Mcs5c: a mammary carcinoma susceptibility locus located in a gene desert that associates with tenascin C expression

Abstract

Genetic factors have been estimated to account for at least 30% of a woman's risk to develop breast cancer. We have developed a rat model using Wistar Furth (WF) and Wistar Kyoto (WKy) strains to genetically identify mammary cancer susceptibility loci. The WKy allele of the mammary carcinogenesis susceptibility locus Mcs5c, was previously shown to reduce carcinoma multiplicity after 7,12-dimethylbenz-[a]anthracene (DMBA) exposure. In this study, Mcs5c was fine-mapped using WF.WKy congenic lines. Mcs5c was located to a region of approximately 176 kb on rat chromosome 5. One of the Mcs5c congenic lines containing a narrow Mcs5c WKy interval displayed a 40% decrease in average carcinoma number compared with WF-homozygous congenic controls after mammary carcinogenesis induction using two different models. As genetically mapped, the Mcs5c locus is located in a gene desert and thus is devoid of genes and annotated RNAs; thus, a genetic element in Mcs5c was hypothesized to regulate the expression of genes outside the locus. Tenascin c (Tnc) was identified as a candidate gene due to its reduced expression in thymus and ovarian tissues of Mcs5c WKy-homozygous congenic females compared with WF-homozygous congenic controls. This allele-specific differential expression is environmentally controlled.

Figure 1. Fine-mapping of Mcs5c

Maps of the congenic lines used to fine-map the Mcs5c locus beginning with the previously defined 4.5 Mb region (20). Microsatellite markers in the Mcs5c region on rat chromosome 5 are shown along the Y-axis. Negative or susceptible congenic lines for which the WKy-homozygous rats develop the same average number of mammary carcinomas as WF-homozygous rats are shown in white. Resistant congenic lines, for which the WKy-homozygous rats average fewer mammary carcinomas compared to WF-homozygous rats, are shown in dark grey. Areas of recombination are shown in light grey. A) Mcs5c was originally localized within a 301 Kb region that is contained in the resistant lines 5C-11 and 5C-18, but does not overlap with the susceptible line 5C-17. B) The Mcs5c interval is further reduced to a 176 Kb region as defined by the overlapping congenic lines 5C-14 (resistant) and 5C-23 (susceptible).

Figure 2. Mcs5c equivalently decreases mammary carcinoma multiplicity following DMBA administration and HER2/neu infusions

Average number of carcinomas per rat ± standard error is shown for congenic line 5C-11 WKy-homozygous animals (black) and WF-homozygous congenic controls (grey) for each of the two treatment groups. P-values were obtained using the non-parametric Mann-Whitney test for the DMBA study and using a generalized linear model for the HER2/neu experiment.

Figure 3. Comparative genomic map of the Mcs5c locus

The gene poor 176 Kb Mcs5c locus ± ~500 Kb and its orthologous regions in the mouse and human are shown. The potential candidate genes, Tnfsf15, Tnfsf8 and Tnc and Pappa are indicated. The dashed line indicates the sequence gap in the rat genome database, which was covered by sequencing the BAC clones. The map coordinates were obtained using the November 2004, July 2007 and Feb 2009 UCSC genome browser assemblies for the rat, mouse and human, respectively.

Figure 4. Relative expression of Tnfsf15, Tnfsf8 and Pappa in the thymus, ovary and mammary glands of Mcs5c WKy-homozygous rats 4 weeks post-DMBA

No differential expression of Tnfsf15, Tnfsf8 and Pappa was observed in a) the thymus, b) the ovary, and c) the mammary gland of WKy-homozygous congenic rats having the Mcs5c region (black) versus the WF-homozygous congenic controls (grey) at 4 weeks post-DMBA administration. Tnfsf8 expression was only quantified in the thymus; Pappa expression could not be quantified in the thymus. Mean relative expression ± standard errors are shown, with 10 or more rats in each group.

Figure 5. Tnc expression is reduced in the ovary and thymus of Mcs5c congenic rats after DMBA treatment and HER2/neu treatment

Tnc expression is reduced in the thymus (a) and ovary (b) but not in the mammary gland (c) of WKy-homozygous resistant congenic rats (black) compared to WF-homozygous congenic controls (grey) at 4 weeks post-DMBA treatment. Differential expression was not seen in any of these tissues of the age-matched rats that did not receive DMBA (a–c). Tnc expression was also decreased in the thymus and ovary of WKy-homozygous resistant congenic rats 12 weeks post HER2/neu infusions (d). P-values were obtained using the Mann-Whitney test. Mean relative expression ± standard errors are shown, with 10 or more rats per group.