Isabelle Van Seuningen

Audrey Vincent

Jean-Pierre Aubert

Mucins: a new family of epigenetic biomarkers in epithelial cancers

Keywords: biomarker, cancer, epigenetics, mucin

Background: Epigenetic regulation of gene expression is a common feature of cancer development and progression. The search for new biomarkers and tools to detect cancer in its early stages has unveiled the usefulness of epigenetics and genes epigenetically regulated as potential targets. Among them, genes encoding mucins has been shown to be regulated by DNA methylation and histone modifications in epithelial cancer cells. These genes encode either secreted glycoproteins necessary for epithelial homeostasis or membrane-bound glycoproteins that participate in tumor progression.

Objective: The important biological functions played by these large molecules in pathophysiology of the epithelia make them key genes to target to propose new therapeutic strategies and new diagnostic and/or prognostic tools in cancer. Results: In that context, the recent data regarding the epigenetic regulation of these genes are reported and their potential as biomarkers in cancer is discussed. Mucin genes are also potentially interesting to study as they may be regulated by miRNAs but also regulate miRNA activity. Conclusion: Epigenetic regulation of mucin genes is at its dawn, but there is great potential in that research to (with new technologies and high-throughput methods) provide quickly new biomarkers (diagnostic and/or prognostic), help tumor identification/classification and propose new therapeutic targets to the clinician and pathologist.

Introduction

The term 'epigenetic' refers to a heritable change in the pattern of gene expression that is driven by mechanisms other than alterations in the primary nucleotide sequence of the gene [START_REF] Russo | Epigenetic mechanisms of gene regulation[END_REF][START_REF] Tost | [END_REF]. Epigenetic changes in the genome include DNA methylation and histone modifications (acetylation, methylation, phosphorylation, ubiquitination, sumoylation), two mechanisms that are often tightly linked in the regulation of gene expression and involved in many cellular processes [START_REF] Robertson | DNA methylation and human disease[END_REF]. For example, DNA methylation is a crucial epigenetic modification that drives embryonic development, X-chromosome inactivation, genomic imprinting and chromosome stability, whereas histone modifications govern rearrangement of chromatin structure.

DNA methylation occurs on CpG dinucleotides and follows a cell-specific profile that is established during development by DNA methyltransferases DNMT3A and DNMT3B [START_REF] Chen | Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b[END_REF] and is maintained through cell division by DNMT1 [START_REF] Wilson | DNA hypomethylation and human diseases[END_REF]. In the human genome, CpG sites are statistically underrepresented but are clustered in GC-rich regions termed CpG islands. These CpG islands are often associated with promoter regions or first exons of half the genes in our genome. In normal cells, 80% of CpG dinucleotides that are not associated with Expert Opin. Med. Diagn. (2009) 3(4)

CpG islands (predominantly in intergenic and intronic regions of DNA, repeat sequences and transposable elements) are heavily methylated, whereas CpG islands are usually unmethylated, whether or not the gene is transcriptionally active [START_REF] Herman | Gene silencing in cancer in association with promoter hypermethylation[END_REF]. On the contrary, the cancer genome is frequently characterized by both global hypomethylation of intergenic regions resulting in chromosomal instability [START_REF] Wilson | DNA hypomethylation and human diseases[END_REF] and specific hypermethylation of CpG islands in promoters resulting in aberrantly silenced tumor suppressor genes [START_REF] Herman | Gene silencing in cancer in association with promoter hypermethylation[END_REF].

DNA methylation by itself does not directly repress transcription. The process of silencing needs the involvement of the epigenetic machinery composed of chromatin modifier enzymes, such as histone deacetylases (HDACs) and histone methyltransferases (HMTs), and proteins that are able to read methylation marks, namely the methylCpG binding proteins (MBPs). MBPs recognize methylated DNA and recruit multiprotein complexes containing HDACs that remove acetyl residues from protruding histone tails, hence tightly compacting chromatin and suppressing promoter access to transcription machinery [START_REF] Razin | CpG methylation, chromatin structure and gene silencing-a three-way connection[END_REF].

In the collaboration between the two epigenetic mechanisms that silences gene expression in cancer it is unclear at the moment whether DNA methylation or histone modification is dominant. Studies using drugs that inhibit epigenetic mechanisms showed that HDAC inhibitors alone cannot induce the expression of hypermethylated genes, indicating that DNA methylation may be preponderant. However, in some cases, histone (de)acetylation can drive DNA (de) methylation [START_REF] Cervoni | Demethylase activity is directed by histone acetylation[END_REF][START_REF] Mutskov | Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9[END_REF], suggesting that DNA methylation may simply be a marker of silent chromatin. Nevertheless, stable gene repression by epigenetics in cancers seems to involve both mechanisms and appears to be a specific mark of cancer cells. The recent discovery of small non-protein-coding RNA, named micro-RNAs (miRNAs), which target 3′-untranslated regions (3′-UTR) of genes and repress either their maturation or lead them to their degradation, added a new layer to epigenetic regulation by means of RNA interference [START_REF] Bartel | MicroRNAs: genomics, biogenesis, mechanism, and function[END_REF][START_REF] He | MicroRNAs: small RNAs with a big role in gene regulation[END_REF][START_REF] Wiemer | The role of microRNAs in cancer: no small matter[END_REF].

As altered patterns of epigenetic modifications are observed in the early stage of carcinogenesis, it was suggested several years ago that aberrant DNA methylation may be used as a biological tool for early detection and prognosis of cancer [START_REF] Shames | Methods for detecting DNA methylation in tumors: from bench to bedside[END_REF][START_REF] Teodoridis | Epigenetic silencing mediated by CpG island methylation: potential as a therapeutic target and as a biomarker[END_REF][START_REF] Palmisano | Predicting lung cancer by detecting aberrant promoter methylation in sputum[END_REF]. Moreover, the potential reversibility of epigenetic modifications gives new opportunities for the clinical treatment of cancer [START_REF] Worm | DNA methylation: an epigenetic pathway to cancer and a promising target for anticancer therapy[END_REF]. Thus, identifying new pharmacological inhibitors of epigenetic modifications has become the focus of research in many laboratories, and both DNMT and HDAC inhibitors have shown promising results in clinical trials [START_REF] Strathdee | Epigenetic cancer therapies: DNA methyltransferase inhibitors[END_REF][START_REF] Yoo | Epigenetic therapy of cancer: past, present and future[END_REF].

