THE ANATOMICAL RECORD 294:1787–1795 (2011)

DNA Methylation and Nonsmall CellLung Cancer

FANG LU AND HONG-TAO ZHANG*

Soochow University Laboratory of Cancer Molecular Genetics,Medical College of Soochow University, Suzhou, Jiangsu Province, People’s Republic of China

ABSTRACTLung cancer is the leading cause of cancer-related death in men and

women worldwide. Owing to the scarcity of effective tools for early detec-tion and therapy strategies, the 5-year survival rate of lung cancer isvery poor. Because the accumulation of multiple genetic and/or epigeneticchanges, including DNA methylation, has been suggested to contribute todevelopment and progression of human cancers, improved understandingof the relationship between DNA methylation and lung cancer will pro-vide new insights for identifying promising biomarkers for diagnosis,prognosis, and treatment of lung cancer. Here, we present a relativelycomprehensive review of DNA methylation and lung cancer, discuss DNAmethylation changes in carcinogenesis and metastasis of lung cancer, andexplore the association of microRNA with DNA methylation. Additionally,we outline the applications of DNA methylation in clinical practice, suchas diagnosis, prognosis, and therapy of lung cancer. Anat Rec, 294:1787–1795, 2011. VVC 2011 Wiley-Liss, Inc.

Keywords: DNA methylation; lung cancer; hypermethylation;hypomethylation;microRNA; diagnostics; prognostics;therapeutics

CURRENT STATUS OF LUNG CANCER

Lung cancer, caused by cigarette smoking in 80%–90%of cases (Hammond and Seidman, 1980), contributes tothe most frequent cancer-related mortality in men andwomen worldwide (Molina et al., 2008). Despite the factthat smoking is a high attributable risk to lung cancer,�20% of lifetime smokers develop the disease (Petoet al., 2000), indicating that genetic susceptibility playsa causal role in lung carcinogenesis. Lung carcinoma isclinically divided into two broad groups, that is, smallcell lung cancer (SCLC) and nonsmall cell lung cancer(NSCLC; Pfeifer and Rauch, 2009). NSCLC is the mostcommon type of lung cancer, accounting for �85% of alllung cancer cases and consisting mainly of adenocarci-noma, squamous cell carcinoma (SCC), and large-cellcarcinoma (Liu et al., 2010).

Surgical resection is usually the most effective therapystrategy for NSCLC patients. To a less extent, even ifthe tumor is surgically resected, lung cancer patientsare still at high risk for recurrence and death. The over-all 5-year survival rate of lung cancer is dismal withmerely <15% in all developed countries and 5% in devel-oping countries (McWilliams et al., 2002), which ismainly due to the scarcity of effective tools for early

detection and therapy strategies (Pfeifer and Rauch,2009). Consequently, developing molecular markers forearly detection, predicting prognosis, and exploiting newtherapy agents of lung cancer are urgently needed.

Grant sponsor: National Natural Science Foundation of China;Grant numbers: 81171894, 30973425, 30672400 (to H.-T. Zhang);Grant sponsor: Program for New Century Excellent Talents inUniversity; Grant number: NCET-09-0165 (to H.-T. Zhang); Grantsponsor: Science and Technology Committee of Jiangsu Province;Grant number: BK2008162 (to H.-T. Zhang); Grant sponsor: SRFfor ROCS, State Education Ministry; Grant number: 2008890 (toH.-T. Zhang); Grant sponsor: Qing-Lan Project of EducationBureau of Jiangsu Province; Grant sponsor: ‘‘333’’ Project ofJiangsu Province Government; Grant sponsor: Soochow ScholarProject of Soochow University (to H.-T. Zhang).

*Correspondence to: Hong-Tao Zhang, Soochow University Lab-oratory of Cancer Molecular Genetics, Medical College of SoochowUniversity, 199 Ren’ai Road, Sino-Singapore Industrial Park, Suz-hou 215123, People’s Republic of China. Fax: þ86-512-65882809.E-mail: htzhang@suda.edu.cn

Received 11 June 2011; Accepted 22 July 2011

DOI 10.1002/ar.21471Published online 28 September 2011 in Wiley Online Library(wileyonlinelibrary.com).

