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Home , Cancer epigenetics, EMP3, Epigenetic therapy, Human epigenome

DNA of DNA Repair in

*1Shilpa V., 2Lakshmi Krishnamoorthy.

1Doctoral Scholar, Department of Biochemistry, Kidwai Memorial Institute of Oncology, Dr. M. H. Marigowda Road, Bangalore-560029. 2Professor and Head, Department of Biochemistry, Kidwai Memorial Institute of Oncology, Dr. M. H. Marigowda Road, Bangalore-560029.

ABSTRACT Introduction

“ has always been all the weird and wonderful Cancer is characterized by uncontrolled cell division things that cannot be explained by genetics” a famous and malignant growth. Cancer cells have a higher quote by Denise Barlow. Coining the term epigenetics proliferation rate than their corresponding normal is credited to Conrad Waddington who describes it as tissue and they often bypass . Furthermore, a “branch of biology which studies causal interactions they can acquire the capability to separate from their between genes and their products, which bring the original tissue and can develop metastatically in other into being”. Epigenetics adds a new stratum regions of the body. of events between genes and their expression. It proposes a control system of “switches” that turn genes on or off The expression of genetic information within an causing heritable effects in . Thousands of DNA individual cell dictates how that cell subsequently damaging events take place every day in our body but behaves. Events at the molecular level can influence efficient DNA repair systems prevent that. Accumulation the expression of certain genes and thereby adversely of DNA damage has been linked to cancer and genetic affect cellular functions to such a degree as to initiate deficiencies in specific DNA repair genes are associated a pathologic process. Cancer cells, for example, must with tumor prone . Epigenetic silencing of DNA undergo a number of molecular events that allow repair genes may promote tumorigenesis. This review them to acquire several distinct pathologic behavioural summarizes current knowledge of the epigenetic inactivation properties. These properties result in deleterious of DNA repair components in cancer. clinical consequences for the host, but paradoxically empower that abnormal cell and its progeny with a survival advantage over normal cells.

Much of the focus of molecular biologic research has concentrated on investigating the role of genetic Key words: Cancer, DNA Repair genes, Epigenetics, changes - that is, direct alterations of DNA base Methylation. sequence through , deletion or insertion

