© Springer International Publishing Switzerland 2017
Wojciech Golusiński, C. René Leemans and Andreas Dietz (eds.)HPV Infection in Head and Neck CancerRecent Results in Cancer Research20610.1007/978-3-319-43580-0_18Predictive Factors for Outcome and Quality of Life in HPV-Positive and HPV-Negative HNSCC
(1)
Department of Otolaryngology, Head and Neck Surgery, University Hospital and Research Group Molecular Mechanisms of Head and Neck Tumors, German Cancer Research Center (DKFZ), Heidelberg, Germany
Abstract
Infection with high-risk types of the human papilloma virus (HPV) is an etiological risk factor for oropharyngeal squamous cell carcinoma (OPSCC) and associated with a better response to therapy and improved survival. A better understanding of the molecular principles underlying the differences in clinical behavior could pave the way to establish more effective and less toxic therapy for HPV-positive OPSCC and their HPV-negative counterparts. Compelling experimental evidence demonstrates that extensive global reprogramming of epigenetic profiles is as important as genetic mutations during neoplastic transformation and malignant progression, including HPV-positive OPSCC. In this chapter, the current knowledge on HPV-related alterations in DNA methylation, histone modification, and chromosome remodeling will be summarized and assessment of cancer-related profiles will be discussed as a valuable tool to gain important diagnostic or prognostic information for therapeutic decision-making and clinical management of HNSCC patients.
Keywords
DNA methylationHistone modificationChromosome remodelingDNMTHDAC1 Background and Clinical Relevance
Infection with high-risk types of human papilloma virus (HPV), predominantly type 16, has been identified as important risk factor in an escalating number of patients with head and neck squamous cell carcinoma (HNSCC) (Gillison et al. 2015). HPV-positive tumors arise mainly in the oropharynx (oropharyngeal squamous cell carcinoma (OPSCC)) and display distinct biological and clinical features as opposed to HPV-negative HNSCC (Mehanna et al. 2013; Ndiaye et al. 2014; Hayes et al. 2015; Network 2015). HPV-positive OPSCCs are associated with a better response to therapy and improved survival, justifying the HPV status as one of the most accurate prognostic biomarkers in primary and progressed tumors (Ang et al. 2010; Chaturvedi et al. 2011; Fakhry et al. 2014; Kang et al. 2015). It is foreseen that a better understanding of the molecular principles underlying the differences in clinical behavior could pave the way to establish more effective and less toxic therapy for HPV-positive OPSCC but also their HPV-negative counterparts.
The manifestation of HNSCC is a multifactorial process, which is characterized by the accumulation of genomic events and somatic mutations affecting tumor-relevant signaling and gene regulatory networks. Recent advances in next-generation sequencing provided a valuable tool to unravel the mutational landscape of HNSCC, including differences between HPV-positive and HPV-negative tumors (Hayes et al. 2015; Network 2015). However, most ongoing clinical trials still focus on therapeutic targets that were already known before cancer genomes were mapped. This is mainly due to the fact that our ability to interpret and translate these complex data sets from bench to bedside is hampered by the vast amount of information generated by global sequencing studies (Edwards et al. 2011).
Clinical and experimental studies of the last decades provide compelling evidence that extensive global reprogramming of epigenetic profiles is as important as genetic mutations during neoplastic transformation and malignant progression. Originally, epigenetics was defined as heritable traits that are not linked to changes in the DNA sequence. Nowadays, the term epigenetics is used to describe the mechanisms by which DNA methylation as well as chromatin-associated proteins and their posttranslational modifications regulate gene transcription. Cell-type-specific epigenetic patterning is essential for the establishment and maintenance of cellular integrity during development and tissue homeostasis, and its deregulation has been reported for all human malignancies (Berdasco and Esteller 2010; Baylin and Jones 2011). The most extensively studied epigenetic marker is DNA methylation, and together with posttranslational histone modifications affecting chromatin remodeling and specific miRNA expression signatures, it defines the epigenetic landscape of human cancers, including HNSCC (Kostareli et al. 2012; Koffler et al. 2014; Le et al. 2014; van Kempen et al. 2014; Anayannis et al. 2015). It is well documented that all of the major classes of cancer-causing agents including viruses elicit alterations in epigenetic patterning (Minarovits et al. 2016). This is of particular clinical relevance as many epigenetic modifications persist or increase during disease progression, and assessment of cancer-related profiles provides a valuable tool to gain important diagnostic or prognostic information for therapeutic decision-making and clinical management of HNSCC patients (Koffler et al. 2014; van Kempen et al. 2014). Moreover, the dynamic and reversible nature of epigenetic reprogramming makes key nodes of its regulatory circuits bona fide drug targets for precision medicine (Azad et al. 2013).