VVC 2011 WILEY-LISS, INC.

As lung cancer could result from the accumulation ofmultiple genetic and/or epigenetic changes, includingDNA methylation, more interests have been attracted tothe potential applications of DNA methylation in lungcancer. Improved understanding of the relationshipbetween DNA methylation and lung cancer will providean exciting new era for identifying promising biomarkersfor diagnosis, prognosis, and treatment of lung cancer.

DNA METHYLATION

Epigenetic alterations, which result in aberrant geneexpression without any concomitant change in DNAsequence, are heritable through cell division (Boumberand Issa, 2011). They have been shown to play impor-tant roles in determining when and where as well aswhether a gene would express or silence. Thus far, DNAmethylation is considered as the best-characterized andmost easily quantifiable epigenetic mechanism underly-ing gene expression or silencing. In mammals, DNAmethylation occurs by transfer of a methyl group from adonor S-adenosyl-methionine to the 5 position of cytosinein the dinucleotide sequence CpG (Kerr et al., 2007).DNA methylation can be completed by catalyze of DNAmethyltransferases, including DNA methyltransferase 1(DNMT1), DNMT3A, and DNMT3B. Of them, DNMT1 isresponsible for maintaining pre-existing DNA methyla-tion patterns in a cell (Jurkowska et al., 2011). DNAmethylation frequently occurs in CpG islands (CGIs)around the 50 untranslated regions (50-UTR) of genes(Sekido et al., 1998). In detail, CGIs, close to transcrip-tional start sites and included in the first exon andintron of a gene (Bird, 1986), are GC-rich (60–70%, CpG/GpC>0.6). CGIs usually range from 0.5 to 5 kb andoccur per 100 kb on average (Belinsky et al., 2003).These CGIs are present in nearly 50% of total genes andare always promoter associated (Suzuki and Bird, 2008).They play significant roles in protecting normal cellsfrom methylation, promoting expression of several keygenes involving cell growth and development, andexcepting for genes on the inactive X chromosome andimprinted genes. Methylation of promoter region withCGIs will result in inactivation of gene expression (Ushi-jima and Okochi-Takada, 2005). With respect to howDNA methylation regulates gene expression, several pos-sible theoretical ways are presented as follows: A possi-bility is that the methylated CpG residues couldinterfere directly with the transcriptional factors thatcan bind to the specific DNA sequence. Second, specificrepression factors, including MeCP1 and MeCP2, maydirectly bind to methylated DNA. Third, active chroma-tin structure could be converted into an inactive form bymethylation. Lastly, epigenetic events can interact withseveral cell cycle regulators (Chuang et al., 1997).Deregulated gene expression via DNA methylation hasalso been recognized as a key factor of aging (Qureshiand Mehler,in press) and many kinds of pathologies (Shiet al., 2007). To date, DNA methylation is emerging asfunctions in not only cancer but also neurological, cardi-ovascular (Frey, 2005), and immunological diseases (Shiet al., 2007; Esteller, 2006). In the following sections, wewill discuss what is known about DNA methylationchanges in lung cancer and how these alterations areapplied potentially to diagnosis and prognosis of thedisease.

