Identification of pathways involved in breast cancer susceptibility - contribution of SuprMam loci

Abstract

Two genomic regions that modify mammary, SuprMam1 and SuprMam2 have been identified in the BALB/c mouse strain in the Trp53+/- mouse model. We studied mammary gland morphology, tumour growth and expression levels of potential candidate genes and pathways in SM09 congenic mice (BALB/c SuprMam loci in C57BL/6 background) in comparison to parental strains, to identify the genes within the SuprMam loci that might be responsible for higher breast cancer susceptibility in BALB/c mice. While the analysis of mammary gland morphology revealed that there is a significant difference between the number of ductal branches between the two parental strains, there was no significant difference between SM09 and control mice. A similar pattern was observed for total epithelial area, suggesting that BALB/c SuprMam alleles alone do not contribute significantly to the morphological differences of the parental strains. The tumour suppressor DMBT1 (deleted in malignant brain tumors) has been identified as a candidate modifier gene within SuprMam1. Using semi-quantitative RT-PCR, Dmbt1 mRNA was found to be significantly lower in mammary glands of susceptible BALB/c mice compared to C57BL/6, while SM09 mice had similar levels to the control C57BL/6. This indicates that the lower level of Dmbt1 expression in BALB/c mice must be due to transcription factor differences outside the region, which are not present in SM09 mice. Assessment of the rate of tumour growth in SM09 and control mice demonstrated no significant difference in the tumour growth rate between the two strains indicating that the BALB/c SuprMam alleles do not affect the growth rate of the tumours in SM09 mice. However, this does not exclude the potential of the BALB/c SuprMam alleles to increase the susceptibility of normal mammary epithelium to tumorigenesis in the SM09 mice. Transcriptional profiling of the mammary glands of SM09 and control mice revealed that the majority of the differentially expressed genes are located within the SuprMam loci, demonstrating a direct effect of the loci. We studied the vitamin D pathway as a potential candidate pathway as a number of genes (Cyp2r1, PTH, Calca, Calcb) involved in the pathway are located within the SuprMam loci. Further, increasing scientific evidence suggests an association of vitamin D and breast cancer risk. While plasma levels of 25(OH)D, calcium and phosphate were not different between the two strains, there was an approximate 3-fold increase in plasma PTH levels in SM09 female mice compared to controls. Cyp2r1 contained potential promoter polymorphisms, but was not differentially expressed in the liver, and Pth, Calca and Calcb genes had no distinct polymorphisms. However, Cyp27b1, which is located outside the SuprMam loci, had a 9-fold reduction of expression in the kidney of the SM09 female mice. Further experiments suggest that reduced Cyp27b1 expression results in lower levels of 1,25(OH)2D leading to increased plasma PTH levels. If this is a significant contributor to BALB/c and human breast cancer susceptibility, then there is a practical opportunity for risk reduction. Identification of the genetic factors within the SuprMam region causing the differential expression of Cyp27b1 will require further analysis.