Corresponding author: Shilpa V., Department of Biochemistry, Kidwai Memorial Institute of Oncology, Dr. M. H. Marigowda Road, Bangalore-560029. Phone: 080-26094072, Fax: 080-26560723, E-mail: [email protected] 239 Shilpa V. and their effect on subsequent expression and Epigenetic features of normal cell behaviour. Recently, alternative mechanisms of cell gene modulation have been observed that affect its expression and remain preserved after cell division Epigenetic mechanisms are used in many different without disrupting the actual sequence at all. ways to regulate . Epigenetic changes never involve a change in the primary DNA sequence C. H. Waddington in 1939 coined the term or a change in base pairing but are reflected primarily “Epigenetics”, which he defined as “the causal in DNA cytosine modification patterns, post- interactions between genes and their products, which translational modifications, or deposition of certain bring the phenotype into being”.1 Epigenetics, later histone variants along specific gene sequences. was defined as heritable changes in gene expression These epigenetic modifications of genes are generally that are not due to any alteration in the DNA reversible, but can get transmitted to the daughter sequence.2 It is increasingly apparent that, in cells.7 , heritable losses of gene function may be mediated by epigenetic as well as by genetic One common and perhaps the most permanent and abnormalities.3,4 The argument as to whether cancer is stable mechanism of epigenetic gene inactivation is an epigenetic or a genetic , in fact emphasizes the methylation of the 5-carbon of the DNA base that synergy between two processes drives tumor cytosine in the 5’-CpG-3’ dinucleotide sequence progression from the earliest to latest stages. Inclusion context of CpG island or promoter regions. These of epigenetic events in our concepts of how tumors methylation reactions carried out by DNA cytosine evolve heightens our need to understand the basic methyltransferases are a main component of the nature of changes that set heritable states epigenetic regulatory mechanisms in mammals.8 of gene function. From a translational standpoint, Although most CpG sites in the human it enriches the potential and suggests new targets are methylated, CpG dense regions known as CpG to consider for cancer prevention and therapeutic islands are typically unmethylated in normal tissue strategies. The methylation of DNA is recognized as which spans the 5’ end of the regulatory region of a key mechanism in the regulation of gene expression many genes. and evidence for its role in the development of a wide variety of cancers is rapidly accumulating.4 DNA methylation plays an essential role in normal development through its effects on gene Research into DNA methylation has been progressing imprinting, condensation of chromatin, stabilization at a furious pace, despite uncertainty about its of , X- inactivation, tissue- origin and physiological function. Consistent with specific silencing of gene expression and transcriptional a resurgence of interest in the idea that cancer silencing of repetitive elements. 5-Methylcytosine is a disease of faulty development, there has been (m5C) was first found in DNA of higher eukaryotes a revived quest in studies to uncover epigenetic by Hotchkiss in 1948. This epigenetic regulation also processes involved in neoplastic development and coordinates gene expression during cell differentiation progression.5, 6 Epigenetic information, after all, is in mammalian embryogenesis. 9, 10 essential for development and it is clear that cancer is ultimately a disease of aberrant gene expression. The methylation of mammalian genomic DNA is catalyzed by DNA methyltransferases (DNMTs) Effective DNA repair is the backbone of cancer-free that can be divided into maintenance and de novo survival. in DNA repair genes such as DNMTs. A methyl (-CH3) group is covalently (BER), excision repair bonded to the 5-carbon on the cytosine base. This (NER), mismatch repair (MMR), DNA crosslink process is mediated by DNMTs. The methyl group repair, and several others is the cause of inherited is provided by S-adenosyl methionine (SAM), and cancer syndromes. As an alternative mechanism to this is converted to S-adenosyl homocysteine (SAH) genetic mutation, epigenetic gene inactivation can in the process. This is recycled back to SAM in a be brought about that either inactivates or reduces folate and cobalamin dependant pathway (Fig. 1).11 efficiency of DNA repair genes. In this review, we will discuss some examples of DNA repair mechanisms The possible existence of methylated cytosines in cancer. within DNA was known since the early part of the

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 12, No. 4, October 2013 pp 239 - 251 240 Promoter DNA Methylation of DNA Repair Genes in Cancer

reversible. Such epigenetic plasticity is an excellent candidate to mediate the dynamic heterogeneity of cell populations inherent to complex tumor traits such as metastasis. In this regard, most epithelial tumors are highly invasive, and the cells relaxed can form metastatic foci, which grow within visceral organs.

Two mechanisms have been proposed to account for transcriptional repression via DNA methylation. In the first mechanism, DNA methylation directly inhibits the binding of factors (TFs) such as AP-2, c-/ Myn, E2F and NFkB to their binding sites within promoter sequence. In this mechanism, CpG dinucleotides have Figure 1: The methylation cycle Adapted from Ref 11. Methylation of cytosine catalysed to be present within the binding site of TFs, which by DNA methyltransferase, which uses methyl group from are sensitive to methylation of CpG dinucleotides 16 SAM. (Fig. 2). The second mode of repression includes binding of proteins specific for m5CpG dinucleotides to methylated DNA. Methylated DNA recruits last century. They were initially described in the m5CpG-binding protein (MeCP) and m5CpG-binding DNA of the tubercle bacillus and subsequently were domain (MBD) proteins. MeCP1 and MeCP2 bind extracted from calf thymus, where they were known as epi-cytosine, producing a different chromotographic profile from normal cytosine.12, 13 Their purpose was not defined until relatively recently. It was speculated that they acted as a primitive host defense mechanism to silence DNA from viral organisms and provided an explanation for the latency of certain viral infections and how such agents can escape detection.14, 15