HYPERMETHYLATION ANDHYPOMETHYLATION IN LUNG CANCER

Cancer, a multistep process disease characterized bythe accumulation of a series of molecular genetic andepigenetic alternations, which generally exhibit genome-wide hypomethylation and gene-specific hypermethylationof DNA that is associated with silencing of gene expres-sion (Esteller, 2007). Methylated DNA is accompanied bycondensed heterochromatin that is transcriptionallyincompetent, while unmethylated DNA is accompanied bycompetent chromatin that is open to transcription. DNAmethylation status significantly alters in tumors whencompared with those in normal cells and is considered tocontribute to tumorigenesis (Suzuki et al., 2002). Further-more, CpG methylation density is very important due to afact that low-density CpG methylation would merelyrepress a weak promoter, while high-density methylationis required for inhibition of a strong promoter (Bird,1992). Global hypomethylation (Esteller and Herman,2002) and regional promoter CGIs hypomethylation(Chilukamarri et al., 2007; Lin et al., 2007b; Kim et al.,2008) may activate proto-oncogenes, loss gene imprinting,and reactivate transposable elements. Nevertheless,hypermethylated CGIs may suppress the expression ofgenes that contribute to tumor suppression, chromatincondensation, and DNA repair (Baylin, 2005). There isaccumulating evidence demonstrating that aberrantmethylation of the promoter regions of multiple genes isa common phenomenon in cancer (Baylin et al., 2001),particularly, it occurs at the early stage during thepathogenesis of lung carcinoma (Suzuki and Yoshino,2010). Numerous reports have shown that there are con-spicuous epigenetic alterations in carcinoma, giving riseto phenotypic alternations combined with genetic changes,and underlie lung tumorigenesis (Costello et al., 2000;Esteller et al., 2001; Feinberg, 2004; Wilson et al., 2006).

DNA hypermethylation, a well-known epigenetic alter-ation at CGIs, is considered as a major mechanismunderlying loss of gene expression. DNA hypermethyl-ation generally contributes to negative regulation of cellgrowth in large amount of ways, and its relationshipwith the transcriptional silencing of genes has beenlargely recognized (Bird and Wolffe, 1999; Rountreeet al., 2001; Wade, 2001). The silencing is mediatedthrough stabilizing the chromatin structure or inhibitingthe binding of transcription factors to response elementsharboring CpG sites (Costello et al., 2000). CGIs areextensively hypermethylated in human cancers (Costelloet al., 2000; Esteller et al., 2001; Jones and Baylin,2002), including lung cancer (Tsou et al., 2002; Digeland Lubbert, 2005). Hundreds of genes, including p16,RASSF1A, DAPK, MGMT, CDH13, CDH1, AdenomatousPolyposis coli (APC), RARb, and so forth. (Virmani et al.,2000; Heller et al., 2010), have been recognized to har-bor dense methylation in promoter CGIs in lung cancer,especially in NSCLC (Table 1). Among these potentialtumor suppressor genes (TSGs), RARb is methylated in40–43% of NSCLC, p16 25–41%, DAPK 16–44%, MGMT16–27%, and RASSF1A 30–40% (Zochbauer-Mulleret al., 2002; Belinsky, 2005). The following sections willreview the previous findings of DNA hypermethylationin NSCLC, especially in the context of gene silencing.

Promoter hypermethylation-based silencing of p16,located at 9p21 and encoding a cell cycle regulatory

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protein (Gopisetty et al., 2006), is always observed inlung cancer (Auerkari, 2006) and plays an importantrole in preventing cyclin-dependent kinases 4 (CDK4)and CDK6 from forming an active complex with cyclin Din early stage of lung cancer and increasing consistentlywith cancer deterioration (Belinsky et al., 1998, 2002).RASSF1A promoter is frequently hypermethylated in avariety of cancers, including lung carcinoma (Dammannet al., 2000, 2001, 2005; Honorio et al., 2001), and its hy-permethylation leads to epigenetic silencing of RASSF1Aby HOXB3-mediated induction of DNMT3B expression(Palakurthy et al., 2009). APC is a negative regulator ofWingless-INT (WNT) pathway, which involves in self-renewal of stem cell (Mazieres et al., 2005), and APCmethylation occurrence rate is up to 94% in NSCLC but20% in normal control group (Brabender et al., 2001).Activation of the WNT pathway is in part attributed tomethylation-based silencing of APC in lung cancer(Toyooka et al., 2004b; Tsou et al., 2005). In contrast,nine out of 11 noncancerous lung specimens presentedlow levels of APC methylation (Kerr et al., 2007). Meth-ylation-mediated blocking of activation of RB promotermay reduce RB expression to 8.0% in vitro (Ohtani-Fujita et al., 1993). CGIs at 50 end of the RB gene werefound hypermethylated in NSCLC (Joseph et al., 2004).Aberrant methylation of TGFBR2, a major inhibitor ofepithelial cell growth and functions as TSG, may beassociated with down-regulation of TGFBR2 expressionat the transcriptional level in NSCLC (Zhang et al.,2004). Although TGFBR1 haplotype was reported to as-sociate with NSCLC risk (Lei et al., 2009), no aberrantDNA methylation was found to correlate with defectiveexpression of TGFBR1 in NSCLC (Zhao et al., 2008).Apart from the function of DNA methylation in carcino-

