IJC International Journal of Cancer
MicroRNA expression profiling in human Barrett’s carcinogenesis Matteo Fassan1, Stefano Volinia2, Jeff Palatini2, Marco Pizzi1, Raffaele Baffa3, Marina De Bernard4, Giorgio Battaglia5, Paola Parente1,5, Carlo M. Croce2, Giovanni Zaninotto6, Ermanno Ancona5,6 and Massimo Rugge1,5 1
Department of Medical Diagnostic Sciences and Special Therapies, Surgical Pathology and Cytopathology Unit, University of Padova, Padova (PD), Italy Comprehensive Cancer Center, Ohio State University, Columbus, OH 3 Thomas Jefferson University, Philadelphia, PA—USA and Research, MedImmune LLC, Gaithersburg, MD 4 Venetian Institute of Molecular Medicine, Padova (PD), Italy 5 Istituto Oncologico Veneto—IOV-IRCCS, Padova (PD), Italy 6 Department of Gastroenterological and Surgical Sciences, University of Padova, Padova (PD), Italy
Barrett’s esophagus (BE) is characterized by the native stratified squamous epithelium (N) lining the esophagus being replaced by a columnar epithelium with intestinal differentiation (Barrett’s mucosa; BM). BM is considered as the main risk factor for esophageal adenocarcinoma (Barrett’s adenocarcinoma; BAc). MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting messenger RNAs and they are reportedly dysregulated in BM. To test the hypothesis that a specific miRNA expression signature characterizes BM development and progression, we performed miRNA microarray analysis comparing native esophageal mucosa with all the phenotypic lesions seen in the Barrett’s carcinogenic process. Specimens were collected from 14 BE patients who had undergone esophagectomy, including: 14 with N, 14 with BM, 7 with low-grade intraepithelial neoplasia, 5 with high-grade intra-epithelial neoplasia and 11 with BAc. Microarray findings were further validated by quantitive real-time polymerase chain reaction and in situ hybridization analyses using a different series of consecutive cases (162 biopsy samples and 5 esophagectomies) of histologically proven, long-segment BE. We identified a miRNA signature of Barrett’s carcinogenesis consisting of an increased expression of 6 miRNAs and a reduced expression of 7 miRNAs. To further support these results, we investigated target gene expression using the Oncomine database and/or immunohistochemical analysis. We found that target gene expression correlated significantly with miRNA dysregulation. Specific miRNAs are directly involved in BE progression to cancer. miRNA profiling significantly expands current knowledge on the molecular history of Barrett’s carcinogenesis, also identifying molecular markers of cancer progression.
Barrett’s esophagus (BE) is defined as the replacement of the native esophageal squamous epithelium by a lining of columnar epithelium with intestinal differentiation [Barrett’s mucosa (BM)].1,2 Epidemiological and clinico-pathological Key words: miRNA, Barrett’s esophagus, gene target, expression signature Abbreviations: BAc: Barrett’s adenocarcinoma; BE: Barrett’s esophagus; BM: Barrett’s mucosa; HG: high-grade intra-epithelial neoplasia; IHC: immunohistochemistry; IM: intestinal metaplasia; ISH: in situ hybridization; LG: low-grade intra-epithelial neoplasia; miRNA: microRNA; N: native stratified squamous epithelium All authors of this research paper participated directly in the planning and execution of the study, and in the analysis of the results. The authors have no competing interests to declare. DOI: 10.1002/ijc.25823 History: Received 30 Jul 2010; Accepted 19 Nov 2010; Online 2 Dec 2010 Correspondence to: Massimo Rugge, Department of Medical Diagnostic Sciences and Special Therapies, University of Padova, Istituto Oncologico Veneto-IRCCS, Via Aristide Gabelli 61, 35121, Padova, Italy, Tel.: þ39-049-8218990, Fax: þ39-049-8272277, E-mail:
[email protected]
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studies have consistently shown BM to be the initial event in a cascade of phenotypic changes that may lead to Barrett’s adenocarcinoma (BAc).3–5 Very little is known as yet about the molecular mechanisms involved in neoplastic transformation. From a prognostic point of view, the histological diagnosis of dysplasia [defined as low- and high-grade intraepithelial neoplasia (IEN or NiN)] is currently the only biomarker considered in the definition of high risk BE populations.6–8 Expanding on such biological information might lead to the identification of new prognostic markers and targeted therapies. MicroRNAs (or miRNAs) are a class of small noncoding RNAs that control gene expression by targeting messenger RNAs (mRNAs).9–11 A growing number of reports point to the involvement of miRNAs in carcinogenesis and/or tumor progression, and miRNA expression profiles have been suggested as a promising new class of biomarkers for tumor diagnosis and prognosis, including the prediction of response to therapy. MiRNAs are reportedly involved in the oncogenic process leading to the onset of both BM and BAc. The first published report by Feber et al.12 described the miRNA expression profiling of esophageal cancers (both adenocarcinoma and
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squamous cell carcinoma) and a small series of BM samples. After this seminal study, Dijckmeester et al. investigated miR-143 and miR-205 expression in neosquamous esophageal epithelium after Argon plasma ablation of BM.13 In our study, miR-143 was found significantly up-regulated in neosquamous mucosa by comparison with the normal squamous epithelium of control subjects.13 In human Barrett’s carcinogenesis, miR-196a has been established as a potential marker of progression by targeting KRT5, SPRR2C and S100A9,14 whereas in BM/BAc-derived cell lines, miRNA expression profiling revealed that miR-106b-25 polycistron is involved in the neoplastic progression via CDKN1A and BCL2L11 suppression.15 In a large series of esophageal adenocarcinoma samples, BAc showed a specific miRNA expression profile by comparison with cancers unrelated to BE.16 In a series of paired, diseased and normal tissues, Yang et al.17 showed that specific miRNA expression signatures are associated with BM cancerization. None of the abovementioned studies, however, provided comprehensive information on the miRNA profile at each step of the natural history of Barrett’s carcinogenesis. We undertook to find a specific miRNA expression signature consistently associated with the whole spectrum of this oncogenic process, also investigating miRNA target gene expression by means of the Oncomine database and/or immunohistochemical (IHC) analyses. Our results strongly support the direct involvement of specific miRNAs in BE progression and their potential as a novel diagnostic tool in the characterization of BAc gene targets.
