Molecular Basis of Epigenetic Regulation in Cancer Diagnosis and Treatment Notes
Molecular Basis of Epigenetic Regulation in Cancer Diagnosis and Treatment
Global cancer cases and mortality are increasing, demanding efficient biomarkers for accurate screening, detection, diagnosis, and prognosis.
Variations in epigenetic mechanisms like aberrant promoter methylation, altered histone modification, and mutations in ATP-dependent chromatin remodeling complexes play an important role in the development of carcinogenic events.
Alterations in epigenetic marks may have clinical utility as potential biomarkers for early cancer detection and diagnosis.
This review discusses key epigenetic mechanisms and their deregulation in cancer etiology, highlighting gaps in epigenetic drug development and emphasizing integrative analysis of epigenetic biomarkers to establish minimally non-invasive biomarkers with clinical applications.
Keywords: epigenetics, cancer, DNA methylation, histone modification, chromatin remodeling, cancer therapies.
Introduction
Cancer is a multifactorial disease resulting from genetic and epigenetic changes.
Epigenetics involves altering gene expression without changing the DNA sequence.
Coined by Conard Waddington in 1942, it means above or over the genome.
Epigenetic modifications involve structural changes in nucleic acids and histones, creating different chromatin structures.
Three molecular mechanisms: DNA methylation, histone modification, and nucleosome modeling patterns.
Epigenetic modifications are induced by enzymes called epigenetic tools or players.
"Writers" add chemical additions to DNA or histones, "erasers" remove chemical tags, and "readers" interpret these modifications.
Epigenetics controls DNA repair, replication, transcription, translation, post-transcriptional, and post-translational regulation.
Aberrant expression patterns or epigenomic alterations can lead to misregulation, culminating in cancers.
Epigenetics is reversible, unlike genetic changes, altering how a DNA sequence is read.
Factors contributing to epigenetic changes include obesity, diet, lifestyle, alcohol, tobacco use, exposure to electromagnetic radiation, and environmental pollutants.
Research focuses on promoter DNA methylation and histone modifications as key mechanisms mediating epigenetic regulation in anticancer therapies and biomarker discovery.
Altered DNA methylation genes or patterns can be potential biomarkers for cancer screening, diagnosis, and prognosis.
Epigenetic biomarker discovery is crucial for early cancer diagnosis, better cancer therapies, precise treatment, and effective clinical outcomes.
Clinical validation of approved biomarkers is still needed.
Challenges in developing reliable biomarkers include incorporating clinical trial data into routine practice at an affordable cost through interdisciplinary collaboration.
The tumor microenvironment (TME) of cancer cells contains aberrant epigenetic marks that favor tumor growth.
This review sheds light on epigenetic changes, integrated network medicine with epigenetics in epimarker/epidrug development, and challenges faced.
Anticancer therapies might reverse epigenetics, benefiting cancer treatment and management.
Mechanisms of Epigenetic Modification
Molecular mechanisms behind epigenetic regulation include DNA and RNA methylation, histone modifications, and ATP-dependent nucleosome remodeling.
DNA and RNA Methylation
DNA methylation is a widely studied epigenetic mechanism in cancer etiopathogenesis.
Aberrant methylation leads to DNA hypermethylation or hypomethylation.
In DNA hypermethylation, methylation occurs at cytosine bases in gene promoter regions by DNA methyltransferases (DNMTs) like DNMT1, DNMT3a, and DNMT3b.
These enzymes convert cytosine residues to 5-methylcytosine, decreasing gene expression via transcriptional suppression.
DNA hypomethylation indicates an overall decrease in methylation levels compared to normal cells, affecting intergenic and intronic regions, resulting in chromosomal instability and increased mutation activities.
Global hypomethylation with hypermethylation of specific gene promoters has been reported in various cancers.
Inappropriate DNA methylation may lead to altered expression of tumor suppressor genes (deregulation) and/or oncogenes (upregulation) in cancer cells.
Differences in methylation patterns exist within CpG islands of ~70% of mammalian promoters, playing an important role in transcriptional and post-transcriptional regulation.
High throughput sequencing confirms that 5–10% of abnormally methylated CpG promoter islands are present in various cancer genomes.
Hypermethylation of CpG islands in several promoters influences the expression of noncoding RNAs (ncRNA) and messenger RNAs (mRNA), which have a role in cancer progression.
Whole genome sequencing data in several cancers have shown somatic mutations in numerous epigenetic regulators.
RNA methylation is a less studied epigenetic process, about seven times greater than DNA methylation, resulting in mRNA localization and transcript degradation.