genesis, it is possible that the inactivation of metastasissuppressor gene, such as CDH11 (Kashima et al., 2003),could play an important role in pulmonary metastasis.Besides the genes described above, there are many genespresenting aberrant methylation in lung cancer, includ-ing FHIT, GSTP1, hOGG1, SEMA3B, and BLU (Table 1;Zochbauer-Muller et al., 2001; Liu et al., 2008).

Taken together, much data support the notion thatmethylation of specific genes occurs merely in lung can-cers but not at all or only in an extremely low proportionof the corresponding noncancerous lung tissues (Toyookaet al., 2001; Zochbauer-Muller et al., 2001; Heller et al.,2006). Most methylation studies focus mainly on NSCLCbut few on SCLC, therefore more methylation informa-tion on SCLC needs to be gathered in the future.

With respect to large amounts of studies showing hy-permethylation in lung cancer, it reminds us the issue ofwhether there is discovery for demethylation of CGIs.Although it occurs much less frequently than hyper-methylation, gene-specific hypomethylation has beenobserved in human cancer (Esteller et al., 2001).Gene-specific hypomethylation occurs at CpG sites inpromoters of specific genes, leading to increased expres-sion of growth factors, proto-oncogenes, genes that areinvolved in cancer cell proliferation, and invasion andmetastasis (Szyf et al., 2004). For instance, hypomethy-lation enhances expression of uPA, which serves as uro-kinase type plasminogen activator in tumor cells thatcould degrade extracellular matrix (Werb, 1997; Andrea-sen et al., 2000). Expression of the MAGE gene wasfound in 70–85% of NSCLC, and its activation was asso-ciated with loss of methylation in 75–80% of tumors(Jang et al., 2001). Hypomethylation of the synuclein-gamma gene is associated with the metastatic potential

TABLE 1. DNA methylation changes of several key genes and their roles in lung cancer

Methylation changes Genea Function Cell type Reference

Hypermethylation P16 Cell-cycle control NSCLC Belinsky (1998), Belinsky (2002)RASSF1A Ras signaling NSCLC, SCLC Damman (2001), Honorio (2001),

Dammann (2005)APC Regulation of cell proliferation,

migration, and adhesionNSCLC, SCLC Brabender (2001)

RB Cell-cycle control NSCLC, SCLC Ohtani-Fujita (1993),Joseph (2004)

TGFBR2 Inhibition of epithelialcell growth

NSCLC Zhang (2004)

DAPK Proapoptotic NSCLC, SCLC Zochbauer-Muller (2001)MGMT DNA repair NSCLC Belinsky (2005)CDH13 Regulation of cell adhesion NSCLC Toyooka (2001)CDH1 Cell-cycle regulation NSCLC, SCLC Toyooka (2001)RARb Regulation of cell differentiation

and proliferationNSCLC, SCLC Virmani (2000)

FHIT Proapoptotic NSCLC Zochbauer-Muller (2001)GSTP1 Detoxification NSCLC Zochbauer-Muller (2001)SEMA3B Regulation of cell motility

and cell adhesionNSCLC Kuroki (2003)

hOGG1 DNA repair NSCLC Liu (2008)BLU Cell-cycle regulation NSCLC Liu (2008)

Hypomethylation MAGE Unknown NSCLC Jang (2001)SNCG Unknown Lung cancer Liu (2005)

aP16, cyclin-dependent kinase inhibitor 2A; RASSF1A, Ras association domain family member 1; APC, adenomatous poly-posis coli; RB, retinoblastoma; TGFBR2, transforming growth factor beta receptor II; DAPK, death associated protein ki-nase; MGMT, O-6-methylguanine-DNA methyltransferase; CDH13, cadherin 13; CDH1, cadherin 1; RARb, retinoic acidreceptor beta; FHIT, fragile histidine triad gene; GSTP1, glutathione S-transferase pi 1; SEMA3B, semaphorin 3B;hOGG1, 8-oxoguanine DNA glycosylase; MAGE, melanoma antigen family A, 1; SNCG, gamma-synuclein.

