Mapping of three genetic determinants of susceptibility to estrogen-induced mammary cancer within the Emca8 locus on rat chromosome 5

Abstract

The ACI rat model of 17β-estradiol (E2)-induced mammary cancer has gained wide use in the study of breast cancer etiology, prevention, and genetics. Emca8, a QTL that determines susceptibility to E2-induced mammary cancer, was previously mapped to rat chromosome 5 (RNO5) in an intercross between resistant Brown Norway (BN) and susceptible ACI rats. In this study, a panel of congenic rat strains, each of which carries BN alleles across a defined segment of RNO5 on the ACI genetic background, was generated and used to map more precisely the Emca8 determinants of mammary cancer susceptibility. Three distinct genetic determinants were localized within Emca8, and two of these were mapped to intervals of less than 15 megabases. Emca8.1 harbors Cdkn2a, Cdkn2b, and other genes and is orthologous to the 9p21 breast cancer locus identified in genome-wide and candidate gene association studies. Emca8.2 harbors Cdkn2c and other genes and is orthologous to the 1p32 locus in humans that is frequently deleted in breast cancers. Both Emca8.1 and Emca8.2 harbor copy number variants that are orthologous to copy number variant regions in humans. Gene expression profiles were defined for mammary tissues from E2-treated ACI and ACI.BN-Emca8 rats to define the impact of Emca8 on gene expression and identify differentially expressed genes residing within Emca8.1 and Emca8.2. This study further illustrates the relevance of the ACI rat model of E2-induced mammary cancer for identifying novel genetic determinants of breast cancer susceptibility and defining the mechanisms through which estrogens contribute to breast cancer development. Cancer Prev Res; 6(1); 59-69. ©2012 AACR.

Figure 1

The ACI.BN-Emca8 congenic rat strain exhibits reduced susceptibility to mammary cancer relative to the parental ACI rat strain. Ovary intact female ACI and ACI.BN-Emca8 rats were treated with E2 beginning at 9 weeks of age. Each animal was thereafter examined at least once per week for the presence of palpable mammary cancer. Latency to appearance of mammary cancer was prolonged in treated ACI.BN-Emca8 rats (n = 41) relative to contemporaneously treated ACI rats (n = 16) or a population of ACI rats (n = 112) merged from other experiments described herein. Vertical ticks on each line represent censored animals.

Figure 2

Emca8 harbors multiple genetic determinants of mammary cancer susceptibility. The Emca8a, Emca8b and Emca8c congenic rat strains harbor BN alleles across distinct overlapping segments of the larger Emca8 interval (Table 1). Ovary intact female Emca8a (n = 30), Emca8b (n = 35), Emca8c (n = 35), and ACI (n = 128) rats were treated with E2 beginning at 9 weeks of age and were thereafter examined at least once per week for the presence of palpable mammary cancer. Latency to appearance of mammary cancer was prolonged in the Emca8a, Emca8b and Emca8c rats relative to the ACI rats, indicating that two or more physically distinct genetic determinants of mammary cancer susceptibility reside on RNO5. Vertical ticks represent censored animals.

Figure 3

Fine mapping of Emca8.1. Ovary intact female Emca8b1 (n = 21), Emca8b3 (n = 13) and Emca8d (n = 33) rats were treated with E2 beginning at 9 weeks of age and were thereafter examined at least once per week for the presence of palpable mammary cancer. Latency to appearance of mammary cancer was significantly prolonged in the Emca8b1 and Emca8b3 rats, but not Emca8d rats, relative to the ACI rats. Vertical ticks represent censored animals.

Figure 4

Fine mapping of Emca8.2. Ovary intact female Emca8c1 (n = 23) and Emca8c2 (n = 20) rats were treated with E2 beginning at 9 weeks of age and were thereafter examined at least once per week for the presence of palpable mammary cancer. Latency to appearance of mammary cancer was significantly prolonged in the Emca8c2, but not Emca8c1 rats, relative to the ACI rats. Vertical ticks represent censored animals.

Figure 5

aCGH analyses reveal CNVs in Emca8.1 and Emca8.2. DNA was isolated from spleen (A. top panel) or liver (A. bottom panel) of two individual ACI and two individual BN rats. The ACI and BN DNA samples were labeled with Cy5 and Cy3, respectively, and were hybridized to NimbleGen tiling path oligonucleotide arrays. The dye labels were swapped in parallel experiments that are not illustrated to eliminate potential labeling bias. The CNVs residing within Emca8.1 and Emca8.2 are illustrated along the X axis relative to the genome coordinates along RNO5. The log2 transformed Cy3/Cy5 ratio is illustrated on the y-axis. Panel B illustrates the locations of annotated genes residing on the two DNA strands in and around the CNVs.