Deciphering the Epigenetic Symphony of Cancer: Insights and Epigenetic Therapies Implications

DNA Methylation

The process of DNA methylation includes the accretion of methyl group to the fifth carbon atom of cytosine present in the CpG cluster. This epigenetic mark is catalyzed by DNA methyltransferases (DMTs) and affiliated with heterochromatin condensation and gene repression. The most common target of DNA methylation, CpG islands, is typically present at the 5’ end of several genes. ¹⁸ In mammals, 80% of CpG islands are methylated, but in normal cells, the region within 2 kb of the islands is known as CpG shores, and some CpG islands are exceptionally hypomethylated. In the mammalian genome, 85% to 100% of regions are methylated, while 0 to 5% are unmethylated. In mammals, 80% of CpG islands are methylated. DNMT3-A and DNMT3-B catalyze the de novo methylation of double-stranded DNA, whereas DNMT1 maintains the DNA methylation pattern of newly synthesized strands. ¹⁸ DNMT3-A and DNMT3-B belong to a protein family DNMT3 that consists of an enzyme domain at the C-terminal region, an ATRX-DNMT3A-DNMT3L (ADD) domain in the central region, and Pro-Trp-Trp-Pro (PWWP) domain at the N terminal region. The regulation of enzymatic activity and the localization of DNMT3A and DNMT3B to their target sites is performed by both the PWWP and ADD domains. The PWWP domain (reader module) is involved in the proper chromatin binding of DNMT3A and DNMT3B and in recognizing histones H3K36me2 and H3K36me3, while the ADD domain (molecular module) recognizes unmodified histone H3 lysine 4. ¹⁹ DNA C5-cytosine methylation (5mC) and RNA N6-adenosine methylation (m6A) are predominant methylation mechanisms; however, apart from these, N6-methyldeoxyadenine is prevalent in both prokaryotes and eukaryotes mediated by 6mA MTases (methyl transferases). ²⁰ In tumor suppressor genes, gene silencing resulting from DNA methylation leads to carcinogenesis. In the mammalian genome, 85% to 100% of regions are methylated, while 0 to 5% are unmethylated. In housekeeping genes, the CpG island promoters are rarely methylated, while this modification occurs in those gene promoters or enhancers that are stabilized in a silenced state. ¹ DNA methylation of repeat regions maintains genome integrity by preventing retrotransposon activity under normal physiological conditions. In females, it helps in the deactivation of one of the copies of the X-X-chromosome and suppressing genes of specific tissues. ²¹ A prime example of a DNA methylation mechanism in normal cells is the activity of the ubiquitous transcription factor, Specificity protein 1 (Sp1). This protein interaction with DNA is controlled by the presence or absence of DNA methylation at CpG islands. Sp1 protein associates with unmethylated DNA to promote gene transcription while binding to methylated CpG sites is inhibited, therefore promoting gene transcriptional silencing in somatic cells. Altered DNA methylation patterns in cancer metastasis consist of a combination of distal enhancers and promoters overlapping transcriptional start sites hypermethylation causing critical gene regulatory elements repression associated with hypomethylation of retro-elements, centromeres, and oncogenes. ²² For example, the deamination of 5-methylcytosine to thymine causes a mutation in the tumor suppressor gene, or proto-oncogene produces an oncogenic point mutation that disrupts standard cellular mechanisms, contributing to cancer metastasis. ²³

Histone Modifications

Histone proteins posttranslational modification are associated with chromatin structure. A nucleosome comprises 147 bp DNA molecule wrapped around a histone octamer composed of 2 copies of 4 histone proteins, H2A, H2B, H3, and H4. All nucleosomes combine through linker DNA and histone H1 to form chromatin. ²⁴ Histone proteins are rich in positive amino acids lysine and arginine. Histone proteins are a part of nucleosomes rich in positive amino acids lysine and arginine. The most prominent histone modifications are phosphorylation, acetylation, ubiquitination, ADP ribosylation, sumoylation, ²⁵ and methylation, resulting in chromosome remodeling. Histone, a prominent part of chromatin, consists of globular domains and tails in which long tails are more susceptible to these covalent modifications that further modulate chromatin structure and gene expression. ²⁶ In histone modification, specific residues are important in maintaining gene expression and genome integrity and avoiding cancer risk. Histone core proteins and N-terminal amino acid tails are hotspots for their posttranslational modifications. A ubiquitination site is represented at histone 2 (2A and 2B), methylation sites at histone 3 and 4 (H3K9, H3K4, H3K36, H3K27, H3K79, and H4K20), phosphorylation areas at H3S10 and acetylation of amino acids including H3K23, H3K18, H3K9, H3K14, H4K5, H4K12, H4K8, and H4K16.

