Despite the enormous research that has been undertaken over the last decades, cancer continues to be a major health concern and one of the deadliest diseases in the 21^st century.¹ In 2022, approximately 9.7 million people died from cancer with nearly 20 million new cases worldwide.¹ The risk factors of cancer are complex and the current evidence suggests innumerable genetic, environmental, socioeconomic, and lifestyle-related factors that lead to carcinogenesis and tumorigenesis.^2,3

Over 100 years ago, Theodore Boveri proposed the somatic mutation theory describing genetic mutations as the key drivers of cancer and uncontrolled division of cancer cells.⁴ Though this theory is not a complete explanation of cancer, it has contributed significantly to explaining the origin of cancer and cancer biology. The theory-driven models have given a good foundation in the understanding of molecular aspects of cancer and in the implications of somatic mutations as stochastic events in cancer development are still relevant, however, the contemporary understanding of cancer goes beyond just somatic mutations.⁵ Studies have unraveled the increasing role of other important factors such as genomic stability, metabolomics, and dysregulated epigenetic patterns in the progression and metastasis of cancer.^6–12

Epigenetics which refers to alteration in DNA without changing the DNA sequence, is a comparatively new but fast growing field of biology. These changes including methylation, histone modification, and non-coding RNA significantly influence gene expression and other cellular and molecular mechanisms including proliferation and differentiation.^13–16 Epigenetics merges the genetic codes in the DNA and genome with various molecular and biochemical signals from intracellular, extracellular, and other environmental sources. In conjunction with the genome, the epigenome guides the specific gene expression of each cell type, shaping its functional identity throughout development and diseases.^17,18

DNA methylation is the process of adding a methyl group to a DNA base, usually a cytosine in a CpG dinucleotide pair. This modification influences how DNA coils around histones and how it impacts the binding of transcription factors, partly by attracting methyl CpG binding proteins.^19,20 Studies have shown that hypomethylation activates oncogenes, leading to the oncogenic properties of cells.^21–23 Likewise, hypermethylation leads to the suppression of genes associated with tumor suppressor activities, and thus promotes tumorigenesis of tumors.^24,25 Histone modification is another hallmark of cancer in which post-translational changes occur to the tails of histone molecules such as H2A, H2B, H3, and H4.²⁶ Research has demonstrated a substantial association of these post-translational modifications with the pathogenesis of cancer.^27,28

In cancer research, a wealth of literature is available that focuses on aberrant epigenetic regulations such as dysregulation of methylation, histone modification, and modulation of non-coding RNAs. The focus of this review is to summarize the epigenetics-related studies that play an important role in the metastasis of cancer. It will also highlight how aberrant epigenetic regulations can serve as potential therapeutic targets for cancer prevention.

Cancers significantly contribute to the public health burden, and projections estimated that this trend will continue to rise over the next couple of decades.^29–32 According to the GLOBOCAN 2022 estimates,¹ the incidence of major cancer types varies significantly, with lung cancer leading at 2.480 million cases followed by breast and colorectal cancers with 2.296 million and 1.926 million respectively. The incidence of prostate and stomach cancers is 1.467 and 0.866 million respectively (Figure 2A). Similarly, the mortality rates also differ, with lung cancer having the highest mortality at 1.817 million deaths followed by colorectal cancer with 0.904 million deaths. Other cancers with high mortality rates reported by GLOBOCAN 2022 are liver and breast cancers with 0.758 and 0.666 million deaths respectively (Figure 2B).

It is often impossible to determine precisely why one person develops cancer while another does not. However, research has identified specific risk factors that can increase the likelihood of developing cancer. For instance, nearly 30% to 50% of all cancers could be reduced by minimizing tobacco smoking and other carcinogenetic factors prevalent in the environment, maintaining a healthy lifestyle, and adhering to the available cancer screenings methods.³³ Early detection and screening help in many cancers such as cervical and colorectal cancers could be prevented by spotting the lesions that can be removed before they progress into cancer.^34–36

A systematic analysis in which the risk factors of cancer were evaluated from 2010–2019, reported obesity, smoking and alcohol consumption as the potential risk factors of cancer development.³⁷ Chronic infections are another risk factor that contributes to approximately 12% of the incidence of cancer globally.³⁸ Other factors that pose a potential risk of cancer include menopausal hormone therapy,^39,40 oral contraceptives,⁴² air pollution,^43,44 exposure to radiation,^45–47 and family history (genetics).^48,49 These risk factors, whether individually or in combination, can affect the progression of cancers.

According to Hanahan and Weinberg, the classical hallmarks of cancer include cell proliferation, angiogenesis, evasion of cell death, invasion, and metastasis.⁵⁰ Studies have revealed significant epigenetic changes such as methylation, histone acetylation, and non- coding RNA expression in all these classical hallmarks of cancer. In the subsequent sections, we will explore how epigenetic modifications influence these molecular events in cancer.

