THE ANATOMICAL RECORD 294:1787–1795 (2011) DNA Methylation and Nonsmall Cell Lung 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 ABSTRACT Lung cancer is the leading cause of cancer-related death in men and women worldwide. Owing to the scarcity of effective tools for early detection and therapy strategies, the 5-year survival rate of lung cancer is very poor. Because the accumulation of multiple genetic and/or epigenetic changes, including DNA methylation, has been suggested to contribute to development and progression of human cancers, improved understanding of the relationship between DNA methylation and lung cancer will provide new insights for identifying promising biomarkers for diagnosis, prognosis, and treatment of lung cancer. Here, we present a relatively comprehensive review of DNA methylation and lung cancer, discuss DNA methylation changes in carcinogenesis and metastasis of lung cancer, and explore the association of microRNA with DNA methylation. Additionally, we outline the applications of DNA methylation in clinical practice, such as diagnosis, prognosis, and therapy of lung cancer. Anat Rec, 294:1787– C 2011 Wiley-Liss, Inc. 1795, 2011. V Key words: 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 to the most frequent cancer-related mortality in men and women worldwide (Molina et al., 2008). Despite the fact that smoking is a high attributable risk to lung cancer, 20% of lifetime smokers develop the disease (Peto et al., 2000), indicating that genetic susceptibility plays a causal role in lung carcinogenesis. Lung carcinoma is clinically divided into two broad groups, that is, small cell lung cancer (SCLC) and nonsmall cell lung cancer (NSCLC; Pfeifer and Rauch, 2009). NSCLC is the most common type of lung cancer, accounting for 85% of all lung cancer cases and consisting mainly of adenocarcinoma, squamous cell carcinoma (SCC), and large-cell carcinoma (Liu et al., 2010). Surgical resection is usually the most effective therapy strategy for NSCLC patients. To a less extent, even if the tumor is surgically resected, lung cancer patients are still at high risk for recurrence and death. The overall 5-year survival rate of lung cancer is dismal with merely <15% in all developed countries and 5% in developing countries (McWilliams et al., 2002), which is mainly due to the scarcity of effective tools for early C 2011 WILEY-LISS, INC. V detection and therapy strategies (Pfeifer and Rauch, 2009). Consequently, developing molecular markers for early detection, predicting prognosis, and exploiting new therapy 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 in University; Grant number: NCET-09-0165 (to H.-T. Zhang); Grant sponsor: Science and Technology Committee of Jiangsu Province; Grant number: BK2008162 (to H.-T. Zhang); Grant sponsor: SRF for ROCS, State Education Ministry; Grant number: 2008890 (to H.-T. Zhang); Grant sponsor: Qing-Lan Project of Education Bureau of Jiangsu Province; Grant sponsor: ‘‘333’’ Project of Jiangsu Province Government; Grant sponsor: Soochow Scholar Project of Soochow University (to H.-T. Zhang). *Correspondence to: Hong-Tao Zhang, Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, 199 Ren’ai Road, Sino-Singapore Industrial Park, Suzhou 215123, People’s Republic of China. Fax: þ86-512-65882809. E-mail: firstname.lastname@example.org Received 11 June 2011; Accepted 22 July 2011 DOI 10.1002/ar.21471 Published online 28 September 2011 in Wiley Online Library (wileyonlinelibrary.com). 1788 LU AND ZHANG As lung cancer could result from the accumulation of multiple genetic and/or epigenetic changes, including DNA methylation, more interests have been attracted to the potential applications of DNA methylation in lung cancer. Improved understanding of the relationship between DNA methylation and lung cancer will provide an exciting new era for identifying promising biomarkers for diagnosis, prognosis, and treatment of lung cancer. DNA METHYLATION Epigenetic alterations, which result in aberrant gene expression without any concomitant change in DNA sequence, are heritable through cell division (Boumber and Issa, 2011). They have been shown to play important roles in determining when and where as well as whether a gene would express or silence. Thus far, DNA methylation is considered as the best-characterized and most easily quantiﬁable epigenetic mechanism underlying gene expression or silencing. In mammals, DNA methylation occurs by transfer of a methyl group from a donor S-adenosyl-methionine to the 5 position of cytosine in the dinucleotide sequence CpG (Kerr et al., 2007). DNA methylation can be completed by catalyze of DNA methyltransferases, including DNA methyltransferase 1 (DNMT1), DNMT3A, and DNMT3B. Of them, DNMT1 is responsible for maintaining pre-existing DNA methylation patterns in a cell (Jurkowska et al., 2011). DNA methylation frequently occurs in CpG islands (CGIs) around the 50 untranslated regions (50 -UTR) of genes (Sekido et al., 1998). In detail, CGIs, close to transcriptional start sites and included in the ﬁrst exon and intron of a gene (Bird, 1986), are GC-rich (60–70%, CpG/ GpC>0.6). CGIs usually range from 0.5 to 5 kb and occur per 100 kb on average (Belinsky et al., 2003). These CGIs are present in nearly 50% of total genes and are always promoter associated (Suzuki and Bird, 2008). They play signiﬁcant roles in protecting normal cells from methylation, promoting expression of several key genes involving cell growth and development, and excepting for genes on the inactive X chromosome and imprinted genes. Methylation of promoter region with CGIs will result in inactivation of gene expression (Ushijima and Okochi-Takada, 2005). With respect to how DNA methylation regulates gene expression, several possible theoretical ways are presented as follows: A possibility is that the methylated CpG residues could interfere directly with the transcriptional factors that can bind to the speciﬁc DNA sequence. Second, speciﬁc repression factors, including MeCP1 and MeCP2, may directly bind to methylated DNA. Third, active chromatin structure could be converted into an inactive form by methylation. Lastly, epigenetic events can interact with several cell cycle regulators (Chuang et al., 1997). Deregulated gene expression via DNA methylation has also been recognized as a key factor of aging (Qureshi and Mehler,in press) and many kinds of pathologies (Shi et al., 2007). To date, DNA methylation is emerging as functions in not only cancer but also neurological, cardiovascular (Frey, 2005), and immunological diseases (Shi et al., 2007; Esteller, 