Epigenetic Modifications in Cancer Etiology, Diagnosis and Therapy

Received 06 Dec, 2024

Accepted 21 Jan, 2025

Published 30 Jun, 2025

Epigenetic modifications are implicated in the etiologies of various diseases, with cancer being a prominent example. Cancer, a debilitating disease, stands to benefit significantly from advances in the field of epigenetics. Unfortunately, epigenetics has not received sufficient attention and has not been integrated into mainstream healthcare. This study aims to raise public awareness about the link between epigenetics and disease etiologies, with a particular focus on cancer therapeutics. Relevant information was retrieved from reputable academic databases, including Embase, PubMed, Scopus and SpringerLink. The results indicate that epigenetic modifications, mainly noncoding RNA silencing, DNA methylation and histone modification, are essential for growth and development. However, aberrant epigenetic modifications or those exceeding normal levels can lead to diseases. Specifically, abnormal epigenetic changes in tumor suppressor genes (e.g., TP53, RB1, NF1, NF2, CDKN2A, WT1, BRCA1, BRCA2, PARP-1, VHL, APC, PTEN, PTCH1 and CDH1), oncogenes (e.g., RAS, EGFR, EML4AK, LINE-1 and SAT2) or DNA repair genes (e.g., MSH2, MSH6, MLH1, PMS1 and PMS2) can result in cancer. Furthermore, some epigenetic changes are reversible, suggesting that therapeutics targeting these changes in predisposed individuals could be more effective. Epigenetic tests such as Epicup^®, Cologuard^® and EpiproColon^®, along with epigenetic drugs like Azacitidine, Belinostat and Tubacin, have been developed for cancer treatment. However, these drugs face challenges related to poor pharmacokinetics and safety due to a lack of specificity and off-target effects. These issues are currently being addressed with epigenomic therapies. Health professionals are encouraged to target epigenetic changes in predisposed individuals to achieve better outcomes.

INTRODUCTION

Epigenetics is an evolving field of biology focused on disruptions in gene expression that are not attributed to changes in the DNA sequence itself but rather to modifications in the chemical tags on the DNA and associated proteins¹. These alterations in gene expression are heritable and influenced by environmental factors^2-4. Epigenetic changes can be classified as direct or indirect. Direct changes refer to alterations occurring during an individual's lifetime as a result of direct interactions with their environment⁵. Indirect changes include modifications that occur in the womb due to events during gestation, as well as changes affecting an individual’s ancestors (such as parents or grandparents), originating from events that took place long before conception⁵. Epigenetic mechanisms are crucial for normal growth and development, homeostasis and cell and tissue differentiation⁶. They also play a critical role in regulating pluripotency genes, which become inactivated during differentiation⁶. In multicellular organisms, epigenetic changes allow different adult cells to express the specific genes required for their unique functions and pass this information on to daughter cells⁷. Genome-wide patterns of DNA and histone modifications are established during early development and remain stable across numerous cell divisions⁸. Epigenetics also provides a means for organisms to integrate and react to environmental cues⁹. Epigenetic modifications influence human appearance, behavior, stress responses, disease susceptibility and even longevity, helping to shape an individual’s phenotype¹⁰. Cellular processes associated with epigenetic mechanisms encompass a variety of phenomena such as bookmarking, paramutation, genomic imprinting, gene silencing, position effects, X chromosome inactivation, variability in disorders or phenotypic outcomes, maternal inheritance patterns, carcinogenesis, reprogramming, teratogenic effects, heterochromatin formation, cloning and regulation of histone modifications⁷.

Incorrect epigenetic modifications or those beyond what is required for normal processes can lead to birth defects, childhood diseases or the emergence of disease symptoms later in life⁷. Abnormal epigenetic changes have been linked to the development of various diseases, including type 1 diabetes¹¹, type 2 diabetes¹², hepatocellular carcinoma¹³, breast cancer¹⁴ and reproductive anomalies¹⁵, among others. Fortunately, some epigenetic modifications are reversible, encouraging many researchers to focus on developing epigenetic therapies⁷. Therefore, targeting the epigenome to treat and prevent diseases represents a promising therapeutic strategy, particularly for debilitating diseases^10,16.

Cancer is among the top debilitating diseases that may benefit from improved understanding and breakthroughs in the field of epigenetics. Cancer is the leading cause of death globally, with mortality rates surpassing those of HIV/AIDS, tuberculosis and malaria combined¹⁷. It ranks as the second leading cause of death in developed countries and is one of the top three causes of death for adults in developing countries¹⁷. In 2022, an estimated 20 million new cancer cases were reported worldwide, along with 9.7 million deaths attributed to cancer¹⁸. Currently, over 53.5 million individuals are living with the disease¹⁸. Approximately one in five people will develop cancer at some point in their lives, with the mortality rate being about one in nine for men and one in twelve for women¹⁸. In Nigeria, annual statistics indicate around 72,000 cancer-related deaths and approximately 102,000 newly diagnosed cases¹⁷.

