Aberrant DNA Methylation in Cutaneous Squamous Cell Carcinoma

Feng-Juan Li,Yi Wu,Qun Lv,Xue-Yuan Yang,Ming-Jun Jiang,Li-Ming Li,?

1Department of Dermatology,The Affiliated Hospital of Qingdao University,Qingdao,Shandong 266003 China,2West China School of Medicine,Sichuan University,Chengdu,Sichan 610041 China,3Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs,Hospital for Skin Diseases(Institute of Dermatology),Chinese Academy of Medical Sciences and Peking Union Medical College,Nanjing,Jiangsu 210042,China.

Introduction

Cutaneous squamous cell carcinoma(CSCC)is the secondmost common non-melanoma skin cancer after basal cell carcinoma,accounting for 20%of all cutaneous malignancies.CSCC originates from epidermal keratinocytes or adnexal structures such as eccrine glands or pilosebaceous units.1According to several studies focused on Caucasian populations in Europe,the USA and Australia,about 15-35 per 100,000 individuals are diagnosed with CSCC each year,and the incidence of CSCC is expected to increase by 2%-4%per year.2The incidence of primary CSCC has increased by 50%-300%globally,especially amongst Caucasian populations in New Zealand,Australia,and North America over the last 3 decades.3

Although early-stage CSCC has a good prognosis,metastatic CSCC cases can be difficult to treat depending on the location of the lesions and the extent of metastasis,with a mortality rate of over 70%in some cases.4The main risk factors for CSCC include sunlight exposure,ultraviolet(UV)radiation,and other genetic and environmental factors such as light skin(Fitzpatrick skin types I-III),immunosuppression,and human papillomavirus.5Accumulating evidence suggests that these predisposing factors lead to a wide range of genetic and epigenetic alterations that promote genomic instability,malignant transformation,and ultimately tumor development and progression.6

Epigenetics refers to the stable regulation of gene expression without alterations of genetic information.Epigenetic modifications comprise DNA methylation,histone modification,and non-coding functional RNA.The most common form of DNA methylation is the post-DNA replication covalent addition of a methyl group onto the 5-carbon of the cytosine ring within CpG dinucleotides by DNA methyltransferases,and it is by far the most well-investigated epigenetic modification.CpG dinucleotides aggregate in genomic regions called CpG islands,which are short interspersed DNA sequences that deviate significantly from the average genomic pattern.Many CpG islands reside in promoter regions and remain unmethylated.7It is hypothesized that CpG methylation is associated with closed chromatin configurations,8preventing the binding of transcription factors in the gene promoter region and resulting in gene silencing.Genespecific CpG island promoter hypermethylation is associated with the silencing of tumor suppressor genes(TSGs)in various types of tumors,including CSCC.9-12Methylation of CpG sequences may also participate in the development of CSCC by creating preferential targets for UV radiation-induced DNA damage associated with polymorphisms of methylenetetrahydrofolate reductase,as the presence of a methyl group in a CpG dinucleotide increases the rate at which C-T mutations are induced by UV radiation by shifting the absorption spectrum for cytosine.13

In the present study,we reviewed the literature for studies on DNA methylation in CSCC,and summarized the DNA methylation alterations reported in SCC and their association with CSCC clinical characteristics.We also discussed the clinical implications of DNA methylation alterations in CSCC and future research directions.

Global DNA hypomethylation

Global DNA hypomethylation activates endogenous retroviral elements,resulting in chromosomal instability and potential oncogene activation.Recent studies of global methylation patterns in cancer suggest that specific DNA hypomethylation directly participates in tumor initiation and progression through gene expression regulation of many signaling pathways.14Yooyongsatit et al.15determined global DNA methylation levels in CSCC by measuring the repetitive long interspersed nuclear elements-1(LINE-1)sequence methylation level using a combined bisulfite restriction analysis,and reported a LINE-1 methylation level of 41.09%in CSCC tissues,which is significantly lower than the level of 46.05%in the normal epidermis.As LINE-1 constitutes approximately 17%of the human genome,16the LINE-1 methylation level could be used as a surrogate marker for global DNA methylation.However,a relatively low level of LINE-1 methylation is also reported in other cancers,17-18and so the specificity of LINE-1 methylation for CSCC remains unclear.Vandiver et al.19reported that global hypomethylation in squamous cell carcinoma(SCC)is associated with age and sun exposure,and such methylation changes are also detected in sun-exposed non-malignant tissues,suggesting that these changes might occur before malignant transformation.Future studies are needed to confirm the association and mechanism of DNA hypomethylation with sun exposure, and to determine whether sun exposure-associated DNA hypomethylation is directly involved in CSCC tumorigenesis.