2006). In the following sections, we will discuss what is known about DNA methylation changes in lung cancer and how these alterations are applied potentially to diagnosis and prognosis of the disease. HYPERMETHYLATION AND HYPOMETHYLATION IN LUNG CANCER Cancer, a multistep process disease characterized by the accumulation of a series of molecular genetic and epigenetic alternations, which generally exhibit genomewide hypomethylation and gene-speciﬁc hypermethylation of DNA that is associated with silencing of gene expression (Esteller, 2007). Methylated DNA is accompanied by condensed heterochromatin that is transcriptionally incompetent, while unmethylated DNA is accompanied by competent chromatin that is open to transcription. DNA methylation status signiﬁcantly alters in tumors when compared with those in normal cells and is considered to contribute to tumorigenesis (Suzuki et al., 2002). Furthermore, CpG methylation density is very important due to a fact that low-density CpG methylation would merely repress a weak promoter, while high-density methylation is 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 of genes that contribute to tumor suppression, chromatin condensation, and DNA repair (Baylin, 2005). There is accumulating evidence demonstrating that aberrant methylation of the promoter regions of multiple genes is a common phenomenon in cancer (Baylin et al., 2001), particularly, it occurs at the early stage during the pathogenesis of lung carcinoma (Suzuki and Yoshino, 2010). Numerous reports have shown that there are conspicuous epigenetic alterations in carcinoma, giving rise to 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 alteration at CGIs, is considered as a major mechanism underlying loss of gene expression. DNA hypermethylation generally contributes to negative regulation of cell growth in large amount of ways, and its relationship with the transcriptional silencing of genes has been largely recognized (Bird and Wolffe, 1999; Rountree et al., 2001; Wade, 2001). The silencing is mediated through stabilizing the chromatin structure or inhibiting the binding of transcription factors to response elements harboring CpG sites (Costello et al., 2000). CGIs are extensively hypermethylated in human cancers (Costello et al., 2000; Esteller et al., 2001; Jones and Baylin, 2002), including lung cancer (Tsou et al., 2002; Digel and Lubbert, 2005). Hundreds of genes, including p16, RASSF1A, DAPK, MGMT, CDH13, CDH1, Adenomatous Polyposis coli (APC), RARb, and so forth. (Virmani et al., 2000; Heller et al., 2010), have been recognized to harbor dense methylation in promoter CGIs in lung cancer, especially in NSCLC (Table 1). Among these potential tumor suppressor genes (TSGs), RARb is methylated in 40–43% of NSCLC, p16 25–41%, DAPK 16–44%, MGMT 16–27%, and RASSF1A 30–40% (Zochbauer-Muller et al., 2002; Belinsky, 2005). The following sections will review the previous ﬁndings of DNA hypermethylation in NSCLC, especially in the context of gene silencing. Promoter hypermethylation-based silencing of p16, located at 9p21 and encoding a cell cycle regulatory 1789 DNA METHYLATION AND LUNG CANCER TABLE 1. DNA methylation changes of several key genes and their roles in lung cancer Methylation changes Hypermethylation Genea Cell type Reference Belinsky (1998), Belinsky (2002) Damman (2001), Honorio (2001), Dammann (2005) Brabender (2001) P16 RASSF1A Cell-cycle control Ras signaling NSCLC NSCLC, SCLC APC Regulation of cell proliferation, migration, and adhesion Cell-cycle control NSCLC, SCLC Inhibition of epithelial cell growth Proapoptotic DNA repair Regulation of cell adhesion Cell-cycle regulation Regulation of cell differentiation and proliferation Proapoptotic Detoxiﬁcation Regulation of cell motility and cell adhesion DNA repair Cell-cycle regulation Unknown Unknown NSCLC Ohtani-Fujita (1993), Joseph (2004) Zhang (2004) NSCLC, SCLC NSCLC NSCLC NSCLC, SCLC NSCLC, SCLC Zochbauer-Muller (2001) Belinsky (2005) Toyooka (2001) Toyooka (2001) Virmani (2000) NSCLC NSCLC NSCLC Zochbauer-Muller (2001) Zochbauer-Muller (2001) Kuroki (2003) NSCLC NSCLC NSCLC Lung cancer Liu (2008) Liu (2008) Jang (2001) Liu (2005) RB TGFBR2 DAPK MGMT CDH13 CDH1 RARb FHIT GSTP1 SEMA3B Hypomethylation Function hOGG1 BLU MAGE SNCG NSCLC, SCLC a P16, cyclin-dependent kinase inhibitor 2A; RASSF1A, Ras association domain family member 1; APC, adenomatous polyposis coli; RB, retinoblastoma; TGFBR2, transforming growth factor beta receptor II; DAPK, death associated protein kinase; MGMT, O-6-methylguanine-DNA methyltransferase; CDH13, cadherin 13; CDH1, cadherin 1; RARb, retinoic acid receptor 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. protein (Gopisetty et al., 2006), is always observed in lung cancer (Auerkari, 2006) and plays an important role in preventing cyclin-dependent kinases 4 (CDK4) and CDK6 from forming an active complex with cyclin D in early stage of lung cancer and increasing consistently with cancer deterioration (Belinsky et al., 1998, 2002). RASSF1A promoter is frequently hypermethylated in a variety of cancers, including lung carcinoma (Dammann et al., 2000, 2001, 2005; Honorio et al., 2001), and its hypermethylation leads to epigenetic silencing of RASSF1A by HOXB3-mediated induction of DNMT3B expression (Palakurthy et al., 2009). APC is a negative regulator of Wingless-INT (WNT) pathway, which involves in selfrenewal of stem cell (Mazieres et al., 2005), and APC methylation occurrence rate is up to 94% in NSCLC but 20% in normal control group (Brabender et al., 2001). Activation of the WNT pathway is in part attributed to methylation-based silencing of APC in lung cancer (Toyooka et al., 2004b; Tsou et al., 2005). In contrast, nine out of 11 noncancerous lung specimens presented low levels of APC methylation (Kerr et al., 2007). Methylation-mediated blocking of activation of RB promoter may reduce RB expression to 8.0% in vitro (OhtaniFujita et al., 1993). CGIs at 50 end of the RB gene were found hypermethylated in NSCLC (Joseph et al., 2004). Aberrant methylation of TGFBR2, a major inhibitor of epithelial cell growth and functions as TSG, may be associated with down-regulation of TGFBR2 expression at the transcriptional level in NSCLC (Zhang et al., 2004). Although TGFBR1 haplotype was reported to associate with NSCLC risk (Lei et al., 2009), no aberrant DNA methylation was found to correlate with defective expression 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 metastasis suppressor gene, such as CDH11 (Kashima et al., 2003), could play an important role in pulmonary metastasis. Besides the