Next to pharmacological inhibitors, growing interest has been shown by researchers in controlling gene expression during the early steps of carcinogenesis and/or using altered pattern of expression of these genes as diagnostic/prognostic markers. Epigenetically marked genes offer great and exciting possibilities in that sense because methylation occurs during the early stages of cancer. Among them, genes encoding secreted and membrane-bound mucins are often aberrantly expressed in tumors and this finding has led to the hypothesis that mucins may be used as diagnostic and/or prognostic factors in epithelial cancers of different origins [START_REF] Liang | Diagnostic and prognostic biomarkers in pancreatic carcinoma[END_REF][START_REF] Van Seuningen | Transcriptional regulation of the 11p15 mucin genes. Towards new biological tools in human therapy, in inflammatory diseases and cancer?[END_REF][START_REF] Mall | Analysis of mucins: role in laboratory diagnosis[END_REF][START_REF] Leteurtre | The epithelial mucins: structure/function Roles in cancer and inflammatory diseases[END_REF]. Alterations of expression include both silencing and overexpression of mucin genes while their promoters share a GC-rich structure and, for most of them, contain a CpG island [START_REF] Van Seuningen | Transcriptional regulation of the 11p15 mucin genes. Towards new biological tools in human therapy, in inflammatory diseases and cancer?[END_REF]. Moreover, the four genes MUC2, MUC5AC, MUC5B and MUC6, encoding the gel-forming mucins, are clustered on the p15.5 region of chromosome 11 (11p15 MUC genes) [START_REF] Pigny | Human mucin genes assigned to 11p15.5: identification and organization of a cluster of genes[END_REF], in an area known to be a hot spot of abnormal methylation in cancer [START_REF] De Bustros | The short arm of chromosome 11 is a 'hot spot' for hypermethylation in human neoplasia[END_REF]. This explains why most of the studies regarding epigenetic regulation of mucin genes focused on the 11p15 MUC cluster.

In this paper, the studies that were conducted by the authors' laboratory are reviewed, as well as others about the epigenetic regulation of the 11p15 MUC genes and those about MUC1 and MUC4 genes that encode two membranebound mucins known to participate in tumor progression and alter cancer cell biological properties [START_REF] Hollingsworth | Mucins in cancer: protection and control of the cell surface[END_REF][START_REF] Jonckheere | The epithelial mucins: structure/function Roles in cancer and inflammatory diseases[END_REF][START_REF] Yonezawa | Expression profiles of MUC1, MUC2, and MUC4 mucins in human neoplasms and their relationship with biological behavior[END_REF]. The second part of the review opens up to the recent development of new technologies to study epigenetic regulation, and the exciting new avenues that mucin gene regulation by miRNAs may provide to the clinicians in terms of therapeutic tools are discussed. No data exist about mucin gene regulation by miRNAs in the literature, so for the first time miRNA databases were searched and the (long) lists of potential regulatory miRNAs targeting 3′-UTRs of mucin genes established. The paper concludes with a discussion of what the detection of mucin gene methylation could bring to the clinician and/or pathologist as a diagnostic/prognostic tool for early detection of cancer and tumor identification/classification.

Mucins and epigenetics

2.1 DNA methylation and histone marks 2.1.1 The 11p15 mucin genes 2.1.1.1 The 11p15. [START_REF] Wilson | DNA hypomethylation and human diseases[END_REF]

chromosomal region

The 11p15 chromosomal region is of particular interest for studying epigenetic mechanisms of regulation. Aberrant DNA methylation in this region is associated with severe developmental abnormalities [START_REF] Gicquel | Epimutation of the telomeric imprinting center region on chromosome 11p15 in Silver-Russell syndrome[END_REF][START_REF] Schonherr | The centromeric 11p15 imprinting centre is also involved in Silver-Russell syndrome[END_REF] and cancer [START_REF] De Bustros | The short arm of chromosome 11 is a 'hot spot' for hypermethylation in human neoplasia[END_REF][START_REF] Weksberg | Beckwith-Wiedemann syndrome demonstrates a role for epigenetic control of normal development[END_REF]. This chromosomal region is characterized by one of the highest concentration of CpG islands in the genome, being separated from each other by 15 -100 kb (Figure 1) [START_REF] Craig | The distribution of CpG islands in mammalian chromosomes[END_REF]. Within this region lies a 1 Mb imprinted domain, containing several genes involved in growth and development, and potential tumor suppressor genes that are aberrantly methylated in cancer [START_REF] De Bustros | The short arm of chromosome 11 is a 'hot spot' for hypermethylation in human neoplasia[END_REF][START_REF] Nakano | Expression profile of LIT1/KCNQ1OT1 and epigenetic status at the KvDMR1 in colorectal cancers[END_REF][START_REF] Sato | Epigenetic down-regulation of CDKN1C/p57KIP2 in pancreatic ductal neoplasms identified by gene expression profiling[END_REF][START_REF] Schwienbacher | Gain of imprinting at chromosome 11p15: a pathogenetic mechanism identified in human hepatocarcinomas[END_REF]. Interestingly, MUC2, one of the 11p15 mucins, was recently proposed to have tumor suppressor activity in colorectal cancer [START_REF] Velcich | Colorectal cancer in mice genetically deficient in the mucin Muc2[END_REF].

The 11p15 MUC cluster is 400 kb in length and is situated ∼ 700 kb from the imprinted domain, between insulin-like growth factor-2 (IGF-2) and Harvey rat sarcoma viral oncogene homolog (H-RAS) genes, within a region containing 15 CpG islands (Figure 1) [START_REF] Pigny | Human mucin genes assigned to 11p15.5: identification and organization of a cluster of genes[END_REF]. Within the cluster, mucin genes are organized as follows, MUC6-MUC2-MUC5AC-MUC5B from telomere to centromere, MUC2, MUC5AC and MUC5B being transcribed from telomere to centromere, whereas MUC6 is transcribed in the opposite direction (Figure 1).

Epigenetic regulation of the 11p15 mucin genes

The first studies that aimed at studying epigenetic regulation of mucin genes focused on MUC2. Based on the observation that MUC2 mucin expression was repressed in non-mucinous colorectal cancers and their metastasis, Hanski and collaborators were the first to investigate the role of DNA methylation in MUC2 silencing [START_REF] Hanski | MUC2 gene suppression in human colorectal carcinomas and their metastases: in vitro evidence of the modulatory role of DNA methylation[END_REF]. To this end, they used the methylationsensitive enzyme HpaII and the methylation-independent enzyme MspI to digest DNA from colon carcinoma cell lines expressing variable levels of MUC2, followed by Southern blotting. They showed hypermethylation of MUC2 promoter in non-mucinous carcinoma cell lines [START_REF] Hanski | MUC2 gene suppression in human colorectal carcinomas and their metastases: in vitro evidence of the modulatory role of DNA methylation[END_REF][START_REF] Gratchev | Regulation of the intestinal mucin MUC2 gene expression in vivo: evidence for the role of promoter methylation[END_REF].

