysis of the entire (ACI x COP)F₂ population, indicating that these regions of the genome may contain loci that modulate E2-induced mammary carcinogenesis (data not shown). The association between genotype at D6Mit10 and tumor multiplicity at sacrifice was confirmed using nonparametric statistical analysis (P < 0.01, data not shown). However, the Breslow test failed to reveal a clear association between genotype at markers in these intervals and tumor latency (data not shown). No evidence for these suggestive loci was observed in the (COP x ACI)F₂ population.

Composite interval mapping was also employed to determine whether the effects of Emca1 limited our power to detect other loci controlling susceptibility to E2-induced mammary carcinogenesis. For this analysis, regression and interval mapping were performed following fixing the peak marker within the Emca1 interval, D5Rat30, as background. This analysis revealed no significant evidence that loci unlinked to Emca1 modulate mammary tumor susceptibility in this cross. However, suggestive evidence for a QTL linked to D6Mit10 that modulates tumor multiplicity in this cross remained, as the LRS value of 14.8 associated with this marker was still suggestive after fixing Emca1 as background.

The effect of genotype at a number of specific additional markers on mammary tumor latency was also assessed. Specifically, we tested the hypothesis that loci known to modulate the ability of the estrogen diethylstilbestrol (DES) to induce proliferation of the prolactin-producing lactotrophs in the pituitary would also modulate susceptibility to E2-induced mammary cancer. The ACI and COP rat strains differ in their sensitivity to DES-induced lactotroph hyperplasia and associated hyperprolactinemia, commonly referred to as pituitary tumors (SPADY et al. 1999). Genetic analysis of (ACI x COP)F₂ and (COP x ACI)F₂ males, siblings of the females described here, indicates that sensitivity to DES-induced pituitary tumorigenesis is controlled by six genetic loci: Ept10 (estrogen-induced pituitary tumor) and Ept13 on RNO1, Ept2 and Ept6 on RNO3, Ept1 on RNO6, and Ept9 on RNO10 (T. E. STRECKER and J. D. SHULL, unpublished data). The region encompassing Ept1 on RNO6 is unlinked to D6Mit10, the peak marker associated with the suggestive locus on RNO6 that may modulate mammary tumor multiplicity. Thus, the linkage analyses described above indicated that these six significant Ept loci had no effect on E2-induced mammary tumor incidence or on multiplicity in the (ACI x COP)F₂ cross. Likewise, genotype at each of these Ept loci had no effect on mammary tumor latency in (ACI x COP)F₂ females (Table 1). Linkage analysis using the (ACI x COP)F₂ and (COP x ACI)F₂ males also identified two suggestive Ept loci that may influence sensitivity to DES-induced pituitary tumorigenesis. One suggestive Ept locus was associated with D4Rat196. The other suggestive Ept locus was linked to D5Rat95, which resides within the Emca1 interval. Thus, of all the significant and suggestive QTL that regulate sensitivity to DES-induced pituitary tumorigenesis in the (ACI x COP)F₂ and (COP x ACI)F₂ males, just one, the suggestive Ept locus on RNO5, colocalizes with a locus influencing susceptibility to E2-induced mammary carcinogenesis in the (ACI x COP)F₂ females. The effect of the Ept loci on E2-induced mammary carcinogenesis was not evaluated in the entire (COP x ACI)F₂ population because only a subset of this population was genotyped at markers in the Ept intervals.

View this table: In this window In a new window   TABLE 1 Effect of genotype in Ept intervals on mammary tumor latency

 

Genetic analysis of E2-induced pituitary tumorigenesis in (ACI x COP) and (COP x ACI) F₂ females:

To assess the effect of each Ept locus on pituitary tumorigenesis in the (ACI x COP)F₂ population, LRS values were evaluated across RNO1, -3, -6, and -10 as a function of log₁₀ transformed pituitary mass. No effect of Ept1, Ept2, Ept6, Ept9, or Ept13 on pituitary mass could be detected in these crosses (data not shown). However, it should be noted that the power to detect loci modulating quantitative differences in pituitary mass, which is strongly influenced by the length of estrogen treatment (WIKLUND et al. 1981), is limited in the (ACI x COP)F₂ females because these rats were sacrificed following varying lengths of E2 treatment rather than following a defined length of treatment.

