Epigenetic and epitranscriptomic changes in colorectal cancer: Diagnostic, prognostic, and treatment implications

Accepted Manuscript Epigenetic and epitranscriptomic changes in colorectal cancer: diagnostic, prognostic, and treatment implications Elisa Porcellini, Noemi Laprovitera, Mattia Riefolo, Matteo Ravaioli, Ingrid Garajova, Manuela Ferracin PII:

S0304-3835(18)30071-5

DOI:

10.1016/j.canlet.2018.01.049

Reference:

CAN 13717

To appear in:

Cancer Letters

Received Date: 6 November 2017 Revised Date:

7 January 2018

Accepted Date: 12 January 2018

Please cite this article as: E. Porcellini, N. Laprovitera, M. Riefolo, M. Ravaioli, I. Garajova, M. Ferracin, Epigenetic and epitranscriptomic changes in colorectal cancer: diagnostic, prognostic, and treatment implications, Cancer Letters (2018), doi: 10.1016/j.canlet.2018.01.049. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Epigenetic and epitranscriptomic changes in colorectal cancer: diagnostic, prognostic, and treatment implications Elisa Porcellini1*, Noemi Laprovitera1*, Mattia Riefolo1,2, Matteo Ravaioli2, Ingrid Garajova1,2 & Manuela Ferracin1 1

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Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna (Italy) 2 Sant’Orsola-Malpighi Hospital, Bologna (Italy) *

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equal contribution

Correspondence to: Manuela Ferracin, [email protected]

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Keywords: epigenetics; epitranscriptomics; colorectal cancer; DNA methylation; Histone modifications; RNA editing Abstract

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A cancer cell is the final product of a complex mixture of genetic, epigenetic and epitranscriptomic alterations, whose final interplay contribute to cancer onset and progression. This is specifically true for colorectal cancer, a tumor with a strong epigenetic component, which acts earlier than any other genetic alteration in promoting cancer cell malignant transformation. The pattern of progressive, and usually subtype-specific, DNA and histone modifications that occur in colorectal cancer has been extensively studied in the last decade, providing plenty of data to explore. For this tumor, it became recently evident that also RNA modifications play a relevant role in the activation of oncogenes or repression of tumor suppressor genes. In this review we provide a brief overview of all epigenetic and epitranscriptomic changes that have been found associated to colorectal cancer till now. We explore the impact of these alterations in cancer prognosis and response to treatment and discuss their potential use as cancer biomarkers.

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ACCEPTED MANUSCRIPT Introduction to epigenetics and epitranscriptomics

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Conrad Waddington originally coined the word “epigenetics” in 1942, to describe the poorly understood phenomena that guided a zygote into developing a very complex organism. Currently, epigenetics is mostly defined as the study of heritable changes in gene expression that cannot be explained by modifications in the DNA sequence [1]. Several mechanisms are responsible for such changes, and their disruption leads to the development of different pathologies, including cancer [2, 3]. In the last few decades, several studies analyzed the extent of all (known) epigenetic changes in human cancers and their active contribution to tumor development and progression. Many researches focused on the identification of cancer aberrant “epigenome”, which includes the global changes in DNA methylation and the wide spectrum of altered histone modifications that eventually determine the transcriptional activity of a specific genetic locus [4].

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DNA methylation, especially promoter methylation, has been extensively studied for its involvement in several vital processes in human tissues, such as embryonic development, imprinting, and tissue differentiation. It occurs by enzymatic activity of DNA methyl transferases (DNMTs) that are responsible for the addition of a methyl group to the 5’ position of cytosine to produce 5-methyl cytosine. The consequence of this addition is the silencing of genes and noncoding genomic regions. DNMTs can have a maintenance activity (e.g. DNMT1), preserving the normal methylation patterns during DNA replication, or a de novo activity (e.g. DNMT3A and DNMT3B), modifying the original methylation pattern [4, 5]. In humans and other mammals, this addition occurs in CpG dinucleotides. Methylated CpG dinucleotides are usually located in gene bodies and large repetitive sequences (e.g. LINE-1 and SINE/Alu), commonly identified in centromeres, and other retrotransposon elements. However, some regions that are extremely CpG rich (>50% CpG content), which are called CpG islands (CpGI), can be usually found in about 60-70% of all gene promoters, mostly oncosuppressor genes. During cancer development, the CpGI methylation pattern is altered, showing genome-wide hypomethylation and site-specific CpG island hypermethylation. Usually, the hypomethylation of regions such as large repetitive elements, retrotransposons or introns, results in genomic instability [6, 7]. In addition to CpG islands, a study by Irizarry et al. revealed a relevant role for CpG island shores in gene expression deregulation in cancer. They analyzed the DNA methylation changes in colon cancer CpG island shores, which are regions located in close proximity (within 2kb) to CpG islands, with a lower CpG density. The study proved that methylation changes in CpG island shores are frequent in colon cancer and strongly associated with gene repression [8]. Moreover, CpG island shores methylation pattern is tissue-specific, regarding mostly regions that are involved in cellular reprogramming [9]. Histone covalent post translational modification (PTM) is the second most common epigenetic mechanism [10]. The core histones, H2A, H2B, H3, and H4, constitute an octameric complex, around which 147 DNA base pairs are wrapped in order to build the nucleosome that packages chromatin, repressing transcription [11]. Several enzymes can modify histone’s N-terminal tails catalyzing the post-translational addition of small chemical modifications, such as acetyl, methyl, 2