Thereafter, arising bisulfite-treated genomic DNA-based methods allowed more precise analysis of methylation profiles of CpG sites involved in MUC2 silencing. These studies showed the involvement of 9 CpG sites situated directly upstream of the transcriptional start site of MUC2, at -289, -274, -246, -192, -160, -126, -118, -6 and -2, in the silencing of the gene in non-expressing normal columnar cells and in the non-mucinous carcinoma tissues of the colon [START_REF] Gratchev | Regulation of the intestinal mucin MUC2 gene expression in vivo: evidence for the role of promoter methylation[END_REF] as well as in pancreatic cancer cell lines [START_REF] Siedow | De novo expression of the Muc2 gene in pancreas carcinoma cells is triggered by promoter demethylation[END_REF]. The use of methylationsensitive single nucleotide primer extension (Ms-SNuPE) allowed Mesquita and co-workers to demonstrate the role of site-specific promoter hypomethylation in aberrant MUC2 mucin expression in mucinous gastric carcinoma [START_REF] Mesquita | Role of site-specific promoter hypomethylation in aberrant MUC2 mucin expression in mucinous gastric carcinomas[END_REF].

In 2005, mapping of the methylation status of the 59 CpG sites of MUC2 promoter lying from position -1989 to position +288 in pancreatic cancer cell lines confirmed the role of site-specific methylation in MUC2 silencing, and authors described methylation of DNA binding sites for AP-2 and Sp1 transcription factors. However, the authors did not study the influence of methylation on MUC2 activation by these two transcription factors [START_REF] Hamada | Mapping of the methylation pattern of the MUC2 promoter in pancreatic cancer cell lines, using bisulfite genomic sequencing[END_REF]. They also showed that histone H3 modification status at MUC2 promoter, and especially demethylation of lysine 4, methylation of lysines 9 and 27 and deacetylation of lysines 9 and 27, tightly related to DNA methylation, played an important role in MUC2 gene silencing [START_REF] Yamada | MUC2 expression is regulated by histone H3 modification and DNA methylation in pancreatic cancer[END_REF].

Very few studies have been conducted concerning the three other genes of the cluster. The first study concerning epigenetic regulation of MUC5B was carried out at the authors' laboratory and showed, using HpaII and MspI enzymes combined with Southern blotting and cell treatment with DNA methylation inhibitor 5-aza-22deoxycytidine (5-aza), that the -1095/-695 region of MUC5B proximal promoter was hypermethylated in gastric MUC5B-expressing KATO-III cells, whereas methylation occurred throughout the 55-flanking region in gastric low-expressing AGS cells [START_REF] Van Seuningen | Transcriptional regulation of the 11p15 mucin genes. Towards new biological tools in human therapy, in inflammatory diseases and cancer?[END_REF][START_REF] Perrais | Aberrant expression of human mucin gene MUC5B in gastric carcinoma and cancer cells. Identification and regulation of a distal promoter[END_REF]. This pattern of methylation corresponded to the downregulation of MUC5B apomucin expression observed in undifferentiated gastric carcinomas [START_REF] Perrais | Aberrant expression of human mucin gene MUC5B in gastric carcinoma and cancer cells. Identification and regulation of a distal promoter[END_REF]. The only study carried out on MUC5AC and more particularly on one CpG site in pancreatic cancer cell lines did not show any correlation between epigenetic regulation of the gene and expression of the apomucin [START_REF] Ho | Methylation status of promoters and expression of MUC2 and MUC5AC mucins in pancreatic cancer cells[END_REF].

To assess whether epigenetic regulation of the four 11p15 MUC genes was concerted and associated with cancer, a simultaneous study was undertaken of the four genes in a wide panel of human epithelial cancer cell lines with different epithelial origins and different status of differentiation [START_REF] Vincent | Epigenetic regulation (DNA methylation, histone modifications) of the 11p15 mucin genes (MUC2, MUC5AC, MUC5B, MUC6) in epithelial cancer cells[END_REF]. The analysis was carried out on large portions of the promoters, that is, 3.3 kb for MUC2, 1.3 kb for MUC5AC, 2.3 kb for MUC5B and 1.8 kb for MUC6. In silico analysis showed that MUC2, MUC5B and MUC6 promoters were particularly rich in G+C nucleotides, up to 70, 80 and 75%, respectively, and contained CpG islands of 115, 104 and 166 bp in length, respectively, situated at -4647/-4533, -976/-873 and -160/+6, whereas relatively few CpG sites were found in MUC5AC [START_REF] Vincent | Epigenetic regulation (DNA methylation, histone modifications) of the 11p15 mucin genes (MUC2, MUC5AC, MUC5B, MUC6) in epithelial cancer cells[END_REF].

The studies indicate that regulation by methylation (after treatment with 5-aza) and by histone acetylation (after treatment with HDAC inhibitor trichostatin A [TSA]) is both gene-and cell-specific. In some cases, TSA induced repression of mucin gene expression, especially in confluent differentiated cells expressing high levels of the mucin gene of interest [START_REF] Vincent | Epigenetic regulation (DNA methylation, histone modifications) of the 11p15 mucin genes (MUC2, MUC5AC, MUC5B, MUC6) in epithelial cancer cells[END_REF]. The same inhibitory mechanism by the HDAC inhibitor sodium butyrate had previously been found in MUC2 high-expressing intestinal cancer cell lines [START_REF] Augenlicht | Repression of MUC2 gene expression by butyrate, a physiological regulator of intestinal cell maturation[END_REF]. This may reflect a mechanism of definitive function of the secretory lineage in order to maintain epithelial homeostasis [START_REF] Mayo | In vitro differentiation of HT-29 M6 mucus-secreting colon cancer cells involves a trychostatin A and p27(KIP1)-inducible transcriptional program of gene expression[END_REF].

Bisulfite-treated genomic DNA sequencing and chromatin immunoprecipitation (ChIP) assays confirmed that MUC2 was regulated by site-specific DNA methylation associated with establishment of a repressive histone code, including histone H3 deacetylation and methylation of lysine 9. Moreover, our study covering the -3269/-1791 region of MUC2 promoter allowed us to identify 4 more relevant CpG sites at -3269, -3199, -2331 and -1912. It was also shown that hypermethylation of MUC5B promoter was the main mechanism responsible for its silencing in epithelial cancer cells and involved a wide region of its distal promoter at -2677/-2163, as well as cytosines at -434 and -421 in the proximal promoter. Thus, we and others have identified key methylated cytosines in MUC2 and MUC5B promoters that should provide useful tools, which are easy to detect, to screen mucin gene repression by methylation in epithelial cancers and identify tumors in which their repression may indicate good or poor prognosis.

