Post-translational modifications (PTMs) of histones, one major type of epigenetic element, can serve as a marker for gene activity. Large-scale epigenomic analysis of normal human cells by NIH Roadmap Epigenomics Consortium ¹ and ENCODE consortium (Encyclopedia of DNA Elements) ² has delineated epigenetic dynamics during lineage specification and cellular differentiation. Aberrant patterns of histone modifications have recently been reported in a few cancer types. ³ Despite the discovery of more than 100 histone modifications ⁴ and availability of ChIP(Chromatin Immunoprecipitation)-grade antibodies for several them, most of the studies related to cancer have been focused toward a specific or couple of histone modifications. ^3,5 Histone code hypothesis ⁶ predicts that it is the combinatorial patterns of these marks, referred as “Chromatin States,” that dictate transcriptional output of the associated loci. Therefore, comprehensive determination of chromatin states is warranted for better understanding of epigenomic aberrations in cancers.

In our recent study ⁷ , we sought to determine pro-tumorigenic chromatin state alterations in melanoma progression with a focus on those that are associated with pre-malignant to malignant transition. To this end, we leveraged an isogenic cell model system derived from primary melanocytes with genetic alterations observed in human melanoma tumors, such as those in BRAF (V-Raf Murine Sarcoma Viral Oncogene Homolog B), p53 (TP53, best known as p53), and TERT (Telomerase Reverse Transcriptase) gene. ⁸ Malignant melanocytes harboured additional loss of PTEN (Phosphatase and Tensin Homolog) whereas pre-malignant melanocytes contained normal levels. This strategy was based on synergy between Braf activation and Pten loss in malignant melanoma formation in a transgenic mouse model. ⁹ The relatively quiet genomic background in this system facilitated identification of those epigenomic alterations that may be driving the phenotypic switch. We profiled 35 histone modification markers using a high-through ChIP-sequencing methodology ¹⁰ that allowed relatively easier profiling of these large set of markers in different cell types. Our results indicated the chromatin state transitions involving loss of multiple histone acetylation marks and H3K4me2/3 (Histone H3 Lysine di- or tri- methylation) methylation occurred during switch from pre-malignant (referred as Non-tumorigenic melanocyte, NTM) to malignant (Tumorigenic melanocyte, TM) phenotypes (Fig. 1). Importantly, changes in histone acetylation patterns were also observed between patient-derived benign nevi (pre-malignant) and melanoma (malignant) tissues demonstrating the relevance of our observation to human disease. Interestingly, although widespread losses of histone acetylation and methylation were seen by ChIP-Seq profiles, it was not observed at the global level suggesting selective locus-specific regulation histone acetylation/methylation marks in TM cells. These chromatin state switches occurred in the gene regulatory regions (promoters and enhancers) of cancer regulatory genes such as cell cycle inhibitors and pro-apoptotic genes. Importantly, we observed their preferential enrichment on regulators of various cell signaling events implicated in melanoma such as Phosphoinositide (PI) 3-kinase, interferon-g, PDGF (Platelet-Derived Growth Factor) and LKB (Liver Kinase B) pathways among others.

Loss of histone acetylation and H3K4 methylation promotes melanocytic malignant transformation

All authors

Ayush T. Raman & Kunal Rai

https://doi.org/10.1080/23723556.2017.1359229

Published online:

26 February 2018

Figure 1. Epigenomic alterations that accompany malignant transformation in melanoma. A model summarizing the results from Fiziev et al. ⁷ Malignant melanocytes, based on co-operativity between BRAFV600E (V-Raf Murine Sarcoma Viral Oncogene Homolog B) and PTEN (Phosphatase and Tensin Homolog) loss, harbour reduced histone acetylation and histone H3K4 (Histone H3 Lysine 4) methylation. This study suggests HDACi (Histone deacetylase inhibitors) as a potentially useful tool to revert malignant phenotype in melanoma.

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Are the chromatin state alterations associated with malignant state functional in imparting proliferative advantage? We reasoned that since histone modification patterns are reversible and observed changes are potentially result of competing histone acetyltransferases (HATs) and histone deacetylases (HDACs) activities; inhibition of HDACs in TM cells should reverse the observed epigenomic alterations and would be more effective in abrogating the proliferative abilities of TM cells. Indeed, we found HDAC inhibitors, Vorinostat and Entinostat, successfully suppressed hyper proliferation of TM cells and restored acetylation losses. Again, relevance to disease was established by observations that human melanoma cells harbouring lower H3K27Ac levels were more sensitive to HDAC inhibitors than those harbouring higher levels of this mark.

Although the pre-malignant and malignant melanocyte system is based on deleterious mutations in PTEN (Fig. 1) as seen in melanoma ⁸ , we found that observed epigenomic alterations were not specific to this specific genetic event. Loss of histone acetyltransferase such as CBP (CREB Binding Protein) or histone methyltransferase such as KMT2D (Lysine Methyltransferase 2D) in pre-malignant NTM melanocytes could also drive the transformation to the malignant state and recapitulate the chromatin state alterations seen in TM cells. These results strongly suggest that tumor-promoting epigenomic alterations contribute to phenotypic plasticity may be established by aberrations in either genetic or epigenetic machinery. We propose that it is the specific epigenomic state of a cell and is crucial for malignant transformation. Further studies will be needed to dissect and determine exactly which chromatin state changes at which loci serve as driver events and their potency in tumor formation.

Another key yet unexpected result was poor correlation between steady-state changes in histone acetylation with transcriptomic changes between NTM and TM despite high correlation between chromatin states and gene expression in individual cells. A plausible reason could be measurement of steady-state RNA levels and possibly, a better correlation may be observed if transient RNA-expression levels were measured by GRO-Seq or other methodologies. Biologically, it suggests important roles of post-transcriptional regulatory processes such as miRNAs or factors affecting transcript stability in determining steady-state RNA levels of these tumor-promoting genes. However, a systematic overlap of acetylation-based gene expression changes to those not involving chromatin changes showed the importance of chromatin-based regulation in preferentially mediating cancer-specific cell signaling events such as MAPK (Mitogen-activated Protein Kinase) pathways, p53-mediated signaling, EGFR (Epidermal Growth Factor Receptor) pathway and Caspase-mediated apoptosis.

Overall, our study provides an in-depth systematic view of epigenomic analysis of pre-malignant to malignant transition and identifies loss of chromatin states enriched in histone acetylation and H3K4 methylation as a key epigenetic alteration associated with malignant melanoma. Systematic efforts focused at determination of chromatin state profiles from large number of tumors are needed for comprehensive understanding of epigenomic alterations.