A tumour suppressor role for FOXP3 and FOXP3-regulated microRNAs in breast cancer cells.

Abstract

During their lifetime, 1 in 9 Australian women will be diagnosed with breast cancer, a disease that arises due to mutations and epigenetic modifications to tumour suppressor genes and cancer-promoting oncogenes. This thesis investigates the tumour suppressive role of a transcription factor called Forkhead box Protein 3 (FOXP3) in breast cancer. Little is known regarding its role in the breast and therefore identification of FOXP3-sensitive pathways has the potential to highlight novel targets for breast cancer diagnosis and therapy. FOXP3 is a “master regulator‟ in immunosuppressive T regulatory cells, where it is essential for both cell development and function. It was previously thought that FOXP3 expression was restricted to these immune cells, however recent studies have identified FOXP3 expression in breast epithelia, where it has potential tumour suppressor properties. FOXP3 is mutated or has reduced expression in a significant proportion of human breast cancer samples, and loss of FOXP3 has been linked to increased mammary tumour formation in animal models. Few targets of FOXP3 in the breast have been identified, but it is known to directly repress the HER2 and SKP2 oncogenes while maintaining expression of the p21 tumour suppressor gene. A number of groups have shown that in T regulatory cells, FOXP3 regulates a number of small, non-coding RNAs called microRNAs (miRs). Importantly, many studies have reported extensive microRNA deregulation in human diseases, including breast cancer, and it was therefore hypothesised that similar regulation of miRs by FOXP3 occurs in breast epithelia. This thesis describes how FOXP3 induces two microRNAs, miR-7 and miR-155, in breast epithelial cells, with these miRs contributing to FOXP3-mediated tumour suppressive activity. One way this is achieved is through co-operation with FOXP3 in a feed-forward regulatory loop to suppress an oncogene called SATB1. SATB1 is highly overexpressed in late-stage breast cancers and promotes metastasis, the final and most fatal stage of breast cancer. This work has established that the SATB1 promoter is a direct target for FOXP3 repression and that miR-7 and miR-155 target the 3'UTR of SATB1 for further suppression. Re-introduction of FOXP3 into breast cancer cells using lentiviral technology results in reduced cell proliferation and invasion potential, supporting a role for FOXP3 as a tumour suppressor. To further understand the physiological importance of FOXP3 loss in cancer development, this work also investigated the role of FOXP3 in normal and immortalised breast epithelial cells, with results suggesting that FOXP3 expression prevents the acquisition of a cancerous phenotype. One way that it may achieve this is by maintaining elevated levels of miR-7 and miR-155. After further investigation, it was found that FOXP3 and miR-7 both have the potential to reduce epidermal growth factor receptor signalling and reduce resistance to apoptosis. In summary, this work describes a role for FOXP3 and the FOXP3-regulated microRNAs miR-7 and miR-155 in preventing the transformation of healthy breast epithelium to a cancerous phenotype. One way this is achieved is through a novel feed-forward mechanism by which FOXP3 and FOXP3-regulated miRs work together to suppress the pro-metastatic oncogene SATB1. This thesis provides important insight into the tumour suppressive role of FOXP3 in breast epithelia and with further investigation, this new knowledge may form the basis for the development of a novel and effective targeted breast cancer therapeutic.