By contrast, suggestive evidence for a locus controlling sensitivity to E2-induced pituitary tumorigenesis was obtained in the Ept10 interval on RNO1. When the entire (ACI x COP)F₂ population was analyzed for linkage to log₁₀-transformed pituitary mass, an LRS value of 12.9 was obtained at D1Rat75 (Figure 6A). This LRS value was above the permutation derived threshold of 10.8 for suggestive evidence of linkage but below the threshold of 18.6 for significant evidence of linkage for this phenotype. The average pituitary mass in rats homozygous for the ACI allele of D1Rat75, a marker near the Ept10 peak, was 54.8 mg. In rats heterozygous for this marker, the average pituitary mass was 84.1 mg, significantly greater than that observed in the ACI homozygotes (P = 0.04). In rats homozygous for the COP allele at D1Rat75 the average pituitary mass was 113.6 mg, significantly greater than that observed in rats either heterozygous (P = 0.04) or homozygous for the ACI allele at D1Rat75 (P < 0.01; Figure 6B). Importantly, genotype in the Ept10 interval had no impact on the duration of survival following initiation of E2 treatment (P = 0.92). These data suggest that the COP allele of Ept10 acts in an incompletely dominant manner to increase pituitary mass. These data are similar to those obtained in the analysis of the (ACI x COP)F₂ and (COP x ACI)F₂ males, which also indicates that the COP allele of Ept10 confers sensitivity to DES pituitary tumorigenesis (T. E. STRECKER and J. D. SHULL, unpublished data). Once again, the (COP x ACI)F₂ females were not evaluated in an analogous manner because only a subset of this population was genotyped at markers in the Ept intervals.

View larger version (30K): In this window In a new window Download PPT slide   FIGURE 6.— Ept10 in the (ACI x COP)F2 population. (A) LRS values across RNO1 are shown as a function of log10-transformed pituitary mass. (B) The effect of genotype at D1Rat75 on pituitary mass at the time of sacrifice is illustrated. A 1 indicates that the pituitary mass is significantly different from that in the ACI homozygotes; 2 indicates that the pituitary mass is significantly different from that in the COP homozygotes.

 
Linkage analysis revealed no significant evidence for linkage of a locus modulating E2-induced pituitary tumorigenesis to any other marker examined in the entire (ACI x COP)F₂ female population, including those within Emca1 (LRS = 4.2) and Emca2 (LRS = 1.4; data not shown). Genetic analysis of pituitary tumorigenesis in the entire (COP x ACI)F₂ female population was assessed exclusively in the Emca1 and Emca2 intervals; LRS values in these intervals did not exceed 3.7 (data not shown). Thus, there is no evidence that either Emca1 or Emca2 modulates pituitary tumorigenesis in the (ACI x COP)F₂ or (COP x ACI)F₂ females.

The data presented in this article significantly enhance our understanding of the genetic bases of susceptibility to mammary cancer in a novel animal model in which mammary cancer is induced by continuous treatment with E2. Here, we map Emca1, which modulates E2-induced mammary tumor incidence, multiplicity, and latency, to a region of 38 cM on RNO5. Emca1 segregates in crosses between the ACI and COP strains regardless of the parental orientation used to generate the F₁ rats. In these crosses, the ACI allele of Emca1 appears to act in a recessive or incompletely dominant manner to increase tumor incidence and multiplicity and to decrease tumor latency. The region of RNO5 encompassing Emca1 also contains a determinant of susceptibility to E2-induced mammary carcinogenesis in crosses between the ACI and unrelated BN rat strain (M. TOCHACEK, B. SCHAFFER and J. D. SHULL, unpublished data).