Cancer comprises a range of diseases characterized by abnormal cell growth that can invade or spread to other areas of the body¹⁹. Conventional treatment approaches for cancer include surgery, chemotherapy and radiotherapy. Recent advancements in treatment options encompass stem cell therapy, targeted therapy, ablation therapy, nanoparticles, natural antioxidants, radionics, chemodynamic therapy, sonodynamic therapy and ferroptosis-based therapy²⁰. However, these treatments face several challenges, including their suitability being limited to certain molecular subtypes of cancer, high costs, limited availability and the development of resistance in tumors²¹. Several research initiatives on cancer therapies are ongoing to find better or complementary strategies, with some focusing on epigenetic regulation. It has been established that various stages of tumor progression-such as tumorigenesis, promotion, progression and recurrence-are associated with epigenetic changes, some of which may be reversed through the use of epigenetic drugs²². To this end, some cancer epigenetic drugs, such as I-BET151, decitabine and azacitidine, have shown promise^23,24. Nevertheless, awareness of the role of epigenetic mechanisms in cancer pathogenesis and treatment remains limited. This review aimed to clarify and highlight the significance of epigenetic modifications in both the causes and treatments of cancer.

EPIGENETIC MECHANISMS

Epigenetic mechanisms provide an extra level of control within cells, influencing which genes are activated or silenced⁸. This regulatory process differs among tissues and plays a vital role in cell differentiation⁸. Additionally, variations in gene expression caused by epigenetic changes are key to the specialized functions of different cell types⁸. Unlike genetic inheritance, epigenetic marks are shaped by factors such as lifestyle, environment and nutritional status¹⁰. There are three primary mechanisms through which epigenetic regulations occur: Noncoding RNA (ncRNA) silencing, histone modification/chromatin remodeling and DNA methylation^25-27. These mechanisms can individually influence gene expression or synergistically regulate it¹⁵.

LINK BETWEEN EPIGENETIC REPROGRAMMING AND CANCER

The connection between epigenetics and cancer has been intensely studied over the past two decades, generating substantial clinical data that attest to the involvement of epigenetics in disease modulation and therapeutics^28-30. The formation of aberrant epigenetic reconfigurations is a hallmark of cancer cells. Aberrant epigenetic modifications in DNA repair genes, tumor suppressor genes (TSGs) or oncogenes, can cause cancer³¹. A TSG, also known as an oncogene suppressor or anti-oncogene, regulates a cell during division and replication, while oncogenes are mutated genes capable of causing cancer. The DNA repair genes repair damaged DNA. Hypermethylation of TSGs and hypomethylation of oncogenes and DNA repair genes can lead to cancer^32,33. Abnormal histone posttranslational modifications can alter the expression of these genes and cause cancer^27,34. Moreover, changes in ncRNA expression play crucial roles in human malignancies³⁵. The ncRNAs can act as oncogenes or suppressors, regulating cancer initiation and progression³⁶. Many miRNAs, in particular, function abnormally in cancer cells, contributing to cancer initiation. For instance, miR-126 overexpression downregulates EGFL7 and p53, causing hepatocellular carcinoma and breast colorectal cancers, respectively^36,37. The miR-155 is highly expressed in many cancers, such as breast, colon, lung, liver and gastric cancers³⁸. Furthermore, MiR-215 is overexpressed in glioblastoma in people undergoing hypoxia³⁹. Some miRNAs, regarded as tumor suppressors, such as miR-34, miR-34a, let-7, miR-200 and miR-15/16, are under-expressed in some cancers, including breast cancer, hepatocellular carcinoma, colon cancer, non-small-cell-lung cancer, pancreatic cancer and prostate cancer^36,40. Dysfunctional expression of lncRNAs plays a vital role in the function of tumor suppressor genes and oncogenes. Approximately 15% of upregulated and 11% of downregulated lncRNAs have been reported in several types of cancer, including breast, gastric, pancreatic, lung, hepatocellular, ovarian and prostate cancer⁴¹. In a study on ovarian high-grade serous carcinoma, lncRNAs such as ADAMTS9-AS2, ACTA2-AS1, HAND2-AS1, CBR3-AS1, LINC00312, IPW, LINC00887, XIST, MEG3, TSIX and NBR2 were under-expressed⁴². Hyperexpression of XIST lncRNA is linked with tumor malignancies in some cancers³⁶. The XIST may act as an oncogene, whose overexpression represses KLN2 in non-small cell lung cancers and causes miR-140/miR-124 overexpression in pancreatic carcinoma⁴¹. Overexpression of KCNQTOT1 lncRNA is implicated in colorectal cancer⁴³. Figure 1 describes the links between epigenetic changes and cancer.

Tumor suppressor genes and cancer: Epigenetic modification of tumor suppressor genes causes loss or reduction of its function, which, combined with some factors, can lead to the cell to growing abnormally⁴⁴. These genes’ loss of function may contribute more to the pathogenesis of human cancers than oncogenes’ activation⁴⁵. The list of tumor suppressor genes is extensive. However, as reviewed by Joyce et al.⁴⁶, the commonly encountered tumor suppressor genes include TP53, RB1, NF1, NF2, CDKN2A, WT1, BRCA1, BRCA2, PARP-1, VHL, APC, PTEN, PTCH1 and CDH1.