All these modifications play a vital role in cell DNA replication, DNA repair, chromosome dynamics, and DNA condensation during mitosis and meiosis. They are involved in the interaction of histones with chaperones, proteins, and DNA, etc. In the methylation process, arginine residue is modified by 1 or 2 methyl groups that can be arranged symmetrically or asymmetrically. At the same time, lysine can also contain 1, 2, or 3 methyl groups that do not affect the positive charge of lysine residue. ²⁴ Deregulation of HMTs and histone methyl demethylases (HDMs) can cause breast, prostate, lung, and brain cancer. ¹ Acetylation modification is carried out by histone acetyltransferases (HATs) and HDAC. Acetylation plays a prominent role in molecule charge and DNA unwinding processes. A single acetyl group can bind in the middle of a lysine residue, affecting its positive charge. Histone PTM comprises the addition or removal of small groups: acetylation (acetyl group in lysine residue), methylation (methyl in lysine, arginine, and glutamine residues), phosphorylation (phosphoryl groups in serine and threonine residues), or entire protein in the case of ubiquitin or small ubiquitin-like modification (SUMO) in lysine residue by specific enzymes. ²⁴

Noncoding RNAs

Approximately 75% of the human genome is transcribed into RNA, out of which only 3% encodes for proteins while 72% is not translated to proteins referred to as noncoding RNA. ²⁷ On the basis of length, shape, and location, there are 4 significant types of noncoding RNA: microRNA (miRNA), long ncRNA (lncRNA), circular RNA (circRNA), and PIWI-interacting RNA (piRNA) that function distinctly in cancer. MicroRNA is composed of 22 nucleotides and are small RNA that cause RNA-induced silencing complex by binding the corresponding sequence in targeted mRNA to degrade it. ²⁸ PiRNA is 24 to 30 nucleotides long in length, first identified in Drosophila, present in germ line cells that mediate in epigenetic regulation of chromatin ²⁹ while both lncRNAs and circRNAs are >200 nucleotides long; however, lncRNAs are linear and circRNAs are ring-like. ³⁰ When transcribed, both can fold into secondary structures and enable their interactions with DNA, RNA, and proteins. LncRNAs and circRNAs are involved in the epigenetic modulation of chromatin to regulate gene expression and act as a scaffold to regulate signaling pathways and protein–protein interactions. At transcriptional and posttranscriptional levels, ncRNAs contribute to the regulation and activity of cyclins, cyclin-dependent kinases, and their inhibitors mainly involved in the cell cycle in normal cells. ³¹ For instance, expression of lncRNA is enhanced in response to DNA damage; however, expression of microRNA is modulated by cell-cycle-dependent transcription factors such interactive patterns, when altered, can initiate and progress cancer by acting as an oncogene or suppressor leading to human diseases. ²⁰ These ncRNAs can help to initiate and progress cancer by acting as an oncogene or suppressor leading to human diseases. ³²

Epigenetic Control of Normal Cellular Processes

In somatic differentiated cells, 2 critical epigenetic modifications, DNA methylation and histone modifications allow the expression of tissue-specific and housekeeping genes to control cell differentiation only (Figure 1). DNA methylation plays a crucial role in X-inactivation, genomic stability, differentiation, and imprinting. ³⁴ A number of genes are methylated in somatic cells but unmethylated in germ-line cells. DNA hyper-methylation of repetitive genomic sequences serves as a mechanism to prevent chromosomal instability, translocations, and gene disruption caused by transposons. It has been suggested that some transposon families are both methylated and modified by histone proteins. To equalize the imbalance of the “extra” X-chromosome gene expression as compared to the one X-chromosome in males, DNA methylation is known to lead to X-chromosome inactivation, which causes monoallelic gene repression in female cells. ³⁵ To study the relationship between histone modifications and developmental expression of genes, the β-globin locus is the best example, as at different stages of development, vertebrates express different globin genes. ³⁶ MicroRNAs (miRNAs) are involved in cellular phenomena such as proliferation, development, differentiation, and apoptosis. They are tissue-specific in their expression. For instance, brain-specific miR-184, an intrinsic modulator of adult neurogenesis, is imprinted and subjected to epigenetic control by methyl-binding protein MeCP2 release. ³³

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Figure 1.