On the contrary, the expression of MUC5AC was rarely influenced by epigenetic mechanisms and it was not possible to identify CpG sites in its promoter which hypermethylation would be associated with its silencing. Hypermethylation of MUC6 promoter was not correlated to its repression because DNMT and HDAC inhibitors had no influence on its expression in the cancer cell lines studied. [START_REF] Vincent | Epigenetic regulation (DNA methylation, histone modifications) of the 11p15 mucin genes (MUC2, MUC5AC, MUC5B, MUC6) in epithelial cancer cells[END_REF].

Interestingly, our study also indicates that 11p15 mucin genes share a common feature regarding the methylation pattern of their promoters, which is that their CpG islands are heavily methylated regardless of their level of expression in the cancer cells studied [START_REF] Vincent | Epigenetic regulation (DNA methylation, histone modifications) of the 11p15 mucin genes (MUC2, MUC5AC, MUC5B, MUC6) in epithelial cancer cells[END_REF]. However, these results will have to be confirmed in normal tissues compared with tumors because it is known that cancer cell lines often harbor an altered methylation pattern of their genome and excessive CpG island hypermethylation [START_REF] Smiraglia | Excessive CpG island hypermethylation in cancer cell lines versus primary human malignancies[END_REF].

DNMT1, by a small interfering RNA (siRNA) approach, was identified as a potent regulator of MUC2 and MUC5B epigenetic regulation. The DNMT1 effect was potentiated by HDAC2. In the same studies it was found that methylation of the two promoters dramatically impaired their activation by Sp1 transcription factor. Finally, one of the most important findings in this study was that MUC2 and MUC5B epigenetic regulation, and particularly the methylation profile of their promoters, was directly dependent on the cell differentiation status, leading to the hypothesis that hypermethylation of MUC2 and MUC5B may be a specific mechanism used by epithelial cancer cells to maintain a proliferating undifferentiated state or by normal cells to maintain pluripotency during development [START_REF] Vincent | Epigenetic regulation (DNA methylation, histone modifications) of the 11p15 mucin genes (MUC2, MUC5AC, MUC5B, MUC6) in epithelial cancer cells[END_REF].

Membrane-bound mucins 2.1.2.1 MUC4

The 5′-flanking region of MUC4 is characterized by a high G+C content, up to 72% concentrated almost exclusively in the proximal promoter and the 5′-UTR, and the presence of two CpG islands of 135 and 146 bp, respectively, both located in the proximal promoter at -2370/-2236 and -738/-593 [START_REF] Vincent | Epigenetic regulation of the human mucin gene MUC4 in epithelial cancer cell lines involves both DNA methylation and histone modifications mediated by DNA methyltransferases and histone deacetylases[END_REF].

The influence of histone deacetylation on MUC4 silencing was first assessed by the authors' laboratory in the pancreatic cancer cell line PANC-1 using cell treatment with TSA [START_REF] Jonckheere | A role for human MUC4 mucin gene, the ErbB2 ligand, as a target of TGF-beta in pancreatic carcinogenesis[END_REF] and later shown in prostate cancer cells treated with sodium butyrate by Singh and co-workers [START_REF] Singh | Aberrant expression of transmembrane mucins, MUC1 and MUC4, in human prostate carcinomas[END_REF]. This group also showed the influence of DNA methylation in regulating MUC4 in these cells by using 5-aza.

The studies in epithelial cancer cells that express variable levels of MUC4 showed that MUC4 proximal and distal promoters, including both CpG islands, were heavily methylated whether or not the gene was expressed in the cells. On the contrary, MUC4 5′-UTR, and particularly cytosines at -81, -93, -102, -113 and -121, showed a methylation profile that was directly correlated to the level of MUC4 expression in the cells [START_REF] Vincent | Epigenetic regulation of the human mucin gene MUC4 in epithelial cancer cell lines involves both DNA methylation and histone modifications mediated by DNA methyltransferases and histone deacetylases[END_REF]. Hypermethylation of MUC4 5′-UTR was associated with the establishment of a repressive histone code, including deacetylation of histone H3 and methylation of lysines 9 and 27 of histone H3 [START_REF] Vincent | Epigenetic regulation of the human mucin gene MUC4 in epithelial cancer cell lines involves both DNA methylation and histone modifications mediated by DNA methyltransferases and histone deacetylases[END_REF]. As four Sp1 binding sites had previously been identified and located, just upstream of and within the 5′-UTR [START_REF] Perrais | Characterization of human mucin gene MUC4 promoter: importance of growth factors and proinflammatory cytokines for its regulation in pancreatic cancer cells[END_REF], the influence of methylation of these Sp1 cis-elements on the regulation of MUC4 by this transcription factor after in vitro methylation of the promoter coupled to site-directed mutagenesis of the Sp1 sites was investigated further. It was found that methylation of Sp1 binding sites located in the proximal promoter and the 5′-UTR at -276/-271 and -166/-156 dramatically impaired MUC4 activation by Sp1, whereas methylation of the adjacent CpG site upstream of the 5′-UTR had no effect. Finally, siRNA approach and ChIP assays allowed us to identify DNMT3A, DNMT3B and HDAC3 as potent regulators of MUC4 endogenous expression that directly bind to its 5′-UTR [START_REF] Vincent | Epigenetic regulation of the human mucin gene MUC4 in epithelial cancer cell lines involves both DNA methylation and histone modifications mediated by DNA methyltransferases and histone deacetylases[END_REF].

From these results, a model was proposed of MUC4 epigenetic regulation in epithelial cells [START_REF] Vincent | Epigenetic regulation of the human mucin gene MUC4 in epithelial cancer cell lines involves both DNA methylation and histone modifications mediated by DNA methyltransferases and histone deacetylases[END_REF]. In normal pancreatic cells that do not express MUC4 [START_REF] Fauquette | Transcription factor AP-2alpha represses both the mucin MUC4 expression Expert[END_REF], MUC4 silencing would be mediated by DNA methylation and histone deacetylation at the 5′-UTR, whereas in early steps of pancreatic carcinogenesis progressive demethylation would allow low expression of MUC4, and finally in adenocarcinomatous high-expressing cells, selective hypomethylation and permissive chromatin would allow binding of essential transcription factors such as Sp1 and full transcription of the MUC4 gene. Recently, Yamada and co-workers showed that the methylation level of cytosines at -170 and -160 was associated with MUC4 gene expression in 10 cancer cell lines using the MassARRAY compact system, and therefore confirmed the critical contribution of promoter CpG methylation in MUC4 silencing [START_REF] Yamada | Promoter CpG methylation in cancer cells contributes to the regulation of MUC4[END_REF].