TP53: The TP53 tumor suppressor gene is called the “guardian of the genome” because it monitors cellular stress, such as inappropriate signaling, anoxia and DNA damage by mutated oncoproteins^46,47. The TP53 gene is located on the short arm of chromosome 17 (17p13.1) and codes for the p53 protein, which regulates the expression and activity of proteins involved in apoptosis, cell cycle arrest, DNA repair and cellular senescence⁴⁶. The loss of TP53 function can lead to perpetual cell replication and failure to initiate apoptosis⁴⁷. The TP53 tumor suppressor is the most often mutated gene in human cancers and so the most studied gene in oncology⁴⁷. It has been reported in over 50% of various forms of cancer, including colon, breast and lung cancers, leukemias, lymphomas, sarcomas and neurogenic tumors⁴⁶. Hypermethylation of TP53TG1 lncRNA has been shown to lead to the loss of TP53 function in humans, resulting in cancer⁴⁸. In acute myeloid leukemia, PRMT5 histone phosphorylation inhibits polo-like kinase 4 (PLK4) gene expression, activating the cGAS-STING pathway and repressing the TP53 gene⁴⁹. The DNA hypermethylation has also been shown to repress the TP53 gene, resulting in hepatocellular carcinoma²⁸.

Fig. 1: Epigenetic etiologies of cancer, showing epigenetic mechanisms and associated genes (Drawn using CorelDraw)

RB1: The RB1 gene is embedded in chromosome 13q14 and is the first identified tumor suppressor gene whose mutations predispose to cancer⁵⁰. It encodes the retinoblastoma protein (pRb) and its loss of function causes retinoblastoma, breast cancer and prostate cancer⁵⁰. In a study of 69 retinoblastoma patients and control groups comprising 26 normal relatives and 18 normal unrelated children, hypermethylation of the RB1 gene and DNMT genes was observed⁵¹. Overexpression of Tri-Methyl-Histone H3 Lys27 (H3K27me3) at the RB1 promoter was demonstrated to cause RB1 gene inactivation, leading to cancer⁵². Downregulation of let-7e, miR-320, miR-21, miR-486-3p and miR-532 has been demonstrated in the plasma of retinoblastoma patients⁵³. The under-expression of ncRNA-RB1, a lncRNA in the promoter region of the RB1, downregulates calreticulin (CALR). The CALR is a protein in the endoplasmic reticulum that translocates to the cell surface during pre-apoptosis and serves as a signal to phagocytic cells. The ncRNA-RB1 depletion inhibits the uptake of tumor cells by macrophages⁵⁴.

NF1 and NF2: The NF1 gene resides on chromosome 17q11.2 and codes for neurofibromin 1, a GTPase that negatively regulates RAS⁵⁵. A germline mutation in this gene causes loss of function, resulting in neurofibromatosis type 1, an autosomal dominant disorder characterized by the development of malignant peripheral nerve sheath tumors, neurofibromas and brain tumors such as optic gliomas⁵⁵. The NF2 gene is embedded in chromosome 22q12.2 and provides instructions for the production of

neurofibromin 2 (otherwise called merlin), a cytoskeletal protein that is involved in contact inhibition⁵⁶. Mutational loss of function in this gene leads to neurofibromatosis type 2, another autosomal dominant disorder characterized by an increased risk of tumors, particularly bilateral schwannomas⁵⁶. Hypermethylation of the NF1 gene disrupts RAS/MAPK signaling, causing cutaneous neurofibromas⁵⁷. Repression of NF1 in sporadic Head and Neck Squamous Cell Carcinomas (HNSCC) by overexpression of miRNA-193b activates ERK and results in tumor progression⁵⁸. Decreased dimethylation and trimethylation of H3K27me3 histones at the NF1 gene cause epigenetic changes via EED or SUZ12 mutations, contributing to MPNST tumorigenesis⁵⁹.

CDKN2A: This gene is situated on chromosome 9p21.3 and codes for p16/INK4a and ARF (both tumor suppressor proteins, which augment both RB and p53 functions⁶⁰. Germline mutational loss of function in this gene causes autosomal dominant familial melanoma⁶⁰. A biallelic mutational loss of function has been reported in several cancer types, including carcinomas, melanomas and leukemias⁶⁰. Downregulation of the CDKN2A tumor suppressor gene encoding the p16INK4a protein has been reported in several cases of cancer⁶⁰. The p16INK4a protein is actively involved in cell cycle regulation and senescence via its regulation of the cyclin D complexes and cyclin-dependent kinase (CDK) 4/6⁶¹. In a study of colorectal cancers, CDKN2A gene promoter region was overmethylated in 61% of tumor samples⁶². Hypermethylation of exon 2 in CDKN2A gene in tumor, tumor-distant and tumor-adjacent tissues predisposes to breast cancer⁶³. Abnormal histone H3K4m3 methylation and DNA methylation in the promoter region of CDKN2A gene have been detected in several cases of melanomas⁶¹. Upregulation of hsa-miR-542-5p, hsa-miR-4519 and hsa-miR-3681-3p represses CDKN2A, causing head and neck squamous cell carcinoma⁶⁴.

