Malignant melanoma: Underlying epigenetic mechanisms

Introduction

There were more than 18 million cancer cases diagnosed worldwide in 2018.^[1] In India, melanoma is relatively rare and the commonest subtype seen clinically is the acral lentiginous type, while a mixed epithelioid and spindle cell type is histologically the most common type.^[2] Cancer arises due to accumulation of genetic and/or epigenetic mutations that force cells to undergo repeated, uncontrolled divisions.^[3] Skin cancers are one of the most prevalent groups of cancers—the two broad groups being melanoma and non-melanoma cancers.^[4],[5],[6] Melanoma is the 19^th most commonly occurring cancer in men and women, with about 300,000 new cases reported in 2018.^[7] Although melanoma accounts for a smaller percentage of all skin cancers when compared to non-melanoma cancers, it is the cause for most skin cancer-related deaths.^[8] Melanoma is a result of a combination of factors, including genetic and epigenetic changes.^[9],[10] Several studies have revealed the role of epigenetic dysregulation in inducing melanoma. A robust association has been established between ultraviolet exposure and epigenetic alterations.^[10] It has been estimated that a 10% decrease in ozone levels will result in an additional 4,500 melanoma cases worldwide every year.^[11]

Genetic causes of melanoma have been extensively investigated. It is well known that melanocytes originate from the neural crest and upon differentiation, it converts to melanoblasts.^[12] Activation of tumor protein p53 (P53), the genome guard, induces the biogenesis of melanosomes that help in protecting the skin.^[13] With prolonged exposure to ultraviolet radiation, DNA damage results in a missense mutation in P53 which is considered an initiating event in the melanoma cascade.

Epigenetic Changes in Melanoma

Epigenetic changes can regulate gene expression without affecting the corresponding sequence of DNA.^[14] These changes can affect not only DNA but also chromatin structure through posttranscriptional modifications of histone along with nucleosome remodeling. Non-coding RNAs are also implicated in epigenetic-based regulation of gene expression. These epigenetic changes play a central role in predisposition to several human diseases, including skin cancer by altering genes related to cell proliferation and apoptosis.^[14],[15] A growing list of genes including, but not limited to, BRAF,^[16] PTEN,^[17] MGMT18 and RARB^[19] are proved to be altered either genetically or epigenetically in melanoma. The fact that melanoma is controlled by a set of genes implies that treating this disease represents a challenge.

Genes hypermethylated in melanoma are presented in [Table - 1].^{[20],[21],[22],[23],[24],[25],[26],[27],[28],[29],[30],[31]}

Table 1: Representative genes hypermethylated in melanoma^{[20],[21],[22],[23],[24],[25],[26],[27],[28],[29],[30],[31]}

