Cancer: From Genomics to Pharmaceutics

92 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCER3.1.1. DNA HypermethylationHypermethylation refers to the methylation gain in CpG islands and intergenic DNAregions in the promoter region responsible for regulating the expression of a gene (104,105).DNA hypermethylation causes inactivation of gene expression by silencing tumor suppressorgenes (105), and is associated with many cancers or altered response to cancer therapies (106).In the literature, many cellular pathways are silencing by this type of DNA hypermethylationfound to be involved in cellular processes such as cell cycle, cell-cell interaction, celladherence, DNA repair, carcinogen metabolism, angiogenesis, and apoptosis which areassociated with RB1 (107,108), VHL (109), p16? ? ?4?(110,111), p15? ? ?4?(111), p14???(111), DAPK (111), hMLH1 (112), and BRCA1 (113, 114), CDH1 (111), CDH13 (111),GSTP1 (111) genes. In addition, in the early stages of cancer development, hypermethylationinactivates DNA repair genes such as hMLH1, BRCA1, and MGMT, preventing the repairof genetic errors and paving the way for malignant transformation of the cell (115,116).Therefore, understanding the mechanism of DNA methylation is very important to understandthe processes of many diseases, including cancer (117).3.1.2. DNA HypomethylationAside from CpG island promoter DNA hypermethylation-based gene silencing, humancancers also display gene body DNA hypomethylation that is also associated withdownregulated gene expression (118). DNA hypomethylation was the first epigeneticabnormality discovered in tumors. Feinberg and Vogelstein reported in 1983 that they observedhypomethylation in the total DNA of colon cancer tissue compared with matched-normalcolon epithelium in two simultaneous and independent studies (119,120). Studies reportedthat patients with metastases are more likely to have hypomethylation than patients withoutcancer. In addition hypomethylation can be observed in the tandem centromeric satellite ?,juxta-centromeric satellite 2, Alu, SINE -1 and LINE -1 repeat sequences in the genome-wideof cancer cells (121-123).Genomic imprinting process plays a role in normal development, however abnormalitiesin genomic imprinting genes are also observed present in tumor development (124). DNAhypomethylation, which negatively affects genomic imprinting, is effective in the developmentof CRC (125) and Wilms tumor (126) by impairing IGF2 suppression. In contrast, studies havereported hypomethylation-induced activation of the PAX2 gene (127) and miRNA let-7a-3(128) in endometrium and colon cancers. DNA hypomethylation has been reported tocause genetic alterations by inducing chromosomal rearrangements through deletion andtranslocation (129).Global hypomethylation is often referred to as the intense methylation responsibilityof repeat sequences in the composition of nearly the entire genome (130). Cancer cells areusually 20-60% less methylated than normal cells and include hypomethylation in their genomeCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 93(131). Hypomethylation also leads to chromosome missegregation (132) and activation oftransposable elements during cell division, resulting in genomic instability (133). GlobalDNA hypomethylation is seen in many types of cancer, including breast, colon, head andneck, bladder, esophagus, liver, prostate, stomach, and lung tumors (131).3.1.3. DNA DemethylationUnlike DNA hypermethylation, DNA demethylation is a chemical modification processthat removes the methyl group from 5mC via dioxygenase-mediated oxidation of ten-eleventranslocation 1 (TET1) and converts it into a permanent cytosine base (134). DNAdemethylation occurs in two ways: active demethylation observed during the base excisionrepair system via the enzyme TET and thymine DNA glycosylase, and passive demethylationobserved in the daughter strand during DNA replication (135). While the loss of TET playsa role in the development of lung cancer (136), its gain seems to prevent the development ofCRC (137).3.1.4. DNA HydroxymethylationHydroxymethylation, which occurs by oxidation of 5mC to 5-hydroxymethylcytosine(5hmC), is catalyzed by members of the ten-eleven translocation family (TET1, TET2 andTET3), ?-ketoglutarate (?? ?ℎ?KG) and Fe2+-dependent dioxygenases (131). TET enzymesalso oxidize 5hmC to 5-formylcytosine (5fC) and 5-carboxycytosine (5cC) (138,139). DNAhydroxymethylation plays a role as an epigenetic marker in physiological processes such ascell differentiation, neuronal system development, and aging, as well as in the developmentof various diseases, especially cancer (135). For example, loss of 5-hmC has been reportedto play a role in melanoma by causing promoter hypermethylation in some tumor-dominantgenes (140).3.1.5. Methyl-Binding Domain ProteinsWith epigenome readers are also known as, methyl-binding domain protein family proteins(MBD) they play an important role in the coordination between DNA methylation, histonemodifications, and chromatin organization, which determine the transcriptional status ofthe epigenome (141,142). The MBD protein family consists of eleven defined proteinscontaining the MBD domain. The MeCP2 protein is involved in the conversion of intragenicmethylation to alternative splicing, which is responsible for histone deacetylase/methylation(141). MBD1 protein provides transcriptional repression by maintaining heterochromatinstructure through histone methylation (143), while MBD2 protein acts as a transcriptionalrepressor by interacting with histone deacetylase complexes (144). MBD3 protein bindsto unmethylated and 5-hydroxymethylated DNA (145), while MBD4 protein can bind tomethylated DNA and is involved in DNA repair (146). In addition MBD family consists,MBD5 protein highly expressed in brain, testis and oocytes, MBD6 protein highly expressedCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

94 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCERin testicles (147), and SETDB1 protein localized to 5-methylcytosines via MBD1 and involvedin heterochromatin formation and transcriptional repression (148), and unmethylated DNA ofBAZ2A protein that binds (149). In the literature, about the relationship of methyl bindingproteins with many cancer types such as breast cancer (150), prostate cancer (144), chroniclymphocytic leukemia (151).3.1.6. CpG IslandsThey are short stretches of palindromic DNA with the sequence ”CpG”, which are usuallycharacterized by a transcriptionally active chromatin structure and encode the same sequencein the DNA complementary helix (152). In addition to the promoter region of the gene,methylated CpG residues are also found in intergenic regions, enhancers, and silencers in thegenome (153). Because the promoters where CpGs are abundant, loss of function of CGIsby modifications such as methylation results in downregulation of gene expression (154).Landolin et al. found that while transcription factors such as Sp1, Nrf-1, E2F and ETS tendto activate gene expression by binding to the promoter regions where CGIs are concentrated,transcription factors such as Nf-?E1, ETV7, Ap4, NRSF tend to suppress it (153). Moreover,CGIs are known to affect chromatin configuration in addition to their role in transcriptioninitiation (155).3.1.7. Methyl DonorsMethyl groups are important in many cellular processes such as DNA methylation,phosphatidylcholine and protein synthesis (156). As natural products, folate, methionine,polyamine, betaine, choline, vitamins B6 and B12 are known to play a role as methyl donorsor cofactors and are present in many foods (156,157). Production of S-adenosylmethionine(SAM) is observed by ”one carbon metabolism” that yields methyl groups, using thesurrounding nutrients to perform functions such as methylation of DNA, proteins andphospholipids (157,158). SAM provides regulation of genes responsible for cell invasionand metastasis through a methylation mechanism (159,160). Methionine is a precursor ofS-adenosylmethionine, a universal methyl donor, and is a compound that cannot be synthesizedin the body and must be consumed through diet (161). An essential amino acid, methionineis abundant in nuts, meat, seafood, eggs and dairy products (162). Betaine, formed by theoxidation products of choline in the inner mitochondrial membrane of the liver and spleen,is generally found in products such as shrimp, spinach, and wheat and barley bran (163).Folate-rich foods are important for various cellular processes such as DNA synthesis, DNAmethylation, and amino acid metabolism. These include legumes such as beans and lentils,bread and cereal products, peas, lemons, and green vegetables, and fruits such as banana andwatermelon. Vitamin B6 is mainly found in red and white meat, avocado, pomegranate, andcereal products; vitamin B12 is found in milk, cheese, eggs, cereals, crab, and red meat (164).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 953.2. RNA MethylationAs a post-transcriptional modification, RNA methylation is commonly found in alltypes of RNA, including mRNAs, tRNAs, rRNAs, and some other non-coding RNAs suchas piwiRNAs and long non-coding RNA (lncRNA) (165,166). There are four types ofRNA methylation according to the methylation site differences: m6A, m6C, m6G, and2-O-methylation (165). Among these modifications, N6- methyladenosine (m6A), whichis methylated at the N6 position of adenosine, is the RNA methylation modification mostcommonly found in mRNA (167). m6A methylation catalyzed by METTL3/14, WTAP,RBM15/15B and KIAA1429 methyltransferases cleaves methyl groups bound by FTO andALKBH5 demethylases (167). Due to its role in RNA production and metabolism, m6A RNAmethylation has an important effect on many diseases, including cancer (168).4. Methylation Analysis Methods in Cancer ResearchThere are many different methods that can measure DNA methylation status in the cellusing genome-wide and/or gene-region-based methods. The most used of these methods arerestriction endonuclease-based analysis, bisulfite-based analysis, immunoprecipitation-basedanalysis, mass-spectrometry analysis, next-generation sequencing analysis and epigenomicsanalysis methods (169). Diagram of commonly used methods and their subgroups inmethylation analysis is shown in Figure1.Figure 1: Diagram of commonly used methods and subgroups in methylation analysisCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

96 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCER4.1. Restriction Endonuclease-Based Analysis Method4.1.1. The Methylation-Sensitive Amplified Polymorphism (MSAP):A restriction enzyme pair is used, consisting of isochimers with the same restriction site,both sensitive and insensitive to methylation (170). In the first step, restriction enzymes digestthe regions where the DNA is not methylated by recognizing HpaII via methyl-sensitiverestriction endonucleases (MSRE) methods (171). In the second step, it is based on bindingand amplifying the ends formed by cutting with HpaII using MspIs (insensitive isoschizomer)(172). While it has advantages such as not requiring bisulfite conversion, low DNA input andeasy primer design, it gives false positives in insufficient digestion (173).4.2. Bisulfite-Based AnalysisThe most commonly used methods based on bisulfite conversion of DNAsamples are Bisulfite sequencing PCR, The Sanger-sequencing of the amplifiedbisulfite-treated DNA (BSP), Pyrosequencing, Combined Bisulfite and RestrictionAnalysis (COBRA), Methylation-Sensitive Single-Nucleotide Primer Extension (MS-SnuPE),Methylation-Sensitive Melting Curve Analysis, Methylation-Sensitive High-ResolutionMelting (MS-HRM), and MethyLight techniques (170,174).4.2.1. Bisulfite Sequencing PCR:The revolutionary bisulfite genomic sequencing developed by Frommer et al. is basedon the recognition of cytosine as thymine in PCR amplification and sequencing afterthe deamination reactions of 5-methylcytosine (5mC) are converted to uracil residues insingle-stranded DNA after sodium bisulfite treatment (175).4.2.2. The Sanger-sequencing of the amplified bisulfite-treated DNA (BSP):This technique detects locus-specific methylation changes, after bisulfite-treated DNA isPCR amplified using bisulfite-matched 5’-tailed primers targeting the region of interest, theamplicons are repeat amplified by PCR using tailless primers and cloned into the plasmidvector. After producing the cloned plasmids, extraction is done and the result obtained afterSanger sequencing is formulated and each CpG methylation level is calculated (176).4.2.3 Pyrosequencing:It is based on the detection of luminescence emitted from pyrophosphate released bybinding of genomic DNA to complementary DNA after PCR amplification of the specificDNA region after bisulfite treatment (177). While the major advantage of this method is thatit allows direct sequencing of amplicons, the quality of data decreases for CpGs far from the3’ end of the forward primer and the number of bases that can be analyzed in the reaction iscertain (178).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 974.2.4. Combined Bisulfite and Restriction Analysis (COBRA):TERT promoter UTSS hypermethylation, hypermethylation of CpG dinucleotides wasvisualized by gel electrophoresis after applying bisulfite and restriction enzyme assayprotocols, respectively (179). This method, which is still used today, has disadvantages suchas time consuming and limitation to the available RE regions and producing false positiveresults due to incomplete digestion (173).4.2.5. Methylation-Sensitive Single-Nucleotide Primary Extension (Ms-SnuPE):After bisulfite treatment and PCR amplification, the amplicons are radiolabelled andused as a template for the second reaction. At the end of the reaction, it is separatedby electrophoresis and visualized with a phosphorimager. While it has the advantageof performing semi-quantitative, multiplex and single cytosine analysis without additionalprocessing, it has the disadvantages that each region requires two parallel reactions and theuse of compounds containing radioactive material (180).4.2.6. Methylation-Sensitive Melting Curve Analysis (MS-MCA):It is based on the study of the melting properties of amplicons at high temperature afterbisulfite treatment. It allows methylated DNA to separate from unmethylated DNA becauseit requires higher temperatures to melt (181).4.2.7. Methylation-Sensitive High-Resolution Melting (MS-HRM):Although it has the same principle as MS-MSC, it is more sensitive than the MS-MSCtechnique (182). The melting profile of the amplicons is obtained by comparing them withthose of methylated standardized DNA samples. It has the advantages of being a repeatable,fast and simple technique (183).4.2.8. The MethyLight:It is a fluorescence-based real-time PCR technique that uses methylation-specific primersand probes, providing quantitative analysis of DNA methylation. The amount of methylatedDNA is proportional to the emitted light intensity (182).4.2.9 Bisulfite Based-Next-Generation Sequencing AnalysisAfter the nucleic acid to be sequenced is isolated, the bisulfite conversion is performed,and the target region is amplified by PCR (184). Later library preparation is complete, it isdetermined by massive parallel sequencing to sequence the DNA fragments and finally basedon bioinformatics interpretation (185). In obtaining whole genome sequence information withnext generation sequencing (NGS), DNA methylation maps can be obtained by evaluatingthe DNA methylome at a single nucleotide resolution with whole genome sequencing afterbisulfite conversion (186). NGS, which is a high-throughput sequencing technique thatprovides a more cost-effective platform for large-scale methylation detection, as well asCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