genes described above, there are many genes presenting aberrant methylation in lung cancer, including FHIT, GSTP1, hOGG1, SEMA3B, and BLU (Table 1; Zochbauer-Muller et al., 2001; Liu et al., 2008). Taken together, much data support the notion that methylation of speciﬁc genes occurs merely in lung cancers but not at all or only in an extremely low proportion of the corresponding noncancerous lung tissues (Toyooka et al., 2001; Zochbauer-Muller et al., 2001; Heller et al., 2006). Most methylation studies focus mainly on NSCLC but few on SCLC, therefore more methylation information on SCLC needs to be gathered in the future. With respect to large amounts of studies showing hypermethylation in lung cancer, it reminds us the issue of whether there is discovery for demethylation of CGIs. Although it occurs much less frequently than hypermethylation, gene-speciﬁc hypomethylation has been observed in human cancer (Esteller et al., 2001). Gene-speciﬁc hypomethylation occurs at CpG sites in promoters of speciﬁc genes, leading to increased expression of growth factors, proto-oncogenes, genes that are involved in cancer cell proliferation, and invasion and metastasis (Szyf et al., 2004). For instance, hypomethylation enhances expression of uPA, which serves as urokinase type plasminogen activator in tumor cells that could degrade extracellular matrix (Werb, 1997; Andreasen et al., 2000). Expression of the MAGE gene was found in 70–85% of NSCLC, and its activation was associated with loss of methylation in 75–80% of tumors (Jang et al., 2001). Hypomethylation of the synucleingamma gene is associated with the metastatic potential 1790 LU AND ZHANG of various human cancers, including lung carcinoma (Liu et al., 2005). Re-expression of tumor antigens may contribute to cancer metastasis in multiple ways, including hypomethylation (Simpson et al., 2005). In addition to gene-speciﬁc hypomethylation, global hypomethylation was reported to bring about genomic instability that may in turn accelerate secondary genetic changes (Eden et al., 2003; Gaudet et al., 2003). DNA short tandem repeats alternation that caused by microsatellite instability has been observed in 22% of NSCLC and 35% of SCLC (Mao, 2001). It is supposed that hypomethylation could induce genomic instability either by chromosomal rearrangement or by transposable elements reactivation (Roman-Gomez et al., 2005; Yamada et al., 2005; Howard et al., 2008). Genomic hypomethylation occurs speciﬁcally at transposable elements and repetitive sequences such as long interspersed nuclear element (LINE), short interspersed nuclear element, and long terminal repeat elements, subtelomeric regions and segmental duplications. Repetitive DNA elements are usually hypomethylated in lung SCCs (Pfeifer and Rauch, 2009). For example, the percentage of hypomethylation at LINE-1 repeat sequences in several types of cancer, including lung cancer, is higher than that in normal tissues (Chalitchagorn et al., 2004). Furthermore, unless altering the levels of gene expression, genomic hypomethylation may facilitate the gain and loss of several chromosomes, which could result in genomic instability that causes development and progression of cancer (Sulewska et al., 2007). But the mechanisms underlying cancer-related DNA hypomethylation of repetitive DNA elements are not yet fully understood. One supposed mechanism is that reactivation of a DNA demethylase that is normally not expressed in adult human cells may make CpG methylation of repetitive DNA sequence lost in tumor cells. Another possible mechanism by which small-RNA could regulate methylation of repetitive DNA sequence is through heterochromatin formation. MICRORNA AND DNA METHYLATION MicroRNAs (miRNAs) are short noncoding RNA molecules with 22 nucleotides long and are found in eukaryotic cells. MiRNA is considered as a novel epigenetic regulator of gene expression (Esquela-Kerscher and Slack, 2006), playing important roles in various biological processes such as development, proliferation, cellular differentiation, and apoptosis through silencing speciﬁc target genes (He and Hannon, 2004). They may serve as negative regulators of gene expression by binding to complementary sequences in the 30 -UTR of target mRNAs or by guiding mRNA degradation (Eulalio et al., 2008). There is growing evidence indicating that miRNAs may function as TSGs or oncogenes as well (Calin and Croce, 2006). Although miRNAs, including Let-7 and miR-128b, are found to aberrantly express in lung carcinoma, investigating expression patterns and functions of miRNAs in lung cancer is just at the early stage. Let-7 is a wellunderstood example of miRNAs, and it is one of the ﬁrst identiﬁed miRNAs and seems to function in a critical role as TSG in lung cancer (Takamizawa et al., 2004). Indeed, over-expression of let-7 may result in various biological processes, including inhibition of Ras protein expression (Johnson et al., 2005) and repression of pro- liferation of lung cancer cells (Johnson et al., 2007). Additionally, over-expression of miR-206 can inhibit migration and invasion of lung cancer cells (Wang et al., 2011). As a direct negative regulator of the EGFR oncogene, miR-128b (located on chromosome 3p) expression is lost in lung cancers (Weiss et al., 2008). Let-7a-3 and miR-17-92, typical examples of oncogenic miRNAs, were observed to up-regulate in lung cancer cells (Hayashita et al., 2005; Volinia et al., 2006), could enhance cell proliferation (Hayashita et al., 2005) and inhibit lung epithelial progenitor cells differentiation in transgenic mice (Matsubara et al., 2007). Next, we will discuss the relationship between DNA methylation and regulation of miRNAs expression in the following two aspects (Fig. 1). First, it is worth noting that aberrant DNA methylation is a mechanism for activation or silencing of the miRNA genes, including let-7a3 and miR-124a (Brueckner et al., 2007; Fazi et al., 2007; Lujambio et al., 2007; Bueno et al., 2008). Hypomethylation of let-7a-3 may cause over-expression of let7a-3 in lung cancer cells, enhancing cancer phenotypes and oncogenic alternations at the transcription level (Brueckner et al., 2007). Hypermethylation of miR-124a was identiﬁed to mediate Rb phosphorylation and CDK6 activation in lung ACs (Lujambio et al., 2007). Although little is known about the accurate mechanisms underlying regulation of miRNAs expression in lung cancer, a few existing literature indicates that DNA methylation might affect the status of miRNAs. Second, it is great of interest that miRNAs are regulators as well as targets of DNA methylation. MiRNA-29 