Transcriptional Networks Controlling Triple-Negative Breast Cancer Progression

Various molecular principles underlying different cancer disease mechanisms have been uncovered, and targetedtreatment is available for many cancers. However, patients with advanced metastatic disease confront a terminal illness that is, with few exceptions, incurable by current therapeutic regimes. Triple-negative breast cancer(TNBC) is a particular aggressive subtype of breast cancer, which represents exceptionally proliferative tumorswith high metastatic potential. The diagnosis of TNBC is based on the absence of molecular markers of theER-positive and HER2-positive breast cancer subtypes, hereby illustrating a problematic lack of known mechanisms driving the pathophysiology of TNBC. Furthermore, as TNBC tumors are highly heterogeneous only fewoverlapping characteristics has been identified. One of these is a particular high expression of the MYC oncoprotein. However, while MYC is associated with >70% of all cancers, it is untargetable and its cancer-specifictranscriptional role remains elusive. Thus, despite the clinical importance, the mechanism underlying TNBCbiology has not been uncovered. Consequently, the standard of care offered these patients is chemotherapy.Thus, there is an urgent need to understand the underlying mechanisms controlling TNBC tumorigenesis.

The work presented in this thesis is divided into two parts. In part I (Appendix I), we aimed to identify and next elucidate mechanistic details about transcriptional regulators in TNBC. We utilized a mass spectrometrybased technique to identify transcriptional regulators associated with active cis-regulatory regions in TNBC
cell lines. We hereafter employed genomics analysis to assess genome-wide changes in DNA-binding patterns, chromatin accessibility, and gene expression upon ligand induction, siRNA mediated knockdown, and/or inhibition of selected candidate proteins. This allowed us to uncover a transcriptional network in TNBC consisting of the glucocorticoid receptor (GR), GATA6, MYC, Fra-1, and c-Jun transcription factors. Despite sharing cisregulatory regions, we demonstrate that these transcription factors regulate largely distinct oncogenic pathways. Importantly, we demonstrate that they all depend on the SWI/SNF chromatin remodeling complex to access DNA and execute their oncogenic functions. Consistently, we show that inhibition of SWI/SNF simultaneously blocks cell proliferation, chemoresistance, and invasion, hereby illustrating the therapeutic potential of targeting
this remodeling complex in TNBC.

In the second part (Appendix II), we aimed to investigate the role of MYC at oncogenic enhancers and elucidate the molecular mechanism underlying MYC action at these regulatory sites. MYC enhancer invasion has been proposed to be spillover from saturated MYC promoters, induced upon high oncogenic levels of MYC expression, and has therefore been proposed to have low gene-regulatory potential. Contrary to this model, we demonstrate that MYC enhancer binding drives cancer type-specific gene programs predictive of poor patient outcomes. This indicates that MYC enhancer invasion plays a clinically important role in regulating tumor progression. We demonstrate that this is mediated though a MYC-induced epigenetic switch from H3K9me1/2 to H3K9ac mediated by KDM3A and GCN5, respectively. We furthermore show that H3K9ac promotes BRD4
binding, which facilitates recruitment of RNAPII to MYC-bound enhancers. Thus, we propose a revised model, where the oncogenic role of MYC is largely driven by MYC enhancer invasion rather than MYC-binding to promoters.