MUC1

The MUC1 gene is characterized by the presence of a CpG island in a large transcribed region that consists of 60-bp tandemly repeated units, each containing 1 SmaI restriction site. The first evaluation of the methylation status of various regions of the MUC1 gene by Zrihan-Licht and co-workers using the methylation-sensitive enzymes SmaI and XmaI showed that, in peripheral blood leukocytes that do not express MUC1, the 60-bp repeat array was completely methylated, whereas the same sequences were entirely unmethylated in DNA from MUC1-overexpressing breast tumor tissue. Hypomethylation within the repeat array was also observed in other epithelial tissues that express MUC1 at much lower levels than those seen in breast cancer tissue, demonstrating that hypomethylation of the tandem repeat array was an absolute requirement for MUC1 gene expression in epithelial tissues [START_REF] Zrihan-Licht | DNA methylation status of the MUC1 gene coding for a breast-cancer-associated protein[END_REF]. The influence of epigenetics in MUC1 silencing was then confirmed in prostate cancer cells by using cell treatment with 5-aza and sodium butyrate [START_REF] Singh | Aberrant expression of transmembrane mucins, MUC1 and MUC4, in human prostate carcinomas[END_REF]. The precise mechanisms of epigenetic regulation at MUC1 promoter had not been deciphered until recently, when Yamada and co-workers found hypermethylation of cytosines at -70, -55, -51, -47, -31, -21, -14, -4 and +20, tightly associated with dimethylation of H3-K9, in most MUC1 non-expressing cell lines, providing the first proof that MUC1 gene expression was regulated by DNA methylation of the promoter and associated histone H3 lysine 9 (H3-K9) modification [START_REF] Yamada | MUC1 expression is regulated by DNA methylation and histone H3 lysine 9 modification in cancer cells[END_REF].

Moving towards using mucin methylation as a diagnostic tool

Bisulfite-treated DNA sequencing of single clones is a gold standard and has been a very useful tool in our comprehensive studies to analyse precisely the methylation profile of mucin gene promoters and to determine the key CpG sites to use mucin gene methylation as a biomarker. However, application of this method in routine procedures for molecular diagnosis is constrained by several issues, as it is complex, time-consuming, expensive, and requires a fair number of sequenced clones to be considered quantitative. Therefore, our laboratory is currently validating the pyrosequencing technology to analyse the methylation status of mucin promoters in human tissues and validate the use of mucin gene methylation as a diagnostic and/or prognostic tool in epithelial cancers. Pyrosequencing is a fast, robust and flexible sequencing-by-synthesis-based method that offers a reproducible quantification of individual, consecutive CpG sites (up to 100 bp amplicon length), enabling discrimination of small changes in methylation levels, from single cytosines to global methylation of a promoter region, in a large number of samples [START_REF] Tost | DNA methylation analysis by pyrosequencing[END_REF].

This switch to pyrosequencing seems very promising, as our first data for MUC5B (this review), MUC5AC and MUC4 (unpublished data) already allowed us to confirm the potent status of CpG methylation as a biological tool in lung, colon and pancreatic cancer, respectively. The high level of methylation of MUC5B distal promoter seen in a MUC5B non-expressing normal lung tissue by bisulfite DNA sequencing is even more important in the corresponding MUC5B non-expressing lung adenocarcinoma (Figure 2A). In another histologic type of lung cancer, a MUC5B nonexpressing squamous cell lung carcinoma, it was also found, using pyrosequencing, that MUC5B distal promoter was hypermethylated at cytosines -2430, -2424, and -2421 when compared with normal lung tissue (Figure 2B). Regarding MUC4, our first analysis of its pattern of methylation in normal and cancerous pancreatic tissues confirmed that demethylation of cytosines at -93, -102 and -113 occurs in pancreatic adenocarcinoma when compared with MUC4-non-expressing normal pancreas (not shown). Although these preliminary data need further confirmation on larger panels of tissues, it is hoped to provide a rapid way of evaluating mucin gene methylation in cohorts and use it as a specific biomarker. As mucin genes show altered expression in various types of epithelial cancer (adenocarcinoma, squamous carcinoma), and also in numerous organs, tissues (respiratory tract, gastrointestinal tract, urogenital tract, etc.) and fluids (tears, saliva, sperm, milk, etc.), a lot of work as yet to be done. Moreover, in the future this method should be of great help to determine the prevalence of mucin gene methylation in genomic DNA released from tumors in biological fluids such as plasma or serum, bronchoalveolar lavages for lung cancer, and pancreatic juice for pancreatic cancer.

Mucins and epigenomics

All the techniques that were used to study epigenetic changes at promoters of candidate genes can now be coupled both with genome-wide approaches, such as microarray technologies, and high-throughput methods, such as ultradeep sequencing, for high-resolution mapping of epigenetic profiles in both normal and cancer cell types [START_REF] Esteller | Cancer epigenomics: DNA methylomes and histone-modification maps[END_REF][START_REF] Zilberman | Genome-wide analysis of DNA methylation patterns[END_REF]. Methods for genome-wide mapping of DNA methylation patterns include methylated CpG island amplification (MCA), followed by hybridization on microarrays [START_REF] Omura | Genome-wide profiling of methylated promoters in pancreatic adenocarcinoma[END_REF], methylated DNA immunoprecipitation (MeDIP)-on-chip [START_REF] Keshet | Evidence for an instructive mechanism of de novo methylation in cancer cells[END_REF], or ultradeep bisulfite sequencing [START_REF] Taylor | Ultradeep bisulfite sequencing analysis of DNA methylation patterns in multiple gene promoters by 454 sequencing[END_REF]. Similarly, histone modification status can be widely studied using chromatin-immunoprecipitation-based methods [START_REF] Shames | Methods for detecting DNA methylation in tumors: from bench to bedside[END_REF][START_REF] Massie | ChIPping away at gene regulation[END_REF] such as ChIP-on-chip or ChIP-seq. These new technologies will allow us to decipher further the mechanisms by which mucin genes are epigenetically regulated as clusters. Indeed, regulation of the 11p15 MUC cluster at the gene level has been studied previously in the authors' laboratory, but has not yet been completely understood at a more global level. Similarly, regulation of mucin gene clusters at 3q29 (MUC4, MUC20) and 7q22 (MUC3, MUC12, MUC17) chromosomal regions [START_REF] Jonckheere | The epithelial mucins: structure/function Roles in cancer and inflammatory diseases[END_REF] remains to be investigated.