The region of RNO5 containing Emca1 is syntenic to human chromosomes 1p and 9p (http://www.ensembl.org/), two regions of the human genome that have been implicated in breast cancer etiology. Loss of heterozygosity (LOH) at chromosome 1p markers, one of the most common genetic aberrations observed in breast cancer, is observed in nearly 40% of breast tumors (CALLAHAN et al. 1992; BIECHE and LIDEREAU 1995; WEITH et al. 1996). Likewise, LOH is also observed at human chromosome 9p markers in nearly 40% of breast cancers (EIRIKSDOTTIR et al. 1995; TIRKKONEN et al. 1998; AN et al. 1999). Interestingly, we observe allelic imbalances suggestive of LOH within Emca1 in a significant fraction of E2-induced mammary cancers in (ACI x COP)F₁ rats. The Cdkn2a tumor suppressor gene maps within the Emca1 interval on RNO5 (LAES et al. 1998). Cdkn2a encodes two proteins, p16^Cdkn2a and p19^Arf (QUELLE et al. 1995). Interestingly, a particular germline mutation in Cdkn2a has been demonstrated to predispose women to breast cancer (BORG et al. 2000). Moreover, a large fraction of human breast cancers do not express detectable levels of p16^Cdkn2a (GERADTS and WILSON 1996). We have observed that both the focal regions of atypical hyperplasia and the mammary carcinoma that develop in E2-treated ACI rats exhibit markedly reduced expression of p16^Cdkn2a, suggesting that loss of p16^Cdkn2a expression may be an early and contributory event in E2-induced mammary carcinogenesis (B. XIE and J. D. SHULL, unpublished data). Because of these similarities between human breast cancer and the ACI rat model of E2-induced mammary carcinogenesis, we consider Cdkn2a to be an attractive Emca1 candidate. Other interesting candidates known to play a role in breast cancer in humans that map within the Emca1 interval include Jak1 and c-Jun (SMITH et al. 1999; LUDES-MEYERS et al. 2001; WELCSH et al. 2002).

A second locus, Emca2, has been mapped to a 20-cM region of RNO18. The ACI allele of Emca2 appears to act in a dominant manner to increase tumor incidence and decrease tumor latency. Surprisingly, Emca2 was mapped in the (ACI x COP)F₂ population but not in the (COP x ACI)F₂ rats, which were generated in crosses in which the parental orientation used to generate the F₁ progeny was reversed. One possible explanation for this difference with regard to genetic mapping may be the subtle phenotypic differences observed between the (ACI x COP)F₂ and (COP x ACI)F₂ populations; although there is no difference in the incidence of E2-induced mammary carcinogenesis between the two populations, there is a significant difference between the two populations with respect to the mean latency: 193 (SD 50) days in the (ACI x COP)F₂ rats vs. 157 (SD 44) days in the (COP x ACI)F₂ rats (SHULL et al. 2001). Similar differences in latency were observed in the (ACI x COP)F₁ and (COP x ACI)F₁ rats [207 (SD 59) vs. 163 (SD 32)], suggesting that the parental orientation of the cross may have a significant impact on susceptibility to E2-induced mammary carcinogenesis. The failure to detect Emca2 in the (COP x ACI)F₂ cross is not due to a lack of power because of small sample size; 146 rats from the (COP x ACI)F₂ population were used for genetic analysis whereas 129 rats from the (ACI x COP)F₂ population were studied. The region of RNO18 containing Emca2 is syntenic to human chromosome 5q (http://www.ensembl.org/), which is reported to exhibit LOH in 25% of human breast cancers (LOVEDAY et al. 2000; RICHARD et al. 2000). Interesting candidates in the Emca2 interval include genes involved in breast cancer development or estrogen metabolism, such as fibroblast growth factor 1, glucocorticoid receptor, 17ß-hydroxysteroid dehydrogenase type IV, and adenomatous polyposis coli (http://www.ensembl.org/). As was observed with Emca1, Emca2 appears to modulate susceptibility to E2-induced mammary carcinogenesis in a cross between ACI and BN (M. TOCHACEK, B. SCHAFFER and J. D. SHULL, unpublished data).

