Epigenetic Mechanisms of Human Papillomavirus-Associated Head and Neck Cancer.
HPV-ASSOCIATED OPSCC DISPLAYS DISTINCT DNA METHYLATION PROFILES
By far, the most common epigenetic event in the human genome is the addition of a methyl group to the carbon-5 position of cytosine nucleotides. This covalent DNA modification occurs predominantly in cytosines immediately preceding guanine nucleotides (the CpG dinucleotide). Evidence has now established that aberrant DNA methyla tion and chromatin remodeling associated with promoters, or first exons of genes, are mechanisms frequently associated with the transcriptional silencing of critical genes in head and neck squamous cell carcinoma (HNSCC) and other cancers. In HNSCC, numerous studies have identified promoter DNA methylation of such genes as CDKN2A (p16), DAP kinase (DAPK), and DNA repair genes MGMT and MLH1 (8) In comparisons of epigenetic profiles of HNSCCs, there is also evidence that epigenetic changes observed in head and neck cancer are subsite dependent. (9) It has also been shown that subsets of epigenetic events associated with individual genes have prognostic value in HNSCC tumors. (9) For example, methylation of the promoter region of MGMT is associated with increased tumor recurrence and decreased patient survival, independent of other factors. (10) It is therefore likely that epigenetic events associated with active HPV infection may influence OPSCC progression.
Recent evidence suggests that HPV-driven HNSCC primary tumors display a unique epigenetic profile. (11) A recent study by our group looking at differential methylation in different anatomic sites of HNSCC revealed that when separating OPSCC by HPV status, almost three times as much differential methylation (tumor to adjacent normal) was observed in HPV-positive cases compared with their HPV-negative counterparts. (7) Similarly, Sartor and colleagues (12) used similar high-throughput array platforms to show that HPV-positive OPSCC cell lines have significantly higher methylation in genic and LINE-1 regions than corresponding HPV-negative cell lines. The methylated loci observed in cell lines correlated with levels observed in OPSCC primary tumors when stratified by HPV status.
More recently, our group has used such differences in DNA methylation to identify a 22-CpG loci panel that showed a statistically significant difference in methylation when comparing HPV-positive with HPV-negative OPSCC. (13) Among these 22 CpGs were 4 CDKN2A-specific loci located downstream of the p16(INK4A) transcription start site. DNA hypermethylation of this CDKN2A-specific region correlated with a significant increase in p14(ARF) and p16(INK4A) expression in HPV-positive OPSCC primary tumors, suggesting a potential mechanism connecting HPV positivity and expression of CDKN2A transcripts that is separate from tumor suppressor proteins p53 and pRb, and may involve direct epigenetic regulation.
Recent studies have shown that HPV-positive OPSCC tumors have higher levels of gene promoter methylation compared with HPV-negative tumors. (14) Kostareli and colleagues (15) used an array-based approach to identify specific alterations in genome-wide promoter methylation in HPV-positive OPSCCs, and they identified a signature that predicted clinical outcome. (15) Similarly, Wilson and colleagues (16) identified 43 hypermethylated promoter regions associated with HPV in HNSCC, including 3 cadherins of the polycomb group target genes.
The process of DNA methylation is catalyzed by a group of enzymes known as DNA methyltransferases (DNMTs), the most well-studied of which is the major maintenance DNA methyltransferase DNMT1. In the case of HPV-driven OPSCC, our work has shown increased DNMT1 expression in HPV-positive OPSCC cancers, based on bead chip expression data. (13) Overexpression of DNMT1 has been reported in cervical cancer, so it may be a common mechanism of HPV-driven cancers. (17) Similarly, work by Sartor and colleagues (12) has shown increased expression of a second DNA methyltransferase [DNMT3A] in HPV-positive cells. In addition to altering expression levels, there is evidence that the HPV16 viral oncoprotein E7 can directly interact with the major DNA methyltransferase enzyme DNMT1 via its CR3 zinc finger domain, and is able to stimulate methyltransferase activity in vitro. (18)
The viral E6 oncoprotein may also drive overexpression of DNMT1 through the Sp1 transcription factor. Sp1 is inactive when bound to p53, but in the presence of E6, p53 can be ubiquitinated and degraded. (19) Additionally, transcription of DNMT1 can be regulated by the E2F/Rb pathway. E7 inactivates Rb and causes release of E2F, which can then act as an initiator of DNMT1 transcription. (20) Overall, these observations suggest that one of the underlying mechanisms by which HPV-driven OPSCC tumors alter their DNA methylation profiles is by the targeting of the enzymes catalyzing this epigenetic modification.