TSGs

CDH1 and CDH13

Cadherins are transmembrane glycoproteins commonly expressed on epithelial cell surfaces that mediate intercellular Ca2+-dependent adhesion.In normal epithelial cells,cadherins are involved in the establishment and maintenance of intercellular junctions,cell polarity,intracellular signaling, and tissue architecture.20In cancer cells,cadherin family members such as E-cadherin(CDH1)and H-cadherin(CDH13)are frequently inactivated by aberrant DNA methylation.21-22Consistent with these findings,recent studies have demonstrated that CDH1 is inactivated by aberrant DNA methylation in 37.5%-95%of CSCC tissues,23-25and CDH1 methylation frequency is higher in invasive CSCC than in CSCC in situ or in normal skin tissues.25Further CDH1 inactivation by DNA methylation is directly linked to increased invasiveness in CSCC by upregulating the expression of messenger RNA(mRNA)and protein and the activities of focal adhesion kinase.26DNA methylation of CDH13 is detected in 19%-42.9%of CSCC tissues,27-28but is rarely detected in perilesional and normal skin tissues. These findings suggest that epigenetic inactivation of CDH1 and CDH13 might play an important role in CSCC progression.

CDKN2A

CDKN2A is a cyclin-dependent kinase inhibitor gene that encodes two proteins,p16INK4a and p14ARF,through alternative splicing of exon 1. p16INK4a negatively regulates cell cycle progression through the retinoblastoma gene.29p16INK4a hypermethylation is detected in 20%-36% of CSCCs,24,30demonstrating significant tumor heterogeneity with p16INK4a inactivation by DNA methylation that only occurs in specific parts of the tumor.p14ARF acts as a TSG in epithelial malignancies by regulating p53-and p21-mediated DNA repair and cell proliferation.31-33In addition,p14ARF regulates tumorinduced angiogenesis.31p14ARF hypermethylation is detected in 42%-60% of CSCC tissues,23,30and is correlated with the differentiation degree,lymph node metastasis,and clinical stage.These findings suggest that aberrant methylation of a CDKN2A promoter promotes CSCC carcinogenesis and progression.

G0S2

The G0/G1 switch gene 2(G0S2)is located on chromosome 1 and encodes a small 12 kDa protein that localizes to mitochondria and endoplasmic reticulum.34G0S2 is silenced by promoter DNA methylation in many types of cancer.35-37A recent study demonstrated that G0S2 is epigenetically silenced in CSCC.38G0S2 was silenced in five SCC cell lines correlated with almost 100%methylation of the promoter CpG island,while G0S2 was expressed in two normal keratinocyte cultures with no methylation of the promoter CpG island.38Treatment of SCC cell lines with a demethylating agent led to reactivation of G0S2 expression and a reduced DNA methylation level.38In clinical samples,G0S2 showed a wide range of methylation levels(0-77.7%)in 17 CSCC samples,but a narrow range of methylation levels(0.1%-7.3%)in six normal epidermis samples.38These data strongly suggest that G0S2 is a TSG in CSCC that is frequently inactivated by promoter DNA methylation.

ID4

Inhibitor of DNA binding/inhibitor of differentiation 4(ID4)is a member of the helix-loop-helix transcription factor family.ID4 lacks the basic DNA-binding domain,and thus behaves as a dominant-negative regulator of gene transcription by forming inactive heterodimeric complexes with other helix-loop-helix transcription activators. ID4 plays a key role in several cellular processes, such as proliferation, differentiation, and apoptosis39;it can act as either a TSG or an oncogene based on tissue type.40In skin tissues,ID4 is associated with keratinocyte differentiation and parakeratosis.Ruchusatsawat et al.41reported that three of seven(42.8%)CSCC tissue samples showed ID4 methylation,while all six microdissected normal skin samples showed no methylation.In addition,the ID4 promoter methylation level was significantly higher in SCC tissues(21.8%)than in normal skin tissues(0).41These findings suggest that reversing epigenetic changes might represent a potential novel therapy for skin diseases characterized by parakeratosis,such as CSCC.