With next-generation sequencing, methyl modifications at the mRNA level may affect cellular processes resulting in human diseases.
Over 150 different RNA modifications have been observed, of which N6-methyladenosine (m6A) modification is the most abundant and recognized by RNA binding proteins affecting mRNA function.
Similar to DNA methylation, alterations at the RNA level affect the epigenetic regulation of gene expression.
Histone Modification
Chromatin structure involves wrapping DNA on histone octamer -2 subunits each of H2A, H2B, H3, and H4 proteins joined together by H1 proteins.
Histone modification occurs at the amino-terminal tail of these histones via acetylation, methylation, phosphorylation, ADP-ribosylation, or ubiquitination.
Histone acetyltransferases (HATs), histone methyltransferases (HMTs), and histone kinases catalyze these processes, adding acetyl, methyl, and phosphate groups to histone amino-terminal tails.
These epigenetic marks, known as "writers," act as transcriptional co-activators.
Erasers [histone deacetylases (HDACs), histone demethylases (HDMs), phosphatases] function as transcriptional co-repressors by removing these groups from the histone end.
These enzymes open and close chromatin structure, necessary for gene expression.
Aberrations in these enzymes may lead to altered gene transcription and post-transcriptional modifications, thereby resulting in cancer.
Image Description:
(A) Histone modification: Histone methyltransferases (HMT) add methyl groups to histones (H4K20Me3). Histone demethylases (HDM)/Lysine demethylase (KDM6/4) remove these methyl groups, associated with both gene expression and silencing. Histone acetylation involves adding an acetyl group on H3K9Ac (lysine 9 histone H3) in the enhancer/promoter region by histone acetylase (HAT) enzyme. Histone deacetylase (HDAC) interacts with the transcriptional repressor (TR) to remove the modifications.
(B) DNA methylation: DNA methyltransferases (DNMTs) add methyl groups in the promoter region of genes. DNA hypomethylation indicates an overall decrease in methylation levels in normal cells and affects intergenic and intronic regions, resulting in chromosomal instability and increased mutation events.
(C) RNA methylation: Indirect translational repression by miRNA causes deadenylation, in which the 3' poly(A) tail of an mRNA is removed, leading to increased mRNA degradation. The miRNA–mRNA interaction can lead to several modes of direct translational repression.
ATP Dependent Chromatin Remodelling
DNA nucleosome interactions can be modified (histone ejection, removal, and incorporation) through chromatin remodeling complexes by ATP hydrolysis.
These chromatin-remodeling complexes can be classified into switching defective/sucrose nonfermenting (SWI/SNF), chromodomain-helicase DNA-binding protein (CHD), imitation SWI (ISWI), and INOsitol requiring mutant 80 (INO80) complexes.
The catalytic subunit of these complexes performs DNA translocation along with the histone core of the nucleosome.
SWI/SNF complexes are one of the most widely studied ATP-dependent chromatin remodeling complexes, mutated in 25% of human cancers, and play an essential role in chromatin remodeling by positioning nucleosomes.
Their catalytic activity is associated with SMARCA4/2 proteins.
SWI/SNF complexes are involved in the regulation of cell progression, cell motility, and nuclear hormone signaling.
The SWI/SNF complex was found to be altered in 33–42% of pancreatic cancer cases by whole-exome sequencing studies.
ISWI mobilizes nucleosomes by helping transcription factors bind a nucleosome-free DNA and is held to nucleosomes by a SANT and a SLIDE domain.
ISWI complexes have a key role in DNA repair and recombination.
In humans, two ISWI subunits—sucrose nonfermenting 2L (SNF2L) and sucrose nonfermenting 2H (SNF2H) ATPases—are identified.
SNF2H suppressed the oncogene ras in human cells.
A tissue microarray study on 78 paraffin wax-embedded prostatic tissues observed a significant increase in ISWI (SNF2L and SNF2H) proteins in prostatic intraepithelial neoplasia and prostate adenocarcinoma.
To date, no clinical trials have been performed to unravel the potential of these small molecules as epigenetic biomarkers in cancer therapies.
Epigenetic Diagnostic Biomarkers
Epigenetic changes like DNA methylation and histone modification detected in early tumorigenesis and cancer progression have been proposed as biomarkers for early cancer detection, tumor prognosis, and treatment response.
They are rarely translated into biomarkers for clinical practice, even though there have been major advances in the characterization of cancer.
Due to stability in body fluids like urine and serum, epigenetic changes act as innovative cancer biomarkers with a great opportunity for assay development to assist in patient’s treatment.