DNA methylation is an alteration that has a potential role in controlling gene expression,⁵¹ transposon silencing,⁵² X chromosome inactivation,⁵³ genomic imprinting,⁵⁴ and genomic stability.⁵⁵ Briefly, DNA methylation refers to the process where a methyl group is added to the cytosine at the 5’ end.⁵⁶ DNA methyltransferases (DNMTs) such as DNMT1, DNMT2, DNMT3a, and DNMT3b are a class of enzymes that have a significant role in DNA methylation. These enzymes transfer a methyl group to the fifth carbon of the cytosine pyrimidine ring.^57,58 Promoter methylation of a specific gene suppresses the expression of that gene by preventing the transcription factor from binding to the promoter. Similarly, loss of methylation leads to transcriptional activation, thereby contributing to the tissue and cell-specific expression during cell differentiation and embryogenesis.⁵⁹

Studies have revealed that the tight balance of methylation is lost in the process of cancer development.^60,61 Daskalos et al. reported that hypomethylation promoted genomic instability, leading to chromosomal aggregation and activation of transposable elements in non-small cell carcinomas.⁶² The hypomethylation- induced genomic instability results in increased expression of oncogenes such as claudin4, mesothelin, and S100A4^63,64 (Figure 3). On the other hand, hypermethylation promotes the inactivation of important tumor suppressor genes including BRCA1, retinoblastoma (Rb), and adenomatous polyposis coli (APC) (Figure 3). An increase in methylation also inactivates key genes such as MGMT, DAPK, and GSTP1 involved in DNA repair, apoptosis, and antioxidation respectively.^64,65

A range of studies have reported that hypomethylation is implicated in the progression of cancer at advanced stages^66–68 however studies have also reported it in the early stages of tumorigenesis.^69–71 Hypermethylation on the other hand is predominantly involved in the processes of DNA repair, cell cycle regulation, and tissue infiltration in cancer. Recent research efforts have identified nearly 100 genes which have high methylation in breast cancer.^72,73

The reversible aspect of DNA methylation makes it an appealing target for cancer therapy. Experimental and clinical studies have successfully targeted DNA methylation as a potent therapeutic target using DNMT inhibitor, 5-aza-2′-deoxycytidine (5-AZA).⁷⁴ Inhibitor 5-AZA has been shown to reduce DNMT3b levels in breast cancer and restore the regulation of tumor suppressor genes such as RASSF1A in hepatocellular carcinoma,⁷⁵ CDKN2B in myelodysplastic syndrome⁷⁶ and P53 in melanoma.⁷⁷ Targeting DNMTs by 5-AZA and correcting methylation dysregulation through personalized epigenetic therapies can provide potential therapeutic avenues for treating different types of human cancers.

The expression of the EZH2 gene serves as an independent diagnostic marker, indicating malignancy in breast, endometrial and prostate cancers. The regulation of the DNA damage repair gene MGMT can reverse the effects of chemotherapy and radiotherapy;⁷⁹ thus, MGMT depletion through hypomethylation can produce a favorable treatment response. Additionally, epigenetic modifications can contribute to tumor formation, which can make them valuable diagnostic markers for assessing disease risk and severity.⁸⁰ In NSCLC, the presence of an unmethylated IGFBP3 promoter indicates a positive response to Cisplatin chemotherapy.⁸¹

The level of methylation could also be used to assess the treatment efficacy and overall disease risk assessment. For instance, methylation at the promoter of PITX2 can serve as a predictor of the primary outcome in people with breast cancer undergoing adjuvant Tamoxifen treatment.⁸² Similarly, patients having bladder cancer, p16 hypermethylation may indicate a lower likelihood of relapse after giving IL2 treatment as compared to those without a hypermethylated p16.⁸³ Because epigenetic mechanisms influence genes and associated pathways, therefore they play a crucial role in finding out the most effective treatment and monitoring strategies for the patients.

Eukaryotic DNA is tightly wrapped around histone proteins to form organized chromatin. Nucleosomes, the basic units of chromatin, consist of 147 base pairs of DNA wrapped around a histone octamer, which includes two molecules each of histones H2A, H2B, H3, and H4.⁸⁴ Posttranslational modifications are critical biological processes by which genes and protein functions are regulated.⁸⁵ Histone modifications are posttranslational modifications in which chemical changes take place at the histone proteins. These modifications could be the removal or addition of certain functional groups such as methyl and acetyl groups (Figure 4). Studies have shown diverse biological functions of these histone modifications including signal transduction, apoptosis, cell cycle, and chromatin structure.^86,87

From a theoretical perspective, histone acetylation decreases the positive charge of histones, weakening their interaction with DNA and thereby enhancing gene expression. Typically, higher levels of histone acetylation are present in the promoters of active genes, influencing both the initiation and duration of transcription (Figure 4). Additionally, histone acetylation modifies chromatin structure, which impacts gene transcriptional activity.⁸⁸