Epigenetic changes in cancer

Promoter hypermethylation for down regulating genes has been known to play a critical role in tumorigenesis. Genes that are unmethylated in normal tissues at all ages are also found to be hypermehtylated quite early in tumorigenesis. These early losses of control, altered regulation Figure 2: Repression of transcription via CpG dinucleotide of gene transcription factors, disruption of cell-cell methylation. and cell-substratum interaction and even multiple Adapted from Ref 16. Promoter sequence binds transctiption factors (TFs) and RNA polymerase II that initiates types of genetic instability are characteristic features transcription. (A) Methylation of CpG within promoter of human cancer. binding site directly inhibits requirement of TFs and represses transcription. (B) Methylated DNA binds m5CpG While promoter hypermethylation and associated binding (MeCPs) and m5CpG-binding domain (MBDs) generally remain very stable in cancer proteins forming spatial obstacle that prevents binding of cells, these changes, unlike mutations are potentially TFs to promoter sequence.

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 12, No. 4, October 2013 pp 239 - 251 241 Shilpa V. specifically to methylated DNA in whole genome and density concentrations of CpG repeat sequences form spatial obstacle that disable binding of TFs to between several hundred to a few thousand base promoter sequences (Fig. 2).16 pairs are noted to exist as islands in the promoter regions of many common genes, and in particular, Methylated cytosine has a greater propensity to genes associated with tumor suppression.17 These are undergo spontaneous deamination and the formation normally unmethylated, but if these regions become of . If this occurs on a tumor suppressor methylated, failure to transcribe the downstream gene gene, then a point mutation develops that can lead occurs, causing silencing of that gene (Fig. 4).11 to uncontrolled cell proliferation (Fig. 3).11 What induces methylation to occur at previously In the , the actual prevalence of unmethylated locations in incipient cancer cells CpG dinucleotide pairs is only about 1% as opposed or, indeed, whether methylation is the primary to the expected 6% (1/16). However, localized high- event responsible for genetic silencing or merely a secondary event is unknown. Theoretically, abnormal methylation patterns could arise as a result of an over active “methylating” factor or the loss of a “demethylating” factor. The observation that DNMTs levels are increased in cancers would tend to suggest this concept, but current investigations indicate that the elevation is likely to be a secondary effect of increased cell proliferation rather than a causal mechanism for the former.18, 19 It is now accepted that promoter methylation of genes involved in the control of cell proliferation results in their inactivation, and this is a fundamental event in the pathway to . Figure 3: Methylation precipitating a point mutation. Adapted from Ref 11. Cytosine to thymine point mutation after deamination of methylated cytosine.

Figure 4: Mechanisms of carcinogenesis induced by methylation events. Adapted from Ref 11. (A) Activation of previously silent protooncogenes after hypomethylation. (B) Silencing of tumor suppressor genes after methylation of gene promoter region.

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 12, No. 4, October 2013 pp 239 - 251 242 Promoter DNA Methylation of DNA Repair Genes in Cancer

Hypermethylation of DNA repair with genetic lesions (Table 1).21 Such interactions genes in cancer can be seen when hypermethylation inactivates the CpG island of the promoter of the DNA repair Hypermethylation of the promoter CpG islands can genes hMLH1, BRCA1, MGMT.6, 22-24 In each case, affect genes involved in the cell cycle, DNA repair, silencing of the DNA repair gene blocks the repair the metabolism of , cell-cell interaction, of genetic mistakes, thereby opening the way to apoptosis, and angiogenesis, all of which are involved neoplastic transformation of the cell. in the development of cancer.20, 6 Hypermethylation occurs at different stages in the development of cancer The profiles of hypermethylation of the CpG islands and in different cellular networks, and it interacts in tumor suppressor genes are specific to the cancer

Table 1. Epigenetic Aberrations among Different Tumor Types.

