Title: Histone and p53 Posttranslational Modifications in Breast Tumours: Exploring Molecular Mechanisms
Funder: Cancer Research UK

Abstract: Tumour cells exhibit widespread alterations in the prevalence of posttranslational modifications (PTMs) of chromatin and chromatin-associated proteins. This results in aberrant gene expression that contributes to the pathogenesis of cancer. Recent tumour microarray (TMA) studies have revealed a reduction in selected histone ‘marks’ in breast and prostate tumours that correlate with poor clinical outcome. The mechanism is likely to involve altered activity of the enzymes that impart PTMs such as the deacetylases, acetyltransferases and methyltransferases. This project will investigate chromatin modifications and expression of histone modifying enzymes and their coregulators in TMAs representing 880 well-characterised tumour samples. We will focus on HAT and HDAC proteins in the H4K16 regulatory axis, i.e. the NAD-dependent HDAC SIRT1 and MYST domain HATs, as well as their coregulators such as AROS, ING and DBC proteins. In parallel, we will assess the effects of small molecule inhibitors of these enzymes on breast cancer cell lines, to generate proof of principle evidence for novel therapeutic strategies. The project will be complemented by screening campaigns to isolate novel small molecule inhibitors that target selected PTms 1. To profile the prevalence of posttranslational modifications (PTMs) of histones and the tumour suppressor p53 in a well-characterised set of breast carcinomas (n=880) by tumour microarray profiling (TMA), using modification-specific antibodies. To evaluate the expression of selected histone variants (H2A.Z, macroH2A) in the same tumour cohort. Correlative analysis of the data with a wide range of clinicopathological factors, include tumour grade, prognostic factors, molecular markers, clinical outcomes etc. Validations using western blots on cell-free extracts or acid-extracted histones from selected tumour samples. 2. To investigate molecular pathways underlying these changes by profiling the expression of enzymes that catalyse PTMs, in particular the AcH4K16 axis of regulators, i.e. MYST acetyltransferases (MOF, TIP60, MOZ and MORF), MYST coregulators (ING4, ING5, proteins), HDACs, sirtuin deacetylases and their coregulators such as AROS and DBC. To validate expression data by western blot and real time PCR in selected tumour samples (e.g. type, grade) as appropriate. 3. To explore whether ‘epigenetic modulators’ e.g. small molecule inhibitors of HATs (HATi) and deacetylases (HDACi) can alter histone /p53 PTMs associated with different tumour phenotypes, using a panel of breast cancer cell lines. To determine how these inhibitors impact on the expression of histone variants, chromatin modifier enzymes and their cofactors (as above). To establish whether ‘epigenetic modulators’ impede cell proliferation or induce apoptosis in breast cancer cell lines, and the mechanisms involved. 4. Further validation by siRNA knockdown of HDACs, HATs and/or coregulators in breast cancer cell lines coupled with assessment of the impact on histone/p53 PTms Targets will be seelcted based on signifciant association with tumour grade/ clinical outcome in TMA data. Cell proliferation flow cytometry analyses of cell cycle/ apoptosis. Analysis of gene-specific effects by chromatin immuno-precipitation.


Research activity codes: 5.1 PHARMACEUTICALS;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: CANCER AND NEOPLASMS