AKAP12/gravin

A kinase anchor protein 12(AKAP12/gravin)is a scaffold protein that organizes both protein kinase A and protein kinase C signaling pathways to regulate the β2-adrenergic receptor complex.42AKAP12 was originally isolated as a protein present in the serum of patients with myasthenia gravis.43Subsequently,it has been found that AKAP12 is frequently inactivated by DNA methylation in many malignancies.42,44-45Wu et al.46reported that AKAP12 is methylated in 89.6%of CSCC tissues,and the AKAP12 methylation level is higher in CSCC(＞10%)than in adjacent normal skin tissues(＜10%),suggesting that AKAP12 methylation might be involved in CSCC carcinogenesis.

IRF6

Interferon regulatory factor 6(IRF6)is a transcription factor that belongs to the interferon regulatory transcription factor family.Contrary to other interferon regulatory transcription factor family members, IRF6 does not regulate interferon gene expression,but controls cellular proliferation and differentiation during epidermal development by interacting with ΔNp63,and through the Notch signaling pathway.47IRF6 induces Notch-dependent differentiation in keratinocytes,and downregulation of IRF6 blocks the in vitro and in vivo differentiation of primary human keratinocytes and promotes Ras-induced tumor formation.48Downregulation of IRF6 by DNA methylation has been reported in melanoma and vulvar SCC.49-50In CSCC,IRF6 methylation has been detected in A431 cell lines and in CSCC tumors with low IRF6 expression. In vitro studies demonstrate that downregulation of IRF6 promotes tumor invasion, while reversing DNA methylation inhibits cell growth.51These data strongly suggest that IRF6 is a TSG in CSCC that regulates cancer cell proliferation and invasion.

S100A4

S100A4 belongs to the S100 gene family that contains two EF-hand calcium-binding motifs;it regulates Ca2+-dependent intra-and extracellular activities,including protein phosphorylation, enzyme activity, and cell motility.52Overexpression of S100A4 is associated with aggressiveness,metastasis,and poor prognosis of many different types of cancer.53-55S100A4 is frequently silenced in CSCC by DNA methylation,which may explain the lower metastatic property of CSCC compared with other types of cancer.56Although S100A4 does not contain CpG islands in its promoter region,methylation of critical CpG sites in the first intron correlates with the transcriptional activity of the S100A4 gene.One study showed that all four CpG sites in the first intron were methylated in colo16 cells and CSCC tissues, and 5-aza-2′-deoxycytidine treatment effectively reactivated its expression in colo16 cells.57These data imply that S100A4 methylation could be used as a negative prognostic biomarker or a metastasis risk factor in human CSCC.

Other genes

Other genes that are hypermethylated in CSCC,but whose role in tumorigenesis is yet to be defined include secreted frizzled-related protein,forkhead box E1(FOXE1),deathassociated protein kinase 1,apoptosis-associated specklike protein containing a CARD(ASC),O6-methylguanine DNA methyltransferase(MGMT),and deleted in split hand/split foot 1(DSS1).Secreted frizzled-related protein genes act as negative regulators of the Wnt/beta-catenin signaling pathway,suggesting a role in the suppression of carcinogenesis.58FOXE1 is a member of a large family of transcription factors that regulate cell growth and differentiation.FOXE1 methylation is directly correlated with transcription inactivation,as FOXE1 expression is reactivated by treatment with the demethylating agent 5-aza-2′-deoxycytidine.59Death-associated protein kinase 1 gene is a pro-apoptotic serine/threonine protein kinase that was originally cloned based on its involvement in interferon-gamma-induced apoptosis.28ASC,also known as TMS1,is a bipartite protein that promotes apoptosis in a caspase-dependent manner;the methylation is not only correlated with downregulation of ASC expression,but also with a lack of differentiation and inflammasome activation. Both ASC expression and inflammasome activation are restored by treatment with the demethylating agent 5-aza-2′-deoxycytidine.60MGMT is a DNA repair protein involved in the reversal of DNA alkylation.Reduced MGMT expression and enzyme activity may result in an increased susceptibility to DNA alkylation and carcinogenesis.61DSS1 is a subunit of the 26S proteasome complex involved in ubiquitin-mediated proteolysis.DSS1 participates in many biological processes, including protein degradation,mRNA export,genome stability,homologous recombination,DNA repair,and neoplastic transformation.62