WT1: The WT1 gene is embedded on chromosome 11 and encodes transcription factors necessary for normal genitourinary tissue development⁶⁵. Germline mutational loss of function of this gene is associated with Wilms tumor, a pediatric kidney cancer⁶⁵. The WT1 mutations also cause sporadic Wilms tumors⁶⁵. The WT1 controls the de novo DNA methyltransferase 3A (DNMT3A) and downregulation of WT1 by short-interfering RNAs, causing the depletion of DNMT3A in human embryonal kidney-derived cell lines and Wilms tumor cells⁶⁶. The DNA hypermethylation of the WT1 gene causes elevated DNMT3A, resulting in Wilms tumor cells⁶⁶. The WT1 is hypermethylated and upregulated in breast cancer molecular subtypes⁶⁷. The WT1 is hyperexpressed in various hematologic malignancies, particularly in myelodysplastic syndromes and acute lymphoblastic leukemia⁶⁸. Hypoexpression of WT1 reduces the proliferation and increases the apoptosis of leukemic cells, indicating that WT1 might play the role of an oncogene in certain contexts⁶⁸. Histone deacetylase inhibitors such as Trichostatin A (TSA) can optimally and actively repress WT1 in several cell lines⁶⁸.

BRCA1, BRCA2 and PARP-1: The BRCA1 and BRCA2 are tumor suppressor genes embedded in chromosomes 17q21 and 13q12, respectively⁶⁹. They code for proteins that play a role in the DNA double-strand breaks repair via the homologous recombination repair pathway⁶⁹. The PARP-1 (located on 1q42.12) encodes a protein that assists in DNA single-strand breaks repair⁷⁰. Lack of functional DNA repair proteins can cause the cell cycle to continue propagating defective and mutated genetic material, resulting in aberrant daughter cells. The BRCA1 gene is commonly associated with breast cancer, induced most often by the depletion or absence of its protein product. Abnormal DNA methylation is suspected as a cause of its inactivation⁶². Overmethylation of the gene is linked with hematologic diseases, particularly leukemia⁶². The BRCA1 promoter hypermethylation is higher in malignant breast tumors than in healthy adjacent tissues and benign breast lesions⁷¹. Inactivation of some miRNAs (miR-9-1, let-7, miR-29a-c, miR-148, miR-17-5p, miR-15/16, miR-27b, miR-125a/b, miR-126, miR-130a, miR-143, miR-155, miR-200c, miR-145, miR-335 and miR-205) and the overexpression of others (miR-18a, miR-10b, miR-21, miR-206 and miR-27a) cause BRCA1-related histone modifications, resulting in breast cancers⁷².

VHL: The Von Hippel-Lindau (VHL) gene resides on chromosome 3p25.3 and codes for a constituent of a ubiquitin ligase that plays a role in the degradation of hypoxia-induced factors (HIFs), which are transcription factors that change gene expression in response to hypoxia⁷³. Von Hippel-Lindau syndrome is an autosomal dominant disorder, mediated by germline mutational loss of function. Inactivation of the gene poses a high risk of developing pheochromocytoma and renal cell carcinoma⁷³. Genetic or epigenetic inactivation of the VHL, which also inactivates HIF1A, is the most suspected in renal cancer cases⁷⁴. In one study, VHL promoter hypermethylation increased the risk of renal cell carcinoma⁷⁵. The VHL inactivation induces global genome over methylation in human kidney cancer cells under normoxic conditions⁷⁴. Histone lactylation triggered by inactive VHL contributes to clear cell renal cell carcinoma development through the activation of the transcription of platelet-derived growth factor receptor β (PDGFRβ). The PDGFRβ signaling in turn induces histone lactylation, creating an oncogenic positive feedback loop in clear-cell renal cell carcinoma⁷⁵. Hyperexpression of miR-21, miR-210 and miR-155 inactivates VHL, resulting in renal cancer⁷⁶.