Type of Cancer Epigenetic Disruption CpG-island hypermethylation (hMLH1, p16INK4a, , RARB2, SFRP1, and WRN), hypermethylation of miRNAs (miR-124a), global genomic hypomethylation, loss of im- Colon cancer printing of IGF2, mutations of histone modifiers (EP300 and HDAC2), diminished monoacetylated and trimethylated forms of CpG-island hypermethylation (BRCA1, E-cadherin, TMS1, and estrogen receptor), Breast cancer global genomic hypomethylation CpG-island hypermethylation (p16INK4a, DAPK, and RASSF1A), global genomic hy- pomethylation, genomic deletions of CBP and the chromatin- remodelling factor BRG1 CpG-island hypermethylation (DNA-repair enzyme MGMT, EMP3, and THBS1) CpG-island hypermethylation (p15INK4b, EXT1, and ID4), translocations of histone modifiers (CBP, MOZ, MORF, MLL1, MLL3, and NSD1) CpG-island hypermethylation (p16INK4a, p73, and DNA-repair enzyme MGMT), di- Lymphoma minished monoacetylated and trimethylated forms of histone H4 CpG-island hypermethylation (p16INK4a and TPEF/HPP1), hypermethylation of miR- Bladder cancer NAs (miR-127), global genomic hypomethylation CpG-island hypermethylation (VHL), loss of imprinting of IGF2, global genomic hy- Kidney cancer pomethylation CpG-island hypermethylation (GSTP1), gene amplification of polycomb histone methyl- cancer transferase EZH2, aberrant modification pattern of H3 and H4 CpG-island hypermethylation (p16INK4b and p14ARF), gene amplification of histone Esophageal cancer demethylase JMJD2C/GASC1 Stomach cancer CpG-island hypermethylation Stomach cancer CpG-island hypermethylation (hMLH1 and p14ARF) Liver cancer CpG-island hypermethylation (SOCS1 and GSTP1), global genomic hypomethylation CpG-island hypermethylation (BRCA1)

BRCA1 denotes breast-cancer susceptibility gene 1, BRG1 BRM/SWI2-related gene 1, CBP cyclic AMP response element – binding protein (CREB) – binding protein, DAPK death-associated protein kinase, EMP3 epithelial membrane protein 3, EP300 E1A binding protein p300, EXT1 exostosin 1, EZH2 of zeste drosophila homologue 2, GSTP1 glutathione S-transferase 1, HDAC2 2, hMLH1 homologue of MutL Escherichia coli, ID4 inhibitor of DNA binding 4, IGF2 insulin-like growth factor 2, JMJD2C/GASC1 Jumonji domain-containing protein 2C, MGMT O6-methylguanine– DNA methyltransferase, MLL1 ixed-lineage leukemia 1, MLL3 mixed-lineage leukemia 3, MORF monocytic leukemia protein–related factor, MOZ monocytic leukemia zinc finger, NSD1 nuclear receptor binding SET domain protein 1, RARβ2 retinoic acid receptor β 2, RASSF1A ras association domain family protein 1, SFRP1 secreted frizzled-related protein 1, SOCS1 suppressor of cytokine signalling 1, THBS1 thrombospondin 1, TMS1 target of methylation-induced silencing 1, TPEF/HPP1 hyperplastic polyposis gene 1, VHL von Hippel–Lindau disease and WRN Werner’s syndrome.

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 12, No. 4, October 2013 pp 239 - 251 243 Shilpa V. type (Fig. 5 and Table 1).21, 25, 26 Each tumor type can be Epigenetic inactivation of DNA repair genes in assigned a specific, defining DNA “hypermethylome”. cancer has been reported for several DNA repair Such patterns of epigenetic inactivation occur not pathways including BER, NER, MMR, amongst several only in sporadic tumors but also in inherited cancer others. Within one DNA repair pathway, specific genes syndromes, in which hypermethylation can be the are often preferentially methylated. The specificity second lesion in Knudson’s two hit model of how by which epigenetic mechanism occurs is yet to be cancer develops.27, 28 Recently devised epigenomic determined and the preference for specific genes is techniques have revealed maps of hypermethylation yet to be understood. of the CpG islands that suggest the occurrence of 100-400 hypermethylated CpG islands in the promoter Epigenetic inactivation processes can result in an regions of a given tumor. increase in genetic instability during tumorigenesis that