family may restore aberrant methylation in lung caner by targeting 30 -UTR of DNMT3A and DNMT3B, which are frequently upregulated in lung cancer and associated with poor prognosis (Fabbri et al., 2007). Increased expression of miRNA-29 normalizes aberrant patterns of methylation in NSCLC, induces re-expression of methylation-silenced TSGs, such as FHIT and WWOX, and inhibits lung carcinogenesis (Fabbri et al., 2007). Taken together, elucidating the association between miRNAs and DNA methylation will provide an improved understanding for development, progression, and novel therapeutic targets of lung cancer. APPLICATIONS OF DNA METHYLATION IN LUNG CANCER As DNA methylation has been identiﬁed at early stage of lung cancer, its potential usefulness as diagnostic or prognostic biomarkers of the disease should be considered as well as its contribution to tumorigenesis. DNA methylation of speciﬁc genes seems to be a powerful molecular marker for early detection, prognosis, disease recurrence, risk assessment, and monitoring response to therapy of lung cancer. DNA METHYLATION—LUNG CANCER DIAGNOSTICS There is rapidly increasing evidence supporting the idea that DNA methylation would become clinically potential molecular markers of cancer. It is understood that the useful molecular marker should associate with cancer development and needs to show not only signiﬁcantly different between normal and tumor tissue but DNA METHYLATION AND LUNG CANCER 1791 Fig. 1. Schematic diagram of the relationship between DNA methylation and regulation of miRNAs expression in lung cancer. also signiﬁcantly different speciﬁc and sensitive for neoplastic transformation. In this section, we will present a brief summary of the previous reports, focusing on DNA methylation that potentially acts as diagnostic biomarkers in preinvasive lung cancer. An exempliﬁed report is that p16 promoter hypermethylation is an early event of lung tumorigenesis (Belinsky et al., 1998; Nuovo et al., 1999). In the following investigation, Palmisano et al. (2000) demonstrated that aberrant methylation of p16 and/or MGMT promoters was detected in 100% of sputum DNA from patients with lung SCC up to 3 years before clinical diagnosis. MGMT, a DNA repair enzyme, is commonly inactivated via aberrant promoter methylation in lung ACs (Pulling et al., 2003). These data suggest that hypermethylation of p16 and MGMT may become valuable biomarkers for early detection 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 effective biomarkers for early detection of lung cancer. Why is DNA methylation a feasible marker to detect lung cancer and superior to other diagnostic biomarkers? First, DNA used for the methylation analysis is chemically more stable than proteins and RNA. Second, the frequency for aberrant methylation of speciﬁc CGIs is high in human cancer (Miyamoto et al., 2003). Third, DNA methylation can be feasibly ampliﬁed and sensitively detected through PCR-based approaches, such as methylation-speciﬁc PCR (Herman et al., 1996) and quantitative methylation-speciﬁc PCR (Laird, 2003). Fourth, as DNA hypermethylation generally occurs in or near cancer-speciﬁc gene promoters, design of the targeted probes to measure this epigenetic change is convenient. Lastly, aberrant DNA methylation can be identiﬁed in early stage of tumorigenesis and even in non-neoplastic tissues (Miyamoto and Ushijima, 2005). Although speciﬁcity and sensitivity of DNA methylation as diagnosis marker are various due to relatively small case–control studies and nonstandard assays from a majority of investigations, a series of candidate DNA methylation markers will still provide promising insights for early detection of lung cancer. DNA METHYLATION—LUNG CANCER PROGNOSTICS Conventionally, tumor properties, such as pathological subtypes, nodal invasion, and metastasis, could roughly predict outcomes of cancers, including lung cancer. Besides these traditional prognostic factors, a wealth of molecular biomarkers, including DNA methylation, may offer reﬁned information to the outcome of cancer and thence guide the selection of speciﬁc therapies. More recently, Brock et al. (2008) found aberrant patterns of promoter methylation of APC, RASSF1A, p16, and CDH13 associated with early recurrence in Stage I NSCLC. In support of this notion, Yamamoto et al. (2009) indicated that synchronous methylation of CDH13 and p16 is more effective as a prognostic biomarker than merely p16 methylation. Combining with hypermethylated FHIT, methylation of p16 is related to poorer prognosis of Stage I NSCLC (Kim et al., 2006). Additionally, hypermethylation of the promoter regions of 14-3-3r, a critical G2-M checkpoint control gene, emerges as an effective prognosis biomarker of NSCLC patients (Fu et al., 2000). The prognostic value of 14-33r methylation was evaluated in serum DNA from 115 cisplatin/gemcitabine-treated advanced NSCLC patients (Ramirez et al., 2005), suggesting that methylation of 14-3-3r is a novel independent prognostic factor for survival in NSCLC patients receiving platinum-based chemotherapy. However, there are still conﬂicting results generating on the relationship between methylation of speciﬁc genes and lung cancer prognosis. For example, methylation of RASSF1A was found to correlate with earlier recurrence 1792 LU AND ZHANG in Stages I and II NSCLC (Endoh et al., 2003), but no association of RASSF1A methylation was conﬁrmed by other investigations (Toyooka et al., 2004a). Advancement in DNA methylation-based prognosis assessment of lung cancer has been limited to some extent, and more information about aberrant DNA methylation as prognosis biomarker of the disease needs to be urgently elucidated. DNA METHYLATION—LUNG CANCER THERAPEUTICS Given the fact that epigenetic alternations that play important roles in tumorigenesis and metastasis could be reversed by the pharmacologic application, DNA methylation represents new potential therapeutic targets for lung cancer. Reactivation of methylation-mediated silencing of speciﬁc genes in lung cancer would cause a series of events, including growth arrest, cellular differentiation and induction of apoptosis, and then result in anticancer activity. Nowadays, inhibition of DNA methylation will be of particular interest as anticancer therapy. Over the past few years, investigators have seen that DNA methylation may be effectively reversed by therapies or drugs, including both adenosine and nonadenosine classes DNMT inhibitors. The adenosine class inhibitors consists of 5-aza-20 -deoxycytidine (5-Aza-CdR, decitabine) and 5-azacytidine which can