Epigenetic phenomenon in normal mammalian cells. These processes control and tune chromatin structure as well as gene expression. (A) DNA methylation, (B) histone modification, and (C) noncoding RNAs.

Altered DNA Methylation Patterns in Cancer

Initiation and advancement of cancer are coexistent with a broad range of epigenetic aberrations; such as changes in basic DNA methylation patterns. These changes can be hypomethylation in repetitive DNA sections, laminin-associated domains, and retrotransposons with lesser CpG sites (Figure 2). Hypomethylation ultimately leads to switching on the transposable elements and genetic instability. ³⁷ Feinberg and Vogelstein first discovered this aberrant methylation changes with a global decline in 5mC content among various cancer types. ³⁸ The higher aneuploidies and mutation rates that resulted from the reduction or deletion of DNA methyl-transferase (Dnmt1) have highlighted the contribution of DNA hypomethylation to induce chromosomal instability and fragility.^39,40 The reduction in methylated DNA activates the transcription of oncogenes ⁴¹ or the regions with transposons, consequently enhancing chromosomal recombination and genome instability. ⁴² The frequent hypomethylation of transposable elements enriched in intragenic and intergenic fragments accelerates gene expression. For instance, a study demonstrated how the oncogene is turned on due to DNA hypomethylation of CpG sites within the L1NE1 promoter and the consequent generation of favorable chromatin structure at the MET promoter. ⁴³ Likewise, hypomethylation of SINE repeats is detected in victims of acute myeloid leukemia. ⁴⁴ The hypomethylation of IGF2, PLAU, CDH3, BCSG1, and FEN1 is detected to be a prominent alteration in breast cancer patients. ⁴⁵ The reduction of methylation in pericentromeric areas of chromosome number 1 or 16 is frequently noted in a variety of carcinomas. ⁴⁰ Studies conducted on metastatic tumors of nonsmall lung cancer patients elucidated the upregulation of cell motility 3 and putative-oncogene engulfment elements due to hypomethylation within their promoter region. ⁴¹ Moreover, promoter hypomethylation is correlated with gain in the expression of Iroquois homeobox-1 in metastatic osteosarcoma. ⁴⁶

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Figure 2.

Comparison of DNA methylome in typical and cancer cells.

The aberrant hypermethylation in 5’ CpG sites of the gene is another central alternated methylation pattern resulting in cancer progression. Usually, CpG islands (CPI) located in 60% of gene promoters are nonmethylated. The deficiency of DNA methylation is accountable to the activated gene expression in open chromatin regions. ⁴⁷ Studies on various types of cancer cells showed that hypermethylation in CPI of genes deactivates expression of tumor-suppressor (Figure 2), DNA repair, and cell cycle regulatory genes.^48–50 Research on colorectal cancer and gliomas has confirmed the promoter's hypermethylation of a DNA repair enzyme named O6-methyl-guanine methyltransferase. Inactivation of this enzyme intern makes KRAS and p53 genes vulnerable to genetic mutations.^51,52

Similarly, malignant renal cell carcinoma is initiated due to the silencing of the VHL tumor such oppressor through the promoter's hypermethylation. ⁵³ The absence of imprinting at the 1GF₂/H₁₉ locus due to abnormal methylation is common in Wilms’ tumor, which in turn contributes to activation and advancement of gastric and colorectal cancer due to overexpressed growth factor IGF2. ⁵⁴ Abnormal hypermethylation of a RASSF4 (involved in negative regulation of RAS pathway) and GPX3 genes (stabilizes reactive oxygen species to suppress growth) lead to the development of multiple myeloma.^55,56 In myelofibrosis patients, silencing of a transcriptional regulator ZFP36L1 (a tumor suppressor gene) takes place owing to aberrant methylation of its enhancer, and deregulation of the gene is responsible for neoplastic transformation. ⁵⁷

Remodeling of methylation landscape is possibly accompanied by mutations in 10-11 translocator protein-2, a methyl-cytosine dioxygenase that oxidizes 5-methylcytosine (5mC) into 5- hydroxy-methyl-cytosine (5hmC), its successive conversion into 5-formylcytosine and 5-carboxymethylcysteine.^58,59 The consequent excision and replacement of 5-carboxymethyl cytosine with cytosine to thymine DNA glycosylase contribute to methylation alteration. ⁶⁰ Impaired hydroxylation is observed in almost 15% of myeloid cancers due to mutations in TET2 or functional losses in DMTs. ⁶¹