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ubiquityl, phosphoryl and SUMO groups [12]. These PTMs can affect gene activity by altering chromatin structure or recruiting histone modifiers, affecting several processes, including apoptosis, DNA replication, DNA damage response [13]. Genome-wide analyses have highlighted the possibility that combinations of several histone PTMs could represent a real code, necessary to modify the chromatin structure and to define the activation or repression of gene expression [14]. Euchromatin, in fact, can be distinguished by its high levels of acetylation (ac) and trimethylation (me) of lysines (K) H3K4, H3K36 and H3K79. On the contrary, heterochromatin has low levels of acetylation and high levels of H3K9, H3K27 and H4K20 methylation. Histone modification levels are predictive of gene activity on the basis of specific PTMs. For instance, actively transcribed genes present high levels of H2BK5ac, H3K27ac, H3K4me3, and H4K20me1 in the promoter and high H3K79me1 and H4K20me1 in the gene body [14, 15]. Different combinations of histone modifications can occur simultaneously to activate or repress gene expression, therefore it is important to investigate the whole pattern of histone marks in a specific genomic region to understand its regulation and activity [16]. Current evidences suggest that histone modifications could be involved in primary events in cancer by altering gene expression and regulating oncogenes and tumor suppressors genes [17]. In addition, it has been demonstrated that specific histone modifications could be associated with unfavorable prognosis and high risk of recurrence [18].

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On the other hand, the term “epitranscriptomics” was coined very recently to describe the functional post-transcriptional modifications that occur on RNA molecules [19, 20]. These include several RNA processing events: RNA editing, splicing and methylation. These modifications affect all types of RNA molecules and different RNA editases participate to this process. To date, with the help of high-throughput technologies, more than one-hundred chemical modifications have been discovered in the transcriptome. The most studied modifications comprise N6-methyladenosine (m6A), 5-methylcytosine (m5C), inosine (I), pseudouridine (Ψ), N1-methyladenosine (m1A) and 5hydroxymethylcytosine (hm5C) [20, 21]. The most frequent and reversible RNA modification in eukaryotic cells is N6-methyladenosine (m6A). This modification is operated by specific RNA methylases (writers) and demethylases (erasers) and specific alterations in m6A levels have been detected in human diseases [22]. In colorectal cancer it has been demonstrated that increased levels of m6A reader and mRNA elicase YTHDC2 can increase the translation of HIF-1α thereby promoting metastases formation [23]. In addition to chemical modifications with an impact on RNA structure, stabilization and RNA-protein interaction, RNA editing performed by adenosine and cytidine deaminases (ADARs and APOBECs respectively) can change the transcript sequence. Double-stranded RNA-specific adenosine deaminase (ADAR) family members catalyze the hydrolytic deamination of adenosine to inosine in both coding and non-coding RNAs, especially in Alu sequences [24]. This modification, since inosine is recognized as guanosine, leads to an A-to-G substitution. Differently, activation-induced cytidine deaminase (AID) and its relative APOBEC cytidine deaminases are a family of evolutionary-conserved Zn-dependent cytidine deaminases that catalyze the C-to-U conversion on mRNA [25, 26]. Recent studies reported a relative abundance of adenosine-to-inosine (A-to-I) RNA editing sites in several cancer types, suggesting their possible involvement in the carcinogenesis process [27]. Indeed, RNA editing has the 3

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potential to generate novel proteins from the same transcripts (RNA recoding) and cancer cells could use this to their advantage. Most RNA editing sites are detected in mRNA regulatory regions, such as introns, 3’-UTRs, 5’-UTR and intronic retrotransposable elements (SINE/Alu and LINE-1 etc.). Rarely, they occur in coding regions. ADAR’s modifications (A-to-I) are very frequently detected in close proximity to Alu elements, their primary target of editing. Emerging data highlighted the possible involvement of RNA changes performed by ADARs or APOBECs in cancer development and progression [28]. Cancer-specific patterns of RNA editing, whose levels can be precisely increased or decreased, have been observed in several tumor types. Furthermore, an increase in RNA editing levels often correlates with increased expression of the ADAR enzyme [28]. In addition, RNA editing can affect non-coding RNAs [24, 29], which in cancer leads to a dysregulated miRNA expression or to changes in miRNA-3’UTR interactions [30, 31]. In Figure 1, we summarized the main epitranscriptomic alterations observed in cancer till now, including their consequences in cancer development. This field is relatively new, meaning that several aspects remain to be elucidated.

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Colorectal cancer

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Colorectal cancer (CRC) is the most frequent gastrointestinal cancer, the third most common neoplasm worldwide and the third leading cause of tumor-associated death in developed countries (source: IARC GLOBOCAN). In the last decade, several studies have demonstrated that CRC arises because of epigenetic as well as genetic alterations in several key tumor-suppressor genes and oncogenes [32]. Classically, three major pathways of genomic instability have been described that contribute to the development of CRC: chromosomal instability (CIN), microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) [33]. CRCs with CIN (about 85% of sporadic tumors) are characterized by chromosomal gains and losses and aneuploidy; those with MSI are identified by the presence of frequent insertion and deletion mutation in repetitive DNA sequences; CIMP-positive CRCs are characterized by a global genome hypermethylation in CpG islands of 8 markers (RUNX3, MLH1, NEUROG1, CDKN2A, IGF2, CRABP1, SOCS1 and CACNA1G). CIMP-positive tumors represent 15% of CRC and are partially overlapped with MSI tumors, although CIMP is significantly associated with a specific gene expression profile [34] and worse prognosis, independently from the MSI status [35]. MSI tumors are caused by defects in the mismatch repair system (dMMR), and the main proteins involved are MLH1/PMS2 and MHS2/MHS6 [36]. Traditionally, in order to define CRC MSI status it was necessary to perform the analysis of five microsatellite markers: BAT25 and BAT26 (mononucleotides) and D2S123, D5S346, D17S250 (dinucleotides). MSI was considered high (MSHH) if ≥ 30% of the repeats were unstable, low (MSI-L) if they were less than 30%, and microsatellite stable (MSS) if no unstable repeats were found [37]. MSI/hypermutated carcinomas show typical features: right-sided location, mucinous and poor differentiation with signet ring cells and high density of infiltrating lymphocytes [38]. MSI status is important because these tumors have a better stage-adjusted survival and do not benefit of adjuvant therapy with 5-fluorouracil [39]. Furthermore, colorectal carcinomas with MSI-H are associated with higher expression of programmed death receptor ligand 1 (PD-L1) and show 4