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of various human cancers, including lung carcinoma(Liu et al., 2005). Re-expression of tumor antigens maycontribute to cancer metastasis in multiple ways, includ-ing hypomethylation (Simpson et al., 2005).

In addition to gene-specific hypomethylation, globalhypomethylation was reported to bring about genomicinstability that may in turn accelerate secondary geneticchanges (Eden et al., 2003; Gaudet et al., 2003). DNAshort tandem repeats alternation that caused by micro-satellite instability has been observed in 22% of NSCLCand 35% of SCLC (Mao, 2001). It is supposed that hypo-methylation could induce genomic instability either bychromosomal rearrangement or by transposable ele-ments reactivation (Roman-Gomez et al., 2005; Yamadaet al., 2005; Howard et al., 2008). Genomic hypomethyla-tion occurs specifically at transposable elements and re-petitive sequences such as long interspersed nuclearelement (LINE), short interspersed nuclear element, andlong terminal repeat elements, subtelomeric regions andsegmental duplications. Repetitive DNA elements areusually hypomethylated in lung SCCs (Pfeifer andRauch, 2009). For example, the percentage of hypome-thylation at LINE-1 repeat sequences in several types ofcancer, including lung cancer, is higher than that in nor-mal tissues (Chalitchagorn et al., 2004). Furthermore,unless altering the levels of gene expression, genomichypomethylation may facilitate the gain and loss of sev-eral chromosomes, which could result in genomic insta-bility that causes development and progression of cancer(Sulewska et al., 2007). But the mechanisms underlyingcancer-related DNA hypomethylation of repetitive DNAelements are not yet fully understood. One supposedmechanism is that reactivation of a DNA demethylasethat is normally not expressed in adult human cells maymake CpG methylation of repetitive DNA sequence lostin tumor cells. Another possible mechanism by whichsmall-RNA could regulate methylation of repetitive DNAsequence is through heterochromatin formation.

MICRORNA AND DNA METHYLATION

MicroRNAs (miRNAs) are short noncoding RNA mole-cules with �22 nucleotides long and are found ineukaryotic cells. MiRNA is considered as a novel epige-netic regulator of gene expression (Esquela-Kerscherand Slack, 2006), playing important roles in various bio-logical processes such as development, proliferation, cel-lular differentiation, and apoptosis through silencingspecific target genes (He and Hannon, 2004). They mayserve as negative regulators of gene expression by bind-ing to complementary sequences in the 30-UTR of targetmRNAs or by guiding mRNA degradation (Eulalio et al.,2008). There is growing evidence indicating that miR-NAs may function as TSGs or oncogenes as well (Calinand Croce, 2006).

Although miRNAs, including Let-7 and miR-128b, arefound to aberrantly express in lung carcinoma, investi-gating expression patterns and functions of miRNAs inlung cancer is just at the early stage. Let-7 is a well-understood example of miRNAs, and it is one of the firstidentified miRNAs and seems to function in a criticalrole as TSG in lung cancer (Takamizawa et al., 2004).Indeed, over-expression of let-7 may result in variousbiological processes, including inhibition of Ras proteinexpression (Johnson et al., 2005) and repression of pro-

liferation of lung cancer cells (Johnson et al., 2007).Additionally, over-expression of miR-206 can inhibitmigration and invasion of lung cancer cells (Wang et al.,2011). As a direct negative regulator of the EGFR onco-gene, miR-128b (located on chromosome 3p) expressionis lost in lung cancers (Weiss et al., 2008). Let-7a-3 andmiR-17-92, typical examples of oncogenic miRNAs, wereobserved to up-regulate in lung cancer cells (Hayashitaet al., 2005; Volinia et al., 2006), could enhance cellproliferation (Hayashita et al., 2005) and inhibit lungepithelial progenitor cells differentiation in transgenicmice (Matsubara et al., 2007).