2 HPV-Related Alterations in DNA Methylation
DNA methylation is a physiologic epigenetic modification, which occurs primarily on the addition of a methyl group to a CpG dinucleotide in the DNA sequence. CpGs are asymmetrically distributed into poor and dense regions (CpG islands). CpG islands are predominantly located in the promoter regions or first exon of approximately half of all genes (Jones and Baylin 2002). DNA methylation is catalyzed by the enzymatic activity of DNA methyltransferases (DNMTs) of which three variants have been identified in humans: DNMT1, DNMT3A, and DNMT3B (Subramaniam et al. 2014). Aberrant DNA methylation is a hallmark of all human malignancies, including HNSCC, and distinct profiles have been attributed to environmental factors, patient habits (e.g., tobacco and alcohol consumption), and viral infection (Kostareli et al. 2012; van Kempen et al. 2014; Minarovits et al. 2016). The cancer methylome displays a characteristic loss of global DNA methylation in repetitive regions and concomitant accumulation of gene promoter methylation. Although the underlying molecular principles and effect of global DNA hypo-methylation remain elusive, it is thought to contribute to chromosomal instability and activation of proto-oncogene expression (Robertson 2005; Jones and Baylin 2007). It is worth noting that several studies reported a HPV-related difference in hypo-methylation of repetitive LINE-1 elements, indicating a more efficient maintenance of global DNA methylation accompanied by reduced genetic instability in HPV-positive HNSCC (Richards et al. 2009; Poage et al. 2011; Sartor et al. 2011). This assumption is supported by recent studies addressing the quality and quantity of genomic aberrations in HPV-positive and HPV-negative HNSCCs (Klussmann et al. 2009; Wilting et al. 2009; Agrawal et al. 2011; Stransky et al. 2011).
2.1 HPV and Gene Promoter Hyper-Methylation
Gene promoter hyper-methylation often causes reduced transcription of tumor suppressor genes involved in cellular processes of DNA damage repair, detoxification, cell cycle regulation, and apoptosis (Rodriguez-Paredes and Esteller 2011). In cancer, transcriptional silencing by gene promoter methylation may occur even more frequently than structural inactivation of genes by deletion or somatic mutation (Rodriguez-Paredes and Esteller 2011; Azad et al. 2013). Numerous studies have explored HPV-related differences in the profile of gene promoter methylation; however, many reports evaluated only a limited number of selected genes and did not focus solely on OPSCC, which is the most common site for HPV-related tumors in the upper aerodigestive tract (Kostareli et al. 2012; van Kempen et al. 2014). More recent studies focused on global analysis of gene promoter methylation in HPV-positive versus HPV-negative HNSCCs with the aim of gaining a detailed view of clinically relevant alterations and unraveling affected signaling and gene regulatory networks (Koffler et al. 2014). Collectively, these studies reported a trend toward a higher level of gene promoter hyper-methylation in HPV-positive tumors (Sartor et al. 2011; Colacino et al. 2013; Lechner et al. 2013; Lleras et al. 2013). The widespread gain of gene promoter methylation raises the question whether HPV-positive HNSCC resembles a CpG island methylator phenotype (CIMP), which was originally discovered in colorectal cancer (Hughes et al. 2013; Suzuki et al. 2014). Combinatorial ectopic expression of the viral oncogenes E6 and E7 in an HPV-negative cell line partially phenocopied the CIMP signature seen in HPV-positive tumors and established E6 as the main viral effector gene (Lechner et al. 2013). It is worth noting that HPV-related tumors with CIMP had a poor clinical outcome with significantly shorter survival (Lechner et al. 2013). An association of CIMP and poor prognosis was also reported for patients with oral cancer, though CIMP was not an independent factor in predicting prognosis (Jithesh et al. 2013).
2.2 Functional Interaction of Viral Proteins with DNMTs
One molecular mode of action by which HPV might alter profiles of gene promoter methylation is due to direct targeting the expression and enzymatic activity of DNMTs by viral oncoproteins (Minarovits et al. 2016). Increased expression of DNMT1 and DNMT3A was evident in HPV-positive tumor cell lines and primary OPSCCs (Sartor et al. 2011; Lechner et al. 2013; Schlecht et al. 2015). Additionally, viral oncoproteins stimulate DNMT activity in vitro and tumor cell lines, which is at least in part due to a direct physical interaction of E7 and DNMT1 (Burgers et al. 2007; Laurson et al. 2010; D’Costa et al. 2012). Chromatin immunoprecipitation assays further confirmed E7-DNMT1 complex formation at the CCNA1 promoter serving as a model for HPV-related gene promoter methylation (Chalertpet et al. 2015).
2.3 HPV-Related Gene Promoter Methylation Patterns and Signaling Pathways
Distinct HPV-related methylation patterns extrapolated from global gene promoter methylation profiling provide a valuable molecular tool for diagnostic and prognostic assessment of HNSCC patients (Colacino et al. 2013; Kostareli et al. 2013). But, they also facilitate an integrative data analysis to infer clinically relevant differences in signaling and gene regulatory networks taking into account the HPV status (Koffler et al. 2014). This knowledge could pave the way to identify promising new drug targets for a more specific and individualized therapy of HNSCC patients.