98 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCERa method that can provide both qualitative and quantitative results, has led to a rapidimprovement in diagnosis and personalized drug studies (187).4.2.10. EpigenomicsMethylation microarrays allow quantitative identification of selected methylation sitesacross the genome (188). In addition to reducing the cost per sample with its high-throughputtechnologies, it provides advantages such as CpG islands, non-CpG and differentiallymethylated regions, enhancers, open chromatin, transcription factor binding sites, miRNApromoter regions (189,190). The basis of the method is the hybridization of hundreds ofthousands of probes fixed to a solid surface with its substrate. The procedure consistsof bisulfite conversion of target DNA, whole genome amplification, enzyme-mediatedfragmentation, probe hybridization washing, fluorescent labeling, bead dissolving, andfluorescence scanning respectively. Green fluorescence indicates methylated sequences, whilered fluorescence indicates unmethylated sequences (190,191).4.3. Immunoprecipitation-Based AnalysisImmunoprecipitation-based methods which are Methylated-CpG Island Recovery Assay(MIRA), Methyl Binding PCR (MB-PCR) and Methylated DNA Immunoprecipitation(MeDIP), are based on enrichment of the methylated part of the genome using methyl bindingproteins or antibodies specific for 5mC (192).4.3.1. Methylated-CpG Island Recovery Assay (MIRA):It is based on the benefit of the capacity of the MBD2b protein to bind methylated DNA,as well as increasing the affinity with the addition of MBD3L1, increasing the formation ofmethylated DNA binding complexes. MIRA can be used for DNA methylation analysis intissues and body fluids, works on double-stranded DNA and has the advantages of being asimple and convenient method that does not require the use of antibodies for single-strandedDNA (193).4.3.2. Methyl Binding PCR (MB-PCR):It is a sensitive, reliable and one-step method using recombinant polypeptides with bindingactivity to methylated DNA that does not require sodium bisulfite conversion (194).4.3.3. Methylated DNA Immunoprecipitation (MeDIP):By using antibodies that bind to single-stranded DNA fragments containing 5-mCmethylation, DNA samples specially enriched for 5-mC DNA methylation are obtained.Unlike other methods, it is an important technique as it provides the separation of 5-mC (195).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 994.4. Mass-Spectrometry AnalysisIt is an analytical technique that ionizes chemical species and ranks the formed ionsbased on the mass/charge ratio, but it is a useful and safe method (196). In order tomeasure methylation in nucleic acids, there are two different uses: Matrix assisted laserdesorption/ionization time of flight (MALDI-TOF) and primer-extended MALDI-TOF massspectrometry (197,198).5. Methylation Biomarkers in Liquid Biopsy Studies in Cancer5.1. Gene-Panel StudiesSome recent studies have focused on the analysis of promoter methylation in panels ofgenes involved in vital cellular mechanisms through the candidate gene strategy approach(199,200) (Table1). In a study of saliva rinses and tumor tissue samples from patientswith head and neck squamous cell carcinoma in the North American population, promoterhypermethylation was detected in the EDNRB and KIF1A genes. It has been reported thatEDNRB and KIF1A are potential biomarkers for head and neck squamous cell carcinoma(HNSCC) in the early diagnosis of premalignant and malignant patients (199). Anotherstudy investigating HNSCC-specific epigenetic changes in a gene panel showed that TIMP3promoter hypermethylation was reduced during treatment in saliva rinse samples from patientswith HNSCC before and immediately after treatment and six months after treatment ended.The decrease in recurrence-free survival time in patients with increased TIMP3 methylationsix months after treatment and the correlation of results with tumor tissue samples suggest thatit can be used as a prognostic biomarker for both reliable and recurrence-free survival (200).Nagatasi et al reported 100% sensitivity and 87.5% specificity for the combination of ECAD,TMEFF2, RAR? and MGMT genes based on promoter hypermethylation status in oral rinsesamples from patients with oral squamous cell carcinoma (OSCC). When the combinationof ECAD, TMEFF2 and MGMT was used, the sensitivity and specificity were 97.1% and91.7%. Gene methylation detection by liquid biopsy in oral cancers has been evaluated asan important tool in the detection of biomarkers for the diagnosis and determination of thedisease (201). In a gene panel study with normal mucosa, tumor tissue and saliva rinsesamples to identify candidate genes in HNSCC, abnormal methylation levels were observedin the IRX1 and NOL4 promoters (202).It has been shown that ZNF582 and PAX1 genes in oral epithelial cell samples taken bymouth rinsing method from patients with oral dysplasia with varying degrees of changes wereless methylated than in cell samples collected by oral scraping method. However, methylationlevels for both ZNF582 and PAX1 genes have been shown to be effective biomarkers indifferentiating oral lesions depending on the severity of oral dysplasia (203). Sun et al. OSCCshowed that the ZNF582 and PAX1 genes were hypermethylated at the lesion sites in tissuesamples, confirming the idea that they could serve as a biomarker for the course of the disease(204).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

100 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCERIn the panel that included APC, FOXA1, RAR?2, HOXD3, RASSF1A, SEPT9, GSTP1 andSOX17 genes for detection of lung cancer, colorectal cancer and prostate cancer from cell-freetumor DNA, when gene promoter methylation levels were analyzed for each cancer type,SEPT9 and SOX17 hypermethylation were determined to be common biomarkers in all threecancer types. In addition, it was concluded that HOXD3 and RASSF1A hypermethylationcould be used to differentiate small cell lung carcinoma from non-small cell lung carcinoma.Therefore, it has been suggested that a single patient material may increase the rate of diagnosisof multiple tumors at an early stage (205).The methylation of MUC1, MUC2, and MUC4 genes in formalin-fixed paraffin-embedded(FFPE) and fresh-frozen tissue samples from patients with pancreatic cancer usingendoscopic ultrasound-guided fine-needle aspiration/biopsy (EUS-FNA/FNB) material wasinvestigated using methylation-sensitive NGS technology and in conclusion, abnormalpromoter methylation was detected in MUC genes. Therefore, it was thought that it couldbe used in the diagnosis and guidance of targeted therapies in patients at risk of invasiveintervention (206).In a study examining epigenetic changes in breast cancer, it was reported that the mostfrequently methylated genes in tumor tissues compared to normal tissues were RASSF1,GSTP1, DAPK1 and CDKN2B genes and could be used in the early diagnosis of breast cancer(207).When OCT3/4, KITLG and MAGEC2 gene hypermethylation were examined in ctDNAsamples taken from tissue, plasma and ejaculate samples collected from healthy volunteersand pre- and post-orchiectomy seminoma patients, it was reported that the methylation of these3 genes was decreased in patients after orchiectomy, and therefore, they could be potentialmethylation biomarker candidates in the treatment follow-up. Therefore, it has been reportedthat these genes can be considered as potential biomarkers for seminoma monitoring and basedon the epigenetic changes detected in ctDNA, blood may be a better material of seminomabiomarkers than ejaculate (208).In the study conducted with sputum samples taken from lung cancer patients, a 1.5-2.2-foldincrease in the rate of promoter hypermethylation was observed in p16, PAX5-?, MGMT,DAPK, GATA5 and RASSF1A genes approximately 18 months before the diagnosis, while 18months after the diagnosis of lung cancer, the genes’ methylation levels were increased as 6.5times in at least half of them. The results suggested that promoter hypermethylation levelsmay reflect different stages of lung cancer development, and that they can also be used asbiomarkers to identify individuals at high risk of developing cancer (209). Pleural fluid canbe collected from the patient with the help of an injector and it has become attractive in theuse of liquid biopsy, as it can be obtained more easily from the tumor tissue that needs to betaken by thoracoscopic biopsy. It is thought that abnormal promoter methylation in RAR?,Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 101RASSF1A and p16???4? genes after DNA isolation from pleural fluid samples may play arole as predictive methylation biomarkers in the differential diagnosis of malignant pleuralmesothelioma and lung cancer (210). When the methylation levels of SOX17, TAC1, HOXA7,CDO1, HOXA9 and ZFP42 genes that are not expressed in healthy lung tissue were comparedin non-small cell lung cancer (NSCLC) tissues, sputum and plasma samples; Abnormalpromoter hypermethylation was observed in NSCLC samples. It was interpreted that sputumsamples gave more sensitive and specific results than plasma samples in methylation analysisand that these genes could be used as a predictive test for the differentiation of malignantand benign neoplasms and for early diagnosis, and also as a screening test for people atrisk of developing lung cancer (211). Bronchoalveolar lavage fluid contains tumor cells,as it is obtained by administering sterile normal saline to the bronchi during bronchoscopy,a minimally invasive method, and then collecting it by aspiration for analysis (212). In astudy in which abnormal methylation was detected in the RASSF1A and SHOX2 genes inbronchoalveolar lavage fluid samples from patients with lung squamous cell cancer and lungadenocarcinoma, the data obtained from methylation-specific RT-PCR and Sanger sequencingmethods were compared and the results were shown to be correlated with each other. It hasbeen predicted that the RASSF1A and SHOX2 genes can be used in the early diagnosis of lungcancer development (213).It was determined that methylation levels in BCAT1 and IKZF1 genes after isolation ofctDNA obtained from CCR patients changed in response to treatment applied according todifferent stages and locations of CRC and were correlated with tumor burden. Therefore, ithas been reported that these two genes can be used as potential biomarkers with methylationmeasurements to monitor treatment success and determine changes in tumor burden, sincethey provide real-time monitoring of the disease in CRC (214). It was stated that in thefollow-up of tumor burden in CRC, at least one of the WIF1 and NPY genes had abnormalmethylation and this abnormal methylation is found at a higher rate in metastatic CRCsthan in localized CRC. Therefore these genes’ methylation can be considered as a promisingbiomarker for tumor follow-up (215). In another study using samples taken from feces andtumor and normal colon tissues, it was thought that hypermethylation of SNCA and FBN1genes were detected in feces with higher sensitivity and specificity than tissues and could beused as a screening test for CRC (216).In a prostate cancer (PCa) study with serum and equivalent tumor tissue, it was shown thatthe promoter hypermethylation of ST6GALNAC3, ZNF660, CCDC181 and HAPLN3 geneswas detected both in tissue and serum, and they have the potential as a reliable biomarker.In addition, aberrant methylation of ST6GALNAC3 and ZNF660 may be effective in thediagnosis of prostate cancer, while abnormal methylation of ZNF660 may be promising byproviding information about the prognosis of the disease (217). It has been shown that geneCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

102 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCERhypermethylation of GSTP1, APC and RAR?2, which has prognostic features in PCa tissue,can be a reliable potential prognostic marker as it can be detected using ctDNA, liquid biopsyin plasma and urine samples, as well as in correlation in tumor tissue samples. It was alsoconcluded that GSTP1 may be a potential therapeutic efficacy marker for chemotherapy inpatients with castration-resistant PCa (218,219).5.2. Single gene studiesSingle gene studies have focused on finding new biomarkers for the diagnosis of differentcancer types and monitoring their prognosis, treatment and relapse using biomarker candidatemethylated genes with the help of non-invasive/minimally invasive methods. Potentialmethylation biomarkers obtained by single gene studies are given in Table 1.ZNF154 gene hypermethylation was detected in an early-stage pan-cancer model consistingof 9 different cancer types (BLCA: Bladder urothelial carcinoma, BRCA: Breast invasivecarcinoma, HNSC: Head and neck squamous cell carcinoma, KIRC: Kidney renal clearcell carcinoma, KIRP: Kidney renal papillary cell carcinoma, LIHC: Liver hepatocellularcarcinoma, LUAD: Lung adenocarcinoma, PAAD: Pancreatic adenocarcinoma, STAD:Stomach adenocarcinoma) in plasma ctDNA with liquid biopsy, and it was thought thatthis gene could be a biomarker that can be used to identify patients at high risk of developingcancer. In addition, ZNF154 has been shown to play a role in the development of pancreaticadenocarcinoma by showing 100% sensitivity and 80% specificity compared to the KRASmutation rate, which is frequently observed in pancreatic cancer (220).In a prospective study that included patients with asymptomatic CRC; it was determinedthat the SEPT9 gene was methylated in plasma samples taken from the patients and SEPT9could be a non-invasive diagnostic biomarker for CRC. However, since the sensitivity of the testis quite low in early tumor stages, it was emphasized that this test should be developed further(35). The fact that methylation biomarkers are detectable in stool, are not invasive comparedto endoscopic methods, and have high sensitivity have led to their use in early diagnosis,recurrence, personalized treatment and monitoring of treatment response in digestive systemtumors (221). When the SDC2 gene methylation levels were compared in stool samples andcolonoscopic biopsies to be used in the early detection and prognosis of CRC, the sensitivityof feces samples to detect cancers in the early stages was found to be higher than colonoscopy(222).When the methylation pattern of the CA125 gene was examined by targeting cfDNAsin patient serum samples to detect ovarian cancer early, abnormal CA125 methylation wasdetected approximately 24 months before the diagnosis, and it was stated that it could be apotential predictive biomarker (223).The presence of ctDNA in the cerebrospinal fluid makes it an attractive candidate forliquid biopsy because of its difficulty in crossing the blood-brain barrier and its proximityCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 103to the primary tumor, as it harbors more ctDNA than blood samples (224,225). In a studywith glioblastoma, one of the aggressive brain tumors, it was reported that MGMT promoterhypermethylation in the cerebrospinal fluid of patients could be used as a predictive biomarkerin predicting response to treatment. He also stated that MGMT can be used as a prognosticbiomarker showing that patients with promoter hypermethylation respond well to treatmentand prolong life (226).Global DNA methylation in ctDNA samples obtained from patients with luminal typeB breast cancer (LBBC) was determined using EPIC array methodology and subsequentlyvalidated from the TCGA database. Promoter hypermethylation of WNT1 gene has beendemonstrated in LBBC primary tumor tissues simultaneously with ctDNA obtained by liquidbiopsy method using peripheral blood material, and it has been reported that it can be definedas a biomarker (227).In a study examining P16? ? ?4? gene methylation and expression in head and neckcancers, promoter methylation was found in 67.5% and 28.6% of primary tumors, whiledownside correlation was reported in 67.5% of gene expression. The methylation-dependentloss of expression of the P16? ? ?4? gene can be evaluated as a potential biomarker in headand neck cancers (228).5.3. Multi-Omics StudiesIn 2013, Demokan et al. in their predictive biomarker candidate discovery studies forthe early diagnosis of HNSCC, they profiled the gene signature in tumor tissue and salivasamples of patients by transcriptomics and compared these results with the results obtainedby transcriptome analysis after the pharmacological demethylation approach to discovercandidate methylated gene, in primary and metastatic HNSCC cell lines. They reportedthat the GNG-7 gene could be a potential methylation biomarker in saliva samples for earlydiagnosis of HNSCC by using non-invasive detection method (229).In the transcriptomic analysis of tumors and adjacent tissues of CRC patients, it wasreported that WDR72, SPTBN2, MORC2 and PARM1 genes have m6A modification. Theresults suggest that m6A modifications can be used to predict tumor prognosis of survivaltime of cancer patients (230).In another study investigating new candidate methylation biomarkers, tumorand adjacent normal tissue samples from patients with NSCLC were analyzed byepigenomic-transcriptomic data integration methods, the PCDH17, IRX1, ITGA5, HSPB6,TBX5, ADCY8, GALNT13, and TCTEX1D1 genes were reported as hypermethylated drivergenes, that may be predictive biomarkers for early detection and anti-cancer drug development(231). (Table 1)Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

104 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCERTable 1: Some most the studied methylation biomarkers Gene Gene name Gene function Material Tumor type Method Association with Cancer Ref. TIMP3 Tissue inhibitor of metalloproteinases 3 Inhibition of angiogenesis and tumor growth Saliva rinse and tissue HNSCC QMSP Prognostic factor for local recurrence-free survival (200)EDNRB Endothelin receptor type B Cell signaling Saliva and tissue HNSCC QMSP The early diagnosis (199)KIF1A Kinesin family member 1A Cell signaling, Extracellular transport Saliva and tissue HNSCC QMSP The early diagnosis (199)ECAD E-cadherin Cell adhesion Oral Rinse OSCC QMSP Diagnosis and detection of oral cancer (201)TMEFF2 Transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains2Cell cycle regulation, cell differentiation, cell signaling Oral Rinse OSCC QMSP Diagnosis and detection of oral cancer (201)MGMT O-6 methylguanine DNA methyltransferase Cell signaling, cell cycle regulation, cell differentiation Diagnosis and detection of oral cancer (201)GSTP1 Glutathione S-transferase pi 1 Glutathione metabolism Plasma, urine and tissue PCa Multiplex QMSP, MethyLight Prognostic and chemotherapy response marker in patients with CRPC (218,219)RAR?2 Retinoic acid receptor beta2 Tumor suppression Urine and tissue PCa MethyLight Prognostic marker (219)APC Adenomatous polyposis coli Tumor suppression Urine and tissue PCa MethyLight Prognostic marker (219)p16 Cyclin dependent kinase inhibitor 2A Cell cycle regulation Sputum LC Identify individuals at high risk of developing cancer, cancer staging (209)PAX5? Paired Box 5 β Cell cycle regulation Sputum LC MSP Identify individuals at high risk of developing cancer, cancer staging (209)MGMT O-6 methylguanine DNA methyltransferase DNA repair Sputum LC MSP Identify individuals at high risk of developing cancer, cancer staging (209)RASSF1ARas association (RalGDS/AF-6) domain family member 1A Apoptosis Sputum LC MSP dentify individuals at high risk of developing cancer, cancer staging (209)DAPK Death associated protein kinase 1 Apoptosis Sputum LC MSP Identify individuals at high risk of developing cancer, cancer staging (209)GATA5 GATA binding protein 5 Signal transduction Sputum LC MSP Identify individuals at high risk of developing cancer, cancer staging (209)SEPT9 Septin9 Cytokinesis and cell cycle control Plasma LC, CRC, PCa Multiplex QMSP Reducing healthcare costs (205)SOX17 SRY-box transcription factor 17 Regulation of embryonic development and in the determination of the cell fate Plasma LC, CRC, PCa Multiplex QMSP Reducing healthcare costs (205)HOXD3 Homeobox D3 Morphogenesis Plasma LC, CRC, PCa Multiplex QMSP LC subtyping and prognostication (205)Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 105Table 1: Continued–1RASSF1A Ras association (RalGDS/AF-6) domain family member 1A Tumor suppression Plasma LC, CRC, PCa Multiplex QMSP LC subtyping and prognostication (205)OCT3/4 POU class 5 homeobox 1 Embryonic development and stem cell pluripotency Plasma + tissue + ejaculate SE Pyrosequencing Epigenetic biomarkers in the screening of seminoma (208)KITLG KIT ligand In utero in germ cell and neural cell development, and hematopoiesis Plasma + tissue + ejaculate SE Pyrosequencing Epigenetic biomarkers in the screening of seminoma (208)MAGEC2 MAGE family member C2promotes growth and tumorigenicity of melanoma cells, phosphorylation of KAP1, and DNA damage repair Plasma + tissue + ejaculate SE Pyrosequencing Epigenetic biomarkers in the screening of seminoma (208)ZNF582 Zinc-finger protein 582 Transcription factor Oral epithelial cells collected by the mouth rinse method + tissue OSCC QMSP Early diagnosis, monitoring of OSCC (203,204)PAX1 Paired-box 1 Fetal development and tumor suppression Oral epithelial cells collected by the mouth rinse method + tissue OSCC QMSP Early diagnosis, monitoring of OSCC (203,204)SEPT9 Septin 9 Cytokinesis and cell cycle control Cytokinesis and cell cycle control CRC MSCC Early tumor detection (35)BCAT1 Branched chain amino acid transaminase 1 Cell growth Plasma CRC MSP Monitoring treatment (214)IKZF1 IKAROS family zinc finger 1 Chromatin remodeling Plasma CRC MSP Monitoring treatment (214)WNT1 Wnt family member 1 Cell signaling Plasma LBBCInfinium MethylationEPIC array (EPIC array) + ddPCR Prognostic marker (227)CA125 Mucin 16, cell surface associated Encodes mucin Serum OC MSP + Illumina’s MiSeq or HiSeq 2500 Potential predictive marker (223)WIF1 WNT inhibitory factor 1 Tumor suppression Plasma CRC dPCR Primary/metastatic tumor differentiation, monitoring tumor (215)Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

106 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCER Table 1: Continued–2 NPY Neuropeptide Y Inhibit adenylyl cyclase, activate mitogen-activated protein kinase (MAPK), regulate intracellular calcium levels, and activate potassium channels Plasma CRC dPCR Primary/metastatic tumor differentiation, monitoring tumor (215) ST6GALNAC3 ST6 N-acetylgalactosami- nide alpha-2,6-sialyltrans- ferase 3 Sialic acids transfer Serum PCa dPCR Prognostic marker (217) CCDC181 Coiled-coil domain containing 181 Microtubule binding Serum PCa dPCR Prognostic marker (217) HAPLN3 Hyaluronan and proteoglycan link protein 3 Hyaluronic acid binding and cell adhesion Serum PCa Prognostic marker (217) ZNF 660 Zinc finger protein 660 Encodes a protein that contains multiple C2H2 zinc finger domains Serum PCa Prognostic marker (217) SOX17 SRY-box transcription factor 17 Regulation of embryonic development and in the determination of the cell fate Plasma + Sputum LC Methylation-on-Beads (MOB) + qMSP Predictive marker (211) TAC1 Tachykinin Precursor 1 Neurotransmitters, vasodilators Plasma + Sputum LC Methylation-on-Beads (MOB)+ qMSP Predictive marker (211) HOXA7 Homeobox A7 DNA-binding transcription factor Plasma + Sputum LC Methylation-on-Beads (MOB) + qMSP Predictive marker (211) CDO1 Cysteine dioxygenase type 1 Cysteine dioxygenase activity and Plasma + Sputum LC Methylation-on-Beads (MOB) + qMSP Predictive marker (211) HOXA9 Homeobox A9 DNA-binding transcription factor Plasma + Sputum LC Methylation-on-Beads (MOB) + qMSP Predictive marker (211) ZFP42 ZFP42 zinc finger protein Sequence DNA binding activity Plasma + Sputum LC Methylation-on-Beads (MOB) + qMSP Predictive marker (211)ZNF154 Zinc finger protein 154 Abnormal cell growth and differentiation BLCA, BRCA, HNSC, KIRC, KIRP, LIHC, LUAD, PAAD, STAD DREAMing Early stage tumors (220)MUC1 Mucin 1 Intracellular signaling EUS - FNA / FNB + FFPE + fresh frozen specimen Pancreatic Cancer NGS Prognostic marker (206)Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 107Table 1: Continued–3MUC2 Mucin 2 Mucous barrier that protects EUS - FNA / FNB + FFPE + fresh frozen specimen Pancreatic Cancer NGS Prognostic marker (206)MUC4 Mucin 4 Protection of the epithelial cells EUS - FNA / FNB + FFPE + fresh frozen specimen Pancreatic Cancer NGS Prognostic marker (206)SDC2 Syndecan 2 Integral membrane protein Feces + tissue CRC LTE-qMSP Predictive and prognostic marker (222)SNCA Synuclein alpha Signaling and membrane trafficking Feces + tissue CRC MSP Predictive marker (216)FBN1 Fibrillin 1 Encodes fibrillin-1 asprosin Feces + tissue CRC MSP Predictive marker (216)RASSF1A RAS association domain family 1, isoform A Apoptosis Bronchoalveolar lavage fluid + plasma LC RT-PCR, Sanger sequencing (213)SHOX2 short stature homeobox gene two Bronchoalveolar lavage fluid + plasma LC RT-PCR, Sanger sequencing (213)RASSF1A RAS association domain family 1, isoform A Apoptosis Pleural fluid MPM QMSP Differential diagnosis (210)?16???4? Cyclin dependent kinase inhibitor 2A Cell cycle regulation Pleural fluid MPM QMSP Differential diagnosis (210)RAR? Retinoik asit resept¨or¨uβ Tumor suppression Pleural fluid MPM QMSP Differential diagnosisMGMT O6-methylguanine-DNA methyltransferase DNA repair CSF MSP Treatment response (226)GNG-7 Guanine nucleotide-binding proteinγ-7 G protein-coupled receptor signaling pathway and regulation of adenylate cyclase activity Tissue + saliva rinse HNSCC Transcriptomics, pharmacological demethylation, QMSP Predictive marker (229)WDR72 SPTBN2 MORC2 PARM1 WD repeat domain 72, Spectrin beta, nonerythrocytic 2 MORC family CW-type zinc finger 2 Prostate androgen-regulated mucin-like protein 1 Multiple transcript variants, Glutamate signaling pathway, DNA damage and play, Telomerase activity Tissue CRC MeRIP-seq and RNA-seq Predict tumor prognosis of survival time (230)Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

108 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCER Table 1: Continued–4 NOL4 Nucleolar protein 4 RNA binding activity Tumor suppression HNSCC QMSP Prognostic marker (202) IRX1 Iroquois homeobox 1 Tumor suppression Tumor suppression HNSCC QMSP Prognostic marker (202)?16???4? Cyclin dependent kinase inhibitor 2A Cell cycle regulation Tissue Head and neck cancer MSP Early tumor detection (228)RASSF1Ras association (RalGDS/AF-6) domain family member 1A Tumor suppression Tissue Breast Cancer MS-MLPA Early tumor detection (207)GSTP1 Glutathione S-transferase pi 1 Glutathione metabolism Tissue Breast Cancer MS-MLPA Early tumor detection (207)DAPK1 Death associated protein kinase 1 Apoptosis Tissue Breast Cancer MS-MLPA Early tumor detection (207)CDKN2B Cyclin dependent kinase inhibitor 2A Cell cycle regulation Tissue Breast Cancer MS-MLPA Early tumor detection (207)PCDH17 Protocadherin 17 Cell-cell connections Tissue NSCLC Pyrosequencing Predictive biomarkers (231)IRX1 Iroquois homeobox 1 Tumor suppression Tissue NSCLC Pyrosequencing Predictive biomarkers (231)ITGA5 Integrin subunit alpha 5 Cell surface adhesion and signaling Tissue NSCLC Pyrosequencing Predictive biomarkers (231)HSPB6 Heat shock protein family B (small) member 6 Smooth muscle relaxation Tissue NSCLC Pyrosequencing Predictive biomarkers (231)TBX5 T-box transcription factor 5 Heart development Tissue NSCLC Pyrosequencing Predictive biomarkers (231)ADCY8 Adenylate cyclase 8 Catalytic subunit Tissue NSCLC Pyrosequencing Predictive biomarkers (231)GALNT13 Polypeptide N-acetylgalacto- saminyltrans- ferase 13 Initial transfer of N-acetylgalactosamine Tissue NSCLC Pyrosequencing Predictive biomarkers (231)TCTEX1D1 Dynein light chain Tctex-type family member 5 Dynein intermediate chain binding activity Tissue NSCLC Pyrosequencing Predictive biomarkers (231) HNSCC: Head and neck squamous cell carcinoma, OSCC: Oral squamous cell carcinoma, PCa: Prostate cancer, CRPC: Castrate-resistant prostate cancer, LC: Lung cancer, CRC: Colorectal cancer, SE:Seminoma, LBBC: Luminal B breast cancer, OC: Ovarian cancer, BLCA: Bladder urothelial carcinoma, BRCA: Breast invasive carcinoma, HNSC: Head and neck squamous cell carcinoma, KIRC: Kidney renal clear cell carcinoma, KIRP: Kidney renal papillary cell carcinoma, LIHC: Liver hepatocellular carcinoma, LUAD: Lung adenocarcinoma, PAAD: Pancreatic adenocarcinoma, STAD: Stomach adenocarcinoma, EUS-FNA/FNB: Endoscopic ultrasound-guided fine-needle aspiration/biopsy, FFEP: Formalin-fixed paraffin-embedded, LTE-qMSP: Linear target enrichment-quantitative methylation-specific real-time PCR, MSP: Methylation-specific polymerase chain reaction, QMSP: Quantitative Methylation-specific polymerase chain reaction, RT-PCR: Real time polymerase chain reaction, MPM: Malignant pleural mesothelioma, CSF: Cerebral spinal fluid, MS-MLPA: Methylation-specific multiplex ligation-dependent probe amplification, NSCLC: Non-small cell lung cancerCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 1096. ConclusionLiquid biopsy, which can detect cancer in the early stage by utilizing methylationbiomarkers, is also an area that continues to evolve, offering great potential for screening,diagnosis, identification of tumor types and prediction of prognosis, determination of treatmentresponse and targeted therapy development. DNA methylation-based tests may be among themost frequently chosen sources for biomarker development due to abnormal tumor-specificmodels, tissue specificity, and ease of evaluation in cfDNA. However, although detection ofDNA biomarkers by liquid biopsy remains promising in early diagnosis, there are no confirmedmarkers yet. Clinical applications of liquid biopsy have been limited by some limitations,such as the current unavailability of DNA methylation biomarkers, lack of optimization ofvarious standardization and isolation procedures, and high cost. Therefore, the developmentof procedures for the use and validation of newly discovered biomarkers is urgently needed.In addition, although there may be a risk of developing the disease in asymptomatic patients,it should not be forgotten that this does not necessarily mean that a lesion will develop in thefuture. For this reason, more research is needed to expand and maintain the studies and toensure optimization.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

CANCER: FROM GENOMICS TO PHARMACEUTICS,REFERENCES1. Klein EA, Richards D, Cohn A, Tummala M, Lapham R, Cosgrove D, et al. Clinicalvalidation of a targeted methylation-based multi-cancer early detection test using anindependent validation set. Ann Oncol. 2021;32(9):1167-77.2. Freund M, Luftner D, Wilhelm M. Benefits and risks of cancer screening. Oncol ResTreat. 2014;37 Suppl 3:1.3. Hamilton W, Walter FM, Rubin G, Neal RD. Improving early diagnosis of symptomaticcancer. Nat Rev Clin Oncol. 2016;13(12):740-9.4. Chen M, Zhao H. Next-generation sequencing in liquid biopsy: cancer screening andearly detection. Hum Genomics. 2019;13(1):34.5. Hasirci I, Sahin A. Importance of the neutrophil-lymphocyte ratio and systemicimmune-inflammation index in predicting colorectal pathologies in fecal occultblood-positive patients. J Clin Lab Anal. 2023:e24878.6. World Cancer Report 2014 2014.7. Curtius K, Wright NA, Graham TA. Evolution of Premalignant Disease. Cold SpringHarb Perspect Med. 2017;7(12).8. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell.2011;144(5):646-74.9. Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nat Med.2004;10(8):789-99.10. Goldberg AD, Allis CD, Bernstein E. Epigenetics: a landscape takes shape. Cell.2007;128(4):635-8.11. Blackadar CB. Historical review of the causes of cancer. World J Clin Oncol.2016;7(1):54-86.12. Cazaly E, Charlesworth J, Dickinson JL, Holloway AF. Genetic Determinants ofEpigenetic Patterns: Providing Insight into Disease. Mol Med. 2015;21(1):400-9.13. Hanahan D. Hallmarks of Cancer: New Dimensions. Cancer Discov. 2022;12(1):31-46.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 11114. Carethers JM, Jung BH. Genetics and Genetic Biomarkers in Sporadic ColorectalCancer. Gastroenterology. 2015;149(5):1177-90 e3.15. Esteller M. Epigenetics in cancer. N Engl J Med. 2008;358(11):1148-59.16. Hamilton JP. Epigenetics: principles and practice. Dig Dis. 2011;29(2):130-5.17. Gibney ER, Nolan CM. Epigenetics and gene expression. Heredity (Edinb).2010;105(1):4-13.18. Liang M. Epigenetic Mechanisms and Hypertension. Hypertension.2018;72(6):1244-54.19. Lakshminarasimhan R, Liang G. The Role of DNA Methylation in Cancer. Adv ExpMed Biol. 2016;945:151-72.20. Baba AI CC. TUMOR CELL MORPHOLOGY. Comparative Oncology: Bucharest(RO): The Publishing House of the Romanian Academy; 2007.21. Desaulniers D, Vasseur P, Jacobs A, Aguila MC, Ertych N, Jacobs MN. Integrationof Epigenetic Mechanisms into Non-Genotoxic Carcinogenicity Hazard Assessment:Focus on DNA Methylation and Histone Modifications. Int J Mol Sci. 2021;22(20).22. Peng M, Chen C, Hulbert A, Brock MV, Yu F. Non-blood circulating tumor DNAdetection in cancer. Oncotarget. 2017;8(40):69162-73.23. Sun K, Lun FFM, Jiang P, Sun H. BSviewer: a genotype-preserving, nucleotide-levelvisualizer for bisulfite sequencing data. Bioinformatics. 2017;33(21):3495-6.24. Wong IH, Lo YM, Zhang J, Liew CT, Ng MH, Wong N, et al. Detection of aberrantp16 methylation in the plasma and serum of liver cancer patients. Cancer Res.1999;59(1):71-3.25. Roadmap Epigenomics C, Kundaje A, Meuleman W, Ernst J, Bilenky M, YenA, et al. Integrative analysis of 111 reference human epigenomes. Nature.2015;518(7539):317-30.26. Lehmann-Werman R, Neiman D, Zemmour H, Moss J, Magenheim J,Vaknin-Dembinsky A, et al. Identification of tissue-specific cell death using methylationpatterns of circulating DNA. Proc Natl Acad Sci U S A. 2016;113(13):E1826-34.27. Sun K, Jiang P, Chan KC, Wong J, Cheng YK, Liang RH, et al. Plasma DNA tissuemapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, andtransplantation assessments. Proc Natl Acad Sci U S A. 2015;112(40):E5503-12.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

112 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCER28. Lawrence MS, Stojanov P, Polak P, Kryukov GV, Cibulskis K, Sivachenko A, et al.Mutational heterogeneity in cancer and the search for new cancer-associated genes.Nature. 2013;499(7457):214-8.29. Liang W, Liu D, Li M, Wang W, Qin Z, Zhang J, et al. Evaluating the diagnostic accuracyof a ctDNA methylation classifier for incidental lung nodules: protocol for a prospective,observational, and multicenter clinical trial of 10,560 cases. Transl Lung Cancer Res.2020;9(5):2016-26.30. Locke WJ, Guanzon D, Ma C, Liew YJ, Duesing KR, Fung KYC, et al. DNA MethylationCancer Biomarkers: Translation to the Clinic. Front Genet. 2019;10:1150.31. Torre LA, Siegel RL, Ward EM, Jemal A. Global Cancer Incidence and Mortality Ratesand Trends–An Update. Cancer Epidemiol Biomarkers Prev. 2016;25(1):16-27.32. Skinner MK, Manikkam M, Guerrero-Bosagna C. Epigenetic transgenerationalactions of environmental factors in disease etiology. Trends Endocrinol Metab.2010;21(4):214-22.33. Coppede F. Genes and the Environment in Cancer: Focus on Environmentally InducedDNA Methylation Changes. Cancers (Basel). 2023;15(4).34. Ruiz de la Cruz M, de la Cruz Montoya AH, Rojas Jimenez EA, Martinez Gregorio H,Diaz Velasquez CE, Paredes de la Vega J, et al. Cis-Acting Factors Causing SecondaryEpimutations: Impact on the Risk for Cancer and Other Diseases. Cancers (Basel).2021;13(19).35. Okugawa Y, Grady WM, Goel A. Epigenetic Alterations in Colorectal Cancer: EmergingBiomarkers. Gastroenterology. 2015;149(5):1204-25 e12.36. Hiroshi SAEKI KS. Carcinogenic Risk Factors. Journal of the Japan Medical Association2001;125(3):297–300.37. Herceg Z, Hainaut P. Genetic and epigenetic alterations as biomarkers for cancerdetection, diagnosis and prognosis. Mol Oncol. 2007;1(1):26-41.38. Das Subhayan KM, Jena Bikash Chandra, Mandal Mahitosh Biomaterials for 3D TumorModeling: Elsevier; 2020.39. Giotopoulos G, McCormick C, Cole C, Zanker A, Jawad M, Brown R, et al. DNAmethylation during mouse hemopoietic differentiation and radiation-induced leukemia.Exp Hematol. 2006;34(11):1462-70.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 11340. Koturbash I, Boyko A, Rodriguez-Juarez R, McDonald RJ, Tryndyak VP, Kovalchuk I,et al. Role of epigenetic effectors in maintenance of the long-term persistent bystandereffect in spleen in vivo. Carcinogenesis. 2007;28(8):1831-8.41. Kumar A, Rai PS, Upadhya R, Vishwanatha, Prasada KS, Rao BS, et al. gamma-radiationinduces cellular sensitivity and aberrant methylation in human tumor cell lines. Int JRadiat Biol. 2011;87(11):1086-96.42. Rider CF, Carlsten C. Air pollution and DNA methylation: effects of exposure in humans.Clin Epigenetics. 2019;11(1):131.43. Plusquin M, Guida F, Polidoro S, Vermeulen R, Raaschou-Nielsen O, Campanella G, etal. DNA methylation and exposure to ambient air pollution in two prospective cohorts.Environ Int. 2017;108:127-36.44. Sailani MR, Halling JF, Moller HD, Lee H, Plomgaard P, Pilegaard H, et al. Lifelongphysical activity is associated with promoter hypomethylation of genes involved inmetabolism, myogenesis, contractile properties and oxidative stress resistance in agedhuman skeletal muscle. Sci Rep. 2019;9(1):3272.45. Swiatowy WJ, Drzewiecka H, Kliber M, Sasiadek M, Karpinski P, Plawski A, et al.Physical Activity and DNA Methylation in Humans. Int J Mol Sci. 2021;22(23).46. Lee KW, Pausova Z. Cigarette smoking and DNA methylation. Front Genet. 2013;4:132.47. Smith CJ, Hansch C. The relative toxicity of compounds in mainstream cigarette smokecondensate. Food Chem Toxicol. 2000;38(7):637-46.48. Jethwa AR, Khariwala SS. Tobacco-related carcinogenesis in head and neck cancer.Cancer Metastasis Rev. 2017;36(3):411-23.49. Gao X, Zhang Y, Breitling LP, Brenner H. Tobacco smoking and methylation of genesrelated to lung cancer development. Oncotarget. 2016;7(37):59017-28.50. Shui IM, Wong CJ, Zhao S, Kolb S, Ebot EM, Geybels MS, et al. Prostate tumor DNAmethylation is associated with cigarette smoking and adverse prostate cancer outcomes.Cancer. 2016;122(14):2168-77.51. Jin F, Thaiparambil J, Donepudi SR, Vantaku V, Piyarathna DWB, Maity S, et al.Tobacco-Specific Carcinogens Induce Hypermethylation, DNA Adducts, and DNADamage in Bladder Cancer. Cancer Prev Res (Phila). 2017;10(10):588-97.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

114 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCER52. Zhang J, Bai R, Li M, Ye H, Wu C, Wang C, et al. Excessive miR-25-3p maturationvia N(6)-methyladenosine stimulated by cigarette smoke promotes pancreatic cancerprogression. Nat Commun. 2019;10(1):1858.53. Mons U, Gredner T, Behrens G, Stock C, Brenner H. Cancers Due to Smoking and HighAlcohol Consumption. Dtsch Arztebl Int. 2018;115(35-36):571-7.54. Mizumoto A, Ohashi S, Hirohashi K, Amanuma Y, Matsuda T, Muto M. MolecularMechanisms of Acetaldehyde-Mediated Carcinogenesis in Squamous Epithelium. Int JMol Sci. 2017;18(9).55. Donroe JH, Edelman EJ. Alcohol Use. Ann Intern Med. 2022;175(10):ITC145-ITC60.56. Jamal A, Phillips E, Gentzke AS, Homa DM, Babb SD, King BA, et al. CurrentCigarette Smoking Among Adults - United States, 2016. MMWR Morb Mortal WklyRep. 2018;67(2):53-9.57. HASSOY Dilek OS. Investigation of Smoking Status and Related Factors of Nurses ¨Working in a State Hospital. Journal of Nursing Science. 2021;4(3) 140–7.58. Parrillo L, Spinelli R, Nicolo A, Longo M, Mirra P, Raciti GA, et al. NutritionalFactors, DNA Methylation, and Risk of Type 2 Diabetes and Obesity: Perspectives andChallenges. Int J Mol Sci. 2019;20(12).59. Bouchard L, Rabasa-Lhoret R, Faraj M, Lavoie ME, Mill J, Perusse L, et al. Differentialepigenomic and transcriptomic responses in subcutaneous adipose tissue between lowand high responders to caloric restriction. Am J Clin Nutr. 2010;91(2):309-20.60. Nettore IC, Franchini F, Palatucci G, Macchia PE, Ungaro P. Epigenetic Mechanisms ofEndocrine-Disrupting Chemicals in Obesity. Biomedicines. 2021;9(11).61. Cabrera-Mulero A, Crujeiras AB, Izquierdo AG, Torres E, Ayers D, Casanueva FF, et al.Novel SFRP2 DNA Methylation Profile Following Neoadjuvant Therapy in ColorectalCancer Patients with Different Grades of BMI. J Clin Med. 2019;8(7).62. Alavian-Ghavanini A, Ruegg J. Understanding Epigenetic Effects of EndocrineDisrupting Chemicals: From Mechanisms to Novel Test Methods. Basic Clin PharmacolToxicol. 2018;122(1):38-45.63. Medjakovic S, Zoechling A, Gerster P, Ivanova MM, Teng Y, Klinge CM, et al. Effect ofnonpersistent pesticides on estrogen receptor, androgen receptor, and aryl hydrocarbonreceptor. Environ Toxicol. 2014;29(10):1201-16.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 11564. Watanabe Y, Yamamoto H, Oikawa R, Toyota M, Yamamoto M, Kokudo N, et al. DNAmethylation at hepatitis B viral integrants is associated with methylation at flankinghuman genomic sequences. Genome Res. 2015;25(3):328-37.65. Zhang C, Huang C, Sui X, Zhong X, Yang W, Hu X, et al. Association between genemethylation and HBV infection in hepatocellular carcinoma: A meta-analysis. J Cancer.2019;10(25):6457-65.66. Zheng Y, Hlady RA, Joyce BT, Robertson KD, He C, Nannini DR, et al. DNA methylationof individual repetitive elements in hepatitis C virus infection-induced hepatocellularcarcinoma. Clin Epigenetics. 2019;11(1):145.67. Zhang C, Zhang W, Yuan Z, Yang W, Hu X, Duan S, et al. Contribution of DNAmethylation to the risk of hepatitis C virus-associated hepatocellular carcinoma: Ameta-analysis. Pathol Res Pract. 2022;238:154136.68. Zhang L, Wang R, Xie Z. The roles of DNA methylation on the promotor of theEpstein-Barr virus (EBV) gene and the genome in patients with EBV-associated diseases.Appl Microbiol Biotechnol. 2022;106(12):4413-26.69. Ka-Yue Chow L, Lai-Shun Chung D, Tao L, Chan KF, Tung SY, Cheong Ngan RK,et al. Epigenomic landscape study reveals molecular subtypes and EBV-associatedregulatory epigenome reprogramming in nasopharyngeal carcinoma. EBioMedicine.2022;86:104357.70. Elagan SK, Almalki SJ, Alharthi MR, Mohamed MS, El-Badawy MF. Role of Bacteriain the Incidence of Common GIT Cancers: The Dialectical Role of Integrated BacterialDNA in Human Carcinogenesis. Infect Drug Resist. 2021;14:2003-14.71. Hattori N, Ushijima T. Epigenetic impact of infection on carcinogenesis: mechanismsand applications. Genome Med. 2016;8(1):10.72. Yangyanqiu W, Shuwen H. Bacterial DNA involvement in carcinogenesis. Front CellInfect Microbiol. 2022;12:996778.73. He Z, Tian W, Wei Q, Xu J. Involvement of Fusobacterium nucleatum in malignanciesexcept for colorectal cancer: A literature review. Front Immunol. 2022;13:968649.74. Umana A, Nguyen TTD, Sanders BE, Williams KJ, Wozniak B, Slade DJ. EnhancedFusobacterium nucleatum Genetics Using Host DNA Methyltransferases To BypassRestriction-Modification Systems. J Bacteriol. 2022;204(12):e0027922.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

116 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCER75. Tahara T, Hirata I, Nakano N, Tahara S, Horiguchi N, Kawamura T, et al. Potentiallink between Fusobacterium enrichment and DNA methylation accumulation in theinflammatory colonic mucosa in ulcerative colitis. Oncotarget. 2017;8(37):61917-26.76. Chen S, Zhang L, Li M, Zhang Y, Sun M, Wang L, et al. Fusobacterium nucleatumreduces METTL3-mediated m(6)A modification and contributes to colorectal cancermetastasis. Nat Commun. 2022;13(1):1248.77. Park HE, Kim JH, Cho NY, Lee HS, Kang GH. Intratumoral Fusobacterium nucleatumabundance correlates with macrophage infiltration and CDKN2A methylation inmicrosatellite-unstable colorectal carcinoma. Virchows Arch. 2017;471(3):329-36.78. Muhammad JS, Eladl MA, Khoder G. Helicobacter pylori-induced DNA Methylationas an Epigenetic Modulator of Gastric Cancer: Recent Outcomes and Future Direction.Pathogens. 2019;8(1).79. Portela A, Esteller M. Epigenetic modifications and human disease. Nat Biotechnol.2010;28(10):1057-68.80. Tsai HC, Baylin SB. Cancer epigenetics: linking basic biology to clinical medicine. CellRes. 2011;21(3):502-17.81. Linton A, Cheng YY, Griggs K, Schedlich L, Kirschner MB, Gattani S, et al. AnRNAi-based screen reveals PLK1, CDK1 and NDC80 as potential therapeutic targets inmalignant pleural mesothelioma. Br J Cancer. 2014;110(2):510-9.82. Yang X, Dai W, Kwong DL, Szeto CY, Wong EH, Ng WT, et al. Epigenetic markers fornoninvasive early detection of nasopharyngeal carcinoma by methylation-sensitive highresolution melting. Int J Cancer. 2015;136(4):E127-35.83. Lacal I, Ventura R. Epigenetic Inheritance: Concepts, Mechanisms and Perspectives.Front Mol Neurosci. 2018;11:292.84. Sharma S, Kelly TK, Jones PA. Epigenetics in cancer. Carcinogenesis. 2010;31(1):27-36.85. Chatterjee A, Rodger EJ, Eccles MR. Epigenetic drivers of tumourigenesis and cancermetastasis. Semin Cancer Biol. 2018;51:149-59.86. Menezo Y, Clement P, Clement A, Elder K. Methylation: An Ineluctable Biochemicaland Physiological Process Essential to the Transmission of Life. Int J Mol Sci.2020;21(23).87. Deng S, Zhang J, Su J, Zuo Z, Zeng L, Liu K, et al. RNA m(6)A regulates transcriptionvia DNA demethylation and chromatin accessibility. Nat Genet. 2022;54(9):1427-37.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 11788. Lyko F. The DNA methyltransferase family: a versatile toolkit for epigenetic regulation.Nat Rev Genet. 2018;19(2):81-92.89. Gereige LM, Mikkola HK. DNA methylation is a guardian of stem cell self-renewal andmultipotency. Nat Genet. 2009;41(11):1164-6.90. Yin Y, Morgunova E, Jolma A, Kaasinen E, Sahu B, Khund-Sayeed S, et al. Impactof cytosine methylation on DNA binding specificities of human transcription factors.Science. 2017;356(6337).91. Moore LD, Le T, Fan G. DNA methylation and its basic function.Neuropsychopharmacology. 2013;38(1):23-38.92. Jin B, Li Y, Robertson KD. DNA methylation: superior or subordinate in the epigenetichierarchy? Genes Cancer. 2011;2(6):607-17.93. Kareta MS, Botello ZM, Ennis JJ, Chou C, Chedin F. Reconstitution and mechanismof the stimulation of de novo methylation by human DNMT3L. J Biol Chem.2006;281(36):25893-902.94. Casalino L, Verde P. Multifaceted Roles of DNA Methylation in NeoplasticTransformation, from Tumor Suppressors to EMT and Metastasis. Genes (Basel).2020;11(8).95. Tan T, Shi P, Abbas MN, Wang Y, Xu J, Chen Y, et al. Epigenetic modification regulatestumor progression and metastasis through EMT (Review). Int J Oncol. 2022;60(6).96. Jeltsch A, Ehrenhofer-Murray A, Jurkowski TP, Lyko F, Reuter G, Ankri S, et al.Mechanism and biological role of Dnmt2 in Nucleic Acid Methylation. RNA Biol.2017;14(9):1108-23.97. Suetake I, Shinozaki F, Miyagawa J, Takeshima H, Tajima S. DNMT3L stimulates theDNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction. J BiolChem. 2004;279(26):27816-23.98. Wang Q, Xiong F, Wu G, Liu W, Chen J, Wang B, et al. Gene body methylation in cancer:molecular mechanisms and clinical applications. Clin Epigenetics. 2022;14(1):154.99. Weber M, Davies JJ, Wittig D, Oakeley EJ, Haase M, Lam WL, et al. Chromosome-wideand promoter-specific analyses identify sites of differential DNA methylation in normaland transformed human cells. Nat Genet. 2005;37(8):853-62.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

118 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCER100. Peters TJ, Buckley MJ, Statham AL, Pidsley R, Samaras K, R VL, et al. De novoidentification of differentially methylated regions in the human genome. EpigeneticsChromatin. 2015;8:6.101. Hotta K, Kitamoto A, Kitamoto T, Ogawa Y, Honda Y, Kessoku T, et al. Identificationof differentially methylated region (DMR) networks associated with progression ofnonalcoholic fatty liver disease. Sci Rep. 2018;8(1):13567.102. Rakyan VK, Down TA, Balding DJ, Beck S. Epigenome-wide association studies forcommon human diseases. Nat Rev Genet. 2011;12(8):529-41.103. Irizarry RA, Ladd-Acosta C, Wen B, Wu Z, Montano C, Onyango P, et al. Thehuman colon cancer methylome shows similar hypo- and hypermethylation at conservedtissue-specific CpG island shores. Nat Genet. 2009;41(2):178-86.104. Brait M, Sidransky D. Cancer epigenetics: above and beyond. Toxicol Mech Methods.2011;21(4):275-88.105. Rauluseviciute I, Drablos F, Rye MB. DNA hypermethylation associated withupregulated gene expression in prostate cancer demonstrates the diversity of epigeneticregulation. BMC Med Genomics. 2020;13(1):6.106. Stone A, Zotenko E, Locke WJ, Korbie D, Millar EK, Pidsley R, et al. DNA methylationof oestrogen-regulated enhancers defines endocrine sensitivity in breast cancer. NatCommun. 2015;6:7758.107. McEvoy JD, Dyer MA. Genetic and Epigenetic Discoveries in Human Retinoblastoma.Crit Rev Oncog. 2015;20(3-4):217-25.108. Singh U, Malik MA, Goswami S, Shukla S, Kaur J. Epigenetic regulation of humanretinoblastoma. Tumour Biol. 2016;37(11):14427-41.109. Robinson CM, Lefebvre F, Poon BP, Bousard A, Fan X, Lathrop M, et al. Consequencesof VHL Loss on Global DNA Methylome. Sci Rep. 2018;8(1):3313.110. Tramontano A, Boffo FL, Russo G, De Rosa M, Iodice I, Pezone A. Methylationof the Suppressor Gene p16INK4a: Mechanism and Consequences. Biomolecules.2020;10(3).111. Esteller M. CpG island hypermethylation and tumor suppressor genes: a boomingpresent, a brighter future. Oncogene. 2002;21(35):5427-40.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 119112. Sabi SH, Khabour OF, Alzoubi KH, Cobb CO, Eissenberg T. Changes at global andsite-specific DNA methylation of MLH1 gene promoter induced by waterpipe smokingin blood lymphocytes and oral epithelial cells. Inhal Toxicol. 2020;32(3):124-30.113. Aref-Eshghi E, McGee JD, Pedro VP, Kerkhof J, Stuart A, Ainsworth PJ, et al. Geneticand epigenetic profiling of BRCA1/2 in ovarian tumors reveals additive diagnosticyield and evidence of a genomic BRCA1/2 DNA methylation signature. J Hum Genet.2020;65(10):865-73.114. Downs B, Wang SM. Epigenetic changes in BRCA1-mutated familial breast cancer.Cancer Genet. 2015;208(5):237-40.115. Al-Moghrabi N, Al-Showimi M, Al-Yousef N, Al-Shahrani B, Karakas B, AlghofailiL, et al. Methylation of BRCA1 and MGMT genes in white blood cells are transmittedfrom mothers to daughters. Clin Epigenetics. 2018;10(1):99.116. Heery R, Schaefer MH. DNA methylation variation along the cancer epigenomeand the identification of novel epigenetic driver events. Nucleic Acids Res.2021;49(22):12692-705.117. Jones PA. Functions of DNA methylation: islands, start sites, gene bodies and beyond.Nat Rev Genet. 2012;13(7):484-92.118. Weisenberger DJ, Lakshminarasimhan R, Liang G. The Role of DNA Methylation andDNA Methyltransferases in Cancer. Adv Exp Med Biol. 2022;1389:317-48.119. Ehrlich M. DNA hypomethylation in cancer cells. Epigenomics. 2009;1(2):239-59.120. Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some human cancersfrom their normal counterparts. Nature. 1983;301(5895):89-92.121. Li J, Huang Q, Zeng F, Li W, He Z, Chen W, et al. The prognostic value of global DNAhypomethylation in cancer: a meta-analysis. PLoS One. 2014;9(9):e106290.122. Pappalardo XG, Barra V. Losing DNA methylation at repetitive elements and breakingbad. Epigenetics Chromatin. 2021;14(1):25.123. Zeggar HR, How-Kit A, Daunay A, Bettaieb I, Sahbatou M, Rahal K, et al. TumorDNA hypomethylation of LINE-1 is associated with low tumor grade of breast cancerin Tunisian patients. Oncol Lett. 2020;20(2):1999-2006.124. Plasschaert RN, Bartolomei MS. Genomic imprinting in development, growth, behaviorand stem cells. Development. 2014;141(9):1805-13.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

120 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCER125. Cui H, Cruz-Correa M, Giardiello FM, Hutcheon DF, Kafonek DR, Brandenburg S,et al. Loss of IGF2 imprinting: a potential marker of colorectal cancer risk. Science.2003;299(5613):1753-5.126. Feinberg AP. Imprinting of a genomic domain of 11p15 and loss of imprinting in cancer:an introduction. Cancer Res. 1999;59(7 Suppl):1743s-6s.127. Wu H, Chen Y, Liang J, Shi B, Wu G, Zhang Y, et al. Hypomethylation-linkedactivation of PAX2 mediates tamoxifen-stimulated endometrial carcinogenesis. Nature.2005;438(7070):981-7.128. Brueckner B, Stresemann C, Kuner R, Mund C, Musch T, Meister M, et al. The humanlet-7a-3 locus contains an epigenetically regulated microRNA gene with oncogenicfunction. Cancer Res. 2007;67(4):1419-23.129. Eden A, Gaudet F, Waghmare A, Jaenisch R. Chromosomal instability and tumorspromoted by DNA hypomethylation. Science. 2003;300(5618):455.130. de Koning AP, Gu W, Castoe TA, Batzer MA, Pollock DD. Repetitive elements maycomprise over two-thirds of the human genome. PLoS Genet. 2011;7(12):e1002384.131. Tajbakhsh J, Mortazavi F, Gupta NK. DNA methylation topology differentiates betweennormal and malignant in cell models, resected human tissues, and exfoliated sputumcells of lung epithelium. Front Oncol. 2022;12:991120.132. Prada D, Gonzalez R, Sanchez L, Castro C, Fabian E, Herrera LA. Satellite2 demethylation induced by 5-azacytidine is associated with missegregation ofchromosomes 1 and 16 in human somatic cells. Mutat Res. 2012;729(1-2):100-5.133. Daskalos A, Nikolaidis G, Xinarianos G, Savvari P, Cassidy A, Zakopoulou R, et al.Hypomethylation of retrotransposable elements correlates with genomic instability innon-small cell lung cancer. Int J Cancer. 2009;124(1):81-7.134. Wu X, Zhang Y. TET-mediated active DNA demethylation: mechanism, function andbeyond. Nat Rev Genet. 2017;18(9):517-34.135. Besaratinia A, Caceres A, Tommasi S. DNA Hydroxymethylation inSmoking-Associated Cancers. Int J Mol Sci. 2022;23(5).136. Xu Q, Wang C, Zhou JX, Xu ZM, Gao J, Sui P, et al. Loss of TET reprograms Wntsignaling through impaired demethylation to promote lung cancer development. ProcNatl Acad Sci U S A. 2022;119(6).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 121137. Yang B, Tang H, Wang N, Gu J, Wang Q. Targeted DNA demethylation of the ZNF334promoter inhibits colorectal cancer growth. Cell Death Dis. 2023;14(3):210.138. Melamed P, Yosefzon Y, David C, Tsukerman A, Pnueli L. Tet Enzymes, Variants, andDifferential Effects on Function. Front Cell Dev Biol. 2018;6:22.139. Zhu H, Zhu H, Tian M, Wang D, He J, Xu T. DNA Methylation and Hydroxymethylationin Cervical Cancer: Diagnosis, Prognosis and Treatment. Front Genet. 2020;11:347.140. Fu S, Wu H, Zhang H, Lian CG, Lu Q. DNA methylation/hydroxymethylation inmelanoma. Oncotarget. 2017;8(44):78163-73.141. Du Q, Luu PL, Stirzaker C, Clark SJ. Methyl-CpG-binding domain proteins: readers ofthe epigenome. Epigenomics. 2015;7(6):1051-73.142. Fournier A, Sasai N, Nakao M, Defossez PA. The role of methyl-binding proteinsin chromatin organization and epigenome maintenance. Brief Funct Genomics.2012;11(3):251-64.143. Fujita N, Watanabe S, Ichimura T, Tsuruzoe S, Shinkai Y, Tachibana M, etal. Methyl-CpG binding domain 1 (MBD1) interacts with the Suv39h1-HP1heterochromatic complex for DNA methylation-based transcriptional repression. J BiolChem. 2003;278(26):24132-8.144. Stirzaker C, Song JZ, Ng W, Du Q, Armstrong NJ, Locke WJ, et al. Methyl-CpG-bindingprotein MBD2 plays a key role in maintenance and spread of DNA methylation at CpGislands and shores in cancer. Oncogene. 2017;36(10):1328-38.145. Cui Y, Irudayaraj J. Dissecting the behavior and function of MBD3 in DNA methylationhomeostasis by single-molecule spectroscopy and microscopy. Nucleic Acids Res.2015;43(6):3046-55.146. Sanders MA, Chew E, Flensburg C, Zeilemaker A, Miller SE, Al Hinai AS, et al.MBD4 guards against methylation damage and germ line deficiency predisposes toclonal hematopoiesis and early-onset AML. Blood. 2018;132(14):1526-34.147. Ichino L, Boone BA, Strauskulage L, Harris CJ, Kaur G, Gladstone MA, et al. MBD5and MBD6 couple DNA methylation to gene silencing through the J-domain proteinSILENZIO. Science. 2021.148. Ropa J, Saha N, Hu H, Peterson LF, Talpaz M, Muntean AG. SETDB1 mediatedhistone H3 lysine 9 methylation suppresses MLL-fusion target expression and leukemictransformation. Haematologica. 2020;105(9):2273-85.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

122 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCER149. Gu L, Frommel SC, Oakes CC, Simon R, Grupp K, Gerig CY, et al. BAZ2A (TIP5)is involved in epigenetic alterations in prostate cancer and its overexpression predictsdisease recurrence. Nat Genet. 2015;47(1):22-30.150. Mian OY, Wang SZ, Zhu SZ, Gnanapragasam MN, Graham L, Bear HD, et al.Methyl-binding domain protein 2-dependent proliferation and survival of breast cancercells. Mol Cancer Res. 2011;9(8):1152-62.151. Subhash S, Kanduri M. Comprehensive DNA Methylation Analysis Using aMethyl-CpG-binding Domain Capture-based Method in Chronic LymphocyticLeukemia Patients. J Vis Exp. 2017(124).152. Illingworth RS, Bird AP. CpG islands–’a rough guide’. FEBS Lett.2009;583(11):1713-20.153. Landolin JM, Johnson DS, Trinklein ND, Aldred SF, Medina C, Shulha H, et al.Sequence features that drive human promoter function and tissue specificity. GenomeRes. 2010;20(7):890-8.154. Mitchell C, Schneper LM, Notterman DA. DNA methylation, early life environment,and health outcomes. Pediatr Res. 2016;79(1-2):212-9.155. Vavouri T, Lehner B. Human genes with CpG island promoters have a distincttranscription-associated chromatin organization. Genome Biol. 2012;13(11):R110.156. Obeid R. The metabolic burden of methyl donor deficiency with focus on the betainehomocysteine methyltransferase pathway. Nutrients. 2013;5(9):3481-95.157. Friso S, Udali S, De Santis D, Choi SW. One-carbon metabolism and epigenetics. MolAspects Med. 2017;54:28-36.158. Mentch SJ, Locasale JW. One-carbon metabolism and epigenetics: understanding thespecificity. Ann N Y Acad Sci. 2016;1363(1):91-8.159. Mahmood N, Cheishvili D, Arakelian A, Tanvir I, Khan HA, Pepin AS, et al.Methyl donor S-adenosylmethionine (SAM) supplementation attenuates breast cancergrowth, invasion, and metastasis in vivo; therapeutic and chemopreventive applications.Oncotarget. 2018;9(4):5169-83.160. Mosca L, Minopoli M, Pagano M, Vitiello F, Carriero MV, Cacciapuoti G, et al. Effectsof S-adenosyl-L-methionine on the invasion and migration of head and neck squamouscancer cells and analysis of the underlying mechanisms. Int J Oncol. 2020;56(5):1212-24.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 123161. Lu SC, Mato JM. S-Adenosylmethionine in cell growth, apoptosis and liver cancer. JGastroenterol Hepatol. 2008;23 Suppl 1(Suppl 1):S73-7.162. Nuru M, Muradashvili N, Kalani A, Lominadze D, Tyagi N. High methionine, low folateand low vitamin B6/B12 (HM-LF-LV) diet causes neurodegeneration and subsequentshort-term memory loss. Metab Brain Dis. 2018;33(6):1923-34.163. Rajaie S, Esmaillzadeh A. Dietary choline and betaine intakes and risk of cardiovasculardiseases: review of epidemiological evidence. ARYA Atheroscler. 2011;7(2):78-86.164. Cappuccilli M, Bergamini C, Giacomelli FA, Cianciolo G, Donati G, Conte D, et al.Vitamin B Supplementation and Nutritional Intake of Methyl Donors in Patients withChronic Kidney Disease: A Critical Review of the Impact on Epigenetic Machinery.Nutrients. 2020;12(5).165. Zhang M, Song J, Yuan W, Zhang W, Sun Z. Roles of RNA Methylation on TumorImmunity and Clinical Implications. Front Immunol. 2021;12:641507.166. Zhou Y, Kong Y, Fan W, Tao T, Xiao Q, Li N, et al. Principles of RNA methylation andtheir implications for biology and medicine. Biomed Pharmacother. 2020;131:110731.167. Chen XY, Zhang J, Zhu JS. The role of m(6)A RNA methylation in human cancer. MolCancer. 2019;18(1):103.168. Zaccara S, Ries RJ, Jaffrey SR. Reading, writing and erasing mRNA methylation. NatRev Mol Cell Biol. 2019;20(10):608-24.169. Li Y. Modern epigenetics methods in biological research. Methods. 2021;187:104-13.170. Harrison A, Parle-McDermott A. DNA methylation: a timeline of methods andapplications. Front Genet. 2011;2:74.171. Guevara MA, de Maria N, Saez-Laguna E, Velez MD, Cervera MT, Cabezas JA. Analysisof DNA Cytosine Methylation Patterns Using Methylation-Sensitive AmplificationPolymorphism (MSAP). Methods Mol Biol. 2017;1456:99-112.172. Fulnecek J, Kovarik A. How to interpret methylation sensitive amplified polymorphism(MSAP) profiles? BMC Genet. 2014;15:2.173. Martisova A, Holcakova J, Izadi N, Sebuyoya R, Hrstka R, Bartosik M. DNA Methylationin Solid Tumors: Functions and Methods of Detection. Int J Mol Sci. 2021;22(8).174. Maruvada P, Wang W, Wagner PD, Srivastava S. Biomarkers in molecular medicine:cancer detection and diagnosis. Biotechniques. 2005;Suppl:9-15.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

124 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCER175. Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, et al. Agenomic sequencing protocol that yields a positive display of 5-methylcytosine residuesin individual DNA strands. Proc Natl Acad Sci U S A. 1992;89(5):1827-31.176. Brisotto G, di Gennaro A, Damiano V, Armellin M, Perin T, Maestro R, et al. Animproved sequencing-based strategy to estimate locus-specific DNA methylation. BMCCancer. 2015;15:639.177. Siqueira JF, Jr., Fouad AF, Rocas IN. Pyrosequencing as a tool for better understandingof human microbiomes. J Oral Microbiol. 2012;4.178. Higashimoto K, Hara S, Soejima H. DNA Methylation Analysis Using BisulfitePyrosequencing. Methods Mol Biol. 2023;2577:3-20.179. Lee S, Borah S, Bahrami A. Detection of Aberrant TERT Promoter Methylation byCombined Bisulfite Restriction Enzyme Analysis for Cancer Diagnosis. J Mol Diagn.2017;19(3):378-86.180. Gonzalgo ML, Liang G. Methylation-sensitive single-nucleotide primer extension(Ms-SNuPE) for quantitative measurement of DNA methylation. Nat Protoc.2007;2(8):1931-6.181. Wang H, Zheng Y, Lai J, Luo Q, Ke H, Chen Q. Methylation-Sensitive MeltCurve Analysis of the Reprimo Gene Methylation in Gastric Cancer. PLoS One.2016;11(12):e0168635.182. Olkhov-Mitsel E, Bapat B. Strategies for discovery and validation of methylated andhydroxymethylated DNA biomarkers. Cancer Med. 2012;1(2):237-60.183. Hattori Naoko UT. Analysis of Gene-Specific DNA Methylation. 2017. In: Handbookof Epigenetics [Internet]. Academic Press; [653-68].184. Masser DR, Stanford DR, Freeman WM. Targeted DNA methylation analysis bynext-generation sequencing. J Vis Exp. 2015(96).185. Qin D. Next-generation sequencing and its clinical application. Cancer Biol Med.2019;16(1):4-10.186. Cokus SJ, Feng S, Zhang X, Chen Z, Merriman B, Haudenschild CD, et al. Shotgunbisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning.Nature. 2008;452(7184):215-9.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 125187. Kamps R, Brandao RD, Bosch BJ, Paulussen AD, Xanthoulea S, Blok MJ, et al.Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction andCancer Classification. Int J Mol Sci. 2017;18(2).188. Schumacher A, Kapranov P, Kaminsky Z, Flanagan J, Assadzadeh A, Yau P, et al.Microarray-based DNA methylation profiling: technology and applications. NucleicAcids Res. 2006;34(2):528-42.189. Moran S, Arribas C, Esteller M. Validation of a DNA methylation microarray for850,000 CpG sites of the human genome enriched in enhancer sequences. Epigenomics.2016;8(3):389-99.190. Noguera-Castells A, Garcia-Prieto CA, Alvarez-Errico D, Esteller M. Validation of thenew EPIC DNA methylation microarray (900K EPIC v2) for high-throughput profilingof the human DNA methylome. Epigenetics. 2023;18(1):2185742.191. Planterose Jimenez B, Kayser M, Vidaki A. Revisiting genetic artifacts onDNA methylation microarrays exposes novel biological implications. Genome Biol.2021;22(1):274.192. Thu KL, Pikor LA, Kennett JY, Alvarez CE, Lam WL. Methylation analysis by DNAimmunoprecipitation. J Cell Physiol. 2010;222(3):522-31.193. Mitchell N, Deangelis JT, Tollefsbol TO. Methylated-CpG Island Recovery Assay.Methods Mol Biol. 2011;791:125-33.194. Gebhard C, Schwarzfischer L, Pham TH, Andreesen R, Mackensen A, Rehli M. Rapidand sensitive detection of CpG-methylation using methyl-binding (MB)-PCR. NucleicAcids Res. 2006;34(11):e82.195. Mohn F, Weber M, Schubeler D, Roloff TC. Methylated DNA immunoprecipitation(MeDIP). Methods Mol Biol. 2009;507:55-64.196. Fernandez AF, Valledor L, Vallejo F, Canal MJ, Fraga MF. Quantification of GlobalDNA Methylation Levels by Mass Spectrometry. Methods Mol Biol. 2018;1708:49-58.197. Tost J, Schatz P, Schuster M, Berlin K, Gut IG. Analysis and accurate quantification ofCpG methylation by MALDI mass spectrometry. Nucleic Acids Res. 2003;31(9):e50.198. Ehrich M, Nelson MR, Stanssens P, Zabeau M, Liloglou T, Xinarianos G, et al.Quantitative high-throughput analysis of DNA methylation patterns by base-specificcleavage and mass spectrometry. Proc Natl Acad Sci U S A. 2005;102(44):15785-90.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

126 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCER199. Demokan S, Chang X, Chuang A, Mydlarz WK, Kaur J, Huang P, et al. KIF1A andEDNRB are differentially methylated in primary HNSCC and salivary rinses. Int JCancer. 2010;127(10):2351-9.200. Rettori MM, de Carvalho AC, Bomfim Longo AL, de Oliveira CZ, Kowalski LP,Carvalho AL, et al. Prognostic significance of TIMP3 hypermethylation in post-treatmentsalivary rinse from head and neck squamous cell carcinoma patients. Carcinogenesis.2013;34(1):20-7.201. Nagata S, Hamada T, Yamada N, Yokoyama S, Kitamoto S, Kanmura Y, et al. AberrantDNA methylation of tumor-related genes in oral rinse: a noninvasive method fordetection of oral squamous cell carcinoma. Cancer. 2012;118(17):4298-308.202. Demokan S, Chuang AY, Pattani KM, Sidransky D, Koch W, Califano JA. Validationof nucleolar protein 4 as a novel methylated tumor suppressor gene in head and neckcancer. Oncol Rep. 2014;31(2):1014-20.203. Cheng SJ, Chang CF, Ko HH, Lee JJ, Chen HM, Wang HJ, et al. HypermethylatedZNF582 and PAX1 genes in mouth rinse samples as biomarkers for oral dysplasia andoral cancer detection. Head Neck. 2018;40(2):355-68.204. Sun R, Juan YC, Su YF, Zhang WB, Yu Y, Yang HY, et al. Hypermethylated PAX1 andZNF582 genes in the tissue sample are associated with aggressive progression of oralsquamous cell carcinoma. J Oral Pathol Med. 2020;49(8):751-60.205. Constancio V, Nunes SP, Moreira-Barbosa C, Freitas R, Oliveira J, Pousa I, et al. Earlydetection of the major male cancer types in blood-based liquid biopsies using a DNAmethylation panel. Clin Epigenetics. 2019;11(1):175.206. Yokoyama S, Iwaya H, Akahane T, Hamada T, Higashi M, Hashimoto S, et al. Sequentialevaluation of MUC promoter methylation using next-generation sequencing-basedcustom-made panels in liquid-based cytology specimens of pancreatic cancer. DiagnCytopathol. 2022;50(11):499-507.207. Buyru N, Altinisik J, Ozdemir F, Demokan S, Dalay N. Methylation profiles in breastcancer. Cancer Invest. 2009;27(3):307-12.208. Raos D, Orsolic D, Masic S, Tomic M, Krasic J, Tomaskovic I, et al. cfDNAmethylation in liquid biopsies as potential testicular seminoma biomarker. Epigenomics.2022;14(23):1493-507.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 127209. Belinsky SA, Liechty KC, Gentry FD, Wolf HJ, Rogers J, Vu K, et al. Promoterhypermethylation of multiple genes in sputum precedes lung cancer incidence in ahigh-risk cohort. Cancer Res. 2006;66(6):3338-44.210. Fujii M, Fujimoto N, Hiraki A, Gemba K, Aoe K, Umemura S, et al. AberrantDNA methylation profile in pleural fluid for differential diagnosis of malignant pleuralmesothelioma. Cancer Sci. 2012;103(3):510-4.211. Hulbert A, Jusue-Torres I, Stark A, Chen C, Rodgers K, Lee B, et al. Early Detectionof Lung Cancer Using DNA Promoter Hypermethylation in Plasma and Sputum. ClinCancer Res. 2017;23(8):1998-2005.212. Franz S. Bronchoalveolar Lavage. Principles and Practice of InterventionalPulmonology. 2012:165–76.213. Ren M, Wang C, Sheng D, Shi Y, Jin M, Xu S. Methylation analysis of SHOX2 andRASSF1A in bronchoalveolar lavage fluid for early lung cancer diagnosis. Ann DiagnPathol. 2017;27:57-61.214. Symonds EL, Pedersen SK, Yeo B, Al Naji H, Byrne SE, Roy A, et al.Assessment of tumor burden and response to therapy in patients with colorectalcancer using a quantitative ctDNA test for methylated BCAT1/IKZF1. Mol Oncol.2022;16(10):2031-41.215. Garrigou S, Perkins G, Garlan F, Normand C, Didelot A, Le Corre D, et al. A Study ofHypermethylated Circulating Tumor DNA as a Universal Colorectal Cancer Biomarker.Clin Chem. 2016;62(8):1129-39.216. Li WH, Zhang H, Guo Q, Wu XD, Xu ZS, Dang CX, et al. Detection of SNCAand FBN1 methylation in the stool as a biomarker for colorectal cancer. Dis Markers.2015;2015:657570.217. Haldrup C, Pedersen AL, Ogaard N, Strand SH, Hoyer S, Borre M, et al. Biomarkerpotential of ST6GALNAC3 and ZNF660 promoter hypermethylation in prostate cancertissue and liquid biopsies. Mol Oncol. 2018;12(4):545-60.218. Mahon KL, Qu W, Devaney J, Paul C, Castillo L, Wykes RJ, et al. MethylatedGlutathione S-transferase 1 (mGSTP1) is a potential plasma free DNA epigenetic markerof prognosis and response to chemotherapy in castrate-resistant prostate cancer. Br JCancer. 2014;111(9):1802-9.219. Moreira-Barbosa C, Barros-Silva D, Costa-Pinheiro P, Torres-Ferreira J, ConstancioV, Freitas R, et al. Comparing diagnostic and prognostic performance of two-geneCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

128 METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCERpromoter methylation panels in tissue biopsies and urines of prostate cancer patients.Clin Epigenetics. 2018;10(1):132.220. Miller BF, Petrykowska HM, Elnitski L. Assessing ZNF154 methylation in patientplasma as a multicancer marker in liquid biopsies from colon, liver, ovarian andpancreatic cancer patients. Sci Rep. 2021;11(1):221.221. Hong L, Ahuja N. DNA methylation biomarkers of stool and blood for early detectionof colon cancer. Genet Test Mol Biomarkers. 2013;17(5):401-6.222. Han YD, Oh TJ, Chung TH, Jang HW, Kim YN, An S, et al. Early detection ofcolorectal cancer based on presence of methylated syndecan-2 (SDC2) in stool DNA.Clin Epigenetics. 2019;11(1):51.223. Widschwendter M, Zikan M, Wahl B, Lempiainen H, Paprotka T, Evans I, et al. Thepotential of circulating tumor DNA methylation analysis for the early detection andmanagement of ovarian cancer. Genome Med. 2017;9(1):116.224. Alix-Panabieres C, Pantel K. Clinical Applications of Circulating Tumor Cells andCirculating Tumor DNA as Liquid Biopsy. Cancer Discov. 2016;6(5):479-91.225. Palanca-Ballester C, Rodriguez-Casanova A, Torres S, Calabuig-Farinas S, Exposito F,Serrano D, et al. Cancer Epigenetic Biomarkers in Liquid Biopsy for High IncidenceMalignancies. Cancers (Basel). 2021;13(12).226. Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, et al.MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med.2005;352(10):997-1003.227. Rodriguez-Casanova A, Costa-Fraga N, Castro-Carballeira C, Gonzalez-Conde M,Abuin C, Bao-Caamano A, et al. A genome-wide cell-free DNA methylation analysisidentifies an episignature associated with metastatic luminal B breast cancer. Front CellDev Biol. 2022;10:1016955.228. Demokan S, Chuang A, Suoglu Y, Ulusan M, Yalniz Z, Califano JA, et al. Promotermethylation and loss of p16(INK4a) gene expression in head and neck cancer. HeadNeck. 2012;34(10):1470-5.229. Demokan S, Chuang AY, Chang X, Khan T, Smith IM, Pattani KM, et al. Identificationof guanine nucleotide-binding protein gamma-7 as an epigenetically silenced gene inhead and neck cancer by gene expression profiling. Int J Oncol. 2013;42(4):1427-36.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Seval TURNA, Semra DEMOKAN 129230. Zhang Z, Wang Q, Zhang M, Zhang W, Zhao L, Yang C, et al. Comprehensive analysisof the transcriptome-wide m6A methylome in colorectal cancer by MeRIP sequencing.Epigenetics. 2021;16(4):425-35.231. Sun X, Yi J, Yang J, Han Y, Qian X, Liu Y, et al. An integrated epigenomic-transcriptomiclandscape of lung cancer reveals novel methylation driver genes of diagnostic andtherapeutic relevance. Theranostics. 2021;11(11):5346-64.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

CANCER: FROM GENOMICS TO PHARMACEUTICSCHAPTER 5THE ROLE OF MIRNAS IN CONTROL OF K-RAS GENEEXPRESSIONBus¸ra KURT G ¨ ULTAS¸LAR ¨ 1,2, Ebru Esin YOR¨ UKER ¨ 31PhD Candidate., ˙Istanbul University Institute of Graduate Studies in Health Sciences, ˙Istanbul, T¨urkiye2˙Istanbul University, Oncology Institute, Department of Basic Oncology, ˙Istanbul, T¨urkiyeE-mail: [email protected]. Prof., ˙Istanbul University, Oncology Institute, Department of Basic Oncology, ˙Istanbul, T¨urkiyeE-mail: [email protected]: 10.26650/B/LSB28LSB48LSB56.2024.019.005ABSTRACTmicroRNA (miRNA) is a single-stranded, 21–23 nucleotide length RNA molecule that regulates gene expression.miRNAs are non-coding RNAs, which means that the genes that code for them are translated from DNA but not intoproteins. K-RAS protein is one of the important molecules involved in normal cell growth. The protein produced bythe K-RAS gene plays an important role in the transmission of signals received from the external environment to thecell nucleus. If a mutation occurs, the cell performs ”aggressive” growth. The mutated K-RAS continuously sends thedivision command even if the cell is not receiving signals from the external environment. Thus, the carcinogenesismechanisms begin. At this point, the division of the cells cannot be controlled. Colorectal, non-small cell lung,pancreatic, and thyroid malignancies frequently have activated point mutations at codons 12, 13, and 61 of the K-RASproto-oncogene. Recent research has revealed that miRNAs can act as tumor suppressors or onco-miRNAs. The factthat miRNAs function as oncogenes or tumor suppressor genes in many cancers shows that miRNAs are regulators intumor progression, metastasis and invasion. In some types of cancer, such as colorectal carcinomas, miRNAs havebeen shown to directly inhibit K-RAS and downstream signaling pathways of K-RAS. In this chapter, we discuss thecontrol mechanism of miRNAs on K-RAS gene expression.Keywords: microRNA, K-RAS, gene expression, cancer, geneticsCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

B¨us¸ra KURT GULTAS¸LAR, Ebru Esin Y ¨ OR¨ UKER ¨ 1311. Introduction1.1. MicroRNAsmiRNAs are small, single-stranded, noncoding RNAs that negatively regulate geneexpression. microRNA (miRNA) was concurrently identified in two different researchesin 1993 as a post-transcriptional regulator of gene expression in Caenorhabditis elegans (C.elegans) (1, 2). The miRNA discovered as Lin-4 was observed to reduce the expression ofthe target gene (1). There are thousands of miRNAs encoded in the human genome, andthe target genes of these miRNAs have a regulatory role in cell development, differentiation,proliferation and apoptosis pathways. However, it has been reported that unregulated miRNAexpression is involved in the pathogenesis of many diseases (3).By the decrease or increase of the expression of their target genes, mature miRNAshelp regulate the protein synthesis. About half of all known miRNAs are intragenic andexpressed from introns and relatively few exons of protein coding genes, while the remainingare classified intergenic, transcribed independently of a host gene. Target genes that havematching nucleotide sequences to miRNAs can be identified. In most cases, miRNAs interactwith the 3’ untranslated region (3’ UTR) of target mRNAs to induce mRNA degradationand translational repression. However, it has also been reported that miRNAs interact with5’ UTR, coding sequences, and gene promoters. In a complex with RNA-induced silencingcomplex (RISC), miRNA attaches to mRNA by base pairing and inhibits either its destructionor the translation to proteins (4, 5). Furthermore, a single mRNA may be targeted by numerousdifferent miRNAs or hundreds of mRNAs may be targeted by a single miRNA (6).Genes encoding miRNAs can be found either as single genes or as clusters in the humangenome. About half of the miRNAs show cluster localization. The human X chromosomecontains 10% of all miRNAs detected so far in the human genome but miRNAs could notbe detected only on the Y chromosome. As seen in Figure 1, the biogenesis of miRNAsis controlled multipl steps that begins in the nucleus of the cell and continues throughoutthe cytoplasm. Briefly, miRNA is transcribed to form primary miRNA (Pri-miRNA). Thenuclear RNase III enzyme Drosha converts pri-miRNA to precursor miRNA (pre-miRNA).The pre-miRNA is exported to the cytoplasm, followed by cleavage to form the mature miRNA.This mature miRNA represses mRNA and induces mRNA degradation and translationalrepression (7).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

132 THE ROLE OF MIRNAS IN CONTROL OF K-RAS GENE EXPRESSIONFigure 1: Production and maturation of miRNAs (Adapted from; Terrinoni, A., et al., The circulatingmiRNAs as diagnostic and prognostic markers. Clinical Chemistry and Laboratory Medicine, 2019.57(7): p. 932-953. (7).1.2. miRNA BiogenesisThe biogenesis of miRNAs is a highly complex involving numerous proteins (Figure 2)(8). In miRNA biogenesis, miRNAs are transcribed from DNA sequences into to the primarymiRNA (pri-miRNA) containing one or more hairpin structures. Following transcription,pri-miRNAs have a 5’ 7-methylguanosine cap and at the 3’ end is the poly A tail. NuclearRNase III enzyme Drosha and its co-factor DiGeorge syndrome critical region 8 (DGCR8)process pri-miRNA into 60-70 nt length pre-miRNA. This pre-miRNA is transported fromthe nucleus to the cytoplasm via Exportin-5 (XPO5) and further processed by a second RNaseIII, DICER, producing ∼22-base pair (bp) miRNA duplexes (9,10). miRNA duplexes are firstloaded into Argonaute (Ago) proteins as double-stranded RNA in an ATP-dependent mannerto form the miRNA-Induced Silencing Complex (miRISC). The relative thermodynamicstability of the miRNA duplexes is a key factor in determining the guide strand. The unloadedstrand is called the passenger strand and the release of ATP-independent. miRISCs represstranslation and promote degradation of miRNA targets (11, 12).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

B¨us¸ra KURT GULTAS¸LAR, Ebru Esin Y ¨ OR¨ UKER ¨ 133Figure 2: miRNA Biogenesis (Adapted from; Pisarello, M.J.L., et al., MicroRNAs in theCholangiopathies: Pathogenesis, Diagnosis, and Treatment. Journal of Clinical Medicine, 2015. 4(9):p. 1688-1712.) (8).1.3. miRNAs and CancerNumerous biological processes, including cell division, proliferation, and death, areinvolved by miRNAs. More than 50% of cancer-associated genomic domains or fragileregions are composed of genes encoding miRNA, as a consequence, they frequently exhibitaberrant expression patterns and suggesting that miRNAs are important in the pathogenesisof cancer. miRNAs control more than one-third of the genes that code for proteins. miRNAscan act as tumor suppressors or oncogenes depending on the genes they target and alteredmiRNA expression has been linked to a variety of malignancies. miRNAs can be releasedinto extracellular fluids, as shown by numerous studies. Biomarkers for various diseases canbe identified using extracellular miRNAs. Target cells may receive extracellular miRNAs,which may then serve as autocrine, paracrine, or endocrine regulators to alter cellular activity.MiRNAs function similarly to hormones in this sense (13, 14, 15).let-7, the first miRNA to be discovered, was also one of the first miRNAs found tohave tumor suppressor functions. The fact that let-7 is found in a chromosomal regionCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

134 THE ROLE OF MIRNAS IN CONTROL OF K-RAS GENE EXPRESSIONthat is usually deleted in cancers and that decreased gene expression causes loss of oncogenicdifferentiation has led to the describe of let-7 as a tumor suppressor miRNA. It was determinedthat miR-15a/16-1 group, which is one of the other miRNAs that function as tumor suppressor,targets the anti-apoptotic gene BCL-2 in chronic lymphocytic leukemia (16).The mechanism of oncogene miRNAs is well-known compared to tumor suppressormiRNAs. These oncogenic miRNAs, also known as “oncomirs” and thereby contributing totumor formation by stimulating proliferation, inhibition of apoptosis, angiogenesis or invasion.One widely studied oncomir is miR-21, which is up-regulated in tumors. miR-17-92 groupincluding miR106a, miR17-5p, miR-19a, miR-25, and miR-93 tend to be overexpressed viathe c-MYC oncogene and the overexpression appears to cause lymphomagenesis in rodentmodels (17, 18).1.4. The Function of miRNAs in Cancer Diagnosis and PrognosisA lack of early detection affects the survival of cancer patients. Reliable biomarkersthat enable the detection of cancer at an early stage can improve patient prognosis, treatmentresponse prediction, and recurrence risk. As a marker of cancer, many studies have investigatedthe potential of miRNAs as invasive and non-invasive biomarkers and their usefulness indiagnosis, prognosis and monitoring therapy. Nowadays, molecular profiling is becomingstandard practice for cancer patients due to the development of targeted treatments andresearch in personalized medicine. Molecular characteristics, such as DNA methylation,gene expression, and miRNA expression, can offer crucial clinical information in additionto specific genetic abnormalities. As prognostic indicators, miRNAs may be more usefulthan mRNAs because of their stability, detectable of body fluids, and expression patternsin clinical samples (19). The presence of miRNAs in 6 solid tumors (breast, colon, lung,pancreas, prostate, stomach) of humans were first reported by using microarray panels in2006 (20). It has been shown that miRNA expression profiling has proven to be useful andsensitive results in determining the embryonic and developmental origins of tumor types, andtherefore it can be used as a biomarker in tumor classification (21). In another study by Png etal, it was shown that the changes in miRNA expression profile in breast cancer is also relatedto the metastatic process and can be used as an effective biomarker in determining cancerprognosis (22). MicroRNA miR-21 overexpression in cancer is associated with advancedclinical stage, lymph node metastasis and patients’ poor prognosis. Oncogenic miR-21 isoverexpressed in many malignancies, including breast cancer, glioblastoma, hepatocellularcarcinoma, lung cancer, gastric cancer, colorectal cancer, and prostate cancer. The diagnosticpotential of circulating miR-21 was shown in numerous studies. On the other hand, reducedlet-7 expression is one of the most studied miRNAs. These miRNAs’ levels direct connectionwith survival of cancer patients (23).In the past decades, several methods have been developed for miRNA analysis. qPCRCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

B¨us¸ra KURT GULTAS¸LAR, Ebru Esin Y ¨ OR¨ UKER ¨ 135is a method that is valid in the quantification of miRNAs since it is both quite inexpensiveand highly accurate method according to microarray. In addition, recent advancements innext-generation sequencing have focused on faster, more accurate making possible to analyzehundreds of genes sequencing and reduced costs. Small RNA-seq enables genome-wideprofiling and offers the power to identify both known and novel miRNAs in any sample. Allthese technical advances reveal new potential molecular biomarkers for cancer diagnosis andprognosis (24).2. K-RAS Gene and K-RAS proteinRas proteins were first identified as oncogene products of Harvey and Kirsten rat sarcomaviruses. It was later discovered that Ras homologous genes are normal cellular homologsknown as proto-oncogenes. Harvey Ras virus (H-RAS), Kirsten Ras virus (K-RAS), andNeural Ras (N-RAS) are present in various mammalian organs as 3 homologous genes andare membrane-associated protein. K-RAS gene is located on the short arm of chromosome 12(12p11.1–12p12.1). K-RAS is a part of the RAS/MAPK (mitogen- activated protein kinase)pathway as GTPase, a class of enzymes which convert the nucleotide guanosine triphosphate(GTP) into guanosine diphosphate (GDP)(25). The K-RAS which encodes protein transmitssignals from the outside of the cell to the nucleus to growth, proliferation or differentiation.KRAS was also shown to be involved in the PI3K-AKT-mTOR pathway, which is thoughtto be crucial for cell life processes such cell division, proliferation, apoptosis, and glucosetransport (26). The K-RAS gene is one of the most frequently mutated oncogenes in humancancers.The activity of Ras is highly regulated by a variety of associated proteins. Guaninenucleotide exchange factors (GEFs) stimulate the release of GDP from inactive Ras, whichfacilitates the binding of GTP. Thus, GEFs increase the activity of Ras. GTP is changed intoguanosine diphosphate by the GTPase activity of the K-RAS protein thereby reducing thefunctional activity of Ras. Hence, the GTP and GDP molecules function as switches thattoggle the K-RAS protein on and off (27,28).2.1. K-RAS and CancerK-RAS protein is one of the most important molecules involved in normal cell growth. Theprotein produced by the K-RAS gene plays a role in the transmission of signals received fromthe external environment to the cell nucleus. If a mutation occurs, the cell gains ”aggressive”phenotype. The mutated K-RAS constantly sends the ”divide” command even if the cell is notreceiving a signal from the external environment. Thus, the cancer process begins, and thecell starts to divide rapidly and uncontrollably. Because of these features K-RAS is consideredto be the most common oncogenic driver in human cancers.The most frequent mutations in RAS/MAPK signaling pathway occur in K-RAS gene,which are detected at a rate of 30-40% in colon and rectum cancers (CRC), 10-30% inCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

136 THE ROLE OF MIRNAS IN CONTROL OF K-RAS GENE EXPRESSIONnon-small cell lung cancer (NSCLC), and 90% in pancreatic cancer. For CRC patients, mostof missense mutations in K-RAS occurring at one of three major hotspot codons 12, 13 and61. Once K-RAS mutations occur, the hydrolysis of GTP is disrupted and K-RAS accumulatesin an active state, contributing to continuous signaling cascade to nucleus thereby promotingtumor cell proliferation. As a result of this, patients with K-RAS-mutant CRC have a poorerprognosis than those with K-RAS-wild-type CRC. In addition, N-RAS gene mutations are seenin 3-4% of patients with colon cancer. K-RAS and K-RAS mutated patient will not respondwell to Anti-EGFR treatments such as tyrosine kinase inhibitor and monoclonal antibodytherapy (29). Therefore, K-RAS and K-RAS mutation analysis play a decisive role in thetreatment of various cancers, especially colon and rectum cancer (Figure 3) (30).Figure 3: Despite treatment with medications that target the upstream EGFR protein, a mutant K-RASgene that is constantly ”on” can encourage cancer growth. (Adapted from; Raponi M., et al. K-RASmutations predict response to EGFR inhibitors. Current Opinion in Pharmacology, 2008. 8(4): p.413-418 (31))2.2. What Happens When The K-RAS Gene Is Mutated?K-RAS is the best-known oncogene with the highest mutation rate in the developmentof several cancers, including colorectal cancer, pancreatic ductal carcinoma, lungadenocarcinoma and leukemia. A single amino acid region is responsible for an activatingmutation. K-RAS mutations are most commonly associated with right-sided colon tumors inCRC patients. Among the mutations, K-RAS G12D (glycine 12 to aspartic acid) and G12VCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

B¨us¸ra KURT GULTAS¸LAR, Ebru Esin Y ¨ OR¨ UKER ¨ 137(glycine 12 to valine) mutations, which are the most common subtypes are present with amajority of 65% in CRC. Whereas, G12C (glycine 12 to cysteine) is the most common subtypein NSCL.Different K-RAS mutant subtypes have different response of targeted therapy. For example,a study that performed high-fidelity CRISPR-based engineering found that in pancreaticcancer, K-RAS (G12D) and K-RAS (G12C) mutants have different therapeutic responsesto EGFR inhibition (32). New studies are needed because of the heterogeneity of K-RASmutations and the importance of individualized treatments.3. K-RAS and miRNA regulationsK-RAS has conventionally been regarded as a crucial signal modulator since it is aproto-oncogene that controls several oncogenic pathways. Understanding the miRNAs thatcontrol K-RAS expression has become a hot topic. To identify miRNA candidates that maybe involved in the regulation of K-RAS, recent research has made use of the expressionprofiles that vary between tumor and normal tissue. For instance, miR-143/145 expressionis negatively correlated with K-RAS protein level in tissues, while miR-143/145 expressionis effective in decreasing Ras signaling, proliferation and migration in vitro. It has beenestablished that direct binding to the miR-143/145 3’ untranslated region (UTR) mediatesthese effects, at least in part (33-35).Similarly, miR-193a was discovered to directly control K-RAS and to be increased intransformed cells in comparison to untransformed cells. miR-193a ectopic expression reducedthe level of K-RAS protein and suppressed tumorigenicity, which is in line with this hypothesis(36).K-RAS can also be indirectly and multistep controlled by miRNA-mediated regulations.It has been demonstrated that miR-18a targets K-RAS directly, although miR-18a synthesisis regulated by hnRNPA1, an RNA-binding protein that in turn is affected by miR-15a-5pand miR-35-3p (37). The ratio of GTP hydrolysis to GDP-to-GTP exchange determinesthe amount of active Ras (Ras-GTP). As a result, miRNAs can indirectly affect K-RASactivity by controlling the amount of GAPs and GEFs. A specific example is the RASA1gene, a well-known Ras-GAP that is a target of both miR-21 and miR-31. Increasedexpression of miR-21 and miR-31 in vivo accelerated tumor growth in xenografts and, incollaboration with K-RAS G12D, respectively, started murine lung adenocarcinomas (Figure4) (38-40). Moreover, miRNAs can indirectly affect the expression of K-RAS. Overexpressionof miR-222-3p stimulates K-RAS expression and increases proliferation, immigration, andinvasion in clear cell renal cell carcinoma cell lines by directly targeting SLC4A4, a K-RASantagonist (41).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

138 THE ROLE OF MIRNAS IN CONTROL OF K-RAS GENE EXPRESSIONFigure 4: miRNA-mediated feedback regulation of K-RAS.(A) A thorough review of the literature reveals 166 human miRNAs as being downstream ofK-RAS since at least one hyperactivation of K-RAS signaling is reported to cause dysregulationof their expression. 39 miRNAs are categorized as K-RAS-targeting miRNAs based onexperimental evidence that these miRNAs directly target K-RAS and cause its decrease in arange of cancer cell lines. Let-7a, miR-127, miR-143, miR-145, miR-16, miR-181, miR-193a,miR-200C, miR-27B, and miR-4689 are ten miRNAs that are demonstrated to function bothupstream and downstream of K-RAS.(B) Three distinct types of feedback regulation are demonstrated when miRNA-mediateddirect or indirect regulation of K-RAS is paired with effects on miRNAs caused by K-RAS. Dueto conflicting data on how miR-16 and miR-200c’s expression varies in response to K-RAShyperactivation, they are not included in this list. When oncogenic K-RAS is expressed,expression of the miR-181a-targeting K-RAS is elevated, creating a negative feedback loop(36, 42, 43). miR-21, miR-31, and miR-30c target RAS GAPs such as RASA1 and NF1(38-40). As a result of their enhanced expression in response to K-RAS hyperactivation, theymay be able to positively feedback onto K-RAS by blocking its inhibitors, thus enhancing thesignaling activation (41, 44). In addition, K-RAS downregulates a number of miRNAs thattarget K-RAS (let-7a, miR-127, miR-143, etc.), creating a mutually suppressive connection(38, 45, 46). K-RAS signaling, and miRNA activity probably create a delicate balance duringCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

B¨us¸ra KURT GULTAS¸LAR, Ebru Esin Y ¨ OR¨ UKER ¨ 139homeostasis, and its collapse in a number of disorders may increase the hyperactivation ofoncogenic K-RAS. GTPase-activating protein is referred to by the acronym GAP. (Adaptedfrom; Shui, B., et al., Interplay between K-RAS and miRNAs. Trends in Cancer, 2022. 8(5):p. 384-396 (47) ).3.1. K-RAS and miRNAs in CancerUnderstanding how miRNAs are controlled could help us better understand how cancerdevelops and how to use them for novel therapeutic strategies. The gain-of-function mutationsin K-RAS alone cause dysplastic alterations in various tissues, resulting hyperactivation of thegene a frequent first event in malignancies of the pancreas, colon, lung, and hematologicalsystem (48-51).miRNAs that target and regulate K-RAS act as tumor suppressors. As an example; miR-96,miR-30c and miR-181a, have been shown to regulate K-RAS in various cancers (Table 1) (43,52, 53). Gastaldi et al. recently profiled miRNAs in cutaneous squamous cell carcinomas(cSCCs) using small RNA sequencing, a large-scale profiling technique, and found that themiR-193b/365a cluster was one of the miRNAs that were most noticeably down-regulated asmurine skin tumors progressed (54). Furthermore, Liao et al. further discovered the functionof miR-30b, one of the recognized miRNAs that is down-regulated in colorectal cancer (CRC).Since miR-30b promotes G1 cell-cycle arrest and death and targets numerous genes, includingK-RAS, its abnormal expression and suppression had an impact on cellular proliferation inCRC cell lines and tumor formation in a xenograft mouse model (55).Table 1: Some miRNAs that regulate K-RAS and play roles in some cancers9hyperactivation, they may be able to positively feedback onto K-RAS by blocking its inhibitors, thus enhancing the signalingactivation (41, 44). In addition, K-RAS downregulates a number of miRNAsthat target K-RAS (let-7a, miR-127, miR-143, etc.),creating a mutually suppressive connection (38, 45, 46). K-RAS signaling, and miRNA activity probably create a delicatebalance during homeostasis, and its collapse in a number of disorders may increase the hyperactivation of oncogenic K-RAS.GTPase-activating protein is referred to by the acronym GAP. (Adapted from; Shui, B., et al., Interplay between K-RAS andmiRNAs. Trends in Cancer, 2022. 8(5): p. 384-396 (47) ).3.1. K-RAS and miRNAs in CancerUnderstanding how miRNAs are controlled could help us better understand how cancerdevelops and how to use them for novel therapeutic strategies. The gain-of-function mutations in K-RASalone cause dysplastic alterations in varioustissues, resulting hyperactivation of the gene a frequent firstevent in malignancies of the pancreas, colon, lung, and hematological system (48-51).miRNAs that target and regulate K-RAS act as tumor suppressors. As an example; miR-96, miR30c and miR-181a, have been shown to regulate K-RAS in various cancers (Table 1) (43, 52, 53).Gastaldi et al. recently profiled miRNAs in cutaneous squamous cell carcinomas (cSCCs) using smallRNA sequencing, a large-scale profiling technique, and found that the miR-193b/365a cluster was oneof the miRNAs that were most noticeably down-regulated as murine skin tumors progressed (54).Furthermore, Liao et al. further discovered the function of miR-30b, one of the recognized miRNAs thatis down-regulated in colorectal cancer (CRC). Since miR-30b promotes G1 cell-cycle arrest and deathand targets numerous genes, including K-RAS, its abnormal expression and suppression had an impacton cellular proliferation in CRC cell lines and tumor formation in a xenograft mouse model (55).Table 1: Some miRNAs that regulate K-RAS and play roles in some cancersmiRNA Cancer Typelet-7 Lung CancermiR-96 Pancreatic CancermiR-30c Hereditary Breast CancermiR-181a Oral squamous cell carcinomamiR-30b Colorectal cancer (CRC)miR-18a Colorectal cancer (CRC)miR-193b/365a Cutaneous squamous cellcarcinoma (cSCC)miR-134 Glioblastoma (GBM)Given the complex miRNA network downstream of K-RAS, miRNAs are probably involved inthe regulation of a number of tumor initiation factors. miR-34 was thought to act as a tumor suppressorand is typically downregulated in K-RAS-driven lung cancer. Delivering miR-34 exogenously vialentivirus before inducing K-RAS G12D expression in rat lungs increased tumor initiation duringcarcinogenesis, highlighting the importance of miR-34 suppression (56). The ablation of Dicer from KRAS-driven pancreatic tumors in mice further demonstrates the diversity of phenotypes regulated bymiRNAs. This procedure accelerated the dedifferentiation of dysplastic cells while promoting apoptosisduring tumor initiation (57).Due to their roles in the initiation, growth, and metastasis of cancer, several miRNAs up anddownstream of K-RAS have been connected to various disease stages, prognosis, and therapeuticresponses. These findings indicate the potential of miRNA monitoring for cancer, but a number ofbarriers must be overcome before miRNA monitoring can be applied in the clinic, including thevariability of study outcomes.Given the complex miRNA network downstream ofK-RAS, miRNAs are probably involvedin the regulation of a number of tumor initiation factors. miR-34 was thought to act as atumor suppressor and is typically downregulated in K-RAS-driven lung cancer. DeliveringmiR-34 exogenously via lentivirus before inducing K-RAS G12D expression in rat lungsincreased tumor initiation during carcinogenesis, highlighting the importance of miR-34suppression (56). The ablation of Dicer from K-RAS-driven pancreatic tumors in micefurther demonstrates the diversity of phenotypes regulated by miRNAs. This procedureCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. 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140 THE ROLE OF MIRNAS IN CONTROL OF K-RAS GENE EXPRESSIONaccelerated the dedifferentiation of dysplastic cells while promoting apoptosis during tumorinitiation (57).Due to their roles in the initiation, growth, and metastasis of cancer, several miRNAsup and downstream of K-RAS have been connected to various disease stages, prognosis, andtherapeutic responses. These findings indicate the potential of miRNA monitoring for cancer,but a number of barriers must be overcome before miRNA monitoring can be applied in theclinic, including the variability of study outcomes.3.1.1. Single Nucleotide Polymorphisms (SNPs) control and modify miRNA bindingto K-RASIn addition to miRNA dysregulation, K-RAS 3’ UTR SNPs affect miRNA-mediatedregulation of K-RAS activity. By sequencing the regions of the 3’ UTR of K-RAS in severalnon-small cell lung cancer (NSCLC) cases, rs61764370 (also known as the K-RAS-variant)was shown to be the first single nucleotide polymorphism (SNP) at a let-7 complementary siteto be a biomarker for NSCLC risk (58). While not as thoroughly verified as the K-RAS-variant(rs61764370), another SNP in the 3’ UTR ofK-RAS, rs712, is being investigated as a biomarkerfor the risk of oral squamous cell carcinoma, gastric, colorectal, and papillary thyroid cancer(Table 2) (59).Table 2: SNPs in K-RAS 3’ UTR that are linked to cancer3.1.1. Single Nucleotide Polymorphisms (SNPs) control and modify miRNA binding to K-RASIn addition to miRNA dysregulation, K-RAS 3′ UTR SNPs affect miRNA-mediated regulationof K-RAS activity. By sequencing the regions of the 3′ UTR of K-RAS in several non-small cell lungcancer (NSCLC) cases, rs61764370 (also known as the K-RAS-variant) was shown to be the first singlenucleotide polymorphism (SNP) at a let-7 complementary site to be a biomarker for NSCLC risk (58).While not as thoroughly verified as the K-RAS-variant (rs61764370), another SNP in the 3′ UTR of KRAS, rs712, is being investigated as a biomarker for the risk of oral squamous cell carcinoma, gastric,colorectal, and papillary thyroid cancer (Table 2) (59).Table 2: SNPs in K-RAS 3′ UTR that are linked to cancerSNP ID Association with cancerrs61764370 (K-RAS-variant) Risk of non small-cell lung cancer, epithelial ovariancancer, triple-negative breast cancer, colorectalcancer. Drug response in metastatic colorectal cancerrs712 Risk of oral squamous cell carcinoma, gastric cancer,colorectal cancer, papillary thyroid cancerExtensive sequencing of the K-RAS 3′ UTR in NSCLC cell lines revealed the presence of SNPswith a substantial enrichment in rs712 and rs9266. Essentially, the K-RAS 3′ UTR's capacity to bind toregulatory miRNAs like let-7 and miR-181 may have been hampered by these SNPs (59).4. ConclusionIn this chapter, we attempt to summarize the function of miRNAsin controlling the K-RAS geneand miRNAs that affect K-RAS expression.The K-RAS gene is one of the most frequently mutated oncogenes in human cancers and also amaster regulator of cell signaling. miRNAs are small RNA molecules, approximately 18-24 nucleotideslong, that are encoded by highly conserved DNA regions but not translated into protein. Recent studieshave revealed that miRNAs are regulators of important biological functions such as cellulardevelopment, apoptosis and metabolism. Depending on the mRNAs they target, miRNAs function astumor suppressors or oncogenes in cancer development. It has been revealed that miRNAs will provideimportant results especially in determining the early diagnosis, treatment and prognosis of cancer, bydetecting their presence in cancerous tissues, changes in their expression pattern and detecting themRNAs they target, and it is thought that they can be a biomarker for cancer. Also, the presence ofmutations in K-RAS means that the patient will not respond well to anti-EGFR treatments such astyrosine kinase inhibitor and monoclonal antibody therapy, which means K-RAS is a biomarker forcancer. In this case, both miRNA and K-RAS can be used as biomarkers for cancer, and it would bebeneficial for the development of clinical treatments to understand the molecular mechanism of miRNAmediated regulation of K-RAS through the identification of tumor suppressive miRNAs. A new aspectof K-RAS signaling was revealed by the identification of miRNA as a new class of translationalregulators. To improve signaling, cells might employ miRNA-mediated feedback regulation of K-RASas a further stage of signaling control (47).The miRNA profiles could inform molecular pathways implicated in diagnosis, disease severity,suggestingtargetsfortherapeuticguidance.Asaresult,numerousstudiesareneededtounderstandtheExtensive sequencing of the K-RAS 3’ UTR in NSCLC cell lines revealed the presence ofSNPs with a substantial enrichment in rs712 and rs9266. Essentially, the K-RAS 3’ UTR’scapacity to bind to regulatory miRNAs like let-7 and miR-181 may have been hampered bythese SNPs (59).4. ConclusionIn this chapter, we attempt to summarize the function of miRNAs in controlling the K-RASgene and miRNAs that affect K-RAS expression.The K-RAS gene is one of the most frequently mutated oncogenes in human cancers andalso a master regulator of cell signaling. miRNAs are small RNA molecules, approximately18-24 nucleotides long, that are encoded by highly conserved DNA regions but not translatedinto protein. Recent studies have revealed that miRNAs are regulators of important biologicalfunctions such as cellular development, apoptosis and metabolism. Depending on the mRNAsthey target, miRNAs function as tumor suppressors or oncogenes in cancer development. Ithas been revealed that miRNAs will provide important results especially in determining theCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

CANCER: FROM GENOMICS TO PHARMACEUTICSearly diagnosis, treatment and prognosis of cancer, by detecting their presence in canceroustissues, changes in their expression pattern and detecting the mRNAs they target, and itis thought that they can be a biomarker for cancer. Also, the presence of mutations inK-RAS means that the patient will not respond well to anti-EGFR treatments such as tyrosinekinase inhibitor and monoclonal antibody therapy, which means K-RAS is a biomarker forcancer. In this case, both miRNA and K-RAS can be used as biomarkers for cancer, and itwould be beneficial for the development of clinical treatments to understand the molecularmechanism of miRNA-mediated regulation of K-RAS through the identification of tumorsuppressive miRNAs. A new aspect of K-RAS signaling was revealed by the identification ofmiRNA as a new class of translational regulators. To improve signaling, cells might employmiRNA-mediated feedback regulation of K-RAS as a further stage of signaling control (47).The miRNA profiles could inform molecular pathways implicated in diagnosis, diseaseseverity, suggesting targets for therapeutic guidance. As a result, numerous studies are neededto understand the relationship between miRNAs and the formation of K-RAS-driven cancers.The identification of role of miRNAs in control of K-RAS gene expression should be a focusof future studies.REFERENCES1. Lee RC, Feinbaum RL, Ambros V. The C-Elegans Heterochronic Gene Lin-4 EncodesSmall Rnas with Antisense Complementarity to Lin-14. Cell 1993; 75(5): 843-854.2. Wightman B, Ha I, Ruvkun G. Posttranscriptional Regulation of the Heterochronic GeneLin-14 by Lin-4 Mediates Temporal Pattern-Formation in C-Elegans. Cell 1993; 75(5):855-862.3. O’Connell RM, Rao DS, Chaudhur AA, Baltimore D. Physiological and pathologicalroles for microRNAs in the immune system. Nat Rev Immunol 2010; 10(2): 111-122.4. Shenouda SK, Alahari SK. MicroRNA function in cancer: oncogene or a tumorsuppressor? Cancer Metastasis Rev 2009; 28(3-4): 369-378.5. Sun W, Li YSJ, Huang HD, Shyy JYJ, Chien S. microRNA: A Master Regulator ofCellular Processes for Bioengineering Systems. Annu Rev Biomed Eng. 2010; 12:1-27.6. Pillai RS, MicroRNA function: Multiple mechanisms for a tiny RNA? Rna 2005;11(12):1753-1761.7. Terrinoni A, Calabrese C, Basso D, Aita A, Caporali S, Plebani M et al. The circulatingCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.