Abstract
Oestrogen receptor-positive (ER+) breast cancer accounts for over three quarters of all diagnosed breast cancer cases globally. While ER+ breast cancer has a better prognosis as compared with other subtypes, resistance to current treatments and recurrence of disease is common. ER+ breast cancer has lower immunogenicity than other breast cancer subtypes, meaning it is less likely to be recognised by the immune system and generate an effective immune response against cancer cells. This means it is a poor candidate for immunotherapy, a treatment that utilises the anti-tumour activity of the immune system and has found success in treating more immunogenic breast cancer subtypes. Therefore, better understanding of key players in the immune system and the development of ER+ breast cancer are needed to further the understanding and design of therapeutics to better treat this subtype.
Signal Transducer and Activator of Transcription 1 (STAT1) has been associated with the development of ER+ breast cancer. STAT1 is involved in tumour suppression by the immune system, especially via the IFNγ-signalling pathway, by anti-proliferative effects, recognition and destruction of cancer cells, and induction of apoptosis. Several studies have shown that mice homozygous negative for STAT1 expression (STAT1-/-) spontaneously develop ER+ mammary tumours. Additionally, downregulation of STAT1 expression is associated with ER+ breast cancer in humans, suggesting a link between STAT1 deficiency and ER+ breast cancer development. However, it is unknown how reconstitution of STAT1 expression affects ER+ breast cancer and if this influences cancer cell immunogenicity.
This project aimed to characterise the role of STAT1 in ER+ breast cancer. This was achieved by overexpressing STAT1 in SSM3 cells, a STAT1-/- murine ER+ breast cancer cell line derived from mammary tumours developed by STAT1-/- mice. Generation of a stable STAT1 overexpressing cell line was unsuccessful, therefore transient transfections were used to express STAT1 in SSM3 cells. Immunofluorescence analysis showed both cytoplasmic and nuclear localisation of STAT1 in transfected cells, indicating activity of the protein. Calculation of transfection efficiency via immunofluorescence showed the Stat1-containing vector efficiency of 12.6%. Analysis of apoptosis via flow cytometry initially showed an increase in apoptosis in Stat1-transfected cells, with IFNγ treatment iii having no effect, however further replicates failed to reproduce this result. Analysis of STAT1 target gene expression showed that immunosuppressive PD-L1 expression, but not MHC-I, was significantly upregulated in cells transfected with Stat1. This suggests selective gene upregulation, however further replicates are needed to confirm this. Increasing transfection efficiency would aid further replicates of experiments carried out above. Optimisation of the method for stable transfection would aid further investigation into the role of STAT1. This would allow for analysis of immunogenicity and tumorigenic characteristics, such as proliferation, migration and invasion via growth assays, of STAT1- expressing SSM3 cells as compared with wildtype SSM3 cells.