TOBACCO SMOKING MAY ALSO HAVE AN IMPACT ON DNA METHYLATION CHANGES IN OPSCC
Tobacco smoke has previously been associated with DNA hypermethylation of CpG sites within promoter regions and exons of tumor suppressor genes, and with global hypomethylation of DNA in HNSCC. (21-24) There have been a number of competing mechanisms proposed for tobaccoinduced changes in DNA methylation. These include DNA damage caused by carcinogens found in tobacco smoke, such as arsenic, chromium, formaldehyde, polycyclic aromatic hydrocarbons, and nitrosamines. These carcinogens also increase expression of the eukaryotic DNA methyltransferases DNMT1 and DNMT3B. Cigarette smoke may also modulate DNA methylation through nicotine effects on gene expression. (25) Furthermore, smoking may act indirectly on DNA methylation through inactivation of folate, vitamin B6 and vitamin Bi2, (26,27) which mediate 1-carbon transfers in the methylation pathway, and are critical for ensuring an appropriate balance in methylation capacity. (28) Local (eg, mucosal) levels of 1-carbon moieties (such as folate) can be depleted by chronic exposure to tobacco smoke. (29) Other proposed mechanisms include the hypoxic effects of carbon monoxide, (30) and de novo methylation during embryogenesis as a result of tobacco exposure in utero. (31) The extent to which these tobacco-induced changes have an impact on HPV-positive OPSCC is unknown.
A number of case-control studies have evaluated the association between DNA methylation and OPSCC as well as other head and neck cancer sites, (14,32-34) but no population-based estimates of tobacco use and DNA methylation in normal oral tissue exist to date. From these case-control studies, a number of candidate genes with CpG-rich regions have been found to be hypermethylated. The strongest evidence for DNA hypermethylation (25%60%) exists for the promoter region of CDKN2A gene. (35-47) CDKN2A serves to inhibit the binding of cyclin D1 to cyclin-dependent protein kinase 4, which may also be hypermethylated in lung cancer and OPSCC, and has been identified as a marker of carcinogenesis. (48,49) Other tumor suppressor genes frequently hypermethylated in OPSCC and other smoking-related cancers include RARb, (50-53) RASSFI, (44,54,55) MGMT, (10,47,50,56,57) and GATA4. (58-60) Hyper-methylation of DAPK has also been reported in saliva and tissue samples of oral carcinoma patients, with significantly higher levels (40%) found in patients who smoked for more than 20 years (40%) compared with 11% in those who smoked for less than 20 years. (61) Other genes that have been found to be hypermethylated in multiple studies of smoking-related cancers include TIMP3, FHIT, RUNX3, MLH1, and CHFR. (50,60,62,63)
In addition to gene-specific hypermethylation events, tobacco smoking has been associated with global DNA hypomethylation. (64,65) Repetitive element sequences, including Alu, LINE-1, and Sat2, have been analyzed (66,67) as a surrogate marker for genome-wide methylation levels because these constitute a substantial portion (more than 30%) of the human genome. Tobacco smoking has been significantly associated with LINE-1 methylation in blood, (24) as well as in noncancerous esophageal biopsies in a dose-response fashion with smoking duration, number of cigarettes a day, and pack-years. (23) Evaluating the changes in DNA methylation in OPSCC that may be caused by tobacco smoking versus HPV will be important in future studies.
EPIGENETIC CHANGES IN HPV-POSITIVE CANCERS EXTEND TO CHROMATIN MODIFICATIONS
Epigenetic regulation of gene expressions requires interplay between DNA methylation, histone covalent modifications, and nucleosomal remodeling. In addition to DNA methyltransferases, the enzymes that contribute to chromatin modifications and nucleosome remodeling include histone deacetylases, histone methyltransferases, and a complex of nucleosomal remodeling factors. Transcriptionally active genes are generally characterized by nonmethylated CpG dinucleotides in promoter regions, and nucleosomes arranged in a more disperse manner that favors access to transcription factors and other regulatory proteins. These nucleosome-depleted regions generally have extensive lysine acetylation of histones and are flanked by nucleosomes marked by the histone modification H3 trimethylated on lysine 4 (H3K4me3). (68) In contrast, transcriptionally inactive genes are generally characterized by the presence of inhibitory polycomb group complex proteins, such as enhancer of zeste 2 (EZH2), a histone-lysine N-methyltransferase that catalyzes the repressive histone modification trimethylation of lysine 27 on histone 3 (H3K27me3).