DNA Methylation in Melanoma

Cytosine DNA methylation is the most commonly studied epigenetic marker in the last three decades.^[32] In this process, DNA methyltransferase adds a methyl group using the S-adenosyl methionine as a methyl donor. These methyl groups are added to the fifth carbon atom in Cs occurring in cytosine phosphate-guanine (CpG) dinucleotides [Figure - 1]. Methylation then recruits several proteins namely methylated DNA binding proteins (MBDs) that form a complex with histone deacetylases and chromatin remodeling factors resulting in a repressive chromatin status.^[9],[33] However, cytosine methylation could be repaired by the dioxygenase family of ten-eleven translocation methylcytosine dioxygenase (TET).^[34] These enzymes are unable to remove the methyl group from the cytosine residue, but instead, it hydroxylates the methyl group to form the 5-hydromethylcytosine. This can be further oxidized and finally removed by demethylation repair proteins.^[35],[36] CpG dinucleotides are distributed in the human genome in a precise manner.^[37] It can either occur as a dinucleotide or in clusters known as CpG islands. These islands commonly reside upstream of the gene promoter, in the first exon, where it epigenetically regulates the expression of the genes it locates within.^[38],[39] Normally, CpG islands are hypomethylated or even unmethylated, but as part of neoplastic transformation, these islands become methylated, and this is a hallmark of cancer initiation and/or progression.^[40] CpG island methylator phenotype status could be used to classify melanoma patients and are also found to be highly associated with mutation in ARID2 and IDH1.^[41],[42] These genes are involved in chromatin remodeling. Both methylation and demethylation are securely orchestrated, where misregulation of either process results in dysregulation of cancer-related gene expression can lead to cancer development.^[43] It is well known that hyper- and hypomethylation can lead to cancer, as these processes chiefly depend on the site in the genome where it takes place.^[44] Generally, cancer cells undergo hypermethylation in its tumor suppressor genes along with other regulatory genes. The hypermethylation occurs mainly in the promoter regions of the specified genes, where it, upon recruiting chromatin remodeling proteins, represses the transcription process.^[45] Another study has shown that dysplastic nevi are affected by promoter methylation of genes often methylated in melanoma. This is not the case with common nevi.^[46] DNA hypomethylation also is found to be associated with melanoma, and this hypomethylation might lead to genomic instability. Several studies have evaluated this phenomenon. A systematic review and meta-analysis by Guo et al. found up to 50 genes, associated with risk of melanoma, in the context of promoter methylation.6 Moreover, hypomethylation-mediated inactivation of CLDN11 is reported in melanoma. Hypermethylation of tumor-related genes such as RASSF1A,^[47] APC,^[48] DAPK^[49] HOXB13,^[50] MGMT,^[18] WIF1,^[51] RARB,^[19] INK4A,^[52] SYK,^[53] TFPI2^[41] and SOCS154 are found to be associated with advanced melanoma and poor prognosis. In addition, the methylation status of LINE1 could serve as a prognostic biomarker for cutaneous melanoma, where patients with hypomethylation in these repeats survive better than those with hypermethylation.^[55]

Figure 1: The mechanism of cytosine methylation. DNA methyltransferase adds methyl group to the fifth carbon atom of the cytosine residue exploiting S-Adenosyl methionine as a methyl donor. TET processes this 5 mC further to finally remove the methyl group

Specifically, it has been indicated that O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation is associated with response to dacarbazine/temozolomide (DTIC/TMZ) in disseminated cutaneous melanoma,^[6] and this makes it a promising predictive marker for temozolomide therapy in a metastatic melanoma patient.^[56] Meanwhile, MGMT promoter methylation may frequently coexist with P53 mutation, and these patients may benefit from treatment with alkylating agents.^[57]

DNA Methytransferase (DNMT) Mutation in Melanoma

DNMTs are a group of proteins that facilitate the insertion of a methyl group to the fifth carbon atom in cytosine. DNMTs are well-characterized proteins, with specific function assigned to each member.^[58] DNMT1 maintains methylation patterns in the already-methylated DNA.^[59] It works mainly in the replication fork using the hemimethylated parent as a template to methylate the newly synthesized DNA strand. Whereas DNMT3A and DNMT3B are involved in the de novo methylation process in the previously unmethylated DNA.^[60] Moreover, the expression of these two enzymes is shown to be associated with overall survival in stage II melanoma patients.^[61] Using multivariate analysis, a study reported that the DNMT1 rs2228612 polymorphism is an independent predictor of poor overall survival in melanoma patients.^[9] Disease progression is found to be an independent prognostic factor in melanoma patients.^[62] Polymorphisms related to DNMT are potential targets for new therapeutic approaches. It is reported also that DNMT3B plays a protumorigenic role in human melanoma, where the lack of this protein suppresses melanoma formation in a mouse melanoma model.^[63] Based on that, DNMT3 could be used as a biomarker for melanoma progression.