Considering the importance of these modifications, substantial research has been carried out to investigate their role in different types of malignancies. Studies have revealed reduced H4K16 acetylation in a range of cancers including liver, breast, brain, colon, and lung cancers.^89,90 Reduction in acetylation accompanied by reduced H4K20me3 methylation has also been observed in breast and lung cancer.^89,90 A recent meta-analysis has revealed that H3K4me2 hypomethylation and H3K4me3 hypermethylation are considered poor treatment outcomes in breast cancer patients.⁹¹ Similarly, reduced levels of H3K4me2, H3K9me3, H3K9ac, and H3K18ac have been revealed to be strongly linked with poor prognosis and reoccurrence of lung cancer.⁹² Furthermore, H3K9me3 has the potential to serve as a biomarker for prognosis of gastric cancer.⁹³

The addition or removal of a methyl or acetyl group to the histone tails is achieved by specialized histone-modifying enzymes including histone methyltransferases (HMTs), histone acetyltransferases (HATs), histone deacetylases (HDACs), and histone demethylases (HDMs).⁹⁴ It is well documented that these HDACs are highly expressed in certain cancers such as gastric and prostate cancers.^95,96 In a study, Brehm et al. revealed that HDAC1 suppresses the transcription of cell-cycle-related protein cyclin E through Rb protein which results in the progression of tumorigenesis.⁹⁷ The aberrant regulation of histone methyltransferases and demethylases in cancer cells leads to abnormal histone modifications. In mice, the deletion of EZH2, a methyltransferase specific to H3K27, was linked to a high incidence of spontaneous T-cell leukemia. Additionally, elevated levels of EZH2 expression have been observed in prostate and breast cancers.^98–100

The nature of histone modifications is reversible and that makes these HDACs one of the key therapeutic targets in cancer treatment.¹⁰¹ For this obvious reason, PRMT1/SMARCA4 inhibitors have been under immense investigation.¹⁰² Valproic acid, an HDAC inhibitor, can produce various effects in both metastatic and non-metastatic cancer cell lines. When used alone or in combination with 5-AZA-DC, a DNA demethylating agent, it may help identify the epigenetic profiles that contribute to the metastatic characteristics of colorectal cancer cells.¹⁰³ GSK126 is another methyltransferase inhibitor that targets both the wild-type and mutant forms of the EZH2 enzyme. Studies conducted in xenograft models have demonstrated that GSK126 tends to act as antitumor agent in diffuse large B-cell lymphoma with EZH2 mutations.¹⁰⁴ Although further research is needed to unravel the complex association of histone modification and their association with cancer, these findings suggest that there is a hope of finding novel epigenetic- based therapeutic targets for cancer.

Nearly 75% of the human genome is transcribed into RNA, but only about 3% is converted into protein-coding mRNAs. The remaining non-coding RNAs (ncRNAs) are categorized into various types such as long non-coding RNA (lncRNA), microRNA (miRNA), and circular RNA (circRNA).¹⁰⁵ ncRNAs are another avenue of cancer therapeutics, especially the lncRNAs, which play an important role in modulating oncogenic molecular networks in carcinogenesis and metastasis¹⁰⁶ (Figure 5).

Studies have demonstrated an increasing role of ncRNAs in the development of certain cancers such as colorectal¹⁰⁷ and breast cancers.¹⁰⁸ In a study, Silva et al. found miR- 126 to be highly expressed in human B-cell acute lymphoblastic leukemia.¹⁰⁹ To validate, another study was conducted which revealed that a forced expression of miR-126 resulted in B-cell leukemia in a mouse model.¹¹⁰ Likewise, lncRNAs also play a key role in the regulation of genes and their associated pathways in cancer. In a recent study, Luo et al. revealed that HOXA gene-derived HOTTIP was abnormally high in acute myeloid leukemia,¹¹¹ suggesting a role in the cancer development.

The integration of epigenetic and genomic data holds promise for advancing personalized cancer medicine. By combining these two layers of information, researchers can develop more precise biomarkers and tailor treatments to individual patient’s unique genetic and epigenetic profiles. This approach could lead to significant improvements in treatment efficacy and reduce side effects, as therapies can be customized to address the specific molecular abnormalities present in each patient’s cancer.

Another exciting avenue for future research is the exploration of epigenetic mechanisms underlying drug resistance and cancer recurrence. Understanding how epigenetic modifications contribute to these challenges could lead to the development of novel therapeutic strategies to overcome resistance and prevent relapse. Furthermore, the role of epigenetics in cancer prevention and early detection is a growing field, with the potential for discovering new biomarkers and preventive interventions.

Cancer remains one of the diseases with the highest incidence and mortality worldwide. Over the last few decades, research has focused on finding the association between somatic mutations and cancers. However, the focus of research is shifting towards finding epigenetics-based solutions. The epigenetic imbalance that comes into play during tumorigenesis and metastasis stems from dysregulation in a range of cellular, biochemical, and molecular pathways. Emerging research in recent years has focused on the aberrant regulation of methylation, histone modifications, and other epigenetic hallmarks such as non-coding RNA expression and dysregulated phosphorylation. A number of tumor suppressor genes that are silenced during cancer are under immense research. Similarly, a range of oncogenes that are activated due to hypomethylation and acetylation are considered potential targets for the prevention and early detection of cancers.