Changes in levels of histone modifications as well as the activity of histone-modifying enzymes have also been identified in HPV-associated cancers. As in the case of DNA methylation, the epigenetic changes appear to favor a more transcriptionally repressed state for many target genes. In addition to the increased expression of DNMT3A, Sartor and colleagues (12) reported that promoter regions of poly-comb repressive complex 2 (PRC2) targets tend to be much more highly methylated in HPV-positive HNSCC cell lines than in HPV-negative cells when compared with promoters of corresponding non-PRC2 targets. PRC2 (poly-comb repressive complex 2) is one of the two classes of polycomb-group proteins, along with a group of proteins comprising polycomb repressive complex 1 (PRC1). In HPV-positive cervical cancer cells, it has been shown that the PRC2 complex protein EZH2 is a direct downstream target of viral oncoproteins, activating EZH2 at the transcriptional level via E7-mediated release of E2F from pocket proteins. (69) PRC2 has also been shown to mediate H3K27me3 trimethylation and recruit the DNMTs, furthering the link between chromatin changes and DNA methylation. (70) In OPSCC, it has already been shown that primary tumors positive for p16(INK4A) had global elevations of H3K27me3, the product of EZH2 catalysis, as well as increased levels of histone H4 monomethylated lysine 20 (H4K20me1). (71) These changes were accompanied by a depletion of H4 trimethylated lysine 20 (H4K20me3). Taken together, the observed chromatin changes point to a global alteration in epigenetic mechanisms associated with regulatory control of gene expression that may contribute to resistance to apoptosis through epigenetic silencing of tumor suppressor genes. Moreover, EZH2 may serve as a novel therapeutic target for the treatment of HPV-positive OPSCC in future studies.
CHANGES IN DNA METHYLATION ARE ALSO OBSERVED IN HPV-ASSOCIATED CERVICAL NEOPLASIA
Viral infections can trigger host defense mechanisms, such as methylation machinery activation and epigenetic events in both the viral and the host genome. (72,73) Human genomes harbor DNA sequences that resemble retroviral long terminal repeats that, under certain situations, can be inappropriately activated and play a role in malignant transformation. (74) Also, some viruses can themselves regulate expression of their genes through modulation of DNA methylation. (72) Thus, a virus may suppress activation of its genes in a manner that favors the establishment of persistent infection while evading host immune defenses. (72,75) Cervical cancer cell line studies demonstrate that hypermethylation occurs in the late genes of HPV, whereas the long control region and the E6 and E7 oncogenes are unmethylated. (76,77)
Cervical intraepithelial neoplasias that contain oncogenic HPV, such as HPV16, are more likely to persist and progress. (78,79) However, the vast majority of low-grade lesions regress spontaneously without treatment.80,81 Although there are many reports demonstrating that tumor suppressor genes belonging to nearly every cancer pathway have diminished expression due to abnormal promoter hypermethylation in cervical cancer, (72,82,83) hypermethylation of only some of these genes has been detected in cervical precancerous lesions, (84,85) and the association of these events with HPV infection is still unknown. (86) For example, a number of studies have found associations between aberrant methylation of CDKN2A with cervical cancer (82) and high-grade cervical intraepithelial neoplasia. (84) Cervical cell line data suggest hypermethylation of CDKN2A occurs heterogeneously during cervical cancer progression. (12,87,88)
To date, evidence of aberrant methylation in host genes in cervical carcinogenesis has come primarily from cross-sectional studies. (89,90) In addition to CDKN2A, (91-93) these studies have described hypermethylation of cancer genes like HIC1, (92,93) APC, (92) MGMT, (91,92) RAR[beta], (91,93) and GSTP1. (91) Other genes that have been shown to be differentially expressed include APC, Fas, MMP-1, and TNF-[alpha]. (94) However, because of the cross-sectional design of the studies it remains unclear whether the identified epigenetic changes preceded or followed the disease. Also, prior studies each assessed a limited number of potential epigenetic loci targeting primarily gene promoter regions normally responsible for silencing genes, with the broad profile of aberrant DNA methylation events within genes associated with cervical tumorigenesis still unknown.
DISTINCT MOLECULAR PROFILES OF HPV-ASSOCIATED HNSCC INCLUDE ALTERATIONS IN MicroRNA EXPRESSION
An additional epigenetic mechanism that has been recently implicated in HPV-associated cancer is the deregulation of microRNA expression. MicroRNAs are small non-protein-coding RNAs that regulate RNA translation and decay. Global alterations in microRNA expression have been observed in multiple human cancers, and specific microRNAs have been identified with oncogenic as well as tumor-suppressive potential. (95,96) For example, in HNSCC including the oropharynx, miR-21 is reported to be one of the most consistently overexpressed microRNAs, and it has been shown to target several tumor suppressor targets for degradation, including PTEN, TPM1, and Bd-2. (97) Micro-RNAs originating from the miR-106b-25 gene cluster were also reported to be overexpressed in HNSCC. In contrast, decreased expression of microRNAs, such as miR-375, let-7d, and miR-205, has also been reported in HNSCC tumors, with low levels of miR-375, miR-205, and let-7d significantly correlating with decreased overall patient survival. (97-99) Such changes are known to alter cancer phenotype at the molecular level. For example, a companion paper by Jimenez and colleagues (100) in this Special Section shows that suppression of tumor cell invasion by overexpression of miR-375 acts via a reduction in extracellular matrix degradation and mature invadopodia formation.