Histone Modifications in Melanoma

Histone is the core protein that plays a central role in terms of DNA organization in nucleosomes.^[64] Nucleosomes are the basic structural unit of the chromatin structure in eukaryotic systems.^[65] Histone is mainly characterized by the presence of N-rich tail areas that are rich in positively charged lysine.^[66] These tails undergo epigenetic modifications that include acetylation, methylation, phosphorylation, ubiquitination and SUMO(small ubiquitin-like modifiers)-ylation.^[67] These histone marks have been extensively studied, and its correlation with cancer formation is established. The most prominent histone modifications are acetylation and deacetylation, which are initiated by histone acetyltransferase and histone deacetylase proteins, respectively.^[68],[69] These histone marks play a central role in the regulation of gene expression.^[70] Histone acetyltransferase adds acetyl group that neutralizes the positively charged histone leading to loosening the tight binding between DNA (the negatively charged) and histone.^[71] This action turns the closed heterochromatin to open euchromatin, allowing the accessibility of transcription factors and hence gene transcription. Histone deacetylase performs the opposite action, where it renders the open chromatin to a closed one, preventing the expression of the corresponding gene.^[72] If these actions take place in the promoter region of cancer-related genes, the histone deacetylase activity can cause cancer to develop. Histone methylation also plays a crucial role in remodeling the chromatin and hence in the regulation of gene expression.^[73] This regulation is not only depending on the methylation but also the position (at which amino acid) and degree (number of methyl groups added) of methylation.^[74],[75] For example, histone H3 trimethylation at lysine 9 (K9) is generally associated with closed chromatin and hence silencing of the corresponding gene, while mono- and dimethylation of the same lysine residue is associated with open chromatin that allows the activation of the corresponding gene.^[76] Furthermore, methylation of K4, K36 and K79 in H3 along with methylation of K20 in H4 is considered an active methylation tag that is associated with activated gene expression.^[77] It is well established that a modified histone is associated with melanoma, where hypoacetylation causes downregulation of p21^Cip1 expression along with downregulation of the proapoptotic genes such as Bim, Bak and Bax.^[78] Meanwhile, histone methylation also has a role in melanoma development and progression.^[79] Upregulation of Ezh2 that functions to add triple methyl groups to the K27 in H3, leads to downregulation of p21^Cip1 expression in human melanoma.^[80] Meanwhile, demethylating enzymes such as H3K4 demethylase JARID1B which demethylate lysine 4 at H3 have a crucial role in the development of melanoma.^[81] Furthermore, lysine-specific histone demethylase demethylates histone 3 on K4 and K9 (H3K4 and H3K9).^[82] It eliminates two methyl groups from H3K4me2 resulting in the formation of H3K4me1/0. This H3K4me1/0 is a modification that tags enhancers in the human genome. It is reported that enforcement of the expression of LSD1in vivo has promoted BRAF^V600E-driven melanomagenesis.^[83] On the other hand, downregulating LSD1 in malignant cells stimulates anti-tumor T cell immunity and inhibits cellular proliferation. H3K27 demethylase plays a vital role in transcriptional elongation and cell differentiation. Demethylating H3K27me3 leads to obliterate transcriptional repression caused by the H3K27me3 mark. UTX, the H3K27-demethylase, stimulates transcription in melanoma at sites where the promoters are tagged with trimethylated H3K27.^[84] In addition, H3K4me2 is detected with high rates in melanoma samples compared to healthy skin samples. This histone mark is found to be less prevailed in metastatic melanoma compared to the primary one.

Microrna and Melanoma

MicroRNAs are endogenous, non-coding RNA transcripts of about 20-22 nucleotides in length that are evolutionarily conserved, and function as regulatory elements.^[85] It is considered an epigenetic mechanism of gene expression regulation. Several types of microRNA dysregulation are found to be associated with the development and progression of melanoma [Figure - 2]. Furthermore, microRNAs could also be used as prognostic biomarkers. It has been indicated that microRNA-211 is downregulated in melanoma cell lines, ⁸⁶ where the ectopic expression of this microRNA significantly inhibited its growth and invasion, suggesting that it might possess a tumor-suppressor activity.^[87] Furthermore, this has been confirmed in a study that reported the location of microRNA-211 within TRPM1, a suppressor of melanoma metastasis.^[88] Meanwhile, microRNA-222 is found to affect c-KIT and p27, disturbing the cell proliferation during melanoma progression. Metastasis of melanoma is further regulated by microRNA-205. MicroRNA-214 is found to interact with several tumor suppressor genes including ITGA3 and TFAP2C, where it triggers melanoma progression via suppressing these genes.^[89],[90] Cancer cells survival depends on the ability to handle microenvironment components.^[91] One such important factor in the tumor microenvironment is hypoxia.^[92] MicroRNA-210 expression inhibits MNT and enhances cell proliferation even in the lack of oxygen.^[93] MicroRNA-210 works also in the normoxic conditions, as it is upregulated in a HIF1α-dependent manner, upregulating both ATF3 and BNIP3, which function to adapt the cells to hypoxic conditions.^[94],[95]