These genome-wide approaches will also be essential and necessary if one wants to give an explanation of the neveranswered hypothesis as to how the tight coordination works and leads to simultaneous expression of a specific panel of both secreted and membrane-bound mucins, which follow two different biosynthetic pathways, in each type of epithelium. Finally, these comprehensive studies will allow us to understand better the specific mechanisms responsible for maintaining epithelium protection, as mucins are key proteins in epithelial homeostasis, and, therefore, the potential impacts of the dysregulation of their genes in epithelial diseases.

Most importantly, as it is now agreed that individual biomarkers are not informative enough for the diagnosis of most tumor types, translational epigenomics will be an essential tool to determine an ideal set of various biomarkers (tumor ID or tumor signature), which will include specific epigenetic modifications of mucin genes, that may define each neoplasia and therefore be used for diagnosis and prognosis of epithelial cancers.

Mucins and regulating RNAs 2.4.1 MicroRNAs

MicroRNAs are endogenous non-coding RNAs, of 18 -24 nucleotides in length, typically excised from 60 to 110 nucleotide foldback RNA precursor structures [START_REF] Calin | MicroRNA signatures in human cancers[END_REF], that play important negative regulatory roles in animals and plants by targeting messenger RNA transcripts for degradation or translational repression, involving the RNA interference silencing complex (RISC) [START_REF] Bartel | MicroRNAs: genomics, biogenesis, mechanism, and function[END_REF]. Recently, Gonzalez and co-workers showed that miRNAs can also trigger transcriptional silencing by means of chromatin remodelling (i.e., trimethylation of H3K9) at DNA regions containing complementary sequences [START_REF] Gonzalez | Mechanistic principles of chromatin remodeling guided by siRNAs and miRNAs[END_REF]. So far, hundreds of unique, mature miRNAs have been identified in humans, with more to be discovered. Their expression levels vary greatly among tissues [START_REF] Wang | Direct and sensitive miRNA profiling from low-input total RNA[END_REF] and especially during pathological processes [START_REF] Wang | MicroRNA epigenetic alterations: predicting biomarkers and therapeutic targets in human diseases[END_REF], including carcinogenesis [START_REF] Wiemer | The role of microRNAs in cancer: no small matter[END_REF][START_REF] Lee | MicroRNAs in Cancer[END_REF][START_REF] Zhang | microRNAs: a new emerging class of players for disease diagnostics and gene therapy[END_REF]. Chromosomal rearrangements, genomic amplifications or deletions, epigenetic regulation and mutations at genes encoding miRNAs lead to their specific overexpression or underexpression in different types of tumor, which contributes to cancer development and progression. Changes in miRNA expression have been reported by various genome-wide techniques (microarray platforms, bead-based flow cytometry) in colorectal neoplasia [START_REF] Asangani | MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer[END_REF], pancreatic cancer [START_REF] Bloomston | MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis[END_REF][START_REF] Lee | Expression profiling identifies microRNA signature in pancreatic cancer[END_REF][START_REF] Szafranska | MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma[END_REF], kidney and bladder cancers [START_REF] Gottardo | Micro-RNA profiling in kidney and bladder cancers[END_REF], esophageal [START_REF] Feber | MicroRNA expression profiles of esophageal cancer[END_REF] and gastric carcinomas [START_REF] Volinia | A microRNA expression signature of human solid tumors defines cancer gene targets[END_REF] and lung cancer [START_REF] Mascaux | Evolution of microRNAs expression during human bronchial squamous carcinogenesis[END_REF], potentially affecting expression levels of numerous putative targets (Table 1). This led to the new concept of miRNA expression signatures [START_REF] Volinia | A microRNA expression signature of human solid tumors defines cancer gene targets[END_REF] associated with cancer diagnosis (tissue type), predisposition, prognosis (differentiation and aggressiveness, [START_REF] Lujambio | A microRNA DNA methylation signature for human cancer metastasis[END_REF]), identification of a primary tumor [START_REF] Rosenfeld | MicroRNAs accurately identify cancer tissue origin[END_REF] and response to therapy [START_REF] Love | Not miR-ly small RNAs: big potential for microRNAs in therapy[END_REF]. Moreover, miRNAs are present in human plasma in a remarkably stable form and therefore represent a potentially useful approach for the biological fluid-based detection of human cancer [START_REF] Mitchell | Circulating microRNAs as stable blood-based markers for cancer detection[END_REF]. For most miRNAs though, identification of their target genes and the biological consequences of their repressive activity on cancer progression remain to be defined.

E S T G T T T T T T A C C C G G G GG A T C G T C E S T G T T T T T T A C C C G G G GG A T C G
No evidence for mucin gene regulation by miRNAs has been reported so far. However, interrogation of MiRbase and MiRanda databases ([81-84]) revealed a large number of miRNAs that could potentially target 33-UTR of mucin genes encoding either the membrane-bound mucins MUC1, MUC4, MUC20, MUC3A/3B, MUC17 and MUC13 or the secreted mucins MUC2, MUC5AC, MUC6, MUC7 and MUC19 (Table 2). Interestingly, although miRNAs might be responsible for mucin gene regulation, a recent study by Ramasamy and co-workers showed, conversely, that N-glycosylation of Asn-36 on MUC1 C-terminal subunit suppresses miR-322 expression in breast and prostate cancer cells, thereby increasing expression of galectin-3 [START_REF] Ramasamy | The MUC1 and galectin-3 oncoproteins function in a microRNA-dependent regulatory loop[END_REF]. This very interesting result suggests that, in turn, mucins may regulate miRNA expression and activity.

Antisense RNA

Natural antisense transcripts are endogenous RNA molecules containing sequences that are complementary to the proteinencoding sense transcript. Cis antisense RNAs are transcribed at the same genomic locus from the opposite strand, whereas trans antisense RNA are transcribed from separate loci. Antisense RNAs are thought to regulate their target genes at the genomic level by triggering chromatin remodelling [START_REF] Yu | Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA[END_REF], or at the transcriptomic level by interfering with mRNA transcription, processing, stability, translocation and translation [START_REF] Lapidot | Genome-wide natural antisense transcription: coupling its regulation to its different regulatory mechanisms[END_REF].