Next, we will discuss the relationship between DNAmethylation and regulation of miRNAs expression in thefollowing two aspects (Fig. 1). First, it is worth notingthat aberrant DNA methylation is a mechanism for acti-vation or silencing of the miRNA genes, including let-7a-3 and miR-124a (Brueckner et al., 2007; Fazi et al.,2007; Lujambio et al., 2007; Bueno et al., 2008). Hypo-methylation of let-7a-3 may cause over-expression of let-7a-3 in lung cancer cells, enhancing cancer phenotypesand oncogenic alternations at the transcription level(Brueckner et al., 2007). Hypermethylation of miR-124awas identified to mediate Rb phosphorylation and CDK6activation in lung ACs (Lujambio et al., 2007). Althoughlittle is known about the accurate mechanisms underly-ing regulation of miRNAs expression in lung cancer, afew existing literature indicates that DNA methylationmight affect the status of miRNAs. Second, it is great ofinterest that miRNAs are regulators as well as targetsof DNA methylation. MiRNA-29 family may restoreaberrant methylation in lung caner by targeting 30-UTRof DNMT3A and DNMT3B, which are frequently up-regulated in lung cancer and associated with poor prog-nosis (Fabbri et al., 2007). Increased expression ofmiRNA-29 normalizes aberrant patterns of methylationin NSCLC, induces re-expression of methylation-silencedTSGs, such as FHIT and WWOX, and inhibits lung carci-nogenesis (Fabbri et al., 2007).

Taken together, elucidating the association betweenmiRNAs and DNA methylation will provide an improvedunderstanding for development, progression, and noveltherapeutic targets of lung cancer.

APPLICATIONS OF DNA METHYLATION INLUNG CANCER

As DNA methylation has been identified at early stageof lung cancer, its potential usefulness as diagnostic orprognostic biomarkers of the disease should be consid-ered as well as its contribution to tumorigenesis. DNAmethylation of specific genes seems to be a powerful mo-lecular marker for early detection, prognosis, disease re-currence, risk assessment, and monitoring response totherapy of lung cancer.

DNA METHYLATION—LUNG CANCERDIAGNOSTICS

There is rapidly increasing evidence supporting theidea that DNA methylation would become clinicallypotential molecular markers of cancer. It is understoodthat the useful molecular marker should associate withcancer development and needs to show not only signifi-cantly different between normal and tumor tissue but

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also significantly different specific and sensitive for neo-plastic transformation. In this section, we will present abrief summary of the previous reports, focusing on DNAmethylation that potentially acts as diagnostic bio-markers in preinvasive lung cancer. An exemplifiedreport is that p16 promoter hypermethylation is an earlyevent of lung tumorigenesis (Belinsky et al., 1998;Nuovo et al., 1999). In the following investigation, Palm-isano et al. (2000) demonstrated that aberrant methyla-tion of p16 and/or MGMT promoters was detected in100% of sputum DNA from patients with lung SCC upto 3 years before clinical diagnosis. MGMT, a DNArepair enzyme, is commonly inactivated via aberrantpromoter methylation in lung ACs (Pulling et al., 2003).These data suggest that hypermethylation of p16 andMGMT may become valuable biomarkers for early detec-tion of lung carcinomas. More recently, Rauch et al.(2008) identified 11 CGIs that were methylated in 80–100% of lung SCC, and many hold promise as effectivebiomarkers for early detection of lung cancer.