APC: The Adenomatous polyposis coli (APC) gene is embedded in 5q22.2⁷⁷. This gene provides instructions for a tumor suppressor protein that regulates the WNT signaling pathway negatively, enhancing the creation of a complex that degrades β-catenin⁷⁷. The β-catenin plays a role in the regulation and coordination of gene transcription and cell-cell adhesion⁷⁷. The APC gene mutation is the most encountered in colon cancer and can initiate familial adenomatous polyposis (FAP)⁷⁷. Loss of a normal allele of APC can cause tumor, resulting in colorectal cancer and hepatocellular carcinoma. The APC hypermethylation has been shown to cause gastrointestinal tumors, including esophageal, colorectal, gastric, pancreatic and hepatic cancer⁷⁷. Overmethylation of the promoter region of APC can also predispose to breast cancer, suggesting that mutation in APC can cause other forms of cancer apart from colorectal neoplasms⁷⁸. Enhanced phosphorylation levels of histones such as S2260 and T1438 in the APC gene have been observed in cancers⁷⁷. The miR-494 is hyperexpressed in colorectal cancer tissues and this overexpression is negatively associated with APC expression⁷⁹. The APC is targeted directly by miR-494 in colorectal cancer, with overexpression of miR-494 inducing Wnt/β-catenin signaling, promoting colorectal cancer cell progression⁷⁹. Additionally, overexpression of hsa-miR-135b-5p causes loss of function of the APC gene, resulting in intestinal and diffuse gastric cancers⁸⁰. The lncRNA-mAK028845 was observed to be significantly upregulated, silencing APC during colorectal cancer initiation⁸¹.

PTEN: The Phosphatase and Tensin Homolog (PTEN) gene codes for a lipid phosphatase that negatively controls the mTOR and phosphoinositide-3-kinase (PI3K)-AKT signaling pathways⁸². The gene is located on chromosome 10q23⁸². Mutations or dysregulations in PTEN can cause various cancers, including follicular thyroid cancer, breast cancer, head and neck squamous carcinoma and prostate cancer⁸². In a study, the PTEN methylation was significantly elevated in breast cancer blood samples than in the control and benign samples⁸³. Additionally, hypermethylation of the PTEN gene has been shown to downregulate it by 30% in hypercoagulable blood samples compared to controls⁸⁴. The PTEN relates to histone H1 and regulates chromatin condensation, suppressing tumor development and metabolism. However, overacetylation of H4K16 disrupts the association between histone H1 and PTEN, constituting regulatory feedback that may deteriorate chromatin stability⁸⁵. Overexpression of miR-130a decreases PTEN levels, activating the PI3K/Akt/eNOS (endothelial nitric oxide synthase) signaling pathway, enhancing injury and inflammatory responses in Human Coronary Artery Endothelial Cells (HCAECs)⁸⁶. In a study, overexpression of miR-301a directly targeted and suppressed PTEN, promoting breast cancer⁸⁷. The miR-21 post-transcriptionally downregulates PTEN expression to promote cell proliferation and cervical cancer cell survival⁸⁸. In laryngeal squamous cell carcinoma, lncRNA HOTAIR interacts with miR-29b and represses PTEN⁸⁹.

PTCH1: The PTCH1 tumor suppressor gene resides on chromosome 9q22.3 and encodes the protein patched homolog 1, which negatively regulates the hedgehog signaling pathway⁹⁰. Germline mutational loss of function of this gene predisposes to Gorlin syndrome, an autosomal dominant disorder that poses a high risk of developing medulloblastoma and basal cell carcinoma⁹⁰. Acquired biallelic loss of function of PTCH1 mutations is often linked with sporadic cases of medulloblastoma and basal cell carcinoma⁹⁰. Overmethylation of the promoter region of the PTCH1 gene is the main cause of reduced PTCH1 expression in gastric cancer AGS cells⁹¹. Hypermethylation of the CpG islands of the PTCH1 gene was observed in AGS gastric cancer cells, suggesting that overmethylation of the promoter region of the PTCH1 gene is involved in the carcinogenesis and development of gastric cancer⁹². The miR-9 is overexpressed in Glioblastoma Multiforme (GBM) patients and causes resistance to temozolomide, a drug used for treating glioblastoma that targets the Sonic Hedgehog receptor PTCH1⁹³. This suggests that overexpression of miR-9 downregulates PTCH1. In a study, miR-9 expression was raised in the tissues of GBM patients and early-passage GBM cell lines of patients with recurrent GBM but not from naïve patients⁹³. Protein phosphorylation and autophosphorylation of the PTCH1 gene, such as protein kinase C phosphorylation and peptidyl-tyrosine, are implicated in poor prognosis and increased recurrence of breast cancer⁹⁴. Histone Deacetylase (HDAC) modification has been shown to decrease PTCH1 gene expression, causing breast cancer⁹⁵.

CDH1: The CDH1 (E-cadherin) gene is a tumor suppressor gene that resides on chromosome 16q22.1⁹⁶. Once in contact with neighboring cells, healthy cells stop replicating, which maintains the structure and architecture of the tissue, a phenomenon called contact inhibition⁹⁶. The E-cadherin binds to β-catenin, an important constituent of the WNT signaling pathway, to regulate contact inhibition⁹⁶. Germline mutational loss of function in this gene is associated with autosomal dominant familial gastric carcinoma⁹⁶. Loss of function mutation and hypermethylation of the CDH1 gene were observed in breast cancer patients in Kashmir⁹⁷. The CDH1 gene promoter hypermethylation has been documented in gastric cancer and chronic gastritis⁹⁸. Underacetylation of histones H4K16Ac and H3 in the exon 8 of the CDH1 gene has been reported in gastric cancer cell lines⁹⁹. Additionally, histone methylation analysis showed elevated H3K36 trimethylation in the exon 8 of the CDH1 gene in gastric cancer cell lines compared to healthy cells⁹⁹. In a study, repression of hsa-miR-383 in association with upregulation of AL356608.1, LINC00337, AL357153.2, MALAT1, HULC and LINC00485 interacted with CDH1 to promote breast cancer¹⁰⁰. The miR-383 functionally suppresses tumor, inhibiting cell replication, metastasis and epithelial-mesenchymal transition (EMT) in breast cancer by targeting ETS1²⁹. Furthermore, miR-205, miR-200a and miR-429 are often hyper-expressed in pancreatic tumors, while CDH1 is under-expressed¹⁰¹. Long non-coding RNA H19 mediates methylation-dependent repression of the CDH1 promoter, contributing to the progression of lung adenocarcinoma¹⁰².

