Introduction
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide, accounting for 25% (1.7 million) of the total new cancer cases and 15% (521.900) of the total cancer-related deaths in 2012 (Torre et al.
2017). Breast cancer remains a significant threat to women all over the world, even though the breast cancer death rates have decreased by 40% between 1975 and 2017 (DeSantis et al.
2019).
Gene expression profiling has had an important impact on the understanding of breast cancer (Bell et al.
2017). For example, the analysis of certain biomarkers such as hormone receptor status, Her2 status and expression of Ki67 has led to the characterization of molecular subtypes of breast cancer that have shown significant differences in terms of their incidence, risk factors, prognosis and sensitivity to treatment (Prat et al.
2015).
Epigenetic alterations, such as DNA methylation and posttranslational modification of histones have been shown to have a considerable influence on the gene expression (Wu et al.
2015). Histones are the central component of the nucleosomes’ subunit. They form an octamer containing the four core histone proteins (H3, H4, H2A, H2B) around which is wrapped a 147-base-pair segment of the DNA (Audia and Campbell
2016). The histones’ N-terminal tails extend from the double-strand DNA and are subject to posttranslational modifications, which include acetylation, methylation, phosphorylation, ADP-ribosylation, glycosylation, sumoylation and ubiquitylation (Zhang et al.
2016). Histone acetylation is primarily associated with gene activation, whereas methylation, depending on its position and state, can either be associated with repression or activation (Wang et al.
2008).
Previous studies have analyzed the impact of specific posttranslational modifications on the gene expression (Lawrence et al.
2016). For example, H3K4 methylation has been intensely studied regarding the enzymes and molecular factors required for methylation (Shilatifard
2008). An association between high H3K4me3 expression and poor prognosis was found in patients with hepatocellular carcinoma and cervical carcinoma (Beyer et al.
2017; He et al.
2012). In cervical cancer, the same observation has been made for H3K9ac (Beyer et al.
2017).
As a thorough investigation regarding the influence of histone modifications on the prognosis of breast cancer patients was lacking, an expression analysis of histone H3 trimethyl K4 (H3K4me3) and histone H3 acetyl K9 (H3K9ac) was performed in this study. 235 tissue samples were examined by immunohistochemical methods and assessed by a semi-quantitative score.
Discussion
This study showed that specific histone modifications are important in breast cancer patients. H3K4me3 expression was correlated with positive estrogen receptor status, while H3K9ac staining was correlated with positive Her2 receptor status. Although results were highly significant, the correlations themselves were weak.
High H3K4me3 and H3K9ac expression were correlated with shorter breast cancer-specific survival as well as shorter progression-free survival. Overall survival was decreased in patients with high nuclear H3K4me3 staining.
Histone modifications have been of great scientific interest in the past years. After synthesis of histones, posttranslational modifications like methylation or acetylation are performed. These can take place in the nucleus or in the cytoplasm (Annunziato and Hansen
2000; Wu et al.
2012). Posttranslational alterations, which can mainly be found at the loose N-termini, but also within the global domain of histones, have been shown to regulate the structure, accessibility and replication of DNA and play an important role in fundamental cellular mechanisms in the cell cycle (Zhang et al.
2016). Aberrant histone modifications have been linked to the pathogenesis of several diseases, including inflammatory diseases and cancer, as they cause a shift in the gene expression and the overall metabolic state of the cell (Shanmugam et al.
2018).
Previous studies have concentrated on the impact of specific histone modifications on the cell: it has been shown that while histone acetylation is mainly associated with gene activation, methylation can be associated with either repression or activation, depending on its position and level (mono/di/trimethylated) (Kimura
2013; Lee et al.
1993; Vakoc et al.
2006). In this study, we analyzed the impact of H3K4me3 and H3K9ac on the survival of breast cancer patients, as these modifications have been described to be associated with active chromatin (Ruthenburg et al.
2007).
H3K4 methylation is a modification occurring at the fourth lysine residue from the N-terminus of Histone H3. It can be mono-, di- and trimethylated, which adds to the complexity of the analysis of its impact on the genome (Takahashi and Shilatifard
2010). H3K4me3 is generally associated with transcriptional activation and has been proposed as a predictive factor of poor prognosis in several types of cancer, such as liver and cervical cancer (Li et al.
2018). Poorer prognosis has also been described for patients with cervical cancer with a high expression of H3K9ac (Beyer et al.
2017).
By its neutralizing acetyl-group, H3K9ac leads to a de-condensation of the DNA structures and to an activation of transcription (Lee et al.
1993). The effect of an acetylated H3 at position 9 depends on the tumor entity: high H3K9ac levels seem to be associated with a poor prognosis in cervical cancer (Beyer et al.
2017), while patients with glioma have better prognosis with high H3K9ac levels (Liu et al.
2010).
There are much more histone modifications beside H3K4me3 and H3K9ac, which are associated to activation of transcription. For repressive modifications like H3K20me3 and H3K9me3, it was shown, that their levels are elevated in breast cancer cells (Leszinski et al.
2012). Another modification with repressive effect on gene transcription, H3K27me3, Healy et al. could show that it was associated to low grading and inversely to Her-2-neu status (Healey et al.
2014). These results fit to our observance that activating modifications have a positive correlation to the Her2neu status.
In the present study, we showed that H3K4 tri-methylation and H3K4 acetylation are negative prognosticators for breast cancer patients. Even though the mechanisms of histone modifications are not fully understood, several epigenetic therapies have shown great results in cancer treatment. One promising substance class are HDAC inhibitors, which prevent histone deacetylases from detaching the acetyl group from a histone, inducing cell cycle arrest, differentiation and cell death (Eckschlager et al.
2017). The HDAC inhibitors Vorinostat, Romidepsin and Belinostat have been approved by the FDA for the treatment of T cell lymphoma (Zhang et al.
2019). Many studies have concentrated on the combination of epigenetic therapy with well-established therapies and have demonstrated synergistic effects (Cao et al.
2015; Gao et al.
2016; Mann et al.
2007; Marks and Breslow
2007; Rettig et al.
2015). The addition of Vorinostat to Tamoxifen in breast cancer treatment resulted in tumor regression or prolonged disease stabilization in patients who had progressed on prior hormonal therapy (Thomas et al.
2011). In preclinical trials, HDAC inhibitors showed the ability to re-sensitize tamoxifen-resistant cells and prevent hormone therapy resistance (Munster et al.
2011), as well as a potentiation of the immune checkpoint inhibitor blockade in triple negative breast cancer in mice (Terranova-Barberio et al.
2017).
Our results show significant correlations of H3K9ac, H3K4me3 to Her2neu and ER as well as to survival data in breast cancer. As we examined the level of histone modifications at a fixed time by immunohistochemistry, we cannot say, if these results are cause or consequence of the cancer phenotype. Further experiments are, therefore, needed.
Further investigation of histone modifications in breast cancer could lead to a deeper understanding of the molecular mechanisms of cancer development. It could result in reliable screening methods, as well as the identification of new therapeutic targets for breast cancer treatment.
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