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durable response to PD-1 blockade [40]. Nivolumab, a PD-1 immune checkpoint inhibitor, therapy have shown promising results in patients with MSI-H metastatic colorectal cancer [41]. In the past few years, two novel molecular classifications of CRC have been proposed. One classification, based on the TCGA consortium results, differentiates CRCs as hypermutated/ultramutated tumors or non-hypermutated/MSS tumors (comprising CIN CRCs) [42]. The second one is based on the results of the Consensus Molecular Subtypes (CMS) Consortium and classifies CRCs in four molecular subtypes: CMS1 (MSI, immune, 14%), CMS2 (MSS, canonical, 37%), CMS3 (MSS, metabolic, 13 %) and CMS4 (MSS, mesenchymal, 23 %), with a residual unclassified group (mixed features, 13 %) [43]. These novel classifications have the potential to provide a better prognostic stratification of CRC patients, both in terms of clinical management and therapeutic choices [44, 45]. Recently, Bramsen et al. presented a framework for CRC stratification to further improve patient prognostication, using both transcriptional and DNA methylation profiles from 1.100 CRCs. In this analysis, they identified five tumor subtypes (archetypes), three of them characterized by specific prognostic biomarkers, suggesting that the tumor microenvironment and microbiome have a strong influence on CRC prognosis [46]. Figure 2 summarizes all the main epigenetic and epitranscriptomic modifications that have been discovered in CRC, whose role in CRC carcinogenesis is further detailed in the following sections. Global methylation profile of colorectal cancer

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Multiple studies highlighted the pivotal role of aberrant DNA methylation and histone posttranslational modifications in CRC onset and development, even though other epigenetic processes (nucleosome occupancy, chromatin remodeling and looping) play a role in this disease [32, 47, 48]. CRC epigenetic alterations seem to be more frequent than genetic alterations and occur early in the carcinogenesis process, representing a promising tool for early diagnosis and prognosis [32]. Indeed, colorectal cancer is one of the first tumor types for which a global DNA hypomethylation was described. Later, several studies reported the site-specific promoter hypermethylation of specific tumor suppressor genes [49, 50]. More recently, global methylation analyses performed with high-throughput technologies provided a better characterization of all epigenetic alterations occurring in CRC [51]. The search for differentially-methylated regions (DMRs) using bisulfite-converted DNA has been done with the use of different techniques, but mainly with Illumina methylation arrays. Ang et al. first described a total of 202 CpG sites differentially methylated between CRC and normal tissue using 27K Illumina methylation arrays. From this study, additional methylation differences were associated to CIMP levels, KRAS and BRAF mutations [52]. In 2011, three different groups published their results on CRC global methylation profile [53-55]. Oster et al. performed a whole-genome methylation analysis to identify novel hypermethylated genes in pre-malignant and malignant colorectal lesions [55]. In this study, the authors identified and validated 15 hypermethylated sites in normal mucosa, adenomas, CRC patient (MSI or MSS). The 15 selected genes hypermethylated in adenomas and carcinomas included FLI1, ST6GALNAC5, TWIST1, ADHFE1, JAM2, IRF4, CNRIP1, NRG1 and EYA4, and in carcinomas only: ABHD9, AOX1 and RERG, or in MSI but not MSS carcinomas: RAMP2, DSC3 and MLH1. The correlation between 5