induce degradation of DNMTs through forming irreversible covalent bonds with them, thus leading to DNA demethylation and reactivation of hypermethylated genes. This kind of drug has demonstrated an activity to promote anticancer in leukemia but little activity in solid tumors (Gofﬁn and Eisenhauer, 2002). As a nonadenosine inhibitors, MG98 is an antisense oligodeoxynucleotide directed against the 30 -UTR of DNMT1 mRNA, and it has shown an ability to inhibit DNMT1 expression without effecting DNMT3 and to cause re-expression of p16 in bladder and colon cancer cells (Gofﬁn and Eisenhauer, 2002). Antisense-mediated depletion of DNMT1 and DNMT3B mediates caspase-dependent and p53-independent apoptosis in lung cancer lines, which is not attributable to induction of TSGs, such as RASSF1A and p16 (Kassis et al., 2006). These ﬁndings further support DNMT inhibition strategies for therapy of lung cancer. Indeed, there is supporting evidence that a Stage IV NSCLC patient after treatment with 5-Aza-CdR has long-term survival (Momparler and Ayoub, 2001). Mithramysin-A may decrease methylation of CGIs and inhibit NSCLC invasion phenotype in vitro by reducing DNMT1 expression (Lin et al., 2007a). Although the anti-DNMTs agents, including hydralazine, procainamide, zebularine, procaine, and epigallocatechin-3-gallate, are clinically measured for lung cancer patients, the preliminary outcome of them is not remarkable (Digel and Lubbert, 2005). Until now, only a few preclinical trials have measured the efﬁcacy of DNMT inhibitors therapy. Decitabine was observed to signiﬁcantly restrain cancer development of ApcMin mice and reduce intestinal adenomas formation by 82% (Laird et al., 1995), and decitabine could inhibit tumor formation of lung in mice treated with a tobaccospeciﬁc carcinogen (Lantry et al., 1999). These studies provide a new era for lung cancer therapy with the use of DNMT inhibitors. Taken together, the improvements made in lung cancer epigenetics will facilitate development of drugs that use DNA methylation as attractive therapeutic target. DNMT inhibitors, such as decitabine and MithramysinA, are potential lung cancer therapy drugs that target DNA methylation. Nevertheless, DNA methylation-based therapy has its limitations in the treatment of lung cancer with the possibility of carcinogenicity and mutagenicity, as DNMT inhibitors could nonspeciﬁcally activate oncogenic genes and transposable elements. Meanwhile, there is a possibility that corrected methylation will restore to the primary status based on its reversible nature. Therefore, proper investigation designs and independent experimental validation are warranted to ensure the translation of them to the clinic. SUMMARY AND FUTURE PERSPECTIVES There is sufﬁcient evidence that DNA methylation is an early event observed in the pathogenesis of lung cancer, suggesting that DNA methylation may become feasible markers for diagnosis, prognosis, and potential targets of therapy. Although there are obviously encouraging results obtained in this ﬁeld, lots of challenges still lie ahead. First, the exact contribution of aberrant DNA methylation to lung cancer etiology is not well known. Second, the mechanisms underlying cancer-speciﬁc methylation alternation remain to be clariﬁed. Third, more investigations on mouse models of epigenetic alterations will be moving toward, and to some extent elucidate the causative signiﬁcance of DNA methylation in initiating lung carcinoma. Additionally, it will be of importance to identify the DNA-methylated genes that undoubtedly contribute to development and progression of lung cancer. Lastly, integrating DNA methylation-based strategies with conventional management of lung cancer will be clinically needed. Although DNA methylation markers have great potential to become speciﬁc and sensitive diagnostic tools for early detection of lung cancer, further preclinical trials are needed before the promising DNA methylation markers are introduced into conventional clinical diagnosis. Developing sensitive and reliable techniques that can detect rare methylated DNA molecules in serum, sputum, and circulating tumor cells will be a top priority in the future. DNA methylation may also offer novel promising indicators for prognosis and treatment that monitor recurrence and response to therapy of lung cancer. In conclusion, although we are still at the very beginning of elucidating the mechanisms of DNA methylation changes in lung cancer and of practicing the translation of it to the clinic, much information on the aberrant DNA methylation has been so far obtained. Therefore, it provides support for the idea that novel methylationbased therapies will clinically cure lung cancer one day. LITERATURE CITED Andreasen PA, Egelund R, Petersen HH. 2000. The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci 57:25–40. Auerkari EI. 2006. Methylation of tumor suppressor genes p16(INK4a), p27(Kip1) and E-cadherin in carcinogenesis. Oral Oncol 42:5–13. DNA METHYLATION AND LUNG CANCER Baylin SB. 2005. DNA methylation and gene silencing in cancer. Nat Clin Pract Oncol 2 (suppl 1):S4–S11. Baylin SB, Esteller M, Rountree MR, Bachman KE, Schuebel K, Herman JG. 2001. Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum Mol Genet 10:687–692. Belinsky SA. 2005. Silencing of genes by promoter hypermethylation: key event in rodent and human lung cancer. Carcinogenesis 26:1481–1487. Belinsky SA, Klinge DM, Stidley CA, Issa JP, Herman JG, March TH, Baylin SB. 2003. Inhibition of DNA methylation and histone deacetylation prevents murine lung cancer. Cancer Res 63:7089– 7093. Belinsky SA, Nikula KJ, Palmisano WA, Michels R, Saccomanno G, Gabrielson E, Baylin SB, Herman JG. 1998. Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis. Proc Natl Acad Sci USA 95:11891–11896. Belinsky SA, Palmisano WA, Gilliland FD, Crooks LA, Divine KK, Winters SA, Grimes MJ, Harms HJ, Tellez CS, Smith TM, Moots PP, Lechner JF, Stidley CA, Crowell RE. 2002. Aberrant promoter methylation in bronchial epithelium and sputum from current and former smokers. Cancer Res 62:2370–2377. Bird A. 1992. The essentials of DNA methylation. Cell 70:5–8. Bird AP. 1986. CpG-rich islands and the function of DNA methylation. Nature 321:209–213. Bird AP, Wolffe AP. 1999. Methylation-induced repression—belts, braces, and chromatin. Cell 99:451–454. Boumber Y, Issa JP. 2011. Epigenetics in cancer: what’s the future? Oncology (Williston Park) 25:220–226, 228. Brabender J, Usadel H, Danenberg KD, Metzger R, Schneider PM, Lord RV, Wickramasinghe K, Lum CE, Park J, Salonga D, Singer J, Sidransky D, Holscher AH, Meltzer SJ, Danenberg PV. 2001. Adenomatous polyposis coli gene promoter hypermethylation in non-small cell lung cancer is associated with survival. Oncogene 20:3528–3532. Brock MV, Hooker CM, Ota-Machida E, Han Y, Guo M, Ames S, Glockner S, Piantadosi S, Gabrielson E, Pridham G, Pelosky K, Belinsky SA, Yang SC, Baylin SB, Herman JG. 2008. DNA methylation markers and early recurrence in stage I lung cancer. N Engl J Med 358:1118–1128. Brueckner B, Stresemann C, Kuner R, Mund C, Musch T, Meister M, Sultmann H, Lyko F. 2007. The human let-7a-3 locus contains an epigenetically regulated microRNA gene with oncogenic function. Cancer Res 67:1419–1423. Bueno MJ, Perez de Castro I, Gomez de Cedron M, Santos J, Calin GA, Cigudosa JC, Croce CM, Fernandez-Piqueras J, Malumbres M. 2008. Genetic and epigenetic silencing of microRNA-203 enhances ABL1 and BCR-ABL1 oncogene expression. Cancer Cell 13:496–506. Calin GA, Croce CM. 2006. MicroRNA signatures in human cancers. Nat Rev Cancer 6:857–866. Chalitchagorn K, Shuangshoti S, Hourpai N, Kongruttanachok N, Tangkijvanich P, Thong-ngam D, Voravud N, Sriuranpong V, Mutirangura A. 2004. Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis. Oncogene 23:8841–8846. Chilukamarri L, Hancock AL, Malik S, Zabkiewicz J, Baker JA, Greenhough A, Dallosso AR, Huang TH, Royer-Pokora B, Brown KW, Malik K. 2007. Hypomethylation and aberrant expression of the glioma pathogenesis-related 1 gene in Wilms tumors. Neoplasia 9:970–978. Chuang LS, Ian HI, Koh TW, Ng HH, Xu G, Li BF. 1997. Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1. Science 277:1996–2000. Costello JF, Fruhwald MC, Smiraglia DJ, Rush LJ, Robertson GP, Gao X, Wright FA, Feramisco JD, Peltomaki P, Lang JC, Schuller DE, Yu L, Bloomﬁeld CD, Caligiuri MA, Yates A, Nishikawa R, Huang HJS, Petrelli NJ, Zhang XL, O’Dorisio MS, Held WA, Cavenee WK, Plass C. 2000. Aberrant CpG-island methylation has non-random and tumour-type-speciﬁc patterns. Nat Genet 24:132–138. 1793 Dammann R, Li C, Yoon JH, Chin PL, Bates S, Pfeifer GP. 2000. Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat Genet 25:315–319. Dammann R, Schagdarsurengin U, Seidel C, Strunnikova M, Rastetter M, Baler K, Pfeifer GP. 2005. The tumor suppressor RASSF1A in human carcinogenesis: an update. Histol Histopathol 20:645–663. Dammann R, Takahashi T, Pfeifer GP. 2001. The CpG island of the novel tumor suppressor gene RASSF1A is intensely methylated in primary small cell lung carcinomas. Oncogene 20:3563–3567. Digel W, Lubbert M. 2005. DNA methylation disturbances as novel therapeutic target in lung cancer: preclinical and clinical results. Crit Rev Oncol Hematol 55:1–11. Eden A, Gaudet F, Waghmare A, Jaenisch R. 2003. Chromosomal instability and tumors promoted by DNA hypomethylation. Science 300:455. Endoh H, Yatabe Y, Shimizu S, Tajima K, Kuwano H, Takahashi T, Mitsudomi T. 2003. RASSF1A gene inactivation in non-small cell lung cancer and its clinical implication. Int J Cancer 106:45–51. Esquela-Kerscher A, Slack FJ. 2006. Oncomirs—microRNAs with a role in cancer. Nat Rev Cancer 6:259–269. Esteller M. 2006. The necessity of a human epigenome project. Carcinogenesis 27:1121–1125. Esteller M. 2007. Cancer epigenomics: DNA methylomes and histone-modiﬁcation maps. Nat Rev Genet 8:286–298. Esteller M, Corn PG, Baylin SB, Herman JG. 2001. A gene hypermethylation proﬁle of human cancer. Cancer Res 61:3225–3229. Esteller M, Herman JG. 2002. Cancer as an epigenetic disease: DNA methylation and chromatin alterations in human tumours. J Pathol 196:1–7. Eulalio A, Huntzinger E, Izaurralde E. 2008. Getting to the root of miRNA-mediated gene silencing. Cell 132:9–14. Fabbri M, Garzon R, Cimmino A, Liu Z, Zanesi N, Callegari E, Liu S, Alder H, Costinean S, Fernandez-Cymering C, Volinia S, Guler G, Morrison CD, Chan KK, Marcucci G, Calin GA, Huebner K, Croce CM. 2007. MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B. Proc Natl Acad Sci USA 104:15805–15810. Fazi F, Racanicchi S, Zardo G, Starnes LM, Mancini M, Travaglini L, Diverio D, Ammatuna E, Cimino G, Lo-Coco F, Grignani F, Nervi C. 2007. Epigenetic silencing of the myelopoiesis regulator microRNA-223 by the AML1/ETO oncoprotein. Cancer Cell 12:457–466. Feinberg AP. 2004. The epigenetics of cancer etiology. Semin Cancer Biol 14:427–432. Frey FJ. 2005. Methylation of CpG islands: potential relevance for hypertension and kidney diseases. Nephrol Dial Transplant 20:868–869. Fu H, Subramanian RR, Masters SC. 2000. 14-3-3 proteins: structure, function, and regulation. Annu Rev Pharmacol Toxicol 40:617–647. Gaudet F, Hodgson JG, Eden A, Jackson-Grusby L, Dausman J, Gray JW, Leonhardt H, Jaenisch R. 2003. Induction of tumors in mice by genomic hypomethylation. Science 300:489–492. Gofﬁn J, Eisenhauer E. 2002. DNA methyltransferase inhibitorsstate of the art. Ann Oncol 13:1699–1716. Gopisetty G, Ramachandran K, Singal R. 2006. DNA methylation and apoptosis. Mol Immunol 43:1729–1740. Hammond EC, Seidman H. 1980. Smoking and cancer in the United States. Prev Med 9:169–174. Hayashita Y, Osada H, Tatematsu Y, Yamada H, Yanagisawa K, Tomida S, Yatabe Y, Kawahara K, Sekido Y, Takahashi T. 2005. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation. Cancer Res 65:9628–9632. He L, Hannon GJ. 2004. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5:522–531. Heller G, Fong KM, Girard L, Seidl S, End-Pfutzenreuter A, Lang G, Gazdar AF, Minna JD, Zielinski CC, Zochbauer-Muller S. 2006. Expression and methylation pattern of TSLC1 cascade genes in lung carcinomas. Oncogene 25:959–968. 1794 LU AND ZHANG Heller G, Zielinski CC, Zochbauer-Muller S. 2010. Lung cancer: from single-gene methylation to methylome proﬁling. Cancer Metastasis Rev 29:95–107. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. 1996. Methylation-speciﬁc PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 93:9821–9826. Honorio S, Agathanggelou A, Astuti D, Dallol A, Schuermann M, Martinsson T, Maher ER, Latif F. 2001. Epigenetic inactivation of RASSF1A from the 3p21.3 region in human cancers and its potential use as a molecular marker for early detection of lung and other cancers. J Med Genet 38:S23. Howard G, Eiges R, Gaudet F, Jaenisch R, Eden A. 2008. Activation and transposition of endogenous retroviral elements in hypomethylation induced tumors in mice. Oncogene 27:404–408. Jang SJ, Soria JC, Wang L, Hassan KA, Morice RC, Walsh GL, Hong WK, Mao L. 2001. Activation of melanoma antigen tumor antigens occurs early in lung carcinogenesis. Cancer Res 61: 7959–7963. Johnson CD, Esquela-Kerscher A, Stefani G, Byrom M, Kelnar K, Ovcharenko D, Wilson M, Wang X, Shelton J, Shingara J, Chin L, Brown D, Slack FJ. 2007. The let-7 microRNA represses cell proliferation pathways in human cells. Cancer Res 67:7713–7722. Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D, Slack FJ. 2005. RAS is regulated by the let-7 MicroRNA family. Cell 120:635– 647. Jones PA, Baylin SB. 2002. The fundamental role of epigenetic events in cancer. Nat Rev Genet 3:415–428. Joseph B, Mamatha G, Raman G, Shanmugam MP, Kumaramanickavel G. 2004. Methylation status of RB1 promoter in Indian retinoblastoma patients. Cancer Biol Ther 3:184–187. Jurkowska RZ, Jurkowski TP, Jeltsch A. 2011. Structure and function of mammalian DNA methyltransferases. Chembiochem 12:206–222. Kashima T, Nakamura K, Kawaguchi J, Takanashi M, Ishida T, Aburatani H, Kudo A, Fukayama M, Grigoriadis AE. 2003. Overexpression of cadherins suppresses pulmonary metastasis of osteosarcoma in vivo. Int J Cancer 104:147–154. Kassis ES, Zhao M, Hong JA, Chen GA, Nguyen DM, Schrump DS. 2006. Depletion of DNA methyltransferase 1 and/or DNA methyltransferase 3b mediates growth arrest and apoptosis in lung and esophageal cancer and malignant pleural mesothelioma cells. J Thorac Cardiovasc Surg 131:298–306. Kerr KM, Galler JS, Hagen JA, Laird PW, Laird-Offringa IA. 2007. The role of DNA methylation in the development and progression of lung adenocarcinoma. Dis Markers 23:5–30. Kim JS, Kim JW, Han J, Shim YM, Park J, Kim DH. 2006. Cohypermethylation of p16 and FHIT promoters as a prognostic factor of recurrence in surgically resected stage I non-small cell lung cancer. Cancer Res 66:4049–4054. Kim SJ, Kang HS, Chang HL, Jung YC, Sim HB, Lee KS, Ro J, Lee ES. 2008. Promoter hypomethylation of the N-acetyltransferase 1 gene in breast cancer. Oncol Rep 19:663–668. Kuroki T, Trapasso F, Yendamuri S, Matsuyama A, Alder H, Williams NN, Kaiser LR, Croce CM. 2003. Allelic loss on chromosome 3p21.3 and promoter hypermethylation of semaphorin 3B in non-small cell lung cancer. Cancer Res 63:3352–3355. Laird PW. 2003. The power and the promise of DNA methylation markers. Nat Rev Cancer 3:253–266. Laird PW, Jackson-Grusby L, Fazeli A, Dickinson SL, Jung WE, Li E, Weinberg RA, Jaenisch R. 1995. Suppression of intestinal neoplasia by DNA hypomethylation. Cell 81:197–205. Lantry LE, Zhang Z, Crist KA, Wang Y, Kelloff GJ, Lubet RA, You M. 1999. 5-Aza-20 -deoxycytidine is chemopreventive in a 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone-induced primary mouse lung tumor model. Carcinogenesis 20:343–346. Lei Z, Liu RY, Zhao J, Liu Z, Jiang X, You W, Chen XF, Liu X, Zhang K, Pasche B, Zhang HT. 2009. TGFBR1 haplotypes and risk of non-small-cell lung cancer. Cancer Res 69:7046–7052. Lin RK, Hsu CH, Wang YC. 2007a. Mithramycin A inhibits DNA methyltransferase and metastasis potential of lung cancer cells. Anticancer Drugs 18:1157–1164. Lin ZW, Thomas NJ, Bibikova M, Seifart C, Wang YH, Guo XX, Wang GR, Vollmer E, Goldmann T, Garcia EW, Zhou LX, Fan JB, Floros J. 2007b. DNA methylation markers of surfactant proteins in lung cancer. Int J Oncol 31:181–191. Liu H, Liu W, Wu Y, Zhou Y, Xue R, Luo C, Wang L, Zhao W, Jiang JD, Liu J. 2005. Loss of epigenetic control of synuclein-gamma gene as a molecular indicator of metastasis in a wide range of human cancers. Cancer Res 65:7635–7643. Liu Z, Li W, Lei Z, Zhao J, Chen XF, Liu R, Peng X, Wu ZH, Chen J, Liu H, Zhou QH, Zhang HT. 2010. CpG island methylator phenotype involving chromosome 3p confers an increased risk of nonsmall cell lung cancer. J Thorac Oncol 5:790–797. Liu Z, Zhao J, Chen XF, Li W, Liu R, Lei Z, Liu X, Peng X, Xu K, Chen J, Liu H, Zhou QH, Zhang HT. 2008. CpG island methylator phenotype involving tumor suppressor genes located on chromosome 3p in non-small cell lung cancer. Lung Cancer 62:15–22. Lujambio A, Ropero S, Ballestar E, Fraga MF, Cerrato C, Setien F, Casado S, Suarez-Gauthier A, Sanchez-Cespedes M, Git A, Spiteri I, Das PP, Caldas C, Miska E, Esteller M. 2007. Genetic unmasking of an epigenetically silenced microRNA in human cancer cells. Cancer Res 67:1424–1429. Mao L. 2001. Molecular abnormalities in lung carcinogenesis and their potential clinical implications. Lung Cancer 34 (suppl 2):S27–S34. Matsubara H, Takeuchi T, Nishikawa E, Yanagisawa K, Hayashita Y, Ebi H, Yamada H, Suzuki M, Nagino M, Nimura Y, Osada H, Takahashi T. 2007. Apoptosis induction by antisense oligonucleotides against miR-17-5p and miR-20a in lung cancers overexpressing miR-17-92. Oncogene 26:6099–6105. Mazieres J, He B, You L, Xu Z, Jablons DM. 2005. Wnt signaling in lung cancer. Cancer Lett 222:1–10. McWilliams A, MacAulay C, Gazdar AF, Lam S. 2002. Innovative molecular and imaging approaches for the detection of lung cancer and its precursor lesions. Oncogene 21:6949–6959. Miyamoto K, Asada K, Fukutomi T, Okochi E, Yagi Y, Hasegawa T, Asahara T, Sugimura T, Ushijima T. 2003. Methylation-associated silencing of heparan sulfate D-glucosaminyl 3-O-sulfotransferase-2 (3-OST-2) in human breast, colon, lung and pancreatic cancers. Oncogene 22:274–280. Miyamoto K, Ushijima T. 2005. Diagnostic and therapeutic applications of epigenetics. Jpn J Clin Oncol 35:293–301. Molina JR, Yang P, Cassivi SD, Schild SE, Adjei AA. 2008. Nonsmall cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc 83:584–594. Momparler RL, Ayoub J. 2001. Potential of 5-aza-20 -deoxycytidine (Decitabine) a potent inhibitor of DNA methylation for therapy of advanced non-small cell lung cancer. Lung Cancer 34 (suppl 4):S111–S115. Nuovo GJ, Plaia TW, Belinsky SA, Baylin SB, Herman JG. 1999. In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis. Proc Natl Acad Sci USA 96:12754–12759. Ohtani-Fujita N, Fujita T, Aoike A, Osifchin NE, Robbins PD, Sakai T. 1993. CpG methylation inactivates the promoter activity of the human retinoblastoma tumor-suppressor gene. Oncogene 8:1063– 1067. Palakurthy RK, Wajapeyee N, Santra MK, Gazin C, Lin L, Gobeil S, Green MR. 2009. Epigenetic silencing of the RASSF1A tumor suppressor gene through HOXB3-mediated induction of DNMT3B expression. Mol Cell 36:219–230. Palmisano WA, Divine KK, Saccomanno G, Gilliland FD, Baylin SB, Herman JG, Belinsky SA. 2000. Predicting lung cancer by detecting aberrant promoter methylation in sputum. Cancer Res 60:5954–5958. Peto R, Darby S, Deo H, Silcocks P, Whitley E, Doll R. 2000. Smoking, smoking cessation, and lung cancer in the UK since 1950: combination of national statistics with two case-control studies. Br Med J 321:323–329. Pfeifer GP, Rauch TA. 2009. DNA methylation patterns in lung carcinomas. Semin Cancer Biol 19:181–187. Pulling LC, Divine KK, Klinge DM, Gilliland FD, Kang T, Schwartz AG, Bocklage TJ, Belinsky SA. 2003. Promoter hypermethylation DNA METHYLATION AND LUNG CANCER of the O6-methylguanine-DNA methyltransferase gene: more common in lung adenocarcinomas from never-smokers than smokers and associated with tumor progression. Cancer Res 63:4842– 4848. Qureshi IA, Mehler MF. 2011. Advances in epigenetics and epigenomics for neurodegenerative diseases. Curr Neurol Neurosci Rep, DOI: 10.1007/s11910-011-0210-2. Ramirez JL, Rosell R, Taron M, Sanchez-Ronco M, Alberola V, de Las Penas R, Sanchez JM, Moran T, Camps C, Massuti B, Sanchez JJ, Salazar F, Catot S. 2005. 