Material and Methods Tissue samples
All cases of BE patients who had undergone total (R0, no residual tumor) esophagectomy for HG-NiN and BAc reported between 2005 and 2009 were retrieved from the archives of the surgical pathology and cytopathology unit of Padova University. Of the 158 cases initially found, 144 were subsequently excluded for the following reasons: (i) prior neoadjuvant chemotherapy, 51 cases; (ii) prior surgical or endoscopic treatments, 40 cases; and (iii) archival tumor samples inconsistent with the aims of the study, 49 cases. Informed consent from involved patients was obtained in all, but not in four cases (which were also ruled out). Thus, 14 patients (mean age: 63.667.9 years; range: 52–81; 12 men, 2 women; all Caucasian) were considered, who had all been surgically treated at the same institution (Department of Gastroenterological and Surgical Sciences of Padova University). All cases were assessed by two pathologists (PP and MF); in cases where their opinions differed, a third GI expert pathologist (MR) was consulted. Two 2-mm cores were obtained from the paraffin blocks from: (i) the proximal native squamous esophageal mucosa (N ¼ 14 cases); (ii) intestinal metaplasia (IM) positive esophageal mucosa (BM ¼ 14 cases); (iii) low-grade
intraepithelial neoplasia (LG ¼ 7 cases); (iv) high-grade intraepithelial neoplasia (HG ¼ 5 cases); and (v) BAc (11 cases) were collected and further analyzed in the microarray study. For the quantitative real-time polymerase chain reaction (qRT-PCR) and the IHC studies, tissue samples were retrospectively collected from the files of the Veneto Region’s multicenter Barrett’s Esophagus Registry (EBRA; Padova Unit),5 selecting consecutive cases of histologically proven long-segment BE. A total of 162 biopsy samples obtained from different biopsy sets were considered, including: N ¼ 40 cases, BM ¼ 40 cases, LG ¼ 31 cases, HG ¼ 26 cases and BAc ¼ 25 cases. For the in situ hybridization (ISH) study, tissues were collected from five further BAc patients who had undergone esophagectomy. All the patients considered in our study gave their written informed consent.
miRNA microarray
Tissue samples were deparaffinized with xylene at 50 C for 3 minutes. Total RNA extraction was done using the RecoverAll kit (Ambion, Austin, TX) according to manufacturer’s instructions. RNA labeling and hybridization on miRNA microarray chips were performed as described elsewhere.10,18 Briefly, 5 lg of total RNA from each sample were reversetranscribed using biotin end-labeled random-octamer oligonucleotide primer. Biotin-labeled complementary DNA was hybridized on an Ohio State University custom miRNA microarray chip (OSU_CCC version 4.0), which contains 1100 miRNA probes, including 326 human and 249 mouse miRNA genes and 10 control genes, spotted in duplicate. The hybridized chips were washed and processed for biotincontaining transcript detection by streptavidin-Alexa 647 conjugate and scanned on an Axon 4000B microarray scanner (Axon Instruments, Sunnyvale, CA).
Statistical and bioinformatic analyses
Microarray images were analyzed using GENEPIX PRO 6.0 (Axon Instruments, Sunnyvale, CA). Average values of the replicate spots of each miRNA were background subtracted, normalized using quantiles enabling a comparison between chips19 and further analyzed. The microarray data are deposited in the Gene Expression Omnibus at the National Center for Biotechnology Information (GEO:GSE20099). The miRNAs that were differently expressed between different esophageal lesions were identified using a random-variance t-test, which is an improvement over the standard separate t-test because it enables information on within-class variation to be shared among genes without assuming that all genes have the same variance.20 Genes were considered statistically significant if their p value was less than 0.001; a stringent significance threshold was used to limit the number of false positive findings.21 Only mature miRNAs that were differently expressed are reported. C 2010 UICC Int. J. Cancer: 129, 1661–1670 (2011) V
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Figure 1. miRNAs differently expressed during human Barrett’s carcinogenesis. Hierarchical clustering of the miRNA genes with a significantly different expression (p < 0.001) during Barrett’s carcinogenesis (a), in Barrett’s mucosa (BM versus normal squamous esophageal epithelium; b) and in Barrett’s adenocarcinoma (BAc versus normal squamous esophageal epithelium; c). Rows represent individual genes; columns represent individual tissue samples. Pseudo-colors indicate transcript levels below, equal to, or above the mean
Quantitative real-time polymerase chain reaction
The NCodeTM miRNA qRT-PCR method (Invitrogen, Carlsbad, CA) was used to detect and quantify mature miRNAs on Applied Biosystems RT-PCR instruments in accordance with manufacturer’s instructions. Normalization was performed with the small nuclear RNA U6B (RNU6B; Invitrogen). All real-time reactions, including no-template controls and real-time minus controls, were run in a GeneAmp PCR 9700 thermocycler (Applied