The COP rat has been used in several genetic studies of susceptibility to mammary cancer induction (ISAACS 1986, 1988; KORKOLA et al. 1997; SHEPEL et al. 1998; HARVELL et al. 2000; SHULL et al. 2001). In crosses between the highly susceptible Wistar-Furth (WF) strain and the resistant COP strain, four loci that determine susceptibility to 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary cancer have been mapped to RNO1, -2, -7, and -8 (HSU et al. 1994; SHEPEL et al. 1998). Data from the present genetic study indicate that none of these loci determine susceptibility to E2-induced mammary cancer in reciprocal crosses between the ACI and COP strains. Emca1 maps to the same region of RNO5 as Mcs5, a determinant of susceptibility to DMBA-induced mammary cancer identified in a cross between the susceptible WF and resistant Wistar-Kyoto rat strains (LAN et al. 2001). Whereas Emca1 is a strong modifier of susceptibility to E2-induced mammary cancer in ACI x COP crosses, Mcs5 did not modify susceptibility to DMBA-induced mammary cancer in crosses between the WF and resistant COP strains (SHEPEL et al. 1998). These data suggest significant differences with regard to the genetic bases of susceptibility in the E2- and DMBA-induced mammary cancer models. Consistent with this hypothesis, the ACI rat strain has been demonstrated to be resistant to DMBA-induced mammary tumorigenesis (ISAACS 1988), but highly susceptible to E2-induced mammary cancer (SHULL et al. 1997, 2001; HARVELL et al. 2000, 2001, 2002). Interestingly, the Emca1 interval on RNO5 is syntenic to a region of mouse chromosome 4 containing Mmtg2, a locus that modulated mouse polyoma middle T-antigen-induced mammary tumor mass but not latency in a cross between the I/LnJ and FVB/N-TgN(MMTVPyMT)^634Mul strain (LE VOYER et al. 2001).

It has been postulated that E2-induced mammary carcinogenesis is dependent upon E2-induced pituitary tumorigenesis and associated hyperprolactinemia (STONE et al. 1979; SUMI et al. 1983; ITO et al. 1984). However, we have previously noted that in the (ACI x COP)F₂ and (COP x ACI)F₂ females used for genetic analysis of susceptibility to E2-induced mammary carcinogenesis, there was no correlation between pituitary mass, which correlates with circulating prolactin levels, and susceptibility to E2-induced mammary carcinogenesis (SHULL et al. 2001). Furthermore, genetic analysis of these females, in conjuction with a genetic analysis of the sensitivity to DES-induced pituitary tumorigenesis in male offspring from these same crosses, is not consistent with this hypothesis. If E2-induced mammary carcinogenesis were dependent upon pituitary tumorigenesis and hyperprolactinemia, we would anticipate that Emca1 and Emca2, which modulate mammary carcinogenesis, would also regulate pituitary tumorigenesis. However, neither locus has any discernible effect on pituitary mass in the (ACI x COP)F₂ and (COP x ACI)F₂ females.

In (ACI x COP)F₂ and (COP x ACI)F₂ males, sensitivity to DES-induced pituitary tumorigenesis is controlled by six genetic loci: Ept10 and Ept13 on RNO1, Ept2 and Ept6 on RNO3, Ept1 on RNO6, and Ept9 on RNO10 (T. E. STRECKER and J. D. SHULL, unpublished data). Just one of these loci, Ept10, was found to modulate sensitivity to E2-induced pituitary tumorigenesis in the (ACI x COP)F₂ females. The observed differences between the (ACI x COP)F₂ males and females with regard to the effect of the Ept loci on pituitary tumorigenesis may be due to the fact that the power to detect these loci is limited because females from this cross, unlike the males, were not sacrificed at a defined time point. Alternatively, differences in gender, the estrogen used, and the length of treatment may also affect the impact of the Ept loci on pituitary tumorigenesis. Importantly, there was no evidence that any of the Ept loci had any effect on E2-induced mammary carcinogenesis in the (ACI x COP)F₂ females. Weak evidence, based on an LRS value that just crossed the permutation-derived threshold for suggestive linkage, was obtained for a locus on RNO5 in the same region as Emca1 that modulates sensitivity to DES-induced pituitary tumorigenesis in the (ACI x COP)F₂ and (COP x ACI)F₂ males. Thus loci regulating estrogen-induced pituitary tumorigenesis in these crosses appear to be largely distinct genetically from those controlling susceptibility to E2-induced mammary carcinogenesis.