Figure 5: Hypermethylation profile of promoter region CpG island of tumor suppressor genes in human cancer. Adapted from Ref 21. Four tumor cells are shown undergoing transcriptional silencing by DNA hypermethylation of the regulatory regions of tumor-suppressor genes. In colon cancer, entrance into the cell cycle occurs by means of p16INK4a methylation. In leukemia cells, p15INK4b methylation initiates proliferation. In breast-cancer cells, defects in DNA repair are related to methylation of BRCA1 and in glioma cells, methylation of O6-methylguanine–DNA methyltransferase (MGMT) initiates defects in DNA repair. Other depicted hypermethylated tumor-suppressor genes are CDH1 (cadherin 1), CDH13 (cadherin 11), CRBP1 (cellular retinol binding protein 1), DKK-1 (dickkopf homologue 1), ER (estrogen receptor), GATA-4 (GATA-binding protein 4), GATA-5 (GATA-binding protein 5), HIC1 (hypermethylated in cancer 1), PR (progesterone receptor), PRLR (prolactin receptor), RARβ2 (retinoic acid receptor β 2), SLC5A8 (solute carrier family 5 iodide transporter member 8), WIF-1 (WNT inhibitory factor 1) and lamin A/C.

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 12, No. 4, October 2013 pp 239 - 251 244 Promoter DNA Methylation of DNA Repair Genes in Cancer can be directly attributed to the deficiencies in DNA In , GSTP1 is observed to be silenced repair. Therefore, inactivation of DNA repair genes by promoter methylation.34-37 GSTP1 promoter can be seen as an important event in cancer initiation methylation has been detected in cancerous as well and/or progression by reducing genomic stability as prostatic intraepithelial neoplasia (PIN) lesions, leading to genetic aberrations at other important gene whereas it has been rarely detected in normal prostate loci. Such a mechanism is proven for inactivation of tissues.38-40 Hypermethylation of GSTP1 was also MMR pathways in colorectal tumors but awaits direct found in a subset of proliferative inflammatory atrophy confirmation for a number of other DNA repair genes (PIA) lesions, which are believed to be precursors that are found methylated in tumors. On the other of tumors.41 hand, diminished DNA repair is expected to lead to reduced cell survival in general and additional events The presence of aberrant CpG island methylation are likely occurring that enable a cell with reduced alone does not signal the presence of an invasive repair capacity to undergo uncontrolled proliferation cancer. This may be the case, but premalignant or instead of (e.g. mutation in Tp53). precursor lesions on their way to full tumorigenesis can also carry this epigenetic culprit. In fact, this One of the most studied genes in ovarian cancer is finding may be useful in early detection screenings, BRCA1, due to its role in both inherited and sporadic especially in those people with a high familial risk of forms of this disease.29 The clinical outcome for developing cancer because they may have patterns of ovarian cancer patients having tumor-hypermethylated CpG island hypermethylation similar to the sporadic BRCA1 has recently been compared with patients with cases.42 BRCA1 mutations or wild-type BRCA1.30 BRCA1 hypermethylation occurs in 10–15% of sporadic The MGMT protein (O6-methylguanine DNA disease cases, associates strongly with loss of BRCA1 methyltransferase) is directly responsible for repairing RNA and protein and significantly correlates with the addition of alkyl groups to the (G) base of poor patient response.29, 31 These studies suggest that the DNA.43 This base is the preferred point of attack BRCA1 hypermethylation, which has been reported in the DNA for several alkylating chemotherapeutic to be observed in ovarian cancer patient serum, may drugs, such as BCNU [1, 3-bis(2-chloroethyl)- represent a minimally invasive approach for predicting 1-nitrosourea], ACNU [1-(4-amino-2-methyl-5- patient response to standard therapies.32 pyrimidinyl) methyl- 3-(2-chloroethyl)-3-nitrosourea], procarbazine, streptozotocin, or temozolamide. Thus Another example is the hypermethylation of the tumours that have MGMT inactivation due to CpG promoter region of the MMR gene MLH1 hypermethylation become sensitized to the action of observed in a subgroup of human colorectal cancers alkylating agents since the repair mechanism is already that show microsatellite instability. Microsatellite impaired which leads to cell death. sequences are polymorphic, short, repeating segments of DNA (about 1 to 4 base pairs) distributed across Similar cases to that described for MGMT can be the genome. Alterations to their pattern frequently cited for other DNA repair and detoxifier genes that occur if there is a deficiency in the cells’ ability to also undergo aberrant DNA methylation where the repair defects in DNA. The methylation of MLH1 methylated status of the DNA repair gene renders results in failure to produce a functional protein and host cell sensitive to chemotherapeutic drugs that impairs the ability of the cell to repair mismatches that would otherwise be chemoresistant. For example, the occur in the genome during proliferation, resulting in response to cisplatin and derivatives may be a direct an increased mutation rate, some 100 times greater function of the methylation state of the CpG island than that in normal cells. Microsatellite instability of hMLH1, the response to adriamycin may be related is noted in approximately 13% of all sporadic cases to the methylation status of GSTP1 and the response of and in nearly all patients with to certain DNA damaging drugs could be a function hereditary nonpolyposis colorectal cancer, which in of the state of BRCA1 hypermethylation.44-47 turn is linked to mutations of the MMR genes hMLH1 and hMSH2. In a significant proportion of tumors In vitro studies of ovarian lines have positive for microsatellite instability, no mutational implicated loss of DNA damage dependent apoptotic abnormality can be shown, but hypermethylation and pathway in acquired resistance to clinically important loss of hMLH1 protein expression does occur.33 cytotoxic drugs.48 Methylation of hMLH1 and loss of a