As an example, functional annotation of a gene panel with HPV-related promoter methylation indicated differential activity of WNT/β-catenin signaling, PPAR regulation, retinoic acid signaling, c-KIT signaling, and cell–cell or cell–matrix adhesion (Worsham et al. 2013). Differences in retinoic acid metabolism and signaling due to gene promoter methylation and based on the HPV status of OPSCC were also suggested by Kostareli and colleagues (Kostareli et al. 2013). In another study, gene-set enrichment analysis identified several targets of polycomb repressive complex 2 (PRC2) that were affected by HPV-related gene promoter methylation, including multiple members of the cadherin superfamily such as CDH8, CDH15, PCDH8, PCDH9, PCDH10, and PCDHB3 (Lechner et al. 2013). Finally, Fertig and colleagues applied integrative data analysis based on DNA methylation and gene expression patterns to infer biologically significant molecular pathways that may be exploited as therapeutic targets (Fertig et al. 2013). This approach revealed specific gene promoter methylation patterns that regulate gene expression in HPV-negative HNSCC and distinguish it from HPV-positive HNSCC. Analysis of these differentially regulated genes indicated that activation of the Hedgehog pathway was specific for HPV-negative HNSCC, which was confirmed by increased levels of GLI1, the primary Hedgehog target, in HNSCC compared to normal mucosa with the highest GLI1 expression in HPV-negative tumors.
3 HPV-Related Alterations in Chromatin Architecture
Epigenetic regulation of gene expression requires a complex interplay between DNA methylation, histone modifications, and nucleosome remodeling. The most commonly studied covalent modifications of histones are posttranslational acetylation, deacetylation, and methylation at the amino-terminal ends, and key enzymes are histone acetyltransferases (HAT), histone deacetylases (HDAC), and histone methyltransferases (HMT). Moreover, large complexes of nucleosome remodeling factors regulate gene expression by modulation of the chromatin architecture.
Despite compelling evidence that cancer-associated chromatin states are of clinical relevance, our knowledge on HPV-related alterations in histone modification and nucleosome remodeling as well as their functional interaction with DNA methylation profiles remains largely elusive. A recent study on global DNA methylation profiles of HPV-positive and HPV-negative HNSCCs suggested that HPV modulates the cancer epigenome through hyper-methylation of PRC2 target genes, which are implicated in tumor progression and metastasis (Lechner et al. 2013). Sartor and colleagues also reported a distinct promoter hyper-methylation of PRC2 target genes in HPV-positive as compared to HPV-negative HNSCC cell lines (Sartor et al. 2011). PRC2 maintains the transcriptional repression of a large number of genes with key regulatory roles in development and differentiation, and PRC2 proteins are required for normal embryonic development and exhibit a well-established role in stem-cell maintenance (Conway et al. 2015). It is worth noting that cancer-specific promoter hyper-methylation is more likely for PRC2 targets than non-targets (Ohm et al. 2007; Schlesinger et al. 2007).
The enzyme enhancer of zeste homolog 2 (EZH2) is the catalytic component of PRC2 and acts as an HMT at H3K27, resulting in gene silencing via chromatin condensation. Dysregulation of the repressive H3K27 trimethylation (H3K27me3) mark in HNSCC contributes to aberrant squamous differentiation (Gannon et al. 2013), and p16INK4A-positive OPSCCs display global elevations of H3K27me3 patterns (Biron et al. 2012). Collectively, these data strongly suggest that viral oncoproteins induce epigenetic regulation of PRC2 target genes during HNSCC pathogenesis by altered expression or activity of HMTs and thereby modulate the chromatin architecture at corresponding gene promoters. Indeed, EZH2 is activated at the transcriptional level in HPV-positive cervical cancer cells by E7-mediated release of E2F from pocket proteins (Holland et al. 2008). In a more recent study, Sharma and colleagues also demonstrated a functional interplay between E7 and HOTAIR, a long noncoding RNA that recruits PRC2 to target gene promoters (Sharma et al. 2015).
4 Conclusion and Perspectives
An increasing body of experimental studies has provided compelling evidence that viral oncoproteins of HPV16 interact with key components of the cellular epigenetic machinery to reprogram the gene expression pattern and thereby alter cellular traits of the infected host cell. Monitoring HPV-related disruption of the epigenetic program represents a powerful tool for diagnosis, prognosis, and treatment decision-making, and due to its reversible nature serves as a bona fide target for a more effective and less toxic treatment of HNSCC patients with HPV-positive tumors. Epigenetic-based therapies for cancer treatment have been approved, and additional inhibitors for key regulators of DNA methylation, histone modification, and chromosome remodeling have shown promise in preclinical trials. Although the biology of epigenetic regulation is complex and our knowledge on the underlying regulatory circuits is incomplete, this new generation of more specific and potent inhibitors will hopefully be available for clinical use in the coming years (Cai et al. 2015). Another promising option for epigenetic-based therapy is its combination with already established or novel treatment regimens. As an example, demethylating drugs improve the efficacy of therapeutic viral DNA vaccines in a variety of HPV-associated malignancies (Lu et al. 2009). However, the lack of reliable molecular biomarkers to predict either clinical activity or resistance of epigenetic-based therapy is a serious problem limiting the translation from bench to bedside (Helin and Dhanak 2013).
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