Oncogenes and cancer: The oncogenes are numerous, but some are frequently implicated in cancer. Several proto-oncogenes are functionally involved in embryogenesis and specifically stimulate cell proliferation and growth during organismal development¹⁰³. Additionally, some negatively regulate cell differentiation¹⁰³. Typically, proto-oncogenes are switched off after completing the developmental processes they regulate¹⁰³. However, if proto-oncogene remains active or is faultily reactivated as the individuals get older, it may induce cancer¹⁰³. Some frequently implicated oncogenes include Ras, EGFR, EML4AK, LINE-1 and SAT2.

Ras gene family: The Ras genes produce proteins that regulate how cells receive signals, differentiate, grow and die. The Ras gene family-KRAS, NRAS and HRAS-are the most suspected oncogenes in human tumors¹⁰⁴. The three genes accounted for about 20-30% of all human malignancies, including 25% of lung carcinomas and about 50% of colon carcinomas¹⁰⁴. Mutational activation of the KRAS gene is suspected in about 25% of various forms of cancers^105,106. The KRAS mutations are responsible for almost 85% of all RAS mutations in human tumors, with NRAS accounting for between 11 and 15% and HRAS for about 1%¹⁰⁶. The KRAS G12V hyperexpression in an isogenic lung model shows more than 50,600 demethylated CpGs compared to non-transformed controls¹⁰⁷. In a study, overexpression of KRAS induced upregulation of miR-30c and miR-21, resulting in lung cancer¹⁰⁸. In another study, overexpression of HRAS in MKN-28 cells by siRNA increased the replication, angiogenesis and metastasis of gastric carcinoma cells, while repressing endogenous HRAS induced the opposite¹⁰⁹. Overexpression of NRAS boosts tumor development by stimulating the secretion of IL8 through JAK2 activation and has been reported to be overexpressed in basal-like breast cancer¹¹⁰. Overexpression of miRNA-708 has been shown to successfully reduce the levels of NRAS protein in lung cancer, leukemia and melanoma cell lines with NRAS mutations, suppressing anchorage-independent growth, formation of reactive oxygen species-induced apoptosis and cell proliferation¹⁰⁴. In a study, specific CpG sites demethylation in the first intron of R-RAS activated over half of gastric cancers¹¹¹. When siRNA was introduced into the R-RAS-expressing cells, the adhered cells disappeared, which suggests that functional blocking of the R-RAS-signaling pathway could be an effective therapeutic strategy for gastric cancer¹¹¹.

EGFR and EML4-ALK genes: The Epidermal Growth Factor Receptor (EGFR) gene is a member of the HER/ERB-B family of transmembrane receptor kinases and resides on chromosome 7p11.2¹¹². The EGFR protein is involved in cell signaling pathways that regulate cell division and survival¹¹². Certain types of cancer cells produce EGFR proteins in higher-than-normal amounts due to mutations in the EGFR gene¹¹². Hyperexpression of EGFR decreases cell proliferation, survival and migration in multiple solid tumors¹¹³. However, in a study, hypermethylation (90%) and a moderate degree of methylation (30-50%) of EGFR were observed in breast cancer cell lines¹¹³. Abnormal histone H3K9 and H3K27 methylation promotes EGFR amplification and has been reported in several cases of pancreatic cancer¹¹⁴. The miR-34a downregulation has been shown to cause overexpression of EGFR, leading to the initiation of several forms of cancers, including non-small cell lung cancer¹¹⁵. In a study involving breast cancer cell lines, miR-218 upregulation downregulated EGFR, leading to the downregulation of p44/42 MAPK signaling¹¹⁶. In another study involving human non-small cell lung cancer cell lines, lncRNA LINC00240 served as a sponge for miR-7-5p and caused upregulation of EGFR¹¹⁷.

