Genetic and epigenetic changes associated with the development of lymph node metastasis in triple negative breast cancer

Breast cancer is the most common cancer in women worldwide with one of the highest cancer-related death rates. The most aggressive breast cancer subtype is triple negative breast cancer (TNBC), which is characterised by the lack of expression for the estrogen receptor, progesterone receptor, and human epidermal growth factor 2. These receptors provide treatment targets in other breast cancer subtypes, which express at least one of these. Not only is there no targeted therapy for TNBC, it is also known to metastasise more frequently and earlier than other subtypes. Metastasis is the cause of most cancer-related deaths. This study aimed to identify genetic and epigenetic changes that are associated to lymph node metastasis in TNBC. In the first part of this study the gene expression of primary TNBC tumours (IDC), matched normal adjacent tissues (NAT) and matched lymph node metastases (LN) were examined using whole genome gene expression arrays. This revealed a novel four-gene signature specific for TNBC as well as an 83-gene signature associated with lymph node metastasis. Additionally, these findings were correlated with microRNAs that had previously been associated with lymph node metastasis in our own laboratory (using whole genome microRNA microarrays). microRNAs are small non-coding RNAs that are able to alter gene expression post-transcriptionally. After correlating genes and microRNAs that were associated with lymph node metastasis, the function of a number of these microRNAs were examined by overexpressing them in TNBC cells. Cell proliferation and migration were measured after transient microRNA mimic transfection. None of the tested microRNAs showed an effect on cell proliferation or migration, which may be due to the transfection method. Finally, the epigenetic mechanism of DNA methylation was investigated in these TNBC IDC, NAT and LN samples (using whole genome DNA methylation arrays). DNA methylation is the process whereby a methyl group is added to a cytosine base in the DNA, which leads to structural changes inhibiting gene transcription and therefore leads to gene silencing. The correlation of DNA methylation changes and gene expression changes provided novel insight into the development and progression of TNBC. Additionally, prognostic methylation changes were identified in this part of this study. Taken together, the results presented in this thesis show genetic (gene expression) and epigenetic (microRNAs and DNA methylation) changes that are associated with the development and progression of TNBC. This has not been done in TNBC on a whole genome level, including matched LN samples. This research has added to our knowledge and understanding of genetic and epigenetic changes that occur during the development and progression of TNBC. Especially, the novel insight in the progression of TNBC to secondary cancers provides potential prognostic indicators or therapeutic targets for this hard-to-treat subtype.