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methylation and gene expression was also assessed. An inverse correlation between methylation and gene expression was observed for 6 (IRF4, ADHFE1, JAM2, NRG1, FLI1 and MLH1) out of 15 selected genes. A genome wide methylation study conducted on 24 CRC patients, including 27K loci from 14K genes was performed by Kibriya’s group [53]. Differential methylation was found in CRC tissue when compared with normal tissue from the same patients. In particular, they found a total of 875 significantly differentially methylated autosomal loci in CRC tissue (vs normal colonic tissue) of which 275 were hypomethylated and 600 were hypermethylated including FLJ25477, ITGA4, DAB2IP, KCNQ5, ZNF625, C1orf165, PRKAR1B, MDFI, C2orf32, RYR2, FLI1, RIC3, TRH, VGCNL1, EYA4. Similarly, Kim at al. performed a global methylation profile of 22 matched normal and tumor pairs, validating in a larger cohort a panel of 10 hypermethylated (ADHFE1, BOLL, SLC6A15, ADAMTS5, TFPI2, EYA4, NPY, TWIST1, LAMA1, GAS7) and 2 hypomethylated (MAEL, SFT2D3) CpG sites [54]. Using a different medium-throughput technology, MethylCap-seq, Simmer and colleagues identified a panel of DMRs in 24 matched CRC/normal colon samples [56]. They compared the methylation profile of CRC with that of embryonic stem cells, discovering that CRC hypermethylated promoters are usually silent in normal adult cells and encompassing bivalent chromatin regions of the human genome. In the following years, more comprehensive studies were performed taking advantage of the release of Infinium 450K Illumina methylation arrays, which was designed to assess 450K CpG islands in both genic and intergenic islands, shores and shelves. A research conducted by Naumov et al. in 22 pairs of CRC tumors and adjacent mucosa and in 19 colon tissue samples obtained from healthy controls [57] identified a DNA methylation profile able to discriminate tumor from healthy colon samples. In particular, 36 genes were found significantly hypermethylated in CRC: 28 also reported by other studies [8, 53] and 8 new genes (SND1, OPLAH, C1orf70, MIR124–3, C9orf50, ZFP64, DPY19L2 and ZNF829). A genome-wide array-based study conducted on 41 normal colon tissues, 42 tubular adenomas and 64 CRC patients, showed a different methylation pattern not only in CRC progression from normal to adenoma to cancer, but also in the adenoma and carcinoma subgroups. These methylation profiles correlated with CIMP and KRAS mutations and seemed to play an important role in influencing the transition from normal colon epithelial cells to CRC [58]. The analysis of Timp and colleagues, instead, was aimed to identify common hyper/demethylated regions across several cancer types, including colon cancers. They discovered the existence of hypomethylated CpG blocks that occur early in cancer development, independently from the cancer type [59]. The last three studies have been recently used in a meta-analysis by Durso et al. [60] in an attempt to identify the driver epigenetic alterations in colorectal tumors. Recently, Hanley et al. combined laser-capture microdissection with bisulfite sequencing to discover cancer-associated DNA methylation changes in human aberrant crypt foci (ACF), the earliest most probable precursor to CRC. In this study, they identified, for both CRC and ACF, several differentially methylated regions (DMRs). In CRC, 75% of them was hypomethylated and 25% was hypermethylated; on the contrary, in ACF the state was reversed (66% hypermethylated and 33% hypomethylated). This outcome suggested that reversing the global methylation status 6

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could be fundamental for CRC progression [61]. The analysis of all DMRs, in CRC and ACF, based on their genomic location provided further information. Promoter regions were mostly hypermethylated in CRC and ACF. On the contrary, DMRs in non-promoter, gene-bodies and intergenic regions, were mainly hypomethylated in cancer and hypermethylated in ACF if compared to a matched normal mucosa. This outcome proves that genome-wide hypomethylation represents a key step in the advancement of CRC [61]. Overall, epigenetic changes seemed to be more abundant in ACF than previously thought. These alterations occur in genes that are implicated in cellular identity and differentiation, and their aberrant methylation could contribute to the establishment of early colonic neoplasia. However, in ACF these methylation changes are insufficient to alter gene expression.

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Less successful has been the attempt to perform the analysis of CRC patients’ blood global methylation profile to identify disease biomarkers. A study based on the methylation pattern of circulating leukocytes from 46 CRC patients and 140 controls was able to identify only two significantly differentially methylated CpGs in the promoter region of KIAA1549L gene to use as disease biomarker [62], but their sensitivity was inferior to other established biomarkers for CRC (see further sections).

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The latest release of methylation data from The Cancer Genome Atlas (TCGA) consortium has provided novel data from colorectal cancers to correlate with clinically relevant features [42]. Analyzing these data, Rokavec and colleagues found that CRC tumor methylation was significantly lower in liver metastases compared with primary tumors, and hypomethylation in regulatory regions was more extensive in primary tumors from patients with liver metastasis compared with patients without metastasis [63]. Many other publications are to be expected from the elaboration of the methylation data collected in the TCGA project.

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Aberrant DNA methylation in CRC and its role in cancer diagnosis and prognosis

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Genome-wide DNA hypomethylation plays a pivotal role in CRC development, since this feature induces genomic instability and global loss of imprinting (LOI) [64-67]. Global hypomethylation generally occurs on repetitive transposable elements, such as LINE-1 or SINE/Alu in many cancer types, including CRC. LINE-1 or L1 retrotransposons comprise about 17% of human genome [68]. L1 CpG sites are extensively methylated in healthy cells in order to suppress transposon activity and consequently protect genomic stability. During the carcinogenesis process, these CpG sites are gradually demethylated [69, 70]. Several studies have demonstrated that L1 hypomethylation occurs widely in CRC patients, is associated with clinically relevant bio-pathological features [71] and correlates with poor prognosis and early onset (< 60 years) [72-75]. In fact, it has been demonstrated that LINE-1 hypomethylation is inversely associated with CIMP high and MSI high phenotypes and directly associated with MSI-low and CIN [75, 76]. In addition, in a meta-analysis involving more than 3600 CRC patients, LINE-1 hypomethylation was found to be significantly correlated with shorter overall survival (OS), disease-free survival (DFS) and cancer-specific survival (CSS), possibly representing a promising tool for prognosis prediction [77]. A shorter OS 7

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connected to L1 hypomethylation was also identified in early-stage colorectal cancers [78]. In agreement with these observations, a large study performed on 1317 colon and rectum carcinoma cases highlighted the association between L1 hypomethylation and higher colorectal cancerspecific mortality, which was stronger in proximal colon cancer if compared to distal or rectal cancer [79]. According to Kawakami et al., the hypomethylation of LINE-1 can be used to identify CRC patients who could benefit of adjuvant chemotherapy with oral fluoropyrimidines after surgery [80], who survived longer than patients only treated with surgery. Furthermore, the evaluation of LINE-1 hypomethylation level in plasma cell-free DNA (cfDNA) was recently proposed as novel biomarker for CRC, particularly for early stage detection [81]. Familiar CRC was found to be associated with a higher frequency of L1 hypomethylation, suggesting a possible heritable predisposition to these epigenetic alterations. Additional studies are needed to validate LINE-1 methylation as a molecular biomarker for familial CRC risk assessment [71, 82]. An interesting study by Jorda et al. developed a specific deep sequencing protocol to provide information about the epigenetic changes in Alu elements, the most abundant SINE in human genome. In normal colonic cells only a small fraction of Alu elements is unmethylated; this fraction can increase up to ten-fold in colon cancer cells. Alu hypomethylation contributes to genomic instability and provides a new element to define the epigenetic landscape of CRC [83].

