Disruption of the breast cancer susceptibility gene I, BRCA1, increases the risk of developing breast cancer. Although the range of BRCA1 mutations associated with pathogenicity have been and continue to be comprehensively catalogued, the mechanism by which a decrease or loss of BRCA1 expression leads to tumourigenesis is not well understood. Using a conditional Brca1 knockout mouse model, which displays aberrant lobular-alveolar development and tumour formation, this thesis aimed to determine how dysregulation of both coding and non-coding genes may contribute to the cellular consequences associated with a reduction in Brca1 expression in the mouse, and if these changes contribute to mammary tumourigenesis.
It was hypothesised that the molecular and cellular changes that occur when Brca1 expression is decreased may be mediated, in part, by the dysregulation of coding genes. To investigate this, data from a previously conducted microarray analysis, profiling the gene changes in the mammary glands of conditional Brca1 knockout mice at day one of lactation, in comparison to control mice, was used to identify candidate genes for further study. Based on this data and functional information from the literature, Aldh1a3, c-Kit and FoxA1, were selected for further analysis. The potential functional consequences of increased expression of these genes were assessed in HC11 cells, a mouse epithelial cell line capable of forming dome structures in a process akin to epithelial differentiation. Knockdown of Brca1 reduced the ability of HC11 cells to form dome structures. Interestingly, c-Kit expression but not Aldh1a3 or FoxA1 was elevated with Brca1 knockdown, suggesting a regulatory link between Brca1 and c-Kit may exist. However, over-expression of c-Kit in HC11 cells did not affect dome formation suggesting that increased c-Kit is not responsible for the reduced dome formation evident in cells with reduced Brca1 expression. However, these results do not preclude the possibility of these genes being involved in other cellular processes, such as proliferation or apoptosis, which may contribute to tumourigenesis.
MiRNAs are powerful regulators of both normal and disease processes. To extend our coding gene analysis, we investigated whether dysregulation of Brca1 in mammary epithelial cells perturbed the expression of miRNAs and assessed the functional consequences of aberrant miRNA expression. A candidate approach, based on functional and expression data from the literature, was used to select miRNAs that have been implicated in mammary gland development and/or breast cancer. The expression of candidate miRNAs was then determined in the mammary glands of lactating conditional Brca1 knockout mice. The majority of miRNAs tested were dysregulated in conditional Brca1 knockout mice, with miR-31, miR-148a, miR-181c, miR-200b and miR-210 being down regulated, and miR-135b, miR-155, miR-205 and miR-206 being over-expressed. Ectopic over-expression of miR-135b, miR-155, miR-205 and miR-206 in HC11 cells affected the ability of HC11 cells to form domes. Interestingly, increased expression of miR-155, miR-205 and miR-206 decreased HC11 dome formation, mimicking the phenotype of reduced Brca1 expression in these cells. This demonstrates that reduced Brca1 levels alter the expression of miRNAs, which may impart functional changes that could lead to tumourigenesis.
The expression of miR-206 was up regulated in both conditional Brca1 knockout mice and HC11 cells with decreased Brca1 expression and was therefore characterised further. Aberrant Brca1 expression has been shown to alter proliferation and migration; however, over-expression of miR-206 in HC11 cells did not affect these processes. As miRNAs primarily function by repressing the expression of coding genes, a bioinformatic analysis was used to identify potential gene targets of miR-206. This suggested miR-206 could target the 3’UTR of Sfrp1, an antagonist of the Wnt signalling pathway. Luciferase assays confirmed that miR-206 over-expression represses the Sfrp1 3’UTR and not a mutated Sfrp1 3’UTR in which nucleotides of the miR-206 binding site were modified. To investigate the potential role of miR-206 in mouse mammary gland development in vivo, a transgenic mouse model over-expressing miR-206 was created. Although several stages of mammary gland development were assessed, there was no detectable difference in the morphology or appearance of miR-206 over-expressing mice to controls. Taken together this suggests that although miR-206 may not affect mammary gland development, it may mediate tumourigenesis by repressing coding genes such as Sfrp1.
BRCA1 associated tumours are difficult to treat and are associated with a poor clinical prognosis. This thesis characterised several coding and miRNA genes that are dysregulated in the mammary glands of lactating conditional Brca1 knockout mice, some of which may mediate the consequences of Brca1 repression. These results further our understanding of how reduced Brca1 expression may predispose to tumour formation and could lead to the identification of potential drug targets which may be used in the future treatment of BRCA1 associated tumours.