Recently, evidence for antisense transcripts has been reported in 2900 -6400 human genes using the asymmetric strand-specific analysis of gene expression (ASSAGE) technology [START_REF] He | The antisense transcriptomes of human cells[END_REF]. The distribution of these antisense RNAs is non-random across the genome and differs among cell types, suggesting a fundamental role in regulation of sense transcription. In this comprehensive study, sense and antisense expression of 32,354 Ensembl genes in three cancerous and two normal cell lines was assessed, including those of MUC1, MUC4, MUC3A/3B, MUC17, MUC16, MUC2, MUC5AC, MUC5B, MUC6 and MUC7. The results show that high expression of MUC5B and MUC6 antisense RNAs (Figure 1) was found in non-epithelial cells that do not express sense transcripts of these genes. These preliminary data concerning expression of mucin antisense RNAs will need further investigation. Moreover, as epigenetic silencing of tumor suppressor genes by their antisense RNA has already been reported, the role of these transcripts in mucin gene silencing by methylation and histone modifications will have to be elucidated.

Expert opinion

It is obvious that epigenetic regulation of mucin genes is still at its dawn, but in vitro studies in cancer cell lines, expression studies on human tissue sections and very recent genome-wide studies using new technologies clearly indicate that DNA methylation of their promoters is a key molecular mechanism regulating their expression in epithelial cancers. Detection of MUC methylation may thus soon provide a useful biological tool for the clinician. The greatest challenge for researchers working in the mucin field is of course related to the huge diversity of these glycoproteins and also their genes (> 20 known until now), the huge size of the genes, their polymorphisms, and the redundancy that such a complex family of molecules may create.

We think that systematic comparison of MUC methylation in normal and carcinomatous tissues will provide useful tools for diagnosis and prognosis of numerous epithelial cancers, including those of lung [START_REF] Belinsky | Gene-promoter hypermethylation as a biomarker in lung cancer[END_REF], pancreas [START_REF] Matsubayashi | DNA methylation alterations in the pancreatic juice of patients with suspected pancreatic disease[END_REF] and colon [START_REF] Schuebel | Comparing the DNA hypermethylome with gene mutations in human colorectal cancer[END_REF], but also esophageal and gastric carcinoma. Alterations in DNA methylation are early events in carcinogenesis and, as such, could become specific and sensitive biomarkers that could be detected in biological fluids such as blood (liver cancer), sputum and bronchoalveolar lavage (lung cancer), feces (colon cancer), urine (bladder cancer) or nipple aspirate (breast cancer) [START_REF] Wilson | DNA hypomethylation and human diseases[END_REF][START_REF] Laird | The power and the promise of DNA methylation markers[END_REF]. However, so far, methylation status for one particular gene as a reliable marker has been described only in rare cases (GSTP1 in prostate cancer [START_REF] Cairns | Molecular detection of prostate cancer in urine by GSTP1 hypermethylation[END_REF] or APC in esophageal adenocarcinoma [START_REF] Kawakami | Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma[END_REF]). Most of the time, analysis of several genes (methylome signature) is necessary to distinguish cancers from normal tissues or other pathologies [START_REF] He | The antisense transcriptomes of human cells[END_REF][START_REF] Issa | CpG island methylator phenotype in cancer[END_REF]. Moreover, as methylation is a tumor-specific change in DNA, identification of panels of complementary biomarkers may help to classify histopathologic tumor types, tumor grades as well as tumors from different tissues [START_REF] Shames | Methods for detecting DNA methylation in tumors: from bench to bedside[END_REF]. Thus, methylation of genes encoding mucins, which are markers of cell differentiation in many epithelial tissues, may provide a very useful tool to clinicians and pathologists. Yet, this potential future promising clinical application depends crucially on the discovery of useful aberrantly methylated sequences. In this context, studying the methylation status of mucin genes, which are aberrantly expressed in cancers, may help to develop these early detection tools. The development of new powerful, reliable and rapid methods, such as pyrosequencing or high-throughput technologies on chips [START_REF] Shames | Methods for detecting DNA methylation in tumors: from bench to bedside[END_REF][START_REF] Shames | A genome-wide screen for promoter methylation in lung cancer identifies novel methylation markers for multiple malignancies[END_REF], to screen methylated genes in tissues should quickly provide new information regarding mucin gene epigenetic status in tissues from cancer patients and at different stages of carcinogenesis.

The studies of epigenetic regulation of mucin genes also point out the opposite role of secreted and membrane-bound mucins in carcinogenesis. Indeed, genes encoding secreted mucins, and especially MUC2 and MUC5B, are often aberrantly silenced in non-mucinous colon [START_REF] Mizoshita | Loss of MUC2 expression correlates with progression along the adenoma-carcinoma sequence pathway as well as de novo carcinogenesis in the colon[END_REF] and lung [START_REF] Copin | Normal respiratory mucosa, precursor lesions and lung carcinomas: differential expression of human mucin genes[END_REF] carcinomas, respectively, whereas genes encoding MUC1 and MUC4 membrane-bound mucins are overexpressed in cancers particularly in breast [START_REF] Rakha | Expression of mucins (MUC1, MUC2, MUC3, MUC4, MUC5AC and MUC6) and their prognostic significance in human breast cancer[END_REF] and pancreatic [100] carcinomas, respectively. Moreover, in cancers, expression of secreted mucins is often associated with better prognosis [101], whereas expression of membrane-bound mucins is associated with tumor cell detachment, invasion and metastasis and worse prognosis [START_REF] Rakha | Expression of mucins (MUC1, MUC2, MUC3, MUC4, MUC5AC and MUC6) and their prognostic significance in human breast cancer[END_REF]102]. These data are in accordance with the findings that MUC2 and MUC5B promoters are hypermethylated, whereas those of MUC1 and MUC4 are demethylated in cancers. Aberrant silencing of specific genes by hypermethylation and ectopic onco-fetal expression of other genes by demethylation are common features of cancer cells and lead to selective advantage in term of growth and survival. Thus, epigenetics provide a new generation of tumor suppressor genes and oncogenes [103]. Therefore, studying epigenetic regulation of mucin genes may help clarify their role in carcinogenesis as candidate tumor suppressor genes, as has already been suggested for Muc2 [START_REF] Velcich | Colorectal cancer in mice genetically deficient in the mucin Muc2[END_REF], or oncogenes, as has already been proposed for MUC1 [104].