Recent studies have identified various epigenetic cancer biomarkers that have already been commercialized; however, further validation studies are required to take it to the clinics.
Epigenetic diagnostic and prognostic biomarkers that are most promising for the most common cancers have been discussed.
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Prostate Cancer: Common in men; early detection challenges. PCA3 and ncRNA are highly expressed, and PROGENSA™ measures PCA3/PSA mRNA ratio for early detection. DNA methylation and GSTP1 hypermethylation show promise as biomarkers.
Glioblastoma: MGMT promoter methylation correlates with favorable temozolomide treatment outcomes.
Colorectal Cancer: Hypermethylated DNA leads to genomic instability. CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS126 methylation identifies CIMP-positive cancers. RASSF1A, FHIT, and MGMT methylation act as diagnostic markers.
Esophageal Cancer: Differential methylation patterns potentially serve as diagnostic biomarkers; promoter methylation increases with higher histological grades.
Bladder Cancer: Lacks accurate biomarkers. VIM, GDF15, and TMEFF2 show high sensitivity and specificity in urine. Histone modification data is limited.
Breast Cancer: Alterations in histone-modifying enzymes and reduced histone expression noted. PRMT1 promotes EMT, SETD7 affects post-translational modification with prognostic implications, and TNBCs are divided into risk groups based on epigenetic subtypes.
Ovarian Cancer: Aberrant histone modifications and tubulin expression, reduced PACE3 expression, and silencing of survivin reported. HDAC3 overexpression and loss of H3K27me3 are associated with prognosis. Hypomethylation of MSX1, DAXX, and TMEM88 is described; ZNF671 methylation may predict early relapse. Epigenetic inhibitors may improve drug response.
Epigenome-Targeted Therapies
Quite a few epidrugs are approved for the treatment of several cancers, including inhibitors of DNA methyltransferase (DNMTi) and histone deacetylase (HDACi) enzymes.
The first US-FDA approved epigenetic drug is 5-azacitidine (Azacitidine), a DNMTi used in the treatment of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML).
Even combination therapies including both DNMTi and HDACi are widely inspected in the treatment of MDS, AML, and chronic myelomonocytic leukaemia (CMML).
However, clinical results for such a combination of inhibitors are controversial due to the lack of large-sized cohort studies.
Research on epidrug development has expanded its boundary to targeted therapy, shifting the focus on the presence of activating mutations in epigenetic players, especially histone methyltransferases.
Another inhibitor of EZH2, Tazemetostat (TAZVERIK, Epizyme, Inc.) was approved by US-FDA in June 2020 for treating adult patients with relapsed or follicular lymphoma with EZH2 positive mutations.
One of the main problems in the application of epidrugs is that the drug binds to other targets rather than its own target; this is called “off-target effects” in epigenetic therapy.
Growing epi-research has shown that the use of synthetic lethal approaches might result in apoptosis.
These epidrugs are delivered to target synthetic lethal partners having genetic mutations in cancer cells.
One such epidrug is the inhibitor of the histone methyltransferase DOT1L (disrupter of telomere silencing 1-like), Pinometostat, which specifically kills the MLL-fusion leukemia cells.
Another epidrug used in the treatment of lung cancer with a specific DNA hypomethylation is GSK2879552, an inhibitor of lysine-specific histone demethylase 1A (LSD1).
GSK2879552 treatment results in the increase of H3K4 methylation, thereby reducing tumor potential.
DNA methylation biomarker technology is being employed in circulating free DNA present in body fluids to detect cancers.
In the year 2017, the “Epi proLung®” assay received the Conformité Européenne (CE) mark as an In Vitro Diagnostic (IVD) test for lung cancer diagnosis and is based on methylation analysis of SHOX2 (Short Stature Homeobox 2) and PTGER4 (the prostaglandin E receptor 4) genes.
Numerous reports have shown increased promoter methylation of SEPT9, Vimentin, and NDRG4 gene in colorectal cancer.
The US- Food and Drug Administration (FDA) has also approved non-invasive DNA methylation tests of these genes for early colorectal cancer screening programs.
Another CE-IVD marked test, the miRpredX-31-3p kit (IntegraGen S.A., France) is based on the quantification of miR-31-3p expression levels and is used to recognize metastatic colorectal cancer patients who can benefit from anti-EGFR (epidermal growth factor receptor) therapy.
An effective evolution of epidrugs in cancer therapeutics can be seen from inhibitors to combination therapies to non-invasive diagnostic assays.
However, the area of epigenetics still needs to be explored in precision oncology for effective cancer treatment and management.