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MMR dependent apoptotic response lead to increased tumor traits such as metastasis. In this regard, most resistance to cisplatin and carboplatin drugs that are epithelial tumors are highly invasive, and the cells the cornerstone amongst treatments for ovarian cancer released can form metastatic foci, which grow within and a wide variety of tumors.49, 50 Restoration of hMLH1 the visceral organs. expression, either by gene transfer or by reversal of epigenetic silencing, leads to increased sensitivity to The combination of genetic and epigenetic events carboplatin and other cytotoxic agents.43, 51 in cancer now provides a mechanism for complete inactivation of both allelic locations. Classification The fact how CpG islands become hypermethylated of tumors into categories based on molecular in some types of cancer but not in others is yet to characteristics and whether they display certain be understood. Inactivation of a particular gene by methylation patterns is possible. Tumours are methylation could give certain tumor types a growth termed either as displaying a CpG island methylator advantage. CpG islands can have a location within phenotype (single gene methylation) or as CpG island a particular nucleotide sequence that allows them to methylator phenotype positive – CIMP+ (multiple become hypermethylated, or they can be located in gene ). Distinguishing between molecularly a chromosomal region that is prone to large-scale heterogenous groups of tumours with the same origin epigenetic dysregulation.51, 52 When combined with in a epigenetic manner may yield clinically useful other profiling techniques, such as gene expression parameters for prognosis.56 profiling, a patient’s DNA methylome may play a crucial role in the development of personalized medicine. Methylation related mutational events