LINE-1: Hypomethylation of long-interspersed nuclear element-1 (LINE-1) gene (locus: 22 q12.1) is linked with cell proliferation and malignant traits in lung adenocarcinoma¹¹⁸. The LINE-1 are "jumping genes" often called L1 retrotransposons and constitute 17% of human DNA. They multiply throughout the genome using a “copy-and-paste” mechanism mediated by RNA, a process termed retrotransposition¹¹⁹. The L1s are switched on in the gamete cells and are actively involved in embryogenesis, but are turned off in somatic cells via epigenetic mechanisms¹¹⁹. However, in cancer cells, L1s are wrongly activated and may induce genome instability, a hallmark of carcinogenesis¹¹⁹. Hypomethylation of LINE-1 is suspected in several cancer types and is linked with a worse prognosis. In a study of 1,211 colorectal cancer patients, LINE-1 hypomethylation was observed¹²⁰. In another study, hypermethylation of LINE-1 DNA was observed in the white blood cells of pancreatic cancer patients¹²¹. Downregulation of let-7 miRNA has been shown to lead to overexpression of LINE-1 in lung cancer patients¹²².

SAT2: Spermidine/spermine N1-acetyl transferase 2(SAT2) gene, a locus mapping within the 1q12 pericentromeric region, codes for an enzyme known as diamine acetyltransferase 2¹²³. This enzyme regulates an important metabolic glutamine/glutamate balance underpinning retrograde signaling by dendritic release of the neurotransmitter glutamate¹²⁴. Loss of function SAT2 gene has been reported to cause cancer. Notably, DNA hypomethylation of the SAT2 gene accelerates tumor progression in ovarian cancer and early onset of tumor in breast cancer¹²⁵. In a study involving several cancer cell lines, upregulation of the SAT2 gene by heat shock demethylation was observed¹²⁶. In another study, hypomethylation of SAT2 DNA was detected in individuals with ovarian cancer¹²⁷.

DNA repair genes and cancer: Exogenous and endogenous DNA-damaging agents, including ionizing radiation, chemotherapeutic agents and ultraviolet light can cause DNA lesions¹²⁸. These lesions include single-strand breaks, mismatches, double-strand breaks, interstrand or intrastrand cross-links and chemical modifications of the bases or sugars¹²⁸. Cells encounter more than 20,000 DNA-damaging events daily¹²⁹. To counteract this damage, cells initiate DNA repair processes that tolerate or remove the damage, contributing to genomic integrity. The repair process takes a variable amount of time, depending on the substrate^130,131. Thus, making DNA repair the first defensive strategy against genotoxic stress, albeit without lasting effects^129,132. Further to its cancer prevention role, DNA repair impacts cancer treatment outcomes with DNA-damaging drugs and radiation¹²⁹. The efficiency of DNA repair is mainly governed by the repair gene’s expression levels, which can be altered by several factors. These factors include mutations in their promoter or coding regions, expression levels of transcription factors, and/or epigenetic factors such as CpG promoter methylation or demethylation and histone modifications¹²⁹. Thus, defects in DNA repair genes are a common cause of cancer¹³².

Over 200 DNA repair proteins have been documented and their roles in DNA repair have been outlined¹²⁹. Five main DNA repair pathways are active throughout different stages of the cell cycle, enabling cells to repair DNA damage. These pathways include nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), DNA crosslink repair (Fanconi anemia genes), homologous recombination (HR) and non-homologous end joining (NHEJ)¹³⁰. The BER pathway repairs single-strand breaks, while the NHEJ and HR pathways repair double-strand breaks¹³². The MMR pathway fixes single nucleotide mismatches, deletion and aberrant nucleotide insertions¹³². The NER pathway repairs helix-distorting lesions such as ultraviolet radiation-induced pyrimidine dimers¹³². The Fanconi anemia (FA) pathway, involving downstream repair nucleases, a core complex and ubiquitin ligase recognizes and repairs interstrand crosslinks. The direct reversal (DR) pathway utilizes O6-methylguanine DNA methyltransferase to remove damaging DNA methylations¹³².

Several epigenetic aberrations in DNA repair genes have been implicated in cancer. Notably, silencing mutations and hypermethylation in MMR genes (e.g., MLH1, MSH2, PMS2, MSH6 and PMS1) result in microsatellite instability and Lynch syndrome, increasing the risk of cancer, particularly colorectal cancer¹³². Germline mutational loss of BRCA1 and BRCA2 involved in FA and HR repair has been reported to increase the risk of ovarian and breast cancer¹³². Aberrations in the ATM and HR genes are suspected in ataxia telangiectasia and malignancy risk, while NER abnormalities in xeroderma pigmentosum are linked with an increased risk of skin cancer¹³².

EPIGENETICS IN THE DIAGNOSIS AND MANAGEMENT OF CANCER

Epigenetic factors interact with genetic and non-genetic factors to influence biological events in the body, including disease development. To this end, epigenetic applications such as epigenetic biomarkers, epigenetic drugs and epigenetic editing have been developed to diagnose and treat diseases in the clinic^133,134. Notably, cancer cells exhibit increased DNA methylation at certain genes but have overall lower DNA methylation levels compared to normal cells¹³⁵. This difference allows for the use of epigenetics to differentiate cancerous cells from healthy ones. Additionally, different types of cancer that appear similar can have distinct DNA methylation patterns; thus, epigenetics can help determine the specific type of cancer and detect hard-to-identify cancers earlier¹³⁵. Furthermore, the expression patterns of non-coding RNAs (ncRNAs) are strongly tissue-specific, particularly in cancer. These ncRNAs can be released from cancer cells into blood or urine, serving as diagnostic markers or prognostic indicators^28,36.