Although expression of microRNAs, such as miR-21 and miR-375, is commonly altered in HNSCC, there are also changes in expression of other microRNAs that are more specific to HPV-associated disease. Previous work by Lajer and colleagues (101) has shown that HPV-positive HNSCCs have a distinct microRNA profile that distinguishes them from HPV-negative disease, and they were able to identify 36 differentially expressed microRNAs when comparing HPV-positive with HPV-negative HNSCC tumors. These include overexpression of microRNAs, such as hsa-miR363_st and hsa-miR-21_st, when comparing HPV-positive and HPV-negative tumors, in conjunction with decreased expression of microRNAs, such as hsa-miR-31_st and hsamiR-193b-star_st. In addition, the authors found significant overlap in differentially expressed microRNAs when comparing HPV-positive HNSCC and cervical squamous cell carcinoma, suggesting common HPV-dependent perturbations in microRNA expression that are irrespective of the anatomic site of the tumor. These groups of HPV-associated microRNAs included increased expression of miR-15a and miR-16, with corresponding decreased expression of miR-195 and miR-497. (101) There is now further evidence in cervical cancer that suggests some of these expression changes (eg, miR-15a and miR-16) maybe driven directly by viral E6 and E7 oncoproteins. (102) Although intriguing, many of these HPV-associated microRNA changes have not been studied in OPSCC, and they may represent an opportunity for further investigation into mechanisms of carcinogenesis in HPV-driven OPSCC.
METHYLATION OF THE VIRAL GENOME MAY ALSO PLAY A ROLE IN HPV-POSITIVE OPSCC
Like the human genome, the HPV genome is subject to epigenetic regulation through alterations in DNA methylation. As described earlier, the E6 and E7 oncoproteins can modulate the activity of DNMT1. It has been proposed that methylation of viral DNA may be a defense mechanism of the host cell, (103) and/or that methylation may be a mechanism used by HPV to establish persistent infection. (77)
Methylation of the viral genome is an emerging area of study. In cervical neoplasia, viral methylation may be a diagnostic or predictive marker, (104,105) but it is unknown whether or not viral methylation in OPSCC has a similar association with malignant disease status. One important difference in comparing OPSCC and cervical cancer is the fact that studies of HPV-associated cervical carcinogenesis have looked at clearly defined stages, including asymptomatic infection, intraepithelial neoplasia stages I to III, and cancer, (106) whereas OPSCC studies conducted to date have focused on primary cancers. (107)
However, recent studies have suggested there may be some similarities between cervical cancers and HNSCCs with respect to viral methylation. In a small study of 3 HPV-positive HNSCCs, Wilson and colleagues (16) detected increased methylation at the boundary of the late viral genes L1 and L2 compared with other viral genes that have also been reported in cervical cancer. Methylation of the HPV16 long control region was also detected in the HPV-positive samples collected. This is significant because the long control region is important for the expression of E6 and E7 viral oncoproteins. (108-110) However, a large study subsequently reported hypomethylation of the long control region and heterogeneous methylation at other regions of the viral genome in OPSCC. (107) Therefore, more research remains to be done to clarify the role of viral methylation in HPV-driven OPSCC.
Unlike in the cervix, where HPV is a necessary cause, HPV is associated with only a subset of HNSCCs. In this review we summarize the existing evidence of the epigenetic mechanisms that may contribute to maintaining a malignant cell phenotype in HPV-associated OPSCC. The association between HPV detection in OPSCC tumors and improved patient prognosis is well established. Given this, many of the epigenetic mechanisms described in this review have potential as novel drug targets or as biomarkers for response to treatment in HPV-positive OPSCC.
With the availability of 2 prophylactic HPV vaccines, it is clear that there will be a change in the landscape of HPV-associated cancers. However, any impact on the incidence of HPV-associated OPSCC will not be observed for several decades yet. Understanding the epigenetic changes that determine which few cases will progress to malignant disease remains a major goal with clear clinical implications.
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Nicole V. J. Anayannis, MSc; Nicolas F. Schlecht, PhD; Thomas J. Belbin, PhD
Accepted for publication December 16, 2014.
Published as an Early Online Release May 15, 2015.
From the Departments of Pathology (Ms Anayannis and Dr Belbin), Epidemiology & Population Health (Dr Schlecht), and Medicine (Oncology) (Dr Schlecht), Albert Einstein College of Medicine, Bronx, New York.
The authors have no relevant financial interest in the products or companies described in this article.
Reprints: Thomas J. Belbin, PhD, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461 (e-mail: firstname.lastname@example.org).