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Hypermethylation in specific tumor suppressor genes has been reported in colorectal cancer and some of these genes have been proposed as colorectal cancer early diagnostic biomarkers, to detect in stool or blood of CRC patients. It is not clear if the aberrant methylation of genes in CRC relies on the primary predisposition of some genes to become hypermethylated or it is a consequence of the clonal outgrowth of cells with specific methylated genes, or a combination of the two hypotheses [84]. Among the genes known to undergo CpGI-hypermethylation-induced repression in CRC, the most extensively studied for their impact in cancer diagnosis or prognosis are MGMT, SEPT9, HLTF, NDRG4, BMP3, CDH13, APC, MLH1, CDKN2A, RASSF1A and RUNX3. Promoter hypermethylation of the tumor suppressor gene O(6)-methylguanine-DNAmethyltransferase (MGMT) has been detected early in CRC [85] [86]. MGMT is a DNA repair protein that catalyzes transfer of methyl groups from O(6)-alkylguanine and other methylated moieties of the DNA to its own molecule, therefore repairing toxic lesions. Defective MGMT function mainly results from its transcriptional silencing by gene promoter methylation. Hypermethylation of MGMT prevents the removal of alkyl groups at the O6 position of guanine, leading to G-A transitions in the genome [87]. MGMT promoter hypermethylation is a validated biomarker of response to temozolomide (TMZ) and other alkylating agents, which are used in the therapy of glioma, melanoma, lymphoma [88], although it is known that not all the tumors with MGMT hypermethylation respond to this treatment [89, 90]. Currently, a clinical trial including temozolomide in a combination therapy for MGMT methylated, RAS mutated advanced colorectal cancer has started (NCT02414009 clinicaltrials.gov). Another gene whose methylation status correlates with tumor size, lymph node metastasis, histological grading and tumor stage is the helicase-like transcription factor (HLTF). Hypermethylation of HLTF in serum of CRC patients was initially found to be associated with an 8

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increased risk of disease recurrence (RR: 2.5, 95% CI: 1.1–5.6, P=0.023) [91] and death (RR: 3.4, 95% CI: 1.4–8.1; P=0.007) [92]. Another study confirmed the positive correlation of serum HLTF and HPP1/TPEF DNA methylation with tumor size, stage, grade and metastatic disease [93]. Additional genes were found associated with CRC clinical features in several studies; hypermethylation of NDRG4 occurred in 70%-86% of CRC tissues compared with 4% in noncancerous colonic mucosa [94] and hypermethylated BMP3 was found in a high percentage of CRC tumors and was correlated with microsatellite instability, CIMP, proximal location and BRAF mutations [95]. CDKN2A/p16 hypermethylation has been associated with worse prognosis in CRC in many studies. Two different meta-analysis, encompassing more than 10 studies and 3000 patients, confirmed the association between CDKN2A/p16 hypermethylation and reduced OS, presence of lymph node metastasis and lymphovascular invasion [96, 97]. Recent studies have shown that DNA hypomethylation in cancer occurs through the activity of teneleven translocation enzymes (TET). TET family member can catalyze the formation of 5hydroxymethylcytosine (5-hmC) from methylated cytosine that, targeted by Base Excision Repair (BER) proteins, are rapidly removed and replaced by an unmethylated cytosine [98-100]. The expression of TET1, TET2 and TET3 was found to be reduced in CRC tissues compared to normal mucosa, but hypermethylation was detected only for TET1 in a subgroup of CRCs [101]. These findings were also confirmed in another study, suggesting the potential role of DNA methylation in regulating TET1 expression only in CIMP positive CRCs [102].

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Non-coding RNA aberrant methylation In the past decade, a dysregulated expression of microRNAs and other longer non-coding RNAs (ncRNA) has been reported in many tumor types, including colorectal cancer [103-106]. In CRC, a proportion of these alterations is due to epigenetic mechanisms acting on miRNA and ncRNA genomic regulatory regions [107]. Specifically, Lujambio et al. reported the hypermethylation of microRNA 124-1/3 in colon cancer and other solid tumors, with consequent reduced expression of mature miR-124a and increase of the target gene CDK6 and Rb protein phosphorylation [108]. In CRC tissues, the hypermethylation of microRNA 124 family genes was observed in more than 70% of the cases. These results were further confirmed in acute lymphoblastic leukemia [109], where the methylation at microRNA 124 loci was associated with higher relapse and mortality risk. In addition, the methylation pattern at specific miRNA genes (microRNA 148a, 34b/c and 9) was demonstrated to be linked to the metastatic process [110]. Other miRNAs were found aberrantly methylated in the early stages of CRC, including miR- 137 [111], miR-200 family [112], miR-129 and miR-9 [113], supporting the importance of epigenetic regulation in tumor suppressor miRNAs expression. Epigenetic biomarkers Early colorectal cancer diagnosis could increase the chances of early intervention and improve patients’ survival rate. Colonoscopy, fecal occult blood testing (FOBT) and fecal immunochemical test (FIT) are the most commonly used screening tests worldwide, but each one presents several 9

ACCEPTED MANUSCRIPT sensitivity limitations and/or compliance disadvantages [114, 115], making the discovery of better CRC biomarkers a research priority. The hypermethylation of specific CpG islands is a promising biomarker that shows high potential for translation into non-invasive CRC detection approaches [116].