Figure 2: Different microRNAs involved in melanoma initiation and progression

In addition, microRNA-210 is detected in patients with metastatic melanoma.^[96] Inversely, microRNA-33a/b and microRNA-18b are found to target HIF1α, where their expression inhibits melanoma cell proliferation.^[97] Let7a has been reported to inhibit G6PD, IMPDH2, AASDHPPT, SCD and FASN, leading to induction of oxidative stress in melanoma cells.^[98] Malignant cells can maneuver to avoid the hypoxic conditions via inducing angiogenesis. In melanoma, this process is found to be regulated by several microRNAs including microRNA-199a-3p, microRNA-1908 and microRNA-199a-5p.

Epigenetic-Based Therapy

The treatment of melanoma, including metastatic melanoma, has advanced in recent years with the development of newer groups such as BRAF, CTLA4 and PD1 inhibitors.^[99] Notwithstanding the emergence of many new targeted therapies and immunotherapy drugs, tumor resistance to these new therapies represents a major hindrance, and hence, more efficient treatment strategies are the need of the hour. Several studies have been published in recent years to clarify the role of histone remodeling in cancer formation and progression. A wide range of histone demethylase and deacetylase inhibitors are presented as potential melanoma treatment options.^[100] The recent understanding of the role of these enzymes specifies that they control the activation or repression of histone at the sites of the target genes. Histone deacetylase and histone acetyltransferase are among the proteins that were proposed as therapeutic agents for melanoma.^[101] These proteins are enrolled in the clinical setting under various commercial names. For example, H3K4 and H3K9 demethylases have been used for this purpose.^[102] Furthermore, JARID1 has been used in preclinical studies, and this might enhance its clinical utilization.^[103] Melanoma also could be treated with mitogen-activated protein kinase inhibitors which increase the portion of JARID1B-positive cells within the whole melanoma cell population.^[104] The main limitation of epigenetic-based therapies for melanoma is that they are inherently not very specific in action. It is therefore important to classify these therapies according to the specific situation.^[105] Epigenetic-based interventions can help improve the sensitization of tumor cells to immunotherapy. For example, the use of histone deacetylase is associated with the upregulation of targets associated with PD1 checkpoint inhibitor therapy, which have been correlated to slower progression and better survival.^[105] One of the major advantages of epigenetic-based therapies is based on the fact that epigenetic variation is more easy to be reversed as compared to genetic variations. However, the interplay between genetics and epigenetics must be explored in detail to formulate more effective treatment options.^[105]

Conclusion

Epigenetic changes including histone modifications and DNA methylation play an essential role in the development of melanoma, and possibly even in its prevention and therapy. Reserving of DNA methylation status and induction of histone acetylation results in the activation of tumor-suppressor gene and silencing of oncogenes, leading to control the proliferation of melanoma cells. Studies conducted at the epigenome level have partially demonstrated the function of clusters of histone-like markers in skin cancer, although the understanding of tumors is still unclear. These studies reveal that in a particular area of DNA, histone can be more active in regulating gene expression.

Epigenetic-based therapies, although only in the initial stages of research, have potential in the treatment of melanoma, especially by increasing sensitivity of the tumor to immunotherapy. The relatively non-specific action of epigenetic-based therapies also necessitates the need to have more clarity regarding the type of treatment protocol to be used in a specific type of melanoma. More studies are needed to explore the interactions between epigenetic modifications and genetic variations in melanoma.

Furthermore, microRNAs represent a proven tool for diagnosis, prognosis, and even treatment of several types of melanoma. Clustered regularly interspaced short palindromic repeat/Cas9 is the newly emerging approach that has been employed to treat melanoma

There are some limitations regarding the use of epigenetic-based therapy for melanoma. The most important drawback of using these drugs is its off targeting that might affect normal healthy skin cells along with cancerous cells. The main challenge here is to find drugs that target the affected tissues/cells without causing unwanted effects.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.