Global histone modification fingerprinting in human cells using epigenetic reverse phase protein array

Abstract

The balance between acetylation and deacetylation of histone proteins plays a critical role in the regulation of genomic functions. Aberrations in global levels of histone modifications are linked to carcinogenesis and are currently the focus of intense scrutiny and translational research investments to develop new therapies, which can modify complex disease pathophysiology through epigenetic control. However, despite significant progress in our understanding of the molecular mechanisms of epigenetic machinery in various genomic contexts and cell types, the links between epigenetic modifications and cellular phenotypes are far from being clear. For example, enzymes controlling histone modifications utilize key cellular metabolites associated with intra- and extracellular feedback loops, adding a further layer of complexity to this process. Meanwhile, it has become increasingly evident that new assay technologies which provide robust and precise measurement of global histone modifications are required, for at least two pressing reasons: firstly, many approved drugs are known to influence histone modifications and new cancer therapies are increasingly being developed towards targeting histone deacetylases (HDACs) and other epigenetic readers and writers. Therefore, robust assays for fingerprinting the global effects of such drugs on preclinical cell, organoid and in vivo models is required; and secondly, robust histone-fingerprinting assays applicable to patient samples may afford the development of next-generation diagnostic and prognostic tools. In our study, we have used a panel of monoclonal antibodies to determine the relative changes in the global abundance of post-translational modifications on histones purified from cancer cell lines treated with HDAC inhibitors using a novel technique, called epigenetic reverse phase protein array. We observed a robust increase in acetylation levels within 2–24 h after inhibition of HDACs in different cancer cell lines. Moreover, when these cells were treated with N-acetylated amino acids in addition to HDACs, we detected a further increase in histone acetylation, demonstrating that these molecules could be utilized as donors of the acetyl moiety for protein acetylation. Consequently, this study not only offers a novel assay for diagnostics and drug screening but also warrants further research of the novel class of inexpensive, non-toxic natural compounds that could potentiate the effects of HDAC inhibitors and is therefore of interest for cancer therapeutics.

Emerging epigenomic landscapes of pancreatic cancer in the era of precision medicine

“…We predict that assays based on these types of markers will soon be part of the arsenal of the molecular pathologist at the bedside. Companion assays for histone-based epigenomic therapies are being developed but not yet applied to pancreatic cancer 54 . Epigenomic assays are increasingly becoming more miniaturized and assisted by robotics and microfluidics 55,56 , which can help to expand the repertoire of assays being developed.…”

Proteomics technologies for cancer liquid biopsies

“…RPPA is super robust in parallel to large sample profiling due to its nature of quantifying all samples in one experimental run, which usually ranges from a few hundreds to over a thousand samples [ 63 , 65 , 66 ]. Depending on antibody availability, RPPA can be broadly used for proteins, protein isoforms, and PTM, including phosphorylation, methylation, and acetylation analysis [ 67 , 68 ]. Additionally, its minimal pre-experimental process in a complete denaturing condition increases detection and quantification accuracy, allowing subtle fluctuations to be captured in biological systems.…”

Advancements in Oncoproteomics Technologies: Treading toward Translation into Clinical Practice

“…Further, the minimal pre-experimental process increases sensitivity and permits subtle fluctuations to be monitored. RPPA assays are particularly suitable for identifying proteins, proteoforms, and PTMs including phosphorylation, methylation, and acetylation within signaling networks [ 265 , 266 ]. The disadvantage of RPPA is that the specificity might be compromised to some degree, as a single detection probe/antibody is used.…”