14-3-3sigma methylation in pretreatment serum circulating DNA of cisplatin-plus-gemcitabine-treated advanced non-small-cell lung cancer patients predicts survival: The Spanish Lung Cancer Group. J Clin Oncol 23:9105–9112. Rauch TA, Zhong X, Wu X, Wang M, Kernstine KH, Wang Z, Riggs AD, Pfeifer GP. 2008. High-resolution mapping of DNA hypermethylation and hypomethylation in lung cancer. Proc Natl Acad Sci USA 105:252–257. Roman-Gomez J, Jimenez-Velasco A, Agirre X, Cervantes F, Sanchez J, Garate L, Barrios M, Castillejo JA, Navarro G, Colomer D, Prosper F, Heiniger A, Torres A. 2005. Promoter hypomethylation of the LINE-1 retrotransposable elements activates sense/ antisense transcription and marks the progression of chronic myeloid leukemia. Oncogene 24:7213–7223. Rountree MR, Bachman KE, Herman JG, Baylin SB. 2001. DNA methylation, chromatin inheritance, and cancer. Oncogene 20: 3156–3165. Sekido Y, Fong KM, Minna JD. 1998. Progress in understanding the molecular pathogenesis of human lung cancer. Biochim Biophys Acta 1378:F21–F59. Shi H, Wang MX, Caldwell CW. 2007. CpG islands: their potential as biomarkers for cancer. Expert Rev Mol Diagn 7:519–531. Simpson AJ, Caballero OL, Jungbluth A, Chen YT, Old LJ. 2005. Cancer/testis antigens, gametogenesis and cancer. Nat Rev Cancer 5:615–625. Sulewska A, Niklinska W, Kozlowski M, Minarowski L, Naumnik W, Niklinski J, Dabrowska K, Chyczewski L. 2007. DNA methylation in states of cell physiology and pathology. Folia Histochem Cytobiol 45:149–158. Suzuki H, Gabrielson E, Chen W, Anbazhagan R, van Engeland M, Weijenberg MP, Herman JG, Baylin SB. 2002. A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer. Nat Genet 31:141–149. Suzuki M, Yoshino I. 2010. Aberrant methylation in non-small cell lung cancer. Surg Today 40:602–607. Suzuki MM, Bird A. 2008. DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet 9:465–476. Szyf M, Pakneshan P, Rabbani SA. 2004. DNA methylation and breast cancer. Biochem Pharmacol 68:1187–1197. Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H, Harano T, Yatabe Y, Nagino M, Nimura Y, Mitsudomi T, Takahashi T. 2004. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res 64:3753–3756. Toyooka KO, Toyooka S, Virmani AK, Sathyanarayana UG, Euhus DM, Gilcrease M, Minna JD, Gazdar AF. 2001. Loss of expression and aberrant methylation of the CDH13 (H-cadherin) gene in breast and lung carcinomas. Cancer Res 61:4556–4560. Toyooka S, Suzuki M, Maruyama R, Toyooka KO, Tsukuda K, Fukuyama Y, Iizasa T, Aoe M, Date H, Fujisawa T, Shimizu N, Gazdar AF. 2004a. The relationship between aberrant methylation and survival in non-small-cell lung cancers. Br J Cancer 91:771–774. 1795 Toyooka S, Suzuki M, Tsuda T, Toyooka KO, Maruyama R, Tsukuda K, Fukuyama Y, Iizasa T, Fujisawa T, Shimizu N, Minna JD, Gazdar AF. 2004b. Dose effect of smoking on aberrant methylation in non-small cell lung cancers. Int J Cancer 110:462–464. Tsou JA, Hagen JA, Carpenter CL, Laird-Offringa IA. 2002. DNA methylation analysis: a powerful new tool for lung cancer diagnosis. Oncogene 21:5450–5461. Tsou JA, Shen LY, Siegmund KD, Long TI, Laird PW, Seneviratne CK, Koss MN, Pass HI, Hagen JA, Laird-Offringa IA. 2005. Distinct DNA methylation proﬁles in malignant mesothelioma, lung adenocarcinoma, and non-tumor lung. Lung Cancer 47:193–204. Ushijima T, Okochi-Takada E. 2005. Aberrant methylations in cancer cells: where do they come from? Cancer Sci 96:206–211. Virmani AK, Rathi A, Zochbauer-Muller S, Sacchi N, Fukuyama Y, Bryant D, Maitra A, Heda S, Fong KM, Thunnissen F, Minna JD, Gazdar AF. 2000. Promoter methylation and silencing of the retinoic acid receptor-beta gene in lung carcinomas. J Natl Cancer Inst 92:1303–1307. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C, Ferracin M, Prueitt RL, Yanaihara N, Lanza G, Scarpa A, Vecchione A, Negrini M, Harris CC, Croce CM. 2006. A microRNA expression signature of human solid tumors deﬁnes cancer gene targets. Proc Natl Acad Sci USA 103:2257–2261. Wade PA. 2001. Methyl CpG binding proteins: coupling chromatin architecture to gene regulation. Oncogene 20:3166–3173. Wang X, Ling C, Bai Y, Zhao J. 2011. MicroRNA-206 is associated with invasion and metastasis of lung cancer. Anat Rec (Hoboken) 294:88–92. Weiss GJ, Bemis LT, Nakajima E, Sugita M, Birks DK, Robinson WA, Varella-Garcia M, Bunn PA, Jr., Haney J, Helfrich BA, Kato H, Hirsch FR, Franklin WA. 2008. EGFR regulation by microRNA in lung cancer: correlation with clinical response and survival to geﬁtinib and EGFR expression in cell lines. Ann Oncol 19:1053– 1059. Werb Z. 1997. ECM and cell surface proteolysis: regulating cellular ecology. Cell 91:439–442. Wilson IM, Davies JJ, Weber M, Brown CJ, Alvarez CE, MacAulay C, Schubeler D, Lam WL. 2006. Epigenomics—mapping the methylome. Cell Cycle 5:155–158. Yamada Y, Jackson-Grusby L, Linhart H, Meissner A, Eden A, Lin HJ, Jaenisch R. 2005. Opposing effects of DNA hypomethylation on intestinal and liver carcinogenesis. Proc Natl Acad Sci USA 102:13580–13585. Yamamoto H, Toyooka S, Mitsudomi T. 2009. Impact of EGFR mutation analysis in non-small cell lung cancer. Lung Cancer 63:315– 321. Zhang HT, Chen XF, Wang MH, Wang JC, Qi QY, Zhang RM, Xu WQ, Fei QY, Wang F, Cheng QQ, Chen F, Zhu CS, Tao SH, Luo Z. 2004. Defective expression of transforming growth factor beta receptor type II is associated with CpG methylated promoter in primary non-small cell lung cancer. Clin Cancer Res 10:2359– 2367. Zhao J, Liu ZY, Li WW, Liu X, Chen XF, Zhang HT. 2008. Infrequently methylated event at sites-362 to-142 in the promoter of TGF beta R1 gene in non-small cell lung cancer. J Cancer Res Clin Oncol 134:919–925. Zochbauer-Muller S, Fong KM, Virmani AK, Geradts J, Gazdar AF, Minna JD. 2001. Aberrant promoter methylation of multiple genes in non-small cell lung cancers. Cancer Res 61:249–255. Zochbauer-Muller S, Minna JD, Gazdar AF. 2002. Aberrant DNA methylation in lung cancer: biological and clinical implications. Oncologist 7:451–457.