Epigenetics and Integrated Network Medicine
The future of epidrug development involves the use of integrated network medicine with epigenetics, where several analytical methods like protein-protein interaction (PPI) networks, correlation-based networks, and gene regulatory networks are utilized to roll out key genes, relevant regulatory and co-regulatory networks in causing disease pathogenesis.
A group at Stanford University, United States has developed the Genomic Regions Enrichment of Annotations Tool (GREAT) for functional enrichment analysis of DNA binding events across the entire genome, which is useful in identifying gene-regulatory networks and subnetworks in epigenomics data analysis.
Another integrative epigenome-transcriptome-interactome tool called Functional Epigenetic Modules (FEM), identified HAND2 methylation as an important epigenetic alteration in the development of endometrium cancer.
Integrative analysis on epigenetic modifications and their effect on gene expression can be performed using an Epigenetic Module based on Differential Networks (EMDN) algorithm.
These frameworks could be utilized directly from epigenomic data to unravel co-regulatory networks responsible for causing the disease.
Zheng et al. (2020) used a deep neural network (DNN) algorithm to predict cancer diagnosis in the DNA methylation data of 7,339 patients of 18 different cancer origins from The Cancer Genome Atlas (TCGA).
Recently, weighted correlation network analysis (WGCNA) of 201 patients in a TCGA prostate cancer dataset revealed that hypermethylation of FOXD1 might promote poor prognosis.
Another study yielded a 13-gene epigenetic signature that stratified breast cancer patients into low- and high-risk groups by using WGCNA analysis and single sample gene set enrichment analysis (ssGSEA).
One more tool named SWItchMiner (SWIM) is being used to identify potential therapeutic targets when applied to a large panel of cancer datasets from TCGA.
Network medicine might advance the field of epigenomics as it is possible to rule out the co-regulatory networks of DNA methylation. However, its clinical application is still lacking, accompanied by the challenges of integration of epigenomics data in multi-omics.
Challenges and Way Forward for Epidrug Development
Epidrug development is accompanied by its own set of challenges that needs to be addressed for the establishment of locus-specific, highly sensitive, and cost-effective biomarkers.
The main obstacle is to comprehend the “casualty” of epigenetics, meaning whether the epigenetic abnormality is a result of malignancy or malignancy itself is caused due to these variations.
It is crucial to decipher the link between epigenetic differences and cancer progression to establish a biomarker with potential utility in the cancer clinics; this is only possible by pursuing special cohort studies where epigenome profiling can be maintained before the start and after the end of the disease.
The high cost of such studies limits their application.
Another challenge is the lack of locus specificity, which might cause epigenome-wide “off-target” effects leading to the loss of important gene function and can be resolved using new epigenome editing approaches.
With the introduction of the proteolysis targeting chimera (PROTAC) drug design approach, epidrugs specific to genetically altered chromatin players can be developed, thereby offering a precise cancer therapies approach in treatment.
It is understandable that the epigenetic changes, especially the methylation patterns, are very informative in establishing both diagnostic and prognostic biomarkers, but the problem lies in the complex assay systems, imprecise reproducibility, inadequate clinical validation, and false discovery of these biomarkers.
Therefore, implementing clinical epigenetics for the benefit of public health is the main goal of epigenetics research. However, it is restricted due to the variable cellular composition of epigenomic profiles of bulk cell populations.
To resolve this issue, single-cell methods should be undertaken to provide resolution of DNA at the single-cell level like 5-methylcytosine or 5-hydroxymethylcytosine, and computational algorithms can be used to correct variable cellular composition by comparisons to reference epigenomes.
Another biological challenge is the limited knowledge of the complex epigenome with small-sized studies.
In addition to the epigenome, the epitranscriptome should be studied to rule out potentially modified RNA molecules in cancer, whose potential as epigenetic marks can be exploited before clinical application.
As epigenetic modifications are dynamic, it is vital to consider all epigenetics layers using multiomics approaches along with integrated network medicine for epidrug development.
Conclusion
It is well established that there is a link between cancer and epigenetics.
Some epigenetic drugs have already been approved by US-FDA, and many more epidrugs are under development for appropriate cancer detection and treatment.
Furthermore, there is a scope for epigenetics-based cancer therapies delineating the tumor heterogeneity in different cancers with precision, that should focus on cell-cell behavior in TME.
Epigenetic research should focus on network and precision medicine approaches for the discovery of novel biomarkers so that they can be safely translated to the clinic after proper clinical trials.
Proper identification of the epigenetic landscape behind cancer progression and establishing therapeutic drugs is the future of epigenetics in cancer without forgetting to overcome the challenges faced in effective epidrug development.