Epigenetic crosstalk limits the reactivation of silenced tumor suppressor genes in cancer
Figure 1. Schematic overview of epigenetic crosstalk – created by BioRender and Canva
Abstract
The DNA hypermethylation of promoter regions of tumor suppressor genes (TSGs) and their consequent repression lead to uncontrolled proliferation and migration. In replicating tumor cells, the inhibition of DNMT1 causes transient accumulation of hemimethylated DNA at CpG islands in regulatory regions, stimulating the deposition of a ubiquitin on H3K18 by UHRF1. This triggers SUV39H1/H2 activity, leading to the formation of new H3K9me3 repressive sites. However, the presence of UHRF1 limits PRC2 repressive activity at TSG promoters, avoiding the trimethylation on H3K27 which acts as a second compensatory repressive mechanism when UHRF1 is depleted. Targeting this previously unknown H3K18ub-H3K9me3 crosstalk, without UHRF1 depletion, provides new strategies to improve the efficacy of DNMT1 inhibition therapy. This study enhances our understanding on cancer-related epigenetic mechanisms potentially targetable on a larger spectrum of tumours.
Review
Cancer cells rewrite epigenetics, dysregulating the expression of both tumor suppressor genes (TSGs) and oncogenes1. Specifically, TSGs’ promoters are hypermethylated leading to their silencing. As a consequence, tumor cells continue to proliferate out of control, lose cell adhesion and gain the capacity to migrate. The methylation is carried out by DNA methyltransferases which are enzymes that catalyze the transfer of a methyl group from S-adenosil methionine (SAM) to the 5’ of cytosines. These modifications mainly occur in CpG dinucleotides ensuring the same methylation pattern within both DNA strands2. Particularly, promoter regions rich in CpGs are known as CpG islands, and their hypermethylation leads to the formation of heterochromatin and the consequent gene repression. In detail, DNMT1 is responsible for the maintenance of altered methylation in the newly formed DNA strand during cell division3. For this reason, current therapeutic strategies involve the use of DNMT1 inhibitors for different tumor types, including colon cancer, which is the focus of this review. However, they can not actually achieve the full reactivation of TSGs probably due to epigenetic compensation. For example, histone modifications, triggered by DNA hypomethylation, can modify the accessibility of chromatin and gene expression. These modifications consist in the addition of one or more different chemical groups to some aminoacids on the histone tails.
H3K18ub–H3K9me3 crosstalk as a DNMT1 inhibition escape mechanism
DNMT1 inhibition causes transient accumulation of hemimethylated DNA at CpG islands during cell division that leads to the increase of H3K18ub, H3K27me3 and H3K9me3 signals. In particular, H3K18ub is a histone modification mediated by UHRF14 which presents a specific domain able to recognize hemimethylated DNA5. In this study, the authors assessed the role and the function of UHRF1 through genetic studies by employing doxycycline-inducible short hairpin RNAs to achieve the knock-down (KD) of UHRF1 in colon cancer (RKO) cells6.
It would be expected that, after UHRF1 KD, only H3K18ub levels decrease, but notably also H3K9me3 levels significantly diminished. The ubiquitination on H3K18 mediated by UHRF1 seems to affect the transfer of methyl groups on other lysines, suggesting a possible crosstalk between these modifications. In vitro methyltransferases activity assays show that only SUV39H1/H2 have enhanced activity specifically on ubiquitinated histone tails, including H3K18ub. When these enzymes are genetically silenced, there is not a significant increase of H3K9me3 levels even after DNMT1 inhibition. To further demonstrate the hypothesis that H3K18ub triggers H3K9me3 deposition, it was observed that the disruption of the ubiquitin interactive motif (UIM) of SUV39H1/H2 prevents the accumulation of H3K9me3 following DNMT1 inhibitors administration. All these findings prove the presence of a crosstalk between H3K18ub and H3K9me3. Interestingly, methylation on H3K9 is usually associated with heterochromatin formation and gene silencing, playing a role in TSGs repression7.
This crosstalk represents the first repressive compensatory mechanism carried out by cancer cells to maintain TSGs’ silencing and it is one of the reasons why DNMT1 inhibition therapy is inefficient.
H3K27me3 Compensation Following DNMT1 and UHRF1 Loss
As said before, inhibition of DNMT1 leads to the increase of H3K27me3 levels. The combination of UHRF1 KD and DNMT1 inhibition results in a greater enrichment of trimethylation levels on H3K27, particularly in the CpG islands of regulatory regions of genes. It can be hypothesized the presence of a second compensatory mechanism that maintains gene repression through H3K27me3 when UHRF1 and DNMT1 are not normally expressed. The trimethylation on H3K27 is mediated by the polycomb repressive 2 complex (PRC2)8 which is mainly recruited when DNA methylation is low. In this context, the activation of PRC2 guarantees the maintenance of gene silencing through H3K27me3 when UHRF1 is absent, while the presence of UHRF1 prevents PRC2 activity; however, the specific molecular mechanism should be further investigated.
Overcoming compensatory silencing through dual epigenetic inhibition
Since DNMT1 inhibition is not sufficient to correctly re-activate TSGs expression and the KD of UHRF1 activates the second PRC2-mediated compensatory mechanism, a suitable solution could be the combination of DNMT1 inhibition with SUV39H1/H2 depletion. Testing this combined treatment in RKO cells resulted in huge transcriptomic differences, finding out the upregulation of many protein-coding genes. In detail, most of them are TSGs which negatively regulate WNT and epithelial mesenchymal transition (EMT) pathways, which are respectively involved in cancer cell proliferation and migration9. To further confirm the beneficial therapeutic potential of the combined treatment, proliferation assays in RKO cells were performed, revealing a significant anti-proliferative effect6. These findings pave the way for new effective therapies to fight cancer.
Conclusions
In conclusion, three distinct repressive mechanisms that lead to TSGs silencing have been identified. The first mechanism that occurs is DNA hypermethylation on CpG islands of promoter regions of TSGs mediated by DNMTs. The second mechanism, at hemimethylated DNA regions, involves the recruitment of UHRF1, which mediates the ubiquitination of H3K18 that, in turn, is recognized by SUV39H1/H2, that deposits methyl groups on H3K9. This crosstalk between histone modifications maintains gene repression. Lastly, the third mechanism occurs in the absence of UHRF1 and in the presence of hemimethylated DNA and it includes the trimethylation on H3K27 mediated by PRC2, ensuring the TSGs repression.
Notably, the third layer of epigenetic silencing of these genes is relevant for the development of innovative therapeutic strategies, which combine SUV39H1/H2 depletion and DNMT1 inhibition. However, the development of an effective therapeutic approach remains a long-term goal, as in vivo validation is still required. Additionally, it is still unclear if this crosstalk is a tumor-type specific mechanism or if it is a general hallmark of cancers.
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