MicroRNAs

MicroRNAs(miRNAs)are a class of small noncoding RNAs that regulate gene expression by hybridizing to complementary target mRNAs,resulting in either mRNA degradation or translation downregulation.63Deregulation of miRNAs has been reported in the development and progression of nearly all tumor types.MiR-204 is an intronic miRNA located between exons 7 and 8 of the transient receptor potential cation channel subfamily M member 3(TRPM3)gene,and is regulated by the same promoter and transcription regulatory motifs.64MiR-204 promoter methylation was detected in two CSCC cell lines(SCC12 and SCC13), as well as in the epidermal carcinoma cell line A431.65MiR-204 promoter methylation was also detected in 11 of 20 (55%) CSCC samples.65Among these 11 CSCC samples with mir-204 promoter methylation,seven(64%)lacked expression of miR-204, and correlated with Signal transducer and activator of transcription 3(STAT3)nuclear translocation and activation.65The downregulation of both miR-124 and miR-214 is controlled by hypermethylation of other promoter regions rather than the miR-124/-214 promotermediated abnormal cell proliferation via the post-transcriptional overexpression of extracellular signal-regulated kinases(ERK)in CSCC.66Thus,the serum miR-124 level is a more sensitive biomarker of the tumor than CSCC antigens.

Challenges and opportunities in CSCC methylation research

In general,most of the studies that have evaluated CSCC methylation are not sufficiently rigorous due to the limited sample sizes and lack of control groups.Furthermore,epigenetic modifications are influenced by location,age,and sex,67which may not be taken into consideration when selecting controls.The occurrence of CSCC in the exposed area is supposed to be closely related to UV radiation.3Therefore,different sample locations may have different causes of CSCC and different methylation patterns or frequencies.In addition,although it is generally believed that the promoter methylation frequency of the promoter region alters with the degree of malignancy of the CSCC,12specific CSCC classifications have not been reported in most existing studies.Due to the influence of the abovementioned factors,most of the research results are inconsistent and even contradictory. Furthermore,most of these studies only reported changes in the methylation status of a single gene,which cannot reflect the overall change in the methylation status of the CSCC gene. Compared with research into other cancers,epigenetic research into CSCC is not very common due to the limited genome-wide DNA methylation data in CSCC.Recent progress in methylation research reveals a deeper understanding of the mechanisms of tumorigenesis and provides biomarkers for the early detection,diagnosis,and prognosis of several common cancer types,68especially the role of circulating cell-free DNA methylation in the early detection and continuous monitoring of the progression and metastasis of lung cancer, colorectal cancer,69and hepatocellular carcinoma.70This may inspire the future direction of CSCC methylation research and promising clinical use.

Conclusions

In this review,we summarized the DNA methylation alterations in SCC and their association with CSCC clinical characteristics in Table 1.Promoter methylation,aubiquitous mechanism of gene silencing,plays an important function in the development of many tumor types.The promoter methylation of a variety of genes has been implicated in CSCC.Current DNA methylation analysis techniques provide high levels of sensitivity,specificity,and speed.Determination of promoter methylation status has shown great promise in cancer diagnosis and prognosis.DNA methylation inhibitors such as 5-aza-2′-deoxycytidine and zebularine might have broad prospects for the treatment of CSCC.Future studies should focus on the identification of the optimal combination of markers for the early detection,prediction of outcomes,and disease status, which will ultimately translate these research outcomes into clinical use.

Table 1Representative hypermethylated genes in cutaneous squamous cell carcinoma.

Acknowledgements

Our work was supported by grants from the Chinese Academy Medical Sciences Initiative for Innovative Medicine(No.2016-I2M-3-021),National Natural Science Foundation of China(No.31470274),Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs (No. 2012ZD006), and Jiangsu provincial SixTalent Peaks(No.2013-WSW-060).