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The methylation status of specific genes could be used as an early marker for CRC. For example, ulcerative colitis (UC) is a chronic inflammation of the colon that is considered a pre-cancerous condition, due to its increased risk of developing CRC. Alteration in the methylation levels of RUNX3, MINT1, and COX-2 were found in the non-neoplastic sections of UC-related CRC colons when compared with UC controls [117]. In addition, methylation changes in CRC-related genes (APC, CDH13, MGMT, MLH1 and RUNX3) has been detected also in the non-tumoral colonic mucosa of CRC patients [118], suggesting their potential use as pre-malignant biomarkers for the potential development of colorectal cancer.

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Carmona et al. proposed to use the methylation of three genes (AGTR1, WNT2, SLIT2) in stool DNA of patients with inflammatory bowel disease, for the early detection of possible CRC with a sensitivity of 78% [119]. Recently, a non-invasive stool DNA test for the detection of CRC has been proposed, which includes the analysis of KRAS mutation, NDRG4 and BMP3 methylation and bactin expression [120]. The test was applied to nearly 10 thousand subjects, including CRC patients and subjects with pre-cancerous lesions. The sensitivity of this DNA test for CRC detection reached 92.3% and 42.4% for advanced precancerous lesions and was more sensitive, although less specific (higher number of false positives), than other non-invasive tests such as FIT.

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The detection of colorectal cancer-specific gene promoter methylation in whole blood or cell free DNA (from serum or plasma) have been historically performed since the early 2000s with methylation-specific PCR-based techniques. Several methylated genes were proposed over the years as potential disease biomarkers, including HLTF [92], CDH4 [121], HPP1/TPEF [92, 122], MLH1 [123]. Among the hypermethylated genes, SEPT9 (septin 9) is one the most promising for non-invasive early colon cancer detection [124, 125]. SEPT9 gene was found to be associated to tumorigenesis and cancer progression in different kinds of cancer [126]. The septin gene family is a conserved set of guanosine-5’-triphosphate–binding proteins that controls vesicle trafficking, apoptosis, and cytoskeletal remodeling [127]. Hypermethylation of this gene’s promoter has been observed in CRC and might also be used as a screening biomarker, since alteration in this gene has been found associated in advanced precancerous colorectal lesions [128]. Methylation alteration of SEPT9 in plasma was first reported by Lofton-Day et al., showing that methylated DNA of SEPT9 could be found in 69% of CRC patients, while unmethylated SEPT9 DNA was detected in 86% of healthy individuals. [129]. A large German and US multicenter study, “The PRospective Evaluation of SEPT in 9 (PRESEPT)”, conducted on more than 1500 CRC patients and normal subjects confirm the potential of SEPT9 hypermethylation detection as a screening tool for CRC. Methylated SEPT9 plasma levels displayed a sensitivity of 35.0% for stage I, 63.0% for stage II, 46.0% for stage III and 77.4% for stage IV and an overall specificity of 91.5% [130]. A blood-based CRC screening test has 10

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been recently approved by The Food and Drug Administration that is entirely based on SEPT9 methylation assessment. Hypermethylation of NPY (neuropeptide Y) and WIF1 (WNT inhibitory factor 1) has been found associated with CRC and detectable in circulating DNA. Specifically, a study by Roperch et al. on 15 paired CRC and adjacent normal tissues found significant differences in the methylation levels of NPY and WIF1 between CRC and normal tissue, and in serum of CRC patients vs. controls [131]. These data were confirmed also in another study where NPY and WIF1 were tested in cfDNA with droplet digital PCR [132].

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Given the contribution of methylation alterations in determining a malignant environment in colonic cells, several studies analyzed the correlation between the methylation status of specific genes and response to therapy and prognosis in colon and rectal cancers [133]. Ebert et al. showed that the hypermethylation of the transcription factor activating protein 2 epsilon (TFAP2E) was associated to the resistance to neoadjuvant chemotherapy in colorectal cancer [134]. Also TIMP3 hypermethylation was associated to neoadjuvant chemoradiotherapy response in rectal cancer [135]. Using an unbiased approach (methylation microarrays), Moutinho et al. compared the global methylation profile of oxaliplatin sensitive and resistant colorectal cancer cell lines [136]. They discovered that the inactivation by hypermethylation of SRBC, which interacts with BRCA1, was linked to oxaliplatin resistance. Indeed, when SRBC methylation was tested in FFPE colorectal cancer samples, its promoter hypermethylation was significantly associated with a shorter PFS in oxaliplatin-treated tumors.