Why is DNA methylation a feasible marker to detectlung cancer and superior to other diagnostic biomarkers?First, DNA used for the methylation analysis is chemi-cally more stable than proteins and RNA. Second, thefrequency for aberrant methylation of specific CGIs ishigh in human cancer (Miyamoto et al., 2003). Third,DNA methylation can be feasibly amplified and sensi-tively detected through PCR-based approaches, such asmethylation-specific PCR (Herman et al., 1996) andquantitative methylation-specific PCR (Laird, 2003).Fourth, as DNA hypermethylation generally occurs in ornear cancer-specific gene promoters, design of the tar-geted probes to measure this epigenetic change is con-venient. Lastly, aberrant DNA methylation can beidentified in early stage of tumorigenesis and even innon-neoplastic tissues (Miyamoto and Ushijima, 2005).

Although specificity and sensitivity of DNA methyla-tion as diagnosis marker are various due to relatively

small case–control studies and nonstandard assays froma majority of investigations, a series of candidate DNAmethylation markers will still provide promisinginsights for early detection of lung cancer.

DNA METHYLATION—LUNG CANCERPROGNOSTICS

Conventionally, tumor properties, such as pathologicalsubtypes, nodal invasion, and metastasis, could roughlypredict outcomes of cancers, including lung cancer.Besides these traditional prognostic factors, a wealth ofmolecular biomarkers, including DNA methylation, mayoffer refined information to the outcome of cancer andthence guide the selection of specific therapies.

More recently, Brock et al. (2008) found aberrant pat-terns of promoter methylation of APC, RASSF1A, p16,and CDH13 associated with early recurrence in Stage INSCLC. In support of this notion, Yamamoto et al.(2009) indicated that synchronous methylation ofCDH13 and p16 is more effective as a prognostic bio-marker than merely p16 methylation. Combining withhypermethylated FHIT, methylation of p16 is related topoorer prognosis of Stage I NSCLC (Kim et al., 2006).Additionally, hypermethylation of the promoter regionsof 14-3-3r, a critical G2-M checkpoint control gene,emerges as an effective prognosis biomarker of NSCLCpatients (Fu et al., 2000). The prognostic value of 14-3-3r methylation was evaluated in serum DNA from 115cisplatin/gemcitabine-treated advanced NSCLC patients(Ramirez et al., 2005), suggesting that methylation of14-3-3r is a novel independent prognostic factor for sur-vival in NSCLC patients receiving platinum-basedchemotherapy.

However, there are still conflicting results generatingon the relationship between methylation of specific genesand lung cancer prognosis. For example, methylation ofRASSF1A was found to correlate with earlier recurrence

Fig. 1. Schematic diagram of the relationship between DNA methylation and regulation of miRNAsexpression in lung cancer.

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in Stages I and II NSCLC (Endoh et al., 2003), but noassociation of RASSF1A methylation was confirmed byother investigations (Toyooka et al., 2004a). Advancementin DNA methylation-based prognosis assessment of lungcancer has been limited to some extent, and more infor-mation about aberrant DNA methylation as prognosisbiomarker of the disease needs to be urgently elucidated.

DNA METHYLATION—LUNG CANCERTHERAPEUTICS

Given the fact that epigenetic alternations that playimportant roles in tumorigenesis and metastasis couldbe reversed by the pharmacologic application, DNAmethylation represents new potential therapeutic tar-gets for lung cancer. Reactivation of methylation-medi-ated silencing of specific genes in lung cancer wouldcause a series of events, including growth arrest, cellulardifferentiation and induction of apoptosis, and thenresult in anticancer activity. Nowadays, inhibition ofDNA methylation will be of particular interest as anti-cancer therapy.

