Characterising the role of TET2 in oestrogen receptor positive breast cancer

Abstract

The activity of the transcription factor oestrogen receptor α (ER) drives tumour development and metastasis in ER positive (ER+) breast cancer. This subtype constitutes up to three-quarters of breast cancer cases, and treatments directly targeting ER are currently the standard of care. ER operates as part of a signalling complex, co-ordinating with several other proteins to regulate gene expression. GATA3 is a transcription factor that has been closely linked to ER function, although its precise contribution to ER biology is not fully understood. The initial aim of this thesis was to further investigate the role of GATA3 in ER signalling. In doing so, GATA3 was shown to regulate the participation of the dioxygenase enzyme TET2 in the ER complex. The remainder of the thesis investigates the contribution of TET2 to ER biology. Quantitative multiplexed rapid immunoprecipitation mass spectrometry of endogenous proteins (qPLEX-RIME) was used to assess changes to the ER complex in response to GATA3 knockdown. Upon GATA3 silencing, and its loss from the ER complex, TET2 stood out as the only additional protein that was significantly depleted as an ER interactor, whilst several proteins were significantly enriched in the ER complex, including the transcription factors LHX4 and ZBTB34. LHX4 and ZBTB34 are thought to be capable of binding methylated DNA, whilst TET2 is also implicated in DNA methylation pathways through its iterative conversion of methylated DNA to several additional DNA modifications, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. These results implied a potential role for GATA3 in modulating reading and writing of DNA modifications as part of the ER complex. In the context of the recently developing focus on TET2 in transcriptional regulation, the association between ER and TET2 was investigated further. As few studies have successfully mapped endogenous TET2-chromatin interactions using chromatin immunoprecipitation (ChIP), the performance of various TET2 antibodies was evaluated using both ChIP and non-quantitative RIME. A commercial antibody was identified that performed robustly in both techniques, and TET2 binding events were subsequently shown to constitute a near-total subset of ER binding events in both ER+ breast cancer cell lines and patient-derived xenograft models. ER was shown to bind at several sites upstream of the TET2 promoter, supporting multiple existing studies proposing TET2 as an ER target gene. A key role for TET2 in the ER complex was further reinforced using non-quantitative RIME, where it was shown that TET2 associates not only with ER and GATA3, but additionally with several other key ER-associated proteins such as FOXA1, GREB1 and RARα. It was hypothesised that TET2, as an ER-regulated component of the ER complex, may help to sustain ER-regulated transcription in breast cancer. Consistent with a role for TET2 in mediating the proper activity of the ER complex, loss of TET2 correlated with a depletion of global ER binding. This was associated with dysregulated expression of both ER and GATA3 target genes. The genes most markedly regulated by loss of TET2 in ER+ breast cancer cells included those related to cell cycle control, and providing functional evidence of this, growth of ER+ MCF7 cells was inhibited by TET2 knockdown. To investigate the TET2-ER relationship in more detail, the role of TET2 in regulating DNA modifications in ER+ breast cancer cells was examined. TET2 knockdown did not appear to induce changes in global DNA methylation, and whilst preliminary data suggested TET2 may modulate DNA methylation levels specifically at ER binding regions, this remains to be fully determined. However, additional studies showed that TET2 robustly regulates oxidation of methylated DNA to 5-hydroxymethylcytosine in ER+ breast cancer cells, indicating a role for this enzyme in the production and maintenance of 5-hydroxymethylcytosine at ER sites. The conclusion to this thesis puts the work presented into perspective and provides an outlook for future studies.