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As a non-invasive response-to-therapy biomarker, MGMT seemed the most suitable to be tested in cell-free DNA. Indeed, MGMT methylation could be detected in plasma of CRC patients. In their study, Barault and colleagues assessed MGMT methylation in cfDNA of CRC patients using a digital PCR approach and observed an association between MGMT methylation and with PFS in metastatic patients treated with alkylating agent decarbazine (2.1 versus 1.8 months for the unmethylated group, P = 0.008) [137]. Recently, a panel of five methylated genes (EYA4, GRIA4, ITGA4, MAP3K14-AS1, MSC) was proposed as a liquid biopsy test to monitor CRC minimal residual disease by digital PCR, using cfDNA [138]. Barault et al. first identified candidate biomarkers of CRC analyzing the methylation profile of CRC cell lines. Then they excluded the candidate genes that were methylated in blood cells, the main source of contaminant DNA in cfDNA, and selected the genes whose methylation was potentially assessable by digital PCR. The proposed markers were tested to be hypermethylated in CRC FFPE tissues vs. normal mucosa and then used as biomarkers of disease in the plasma of metastatic CRC (mCRC) patients compared to healthy subjects. Given the strength of the proposed panel, they validated its use in monitoring mCRC response to therapy, including chemotherapy, targeted therapy and temozolomide in a longitudinal study. The 5-gene methylation panel proved to be positively correlated with tumor burden and could be used to monitor therapy efficacy. 11

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Histone tails covalent modifications are fundamental for the regulation of the chromatin state. The first evidence of histone alterations in human cancers was described by Fraga et al. in 2005 [139], when they observed a global reduction of the trimethylation of H4K20 (H4K20me3) and acetylation of H4K16 (H4K16Ac) in human cancers. Furthermore, the deregulation of the enzymes responsible for histone N-terminal tail modifications has been observed in CRC, which in turns generates plenty of aberrant histone modifications (see [140] for a comprehensive review). Regarding histone acetylation, we know that specific lysine residues of histones N-terminal tails are acetylated, neutralizing their positive charge and decreasing their affinity for the DNA [141]. The histone acetyltransferase EP300 is mutated in epithelial cancers, including colorectal cancer [142]. This mutation generates a truncated and non- functional protein. In fact, additional studies confirmed the positive correlation between EP300 expression and favorable prognosis in colorectal tumors [143]. Histone deacetylase activity is supposed to increase in cancer and to be the mechanism by which tumor-suppressor genes are transcriptionally silenced [144, 145]. Controversially, the upregulation of histone deacetylases (HDACs and sirtuins) has been observed in CRC and associated with Wntsignaling pathway activation [140]. But loss of expression of HDAC2 was reported in MSI colorectal cancers, due to truncating mutations [146]. On the contrary, a recent study reported increased nuclear expression of HDAC2 and decreased nuclear expression of SIRT1 and H4K16Ac in 254 colorectal cancer compared with 50 normal mucosae and a better patient survival when more markers (comprising SIRT1, HDAC1 and HDAC2 expression and the histone modifications H4K16Ac and H3K56Ac) showed high nuclear expression [147]. Particularly interesting, also, is the observation of HDAC2 upregulation as a pre-neoplastic alteration predisposing to colon carcinogenesis in animal models [148], and Sirtuin 1 overexpression in CIMP-high MSI CRC [149].

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Methylation on lysine or arginine residues of histone tails can be associated with both condensation and relaxation of chromatin. A dysregulated activity of histone methyltransferases (HMTs) and demethylases (HDMs), due to chromosomal translocations, amplification, deletion, overexpression or silencing, has been described in several human cancers, including CRC. The H3K4 HMT SMYD3 was found upregulated in colorectal cancer cell lines, inducing proliferation [150]. The modification H3K9me2 was found to increase progressively in the transition from normal mucosa to adenoma and then to adenocarcinomas [151]. Furthermore, low H3K27me2 is usually associated with poor survival rates and its methylation is further reduced in metachronous liver metastases [152]. In a recent study, Benard et al. observed that the combination of low H3K4me3, high H3K9me3 and high H4K20me3 was associated with the best prognosis in early stage colon – but not rectal – cancers [153]. Furthermore, the methylation of H3K4, H3K27 and H3K79 seems to play a pivotal role in the epigenetic regulation of CpGIs of microRNAs involved in colorectal cancer [154]. The upregulation of HMTase G9a has been documented in CRC [155], with a role in inducing cell proliferation. Indeed, Paschall et al. reported that H3K9me3-mediated FAS transcriptional silencing is a mechanism used by colon cancer cells to 12

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evade immune surveillance and induce chemoresistance. Verticillin A, a selective inhibitor of HMTase SUV39H1, SUV39H2 and G9a/GLP, was demonstrated to reduce H3K9me3 levels in metastatic human colon carcinoma cells and restore FAS expression [156]. The H3K27-specific HMT EZH2 overexpression in solid tumors has been documented in many studies, with some controversial findings about EZH2 prognostic role. A meta-analysis was recently performed on CRC, including data from 8 studies and more than 1000 patients, and it was confirmed that EZH2 overexpression indicates a better prognosis for colorectal cancer [157].

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The hypothesis to use HDAC inhibitors in the treatment of colorectal cancer was initially explored, in virtue of positive results observed in in-vitro experiments [158]. Nowadays, approved epigenetic therapies to treat cancer patients include the use of DNA methyltransferase (DNMT) inhibitors azacitidine (AZA) and decitabine (DAC) for the treatment of myelodysplastic syndrome and the histone deacetylase (HDAC) inhibitors vorinostat (SAHA) and romidepsin (depsipeptide) for the treatment of cutaneous T-cell lymphoma, but there is no epigenetic drug approved for the treatment of solid tumors, although some promising new molecules and drug combinations are now being explored [159] that could finally lead to the identification of more effective – and selective – epigenetic drugs. Epitranscriptomic changes in colorectal cancer