Differences between loss of Methylated cytosine residues show a high propensity gene function versus epigenetic to undergo deamination to form thymine.57 In this changes way, a C-to-T point mutational event occurs. Because thymine is a normal component of human DNA, this There are some fundamental differences between mutation may not be correctly recognized by the DNA genetic and epigenetic silencing that are potentially repair mechanisms. Instead of repairing the mutated very significant for tumor biology. First, when genetic thymine, the complementary strand guanine may be events are responsible for disruption of both alleles substituted for adenine to form the normal T–A in the classic two-hit paradigm for loss of tumor opposition. Hence, a G-to-A point mutation occurs. suppressor gene function, each event produces a fixed level for loss of gene dosage. First genetic hit The transformation of C- to- T can occur either may potentially result in phenotypically functional through spontaneous deamination of 5mC or by an haplo- insufficiency states.53 The onset of selective enzyme-mediated mechanism where methyltransferase cell advantage does not occur without the complete binding results in deamination before the methyl loss of gene dosage produced by the second hit. In transfer to form uracil, which is then substituted contrast, the loss of gene transcription associated by thymine after two rounds of DNA replication. with aberrant promoter CpG island methylation is The major cause of the high mutation rate at CpG mediated by the density of methylation within the dinucleotides is likely to be spontaneous deamination region. Loss in gene function in association with of 5mC.58 aberrant promoter methylation may manifest in a more subtle selective advantage than gene mutations during tumor progression.54 Clinical relevance of DNA methylation Second, while promoter hypermethylation and associated gene silencing generally remain very stable An understanding of the molecular events that lead in cancer cells, these changes unlike mutations, are to the evolution of cancer and are responsible for potentially reversible.55 Such epigenetic plasticity the heterogeneity of tumors in individual patients is an excellent candidate to mediate the dynamic can be of benefit to the clinician for at least three heterogeneity of cell populations inherent to complex reasons: it can improve the accuracy and timing of

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 12, No. 4, October 2013 pp 239 - 251 246 Promoter DNA Methylation of DNA Repair Genes in Cancer the diagnosis of cancer, it can provide prognostic regulation of specific vital cell proliferation events information about the cancer and it can offer a would be associated with a particular behavioural potential means for cancer therapy. trait of a tumor, such as the ability to form distant metastases.

Diagnosis of Early Cancers The presence of free tumor DNA in the serum of patients has been recognized as a potential means of One key to improving the clinical outcome in monitoring the efficacy of cancer therapy.60 Although patients with cancer is the urgent need to diagnose genetic defects in the DNA specific to the tumor the disease at its earliest possible stage, which of origin can be identified and sometimes correlated translates into a survival benefit for the patient. If with clinical parameters, the process is expensive and diagnosis is possible before extensive local invasion, time-consuming and may not be a reliable reflection of spread, or disseminated disease, then the state of the disease.61-63 Abnormal gene promoter the surgical resection can be less radical, with fewer region methylation patterns within circulating serum complications and side effects. This forms the rationale tumor DNA from patients with breast, lung, liver for population screening and the surveillance of high- and head and neck tumors have recently been risk patients. identified.64-67 This provides a rapid, quantitative and less expensive biologic marker. Subsequent clinical If methylation of gene promoter regions does prove correlation will determine whether this approach has to be a consistent and early event in the incipient the sensitivity to be a useful molecular serum marker. cancer cells, perhaps with specific gene combinations If so, methylation patterns of circulating DNA released for different cancers, a potential tool for diagnosing by the tumor may provide a means for monitoring premalignant lesions could become available. This may the progress of a tumor and its response to therapy. provide the means for a more accurate screening and surveillance rationale by identifying higher-risk patients on a molecular basis. It would also provide justification Tailored Therapeutic Options for more definitive treatment of patients who have molecular but not yet all the typical pathologic or Information about how a cancer develops through microscopic features associated with frank malignancy. molecular events could allow a clinician to predict more accurately how such a cancer is likely to respond The position of CpG island promoter methylation is to specific chemotherapeutic agents. In this way, a constant within an individual gene. Potentially then, regimen tailored to the individual patient and based for all patients, a single primer strategy can be used to on knowledge of the tumor’s chemosensitivity could detect tumor-specific methylation changes in a given be designed. gene by methylation-specific PCR procedures.59 Such assays could be applied to DNA obtained from distal As our understanding of tumor biology increases, the sites such as serum, urine or sputum, even without genes involved in different intracellular biochemical knowing methylation status of the marker directly in reactions specific to individual cancers will be primary tumor DNA. These characteristics of promoter identified. The methylation profile of such genes could hypermethylation renders this to be a valuable DNA be used to predict the efficacy of therapy designed marker for early tumour detection or identification to interrupt these pathways. Reversal of abnormal of high risk individuals. methylation patterns would seem an attractive and logical therapeutic means of arresting cancer growth or spread or even obliterating a . Predicting Outcome and Monitoring Several molecular study groups are investigating Progress the transcriptional failure that accompanies DNA methylation and with this understanding will come Staging of tumors based on the levels and pattern several potential targets for engineering novel distribution of DNA methylation may provide pharmacologic weapons against cancer. a convenient way to assess a tumor’s biologic aggressiveness and to predict patient response. Even before the methylation changes in cancer were Methylation and inactivation of genes essential to known to involve gene promoters, demethylating