The hypothesis that distinct genes control susceptibility to estrogen-induced mammary carcinogenesis and sensitivity to estrogen-induced pituitary tumorigenesis is supported by the fact that certain experimental conditions inhibit the rapid development of E2-induced mammary cancer but have no impact on sensitivity to E2-induced pituitary tumorigenesis (SHULL et al. 1997; HARVELL et al. 2001). For example, ovariectomy inhibited the rapid development of mammary cancers in ACI females treated with E2 for 20 weeks, but had no impact on sensitivity to E2-induced pituitary tumorigenesis in these same rats (SHULL et al. 1997). Likewise, dietary energy restriction reduced mammary tumor incidence and tumor number but had no impact on E2-induced pituitary tumorigenesis or associated hyperprolactinemia in ACI females treated with E2 for 12–31 weeks (HARVELL et al. 2001). Thus, in experiments in which gender, the estrogen used, and, in the case of the ovariectomy study, the length of treatment, are constant, modulation of experimental conditions reveals that susceptibility to E2-induced mammary carcinogenesis and sensitivity to E2-induced pituitary tumorigenesis are likely to be largely genetically distinct.

The mapping of Emca1 and Emca2 significantly expands our understanding of estrogen action in the mammary gland and genetic susceptibility to mammary carcinogenesis. Furthermore, this linkage analysis contributes significantly to our overall understanding of genetic control of estrogen action. It is becoming clear that genetic factors play a significant role in controlling the responses of many tissues to estrogens. In addition to Emca1 and Emca2, loci have been mapped that regulate E2-induced eosinophil infiltration into the uterus in the mouse (GRIFFITH et al. 1997; ROPER et al. 1999) as well as DES-induced pituitary tumorigenesis (WENDELL and GORSKI 1997; T. E. STRECKER and J. D. SHULL, unpublished data), susceptibility to E2-induced uterine inflammation and pyometra (K. A. GOULD and J. D. SHULL, unpublished data), and sensitivity to DES-induced thymic atrophy (K. A. GOULD and J. D. SHULL, unpublished data) in the rat. The loci controlling these estrogen-induced phenotypes map to many distinct regions of the genome, suggesting that a substantial number of genes may control estrogen action in these tissues.

In summary, we have mapped two loci that modify susceptibility to E2-induced mammary cancer in genetic crosses between the highly susceptible ACI rat strain and the genetically related but resistant COP strain. On the basis of the fact that genotype at markers linked to Brca1 and p53, which reside on RNO10, as well as to Brca2, which resides on RNO12, showed no correlation with any of the mammary tumor phenotypes assessed in these crosses, we can exclude these genes as determinants of the susceptibility of the ACI rat to E2-induced mammary cancer. Identification of the genes within Emca1 and Emca2 that confer susceptibility should provide invaluable insights into the mechanisms through which estrogens contribute to breast cancer development.

The authors gratefully acknowledge members of the Shull laboratory, particularly Lois Bartsch, Linda Buckles, and Tracy Strecker, for their helpful discussions and critical review of the manuscript. This work was supported by grants R01-CA68529 (J.D.S.), R01-CA77876 (J.D.S), and R01-CA64364 (M.A.N.) from the National Institutes of Health and grant DAMD17-98-1-8217 (J.D.S.) from the U. S. Army Breast Cancer Research Program. M.T. and B.S.S. were supported in part by training grant DAMD17-00-1-0361 from the U. S. Army Breast Cancer Training Program. B.S.S. was supported in part by DAMD17-03-1-0477 from the U. S. Army Breast Cancer Training Program. Cancer Center support grant P30-CA36727 supported shared resources in the University of Nebraska Medical Center Eppley Cancer Center.

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