It has been demonstrated through previous studies that chromosome remodeling, transcription factors, ⁶² histone modification, ⁶³ and lncRNA ⁶⁴ play significant roles in regulating DNA methylation in various carcinomas. It has been conclusively demonstrated through studies on colon and esophageal cancer that lncRNA-mediated recruitment of DNMT1 ⁶⁵ in cancer cells and direct recruitment of DNMTs at the promoter regions through LINC01270/HOTAIR, respectively, are the predominant mechanisms.^66,67 Whereas indirect recruitment of DNMTs through the association of lncRNA with enhancer of zest-homolog-2 is a common phenomenon in several carcinomas. ⁶⁸ Another study conducted on digestive cancer patients showed the contribution of lysine methyl transferases, protein-arginine methyl transferases, and lysine-demethylases; transformers of histone methylation; in metabolic reprograming, carcinogenesis, invasion, and epithelial–mesenchymal transition of digestive tissues ⁶⁹ thus provided new aspects as therapeutic agents to administer such cancer.

Aberrant Histone Modification

Covalent modifications of histones, such as phosphorylation, acetylation, and methylation, are involved in reshaping the genome, nucleosome dynamics, chromatin compaction, and transcription of genes ⁷⁰ ; thus, their aberrant patterns are frequent in several types of cancers (Figure 3). These modifications are controlled by HMTs, HATs, HDMs, and HDACs. ⁷¹ Under normal conditions, histones located at enhancers and promoters of activated genes were found to be enriched by H₃K₂₇ acetylation. The genome-wide analysis confirmed the accumulation of aberrant H₃K₂₇ acetylation, which activates tumorigenic enhancers, thus resulting in the advancement of cancer. ⁷² Aberrant histone methylation, such as H₃K₂₇me₃ or H₃K₄me₃, is proposed to enhance the adaptation and plasticity of the tumors to their surroundings. ⁷³ Colorectal, prostate, and hepatocellular cancer progression occurs due to the upregulation of H₃K₄ HMT encoded by SMYD₃ ⁷⁴ and H₃K₄ HMT encoded by EZH₂ ⁷⁵ . Mutations of KMD₅C, KMD₅A (influencing methylation of H₃K₄), and KMD₆A (influencing methylation of H₃K₂₇) are prevalent in several carcinomas. ⁷⁶ Studies on hematological cancer have confirmed CREBBP and EP300 chromosomal translocation in genes encoding HATs. ⁷⁷ Similarly, EP300 chromosomal translocation is commonly observed in breast, colorectal, pancreatic, and gastric tumors. ⁷⁸

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Figure 3.

A comparison of general histone modifications in normal and cancer cells.

Histone phosphorylation controls several phenomena, namely DNA repair, mitosis, and gene regulation, and aberrant phosphorylation patterns of histones are frequently detected in prostate, colorectal, and breast cancer. ⁷⁹ Interestingly, overexpressed protein phosphatase 2A is diagnosed as a risk factor for the progression of hepatocellular cancer in patients with chronic hepatitis. ⁸⁰ Correspondingly, the advancement of hepatocellular carcinoma is enhanced due to a decrease in histone-H2A ubiquitination. ⁸¹ The overexpression of a SUMO conjugated enzyme Ubc9 is customarily accompanied by the expansion of myeloma, lung cancer, ovarian cancer, ⁸² and hepatocellular carcinoma. At the same time, its downregulation enhances levels of cleaved-Caspase-3. ⁸³ Similarly, inhibiting ADP-ribose polymerases binding to Sirt6 by a viral protein induces hepatocellular carcinoma. ⁸⁴

