Inhibitor of Differentiation 4: a new player in the DNA damage repair pathway in basal-like breast cancer

Abstract

Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Poor survival is compounded by a lack of targeted treatments. Detailed understanding of the molecular mechanisms underpinning this subtype is essential to the design of better therapies. Inhibitor of Differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator that is highly expressed in mammary stem cells and required for cell proliferation and luminal commitment during mammary gland development. ID4 gain (through overexpression and amplification) in a subset of BLBC associates with a stem-like poor prognosis phenotype. ID4 is necessary for the growth of BLBC cell lines. However the molecular function of ID4 in breast cancer is unknown. In this dissertation, I have used a multi-faceted approach to define the molecular mechanism of action of ID4 in BLBC and the related high-grade serous ovarian cancer (HGSOV). RIME (Rapid Immunoprecipitation and Mass spectrometry of Endogenous proteins) analysis of ID4 binding partners revealed novel interaction with DNA damage response and splicing proteins, in particular MDC1; known to be required for DNA damage sensing and repair. Through MDC1, ID4 interacts with γH2AX and BRCA1 and alters DNA damage foci formation or resolution. Using Chromatin Immunoprecipitation and sequencing, we have shown ID4, MDC1 and γH2AX co-localise at highly transcribed genes that are frequently mutated in cancer. ID4 binding is induced at these sites following ionising-radiation induced DNA damage. Using RNA-Sequencing following ID4 loss of function, we observe numerous changes in RNA splicing, potentially resulting from loss of ID4 s interaction with the splicing proteins SF3A1 and SF3B1 identified in RIME. A subsequent high-throughput siRNA screen of ID4 interactors and targets revealed a requirement for these splicing factors in the maintenance of cell viability and DNA damage foci formation. Clinical analysis demonstrates ID4 gain may occur early in the tumorigenesis of BRCA1-mutant BLBC and occur at a higher frequency, providing genetic evidence for an interaction of ID4 with BRCA1. These data link the interactions of ID4 with MDC1 and splicing factors to DNA damage repair and cell viability in the aetiology of BLBC and HGSOV.