Finally, the findings that mucin genes may be epigenetically regulated in cancers prompt us to consider them as attractive targets for new therapeutic strategies. As epigenetic changes are potentially reversible, an increasing number of specific DNA methylation or histone deacetylation pharmacological inhibitors have been developed over the past few years. Among them, the DNMT inhibitor 5-aza (decitabine) is the most extensively studied and has been shown to reactivate MUC2 and MUC5B in vitro in numerous cancer cell lines [START_REF] Vincent | Epigenetic regulation (DNA methylation, histone modifications) of the 11p15 mucin genes (MUC2, MUC5AC, MUC5B, MUC6) in epithelial cancer cells[END_REF]. On the other hand, the HDAC inhibitor TSA, which is known to induce apoptosis and enhance the response to chemotherapeutic agents in pancreatic cancer [105], has been shown to induce a severe decrease of MUC4 expression in pancreatic cancer cells [START_REF] Vincent | Epigenetic regulation of the human mucin gene MUC4 in epithelial cancer cell lines involves both DNA methylation and histone modifications mediated by DNA methyltransferases and histone deacetylases[END_REF]. Hence, in the future, potential application of epigenetic drugs in either reactivating expression of secreted mucins or inhibiting membrane-bound mucins will have to be investigated. Adapted from Gottardo et al. [START_REF] Gottardo | Micro-RNA profiling in kidney and bladder cancers[END_REF], Volinia et al. [START_REF] Volinia | A microRNA expression signature of human solid tumors defines cancer gene targets[END_REF], Feber et al. [START_REF] Feber | MicroRNA expression profiles of esophageal cancer[END_REF] and Calin and Croce [START_REF] Calin | MicroRNA signatures in human cancers[END_REF]. Target prediction is based on miRBase, TargetScan and MiRanda. The listed miRNAs are predicted by at least two of these prediction programs The most likely tumor-related genes are listed. Deciphering the epigenetic mechanisms regulating mucin gene expression has been very helpful in understanding mucin gene biology better, but in the future it will provide clinicians and pathologists with new biological tools to screen patients and detect cancer at an early stage as well as classify tumors and propose new therapeutic strategies.

Figure 2 .

 2 Figure 2. Methylation profile of MUC5B distal promoter in MUC5B non-expressing lung cancers. A. Mapping of methylated cytosines was carried out by bisulfite-treated DNA sequencing in normal lung (upper panel, N) and in a MUC5B-negative lung adenocarcinoma (lower panel, T). On the right-hand side is shown immunohistochemical staining of MUC5B apomucin in the corresponding tissues (magnification × 100). Partial sequence of MUC5B promoter is represented by the horizontal bar. Numbering refers to the proximal transcription start site of MUC5B (+1, broken arrow) [42,106]. Vertical bars indicate CpG sites. More than four individual clones were analysed per CpG site and type of tissue (normal lung or lung adenocarcinoma). Black circles represent methylated cytosines and open circles represent unmethylated cytosines. Key CpG methylated sites previously identified in cancer cell lines [44] are topped with a star and their position in the promoter is indicated. The horizontal thick bar indicates the promoter region analysed further using the pyrosequencing technology. B. Mapping of methylated cytosines in MUC5B promoter by pyrosequencing in normal lung (upper panel, N) and in a MUC5B-negative lung squamous cell carcinoma (lower panel, T). On the right-hand side is shown immunohistochemical staining of MUC5B apomucin in the corresponding tissues (magnification × 100). The pyrograms show the methylation percentages of the four CpG sites of interest (-2430, -2424, -2421 and -2414) in MUC5B distal promoter using the pyrosequencing technology (PyroMark ™ MD, Biotage).Av: The average methylation content of the promoter in normal lung compared with lung squamous cell carcinoma.

3 Figure 1. The 11p15 chromosomal region. Positions of the

	imprinted domain, the two imprinting control regions (ICR) and the mucin gene cluster (MUC) are indicated	(black boxes). Full arrows indicate the transcription orientation for each gene. Dashed arrows indicate an antisense transcript (MUC5B, MUC6). Imprinted genes are boxed in gray and	bold. Other known designations for each gene are indicated in the same box. Allele-specific methylation of imprinted genes is indicated by half-filled rectangles, p: paternal allele,	m: maternal allele. Epigenetic abnormalities frequently described in Beckwith-Wiedemann Syndrome (BWS) and Silver-Russel Syndrome (SRS) are indicated. Triangles represent putative	target regions for post-transcriptional regulation of mucin transcripts by miRNAs (MUC2, MUC5AC, MUC6). The number of putative miRNAs targeting these regions is indicated and	is detailed in

Table 2 .

 2

Expert Opin. Med. Diagn. (2009) 3(4)

Table 1 . Putative target genes of differentially expressed miRNAs identified in aerodigestive cancers.

 1 Adapted from Gottardo et al.[START_REF] Gottardo | Micro-RNA profiling in kidney and bladder cancers[END_REF], Volinia et al.[START_REF] Volinia | A microRNA expression signature of human solid tumors defines cancer gene targets[END_REF], Feber et al.[START_REF] Feber | MicroRNA expression profiles of esophageal cancer[END_REF] and Calin and Croce[START_REF] Calin | MicroRNA signatures in human cancers[END_REF]. Target prediction is based on miRBase, TargetScan and MiRanda. The listed miRNAs are predicted by at least two of these prediction programs The most likely tumor-related genes are listed.

	495
	496
	497
	498
	499
	500
	501
	502
	503
	504
	505
	506
	507
	508
	509
	510
	511
	512
	513
	514
	515
	516
	517
	518
	519
	520
	521
	522
	523
	524
	525
	526
	527
	528
	529
	530
	531
	532
	533
	534
	535
	536
	537
	538
	539
	540
	541
	542
	543
	544
	545
	546
	547
	548
	549

§ Prognostic and/or diagnostic markers. Expert Opin. Med. Diagn. (2009) 3(4)

Table 1 . Putative target genes of differentially expressed miRNAs identified in aerodigestive cancers (continued).

 1

	605
	606
	607
	608
	609
	610
	611
	612
	613
	614
	615
	616
	617
	618
	619
	620
	621
	622
	623
	624
	625
	626
	627
	628
	629
	630

‡

Predicted only by miRBase.

Target prediction is based on miRBase and miRanda. The listed miRNAs are predicted by the two prediction programs.

Table 2 . Predicted miRNA targeting mucin genes (continued).

 2 Predicted only by miRBase.Target prediction is based on miRBase and miRanda. The listed miRNAs are predicted by the two prediction programs.

	Mucin gene	Chromosomal localization	Number of predicted miRNA	List of iRNA
				miR-765
				miR-890
				miR-936
	Other secreted mucins
	MUC7	4q13-q21	2	miR-199a-3p
				miR-544
	MUC19	12q12	9	miR-140-5p
				miR-146a*,
				miR-146b-5p
				miR-29b
	‡

Expert Opin. Med. Diagn. (2009) 3(4)

Table 2 . Predicted miRNA targeting mucin genes (continued).

 2

Expert Opin. Med. Diagn. (2009) 3(4)

Seuningen & VincentExpert Opin. Med. Diagn. (2009) 3(4)

Acknowledgment

The authors thank F Renaud for tissue section selection.

Seuningen & Vincent

Expert Opin. Med