Noncoding RNAs and Cancer

Being the regulators of genes, alteration in noncoding RNAs (ncRNAs), including deletions and insertions, are associated with the initiation and advancement of several carcinomas. A study on chronic lymphocytic leukemia showed that a 13q14 deletion in genes encoding miR-15 and miR-16 RNAs is responsible for the progression of the disease. ⁸⁵ Inactivation of microRNAs through promoter hypermethylation is often detected in cancer. For example, oncogenic genes comprising CDK6, E2F3, C-MYC, and TGIF2 are activated due to silencing regulatory miR-9-3, miR-148, and miR-34b/c via methylation. ⁸⁶ Proteins and peptides such as FBXW7-185aa, SHPRH-146aa, HOXB-AS3, and PINT-87aa encoded by ncRNAs regulate epithelial-to-mesenchymal transition, glucose metabolism, and ubiquitination pathway lead to tumorigenesis. ⁸⁷ An m6A mutation in various circular RNAs is found to be responsible for initiating a variety of cancers, such as circ-NSUN2, modified by m6A, which establishes colorectal cancer, ⁸⁸ and circE7, modified by m6A, which establishes cervical cancer. ⁸⁹

Similarly, modifications in another class of ncRNAs called long-ncRNAs are prevalent in several carcinomas. For example, an m6A-altered LINC00958 (IncRNA) interns upregulate HDGF and are thus involved in the progression and lipogenesis of hepatocellular cancer. ⁹⁰ Moreover, research on HCC patients showed a miR-29a mediated 5hmC modification that promotes tumorigenesis via the TET-SOCS1-MMP9 pathway.^{91, 92} Figure 4 shows a general mechanism by which ncRNAs induce oncogenesis. In a nutshell, some epigenetic modifications and their mechanism leading to cancer are demonstrated in Table 1.

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Figure 4.

Contribution of ncRNAs in oncogenesis. (A) IncRNAs can either interact with the promotor or repressor and activate or silence a gene, interact with the chromatin-remodeling complex, and sequester miRNAs to inhibit their binding on mRNA. Deregulation of IncRNAs intern represses tumor suppressor genes or upregulates oncogenic signaling. ⁹¹ (B) miRNAs modulate genome expression by binding with mRNA. The silencing of tumor-suppressing genes via abnormal levels of oncogenic miRNAs and activation of oncogenes due to lesser concentration of tumor-suppressing miRNAs is the major phenomenon stimulating metastasis. ¹⁰⁸ (C) circRNAs control transcription by acting as a sequestering agent for miRNAs and transcription regulatory proteins. ¹⁰⁹ (D) Enhanced expression of oncogenic miRNAs by gain-in-function mutations and corresponding silencing of tumor suppressor genes. (E) Sequestering of tumor-suppressing miRNAs by mutated IncRNAs/circRNAs or its down-regulation induced oncogenesis.

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Table 1.

List of Some Epigenetic Alterations and Their Mechanism Leading to Cancer.

Aberrant epigenetic phenomenon	Gene, enzyme, or other elements mutated	Pathways	Outcome	References
DNA methylation	N6AMT1	N6-methyladenosine modulations of DNA, the transcriptional activity of genes, and deregulation of cell cycle inhibitors such as REST, RB1, P21, and TP53.	Breast cancer	93
	hMLH1	Mismatch repair mechanism and microsatellite instability	Colorectal cancer	94
	TERT	Immortalization of cancer cell	Lung cancer	95
	METTL7A	Metabolic processes such as lipid metabolism	Thyroid cancer	96
	CARD11	Inhibits or down regulates autophagy	Lung adenocarcinoma and renal cell carcinoma	97
Histone modifications	GCN5L2	Cell proliferation, cyclin E1 and D1 expression, apoptosis and promotion of E2F1	Breast, prostate, and colorectal cancer	98, 99
	WHSC1	Epithelial-mesenchymal transition, cell invasive character, and metastasis	Prostate cancer	100
	PRMT7	Epithelial–mesenchymal transition in cancer cell and metastasis	Breast cancer	101
	JARID2	Epithelial–mesenchymal transition in cancer cell, stem cell sustainability and poor survival.	Bladder cancer	102
	SET7/9	Worse prognosis, clinical aggressiveness, production, and proliferation of cancer stem cells	Lung cancer and acute myeloid leukemia	103
Noncoding RNA	HOXB13-AS1	Targets HOXB13 and enhances cell proliferation	Glioma	68
	MROS-1	Targets PRUNE2 to promote nodal metastasis	Oral cancer	104
	let-7	Enhances cancer invasiveness and multidrug resistance via oncogene activation	Ovarian cancer	105
	ASAP1	Activates oncogene, proliferation, and metastasis	Liver cancer	106
	FGFR3	Promote epithelial-mesenchymal transition and cancer proliferation by regulating miR-29a-3p/E2F1 axis	Ovarian cancer	107