Currently, DNA methylation of specific genes or the relative expression of microRNAs are the most common epigenetic-based in vitro diagnostic tests. Both are often carried out using pyrosequencing technologies and RT-qPCR-based methods (e.g., methylation-sensitive high-resolution melting, methyl-specific PCR and methyLight)¹³⁶. Commercially available epigenetic tests include the EPICUP^® assay, the EpiproColon^® test (Epigenomics AG, Berlin, Germany), Beadchip 450K (Illumina) and Cologuard^® Stool DNA-Based Test (Exact Sciences Corp., Madison, WI), among others¹³⁷. Epigenetic

biomarkers aid in disease progression monitoring, early diagnosis, patient selection and stratification by risk, disease outcome prediction, evaluation of therapeutic interventions in specific patient subsets and prediction of future comorbidities¹³⁷. Furthermore, microRNAs and DNA methylation are very stable compared to proteins and RNA, making these epigenetic biomarkers more viable and practical in clinical procedures¹³⁷. Epigenetic biomarkers are dynamic in nature, they provide information about gene function and specific genetic programs altered during disease development, all of which make them preferable over protein-based or genetic biomarkers¹³⁷. This suggests that epigenetics has great potential to improve predictive and precision medicine¹³⁷. However, epigenetics alone cannot diagnose cancer; additional screening tests are needed to confirm cancer¹³⁵.

Some epigenetic drugs are currently in clinical trials, while others have been tested and found safe and effective¹³⁸. Numerous small-molecule inhibitors that target chromatin- and histone-modifying enzymes to reverse abnormal epigenetic changes in tumors have passed clinical trials as cancer therapeutics. These include Azacitidine, Nanaomycin A, Belinostat and Tubacin, among others²². Pharmacological inhibitors of DNA methylation, such as azacitidine and decitabine, have also been developed to reverse epigenetic changes via DNA methylation inhibition²². Anti-miRNA oligonucleotides, otherwise called Anti-miRs, are commercially available and include Eteplirsen (Exondys 51) and Golodirsen (Vyondys 53 TM)^139,140. However, some of these drugs exhibit poor pharmacokinetic and safety/tolerability issues due to a lack of specificity and off-target effects¹⁴¹. Additionally, their therapeutic outcomes varied in solid tumors due to the lack of biomarker-driven targeted therapies, resulting in a highly heterogeneous response²². Recently, significant progress has been made in this regard, with the formulation of epigenomic therapies that directly address these issues by targeting defined loci with highly precise, durable and tunable approaches¹⁴¹.

CONCLUSION

The findings of this review demonstrate that epigenetic changes, such as DNA methylation, histone post-translational modifications and noncoding RNA silencing, are important for biological processes, particularly growth and development. These changes are influenced by lifestyle, environmental factors and nutritional status, which can lead to aberrant epigenetic reconfigurations and result in diseases. Specifically, cancer is most often mediated by abnormal epigenetic alterations in tumor suppressor genes (e.g., TP53, RB1, NF1, NF2, CDKN2A, WT1, BRCA1, BRCA2, PARP-1, VHL, APC, PTEN, PTCH1 and CDH1), oncogenes (e.g., Ras, EGFR, EML4AK, LINE-1 and SAT2), or DNA repair genes (e.g., MSH2, MSH6, MLH1, PMS1 and PMS2). Epigenetic changes are reversible, making drugs that target these changes potentially more effective in predisposed individuals. Some drugs, known as epigenetic drugs, such as Azacitidine, Belinostat and Tubacin, have shown promise but face challenges related to pharmacokinetics and safety. However, these issues are currently being addressed. More studies are needed to improve the precision of these drugs and ensure that non-target genes or epigenomes are not affected. Additionally, because epigenetic therapeutic procedures are complex, further research is required to simplify them.

SIGNIFICANCE STATEMENT

Epigenetic reprogramming plays a critical role in the increasing prevalence of diseases, particularly cancers, yet its significance in disease development remains underrecognized. This synthesis of existing literature underscores the links between epigenetic modifications and cancer etiology, highlighting their potential as therapeutic targets. Abnormal epigenetic changes in tumor suppressor genes, oncogenes and DNA repair genes are strongly associated with cancer development. Notably, the reversibility of some epigenetic aberrations using approved drugs, such as Azacitidine, Belinostat and Tubacin, underscores the promise of epigenetic therapies. Clinical trials reveal that these therapies improve outcomes in specific cancer patient populations, suggesting their integration into standard treatment regimens could yield significant benefits. However, challenges such as poor drug specificity and off-target effects highlight the need for further research to improve the precision and safety of epigenetic interventions, advancing the development of targeted and effective cancer therapies.

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