SILAC-based proteomic analysis to dissect the “histone modification signature” of human breast cancer cells
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In living cells, the N-terminal tails of core histones, the proteinaceous component of nucleosomes, are subjected to a range of covalent post-translational modifications (PTMs), which have specific roles in modulating chromatin structure and function. A growing body of evidence suggests that deregulation of histone modification patterns, upstream or downstream of DNA methylation, is a critical event in cancer initiation and progression. However, a comprehensive description of how histone modifications, singly or in combination, is disrupted in transformed cells is missing; consequently the issue whether and how specific changes in histone PTMs patterns correlate to particular tumor features is still elusive. In the present study, we focused on human breast cancer and comprehensively analyzed PTMs on histone H3 and H4 from four cancer cell lines (MCF7, MDA-MB231, MDA-MB453 and T-47D), in comparison with normal epithelial breast cells. We performed high-resolution mass spectrometry analysis of histones, in combination with stable isotope labeling with amino acids in cell culture (SILAC), to quantitatively track the modification changes in cancer cells, as compared to their normal counterpart. Our investigation focuses on lysine acetylation and methylation on fourteen distinct sites in H3 and H4. We observed significant changes for several modifications in cancer cells: while in a few cases those modifications had been previously described as a hallmark of human tumors, we could identify novel modifications, whose abundance is significantly altered in breast cancer cells. Overall, these modifications may represent part of a “breast cancer-specific epigenetic signature”, with implications in the characterization of histone-related biomarkers. This work demonstrates that SILAC-based proteomics is a powerful tool to study qualitatively and quantitatively histone PTMs patterns, contributing significantly to the comprehension of epigenetic phenomena in cancer biology.
KeywordsMass spectrometry Quantitative proteomics Epigenetic mark Histones Breast cancer
Liquid chromatography coupled to tandem mass spectrometry
Stable isotope labeling with amino acid in cell culture
Histone post-translational modifications
TB work is supported by grants from the Giovanni Armenise-Harvard Foundation Career Development Program, the Association of International Cancer Research (AICR), the Associazione Italiana Ricerca sul Cancro (AIRC) and the Fondazione Cariplo. Work in SMi lab is supported by EEC (Epitron) and AIRC. We would like to thank Pietro Spinelli for technical support in cell culture and David Cairns for critical reading of the manuscript.
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