Over the past few years, investigators have seen thatDNA methylation may be effectively reversed by thera-pies or drugs, including both adenosine and nonadeno-sine classes DNMT inhibitors. The adenosine classinhibitors consists of 5-aza-20-deoxycytidine (5-Aza-CdR,decitabine) and 5-azacytidine which can induce degrada-tion of DNMTs through forming irreversible covalentbonds with them, thus leading to DNA demethylationand reactivation of hypermethylated genes. This kind ofdrug has demonstrated an activity to promote anticancerin leukemia but little activity in solid tumors (Goffinand Eisenhauer, 2002). As a nonadenosine inhibitors,MG98 is an antisense oligodeoxynucleotide directedagainst the 30-UTR of DNMT1 mRNA, and it has shownan ability to inhibit DNMT1 expression without effectingDNMT3 and to cause re-expression of p16 in bladderand colon cancer cells (Goffin and Eisenhauer, 2002).Antisense-mediated depletion of DNMT1 and DNMT3Bmediates caspase-dependent and p53-independent apo-ptosis in lung cancer lines, which is not attributable toinduction of TSGs, such as RASSF1A and p16 (Kassiset al., 2006). These findings further support DNMT inhi-bition strategies for therapy of lung cancer. Indeed,there is supporting evidence that a Stage IV NSCLCpatient after treatment with 5-Aza-CdR has long-termsurvival (Momparler and Ayoub, 2001). Mithramysin-Amay decrease methylation of CGIs and inhibit NSCLCinvasion phenotype in vitro by reducing DNMT1 expres-sion (Lin et al., 2007a). Although the anti-DNMTsagents, including hydralazine, procainamide, zebularine,procaine, and epigallocatechin-3-gallate, are clinicallymeasured for lung cancer patients, the preliminary out-come of them is not remarkable (Digel and Lubbert,2005).

Until now, only a few preclinical trials have measuredthe efficacy of DNMT inhibitors therapy. Decitabine wasobserved to significantly restrain cancer development ofApcMin mice and reduce intestinal adenomas formationby 82% (Laird et al., 1995), and decitabine could inhibittumor formation of lung in mice treated with a tobacco-specific carcinogen (Lantry et al., 1999). These studiesprovide a new era for lung cancer therapy with the useof DNMT inhibitors.

Taken together, the improvements made in lung can-cer epigenetics will facilitate development of drugs thatuse DNA methylation as attractive therapeutic target.DNMT inhibitors, such as decitabine and Mithramysin-A, are potential lung cancer therapy drugs that targetDNA methylation. Nevertheless, DNA methylation-basedtherapy has its limitations in the treatment of lung can-cer with the possibility of carcinogenicity and mutagenic-ity, as DNMT inhibitors could nonspecifically activateoncogenic genes and transposable elements. Meanwhile,there is a possibility that corrected methylation willrestore to the primary status based on its reversiblenature. Therefore, proper investigation designs andindependent experimental validation are warranted toensure the translation of them to the clinic.

SUMMARY AND FUTURE PERSPECTIVES

There is sufficient evidence that DNA methylation isan early event observed in the pathogenesis of lung can-cer, suggesting that DNA methylation may become feasi-ble markers for diagnosis, prognosis, and potentialtargets of therapy. Although there are obviously encour-aging results obtained in this field, lots of challengesstill lie ahead. First, the exact contribution of aberrantDNA methylation to lung cancer etiology is not wellknown. Second, the mechanisms underlying cancer-spe-cific methylation alternation remain to be clarified.Third, more investigations on mouse models of epige-netic alterations will be moving toward, and to someextent elucidate the causative significance of DNA meth-ylation in initiating lung carcinoma. Additionally, it willbe of importance to identify the DNA-methylated genesthat undoubtedly contribute to development and pro-gression of lung cancer. Lastly, integrating DNA methyl-ation-based strategies with conventional management oflung cancer will be clinically needed.

Although DNA methylation markers have great poten-tial to become specific and sensitive diagnostic tools forearly detection of lung cancer, further preclinical trialsare needed before the promising DNA methylationmarkers are introduced into conventional clinical diag-nosis. Developing sensitive and reliable techniques thatcan detect rare methylated DNA molecules in serum,sputum, and circulating tumor cells will be a top priorityin the future. DNA methylation may also offer novelpromising indicators for prognosis and treatment thatmonitor recurrence and response to therapy of lungcancer.

In conclusion, although we are still at the very begin-ning of elucidating the mechanisms of DNA methylationchanges in lung cancer and of practicing the translationof it to the clinic, much information on the aberrantDNA methylation has been so far obtained. Therefore, itprovides support for the idea that novel methylation-based therapies will clinically cure lung cancer one day.

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