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The importance of epitranscriptomic changes came recently to light, and its impact in cancer development and progression is now being explored [160]. In colonic tissue, ADAR1 was found to have a key role in the maintenance of intestinal homeostasis and for pluripotency of stem cells [161]. Its depletion in human embryonic stem cells (hESCs) is able to abrogate pluripotency and induce expression of transcripts that cause differentiation. From this and other evidences, it can be expected that the activation of ADAR1 could have a significant role in tumors, especially to acquire aberrant stem cell self-renewal capacity. Indeed, a global activation of RNA editing processes have been reported in cancer [162, 163]. Jiang et al. suggested ADAR1 as a therapeutic target, since the inhibition of ADAR1 expression or activity could reduce self-renewal of cancer stem cells (CSCs) that responsible of disease relapse and drug resistance [19]. Lee et al. have recently compared 39 CRC transcriptome data and their matched normal mucosa from TCGA database in order to discover RNA editing patterns in CRC able to be used as novel biomarkers. In particular, in this study they focused on nonsynonymous RNA editing patterns, especially A-to-I. From their analysis, they found a wide variety of RNA editing events in every cancer (the total number ranging from 12 to 42), and observed both hypo- and hyper- RNA editing patterns and nonsynonymous RNA editing. They also highlighted the presence of ten recurrent nonsynonymous RNA editing sites in nine genes (PDLIM, NEIL1, SRP9, GLI1, APMAP, IGFBP7, ZNF358, COPA, and ZNF587B), whose editing was proved to be performed by ADAR1. Most of these genes are hypo-edited in CRC compared to normal tissues, apart from GL1 that resulted hyper-edited in CRC [164].

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Editing in mRNA could produce alternative isoforms of some proteins. An example is represented by the heterogeneous nuclear ribonucleoprotein K (hnRNPK) that was found to be edited with a single G-to-A base substitution at position 274 in tumors and surrounding mucosa only of patients affected by colorectal cancer [165]. On the contrary, for some genes it was observed a general decrease in editing levels in cancer compared to normal tissues. For instance, human transcripts of bladder cancer-associated protein (BLCAP), thanks to the action of both ADAR1 and ADAR2, normally undergo multiple A-to-I editing events creating alternative protein isoforms. However, BLCAP editing levels were found to be significantly lower in astrocytomas, bladder cancer and colorectal cancer if compared with their related normal tissues [166]. By comparing whole-genome and transcriptome sequence data of a CRC sample, Han et al. discovered novel tumor-associated RNA editing event in RAS homologue family member Q (RHOQ) transcripts. In this case, A-to-I editing causes a N136S substitution. A higher editing level of RHOQ RNA was detected in CRC compared with matched normal tissue. This particular mRNA editing results in an increased activity of RhoQ associated with invasiveness. The concurrence of edited RHOQ transcripts and mutant KRAS further increase its invasion potential in vitro and in CRC patients their frequency of recurrence. This suggest a potential role of RNA editing in CRC progression and invasion [167]. In addition, Zheng et al. analyzed four normal and cancerous colon tissues using smallRNA sequencing. They found that many miRNAs are edited in colon tissues, with 3’-A and 3’-U as predominant editing events in both colon cancerous and adjacent normal tissues. A substantial fraction of mature miRNAs also presents 5’-U addition [168]. The editing event 3’-A was also found in let-7 family members, occurring before the formation of RISC complex, as was previously reported in another study [169]. 3’-U and 3’-A editing events seem to occur on both 5’ and 3’ mature miRNAs in several miRNAs, such as hsa-miR-143-3p/hsa-miR-143-5p. In some cases, 3’-U and 3’-A were found to recur in the same position of same miRNAs providing evidences that the same enzyme could catalyze both uridine and adenosine addition, as was previously reported for PAPD4 [169, 170].Furthermore, Zheng et al. found some miRNAs with 3’-G or 3’-C edits. One example of 3’-C editing is represented by has-miR-21. These modifications were also found in other tissues, but their clinical relevance need to be elucidated [170-172]. Four A-to-I editing sites, previously described in brain tissues, on hsa-miR-376a-2, hsa-miR-376a-1, hsa-miR-411 and hsamiR-376c were also found in colon tissues [29, 173, 174]. This analysis also identified other editing sites, but their mechanism need to be clarified [168]. N6-methyladenosine (m6A) is the most prevalent modification in mRNA and lncRNAs, and was recently found to be very frequent in different cancer types, even if its role in cancer biology and cancer stem cells needs to be further elucidated [175]. Yang et al. in their study on CRC provide evidences of a cancer cell migration promotion mechanism mediated by activated Proteinase activated-receptor 2 (PAR2) through the down-regulation of miR-125b via a m6A-related mechanism [176]. These findings could potentially be useful for future studies on selectively edited mRNAs, to explore their use as diagnostic tool for different cancer types.

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ACCEPTED MANUSCRIPT Aknowledgements

Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

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This work was supported by grants from the Italian Association for Cancer Research (AIRC) to M. Ferracin (IG 18464) and Fondazione Pallotti (University of Bologna) to M. Ferracin. Dr. M. Riefolo is a Fondazione Famiglia Parmiani fellow.

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Figure 1 – Mechanisms of dysregulated RNA editing in cancer. Main mechanisms through which cancer cells take advantage of specific transcriptomic alterations. Chemical modifications (e.g. m6A) and A to I or C to U editing in coding and non-coding RNAs can lead to increased expression of oncogenes expression, reduced expression of tumor suppressor genes or generation of novel oncogenic proteins through RNA recoding. Global changes in RNA editing can be observed in cancer cells due to the increased expression of RNA modifying enzymes such as ADARs, APOBECs and others.

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Figure 2 – Summary of epigenetic and epitranscriptomic alterations in colorectal cancer. The three panels represent the summary of the main alterations in DNA methylation, histone modifications and RNA editing that have been associated with colorectal cancer diagnosis (yellow panel), response to therapy (green panel) and prognosis (blue panel).

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