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 12, No. 4, October 2013 pp 239 - 251 247 Shilpa V. agents such as 5-azacytidine was being tried as mortality rate for ovarian cancer patients due to agents. Several years of study have difficulties in early detection and recurrence after documented some efficacy in treating hematopoietic current chemotherapy, the identification of promising malignancies, as shown by studies done by Lubbert therapeutic targets for molecular targeted therapy and Momparler et.al.68, 69 as well as the identification of relevant for early detection is an immediate and crucial The recurrence of malignancy or the resistance goal. Growing evidence supports the importance of to current therapies is one of the greatest concerns epigenetic changes in tumorigenesis as much as the in the treatment of ovarian cancer. In particular, classical and better known, genetic changes. epigenetic inhibitors hold promise for overcoming chemoresistance in ovarian cancer through the With the development of high-throughput genomic/ restoration of drug response genes and pathways.70 epigenomic approaches, it is now possible to identify It was shown that the DNMT inhibitor, , the global differences between cancers and normal decreased cisplatin resistance in both ovarian cancer tissues. Thus, a comprehensive understanding of both cells and a mouse xenograft through demethylation of the genomics and of ovarian cancer the hMLH1 promoter.43 A combination of decitabine through the use of integrated approaches should and belinostat treatments showed greater cisplatin make it possible to identify epigenetically activated sensitization of a platinum-resistant mouse xenograft . This will facilitate the identification than either single treatment alone.71 Based on the of more significant biomarkers and promising preclinical results of DNMT inhibitors or HDAC novel therapeutic targets for ovarian cancer and inhibitors, epigenetic drugs are undergoing clinical consequently, will contribute to the improved detection trial investigations for the treatment of recurrent of ovarian cancer and its treatment. resistant ovarian cancer.72 The profile of CpG island hypermethylation is specific to the tumor type, opening the avenue for Conclusion its use as a biomolecular marker of the disease. An issue strengthened by the development of automatic Cancer is a polygenetic disease, but it is also a PCR-based technologies is the easy detection of cancer polyepigenetic disease. We cannot understand the lesions. But more questions continue to arise: What dynamics and plasticity of cancer cells if we do is the real contribution of DNA hypermethylation to not invoke epigenetic changes. Given the high tumorigenesis? Why are some tumor suppressor genes

Figure 6: Diagnostic, Prognostic and Pharmacodynamic Biomarkers Adapted from Ref 73. A) Normal cell . B) Cancer cell epigenome.

Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 12, No. 4, October 2013 pp 239 - 251 248 Promoter DNA Methylation of DNA Repair Genes in Cancer more prone to be hypermethylated than others? Are pyrimidines and nucleosides by paper chromatography. J Biol Chem 1948; 175: 315–332. there any genetic disruptions prompting some of the 14. Laird P W, Jaenisch R. The role of DNA methylation in DNA methylation changes observed or is it is the cancer genetic and epigenetics. Annu Rev Genet 1996; 30: other way around? Will we ever find/create a DNA 441– 464. demethylating agent specific for the hypermethylated 15. Robertson K D, Ambinder R F. Methylation of the Epstein- Barr virus genome in normal lymphocytes. Blood 1997; 90: tumor suppressor genes? With the realization of the 4480–4484. Human Epigenome Project, we stand a good chance 16. Michał W. Łuczak and Paweł P. Jagodzinski. The role of DNA at answering the above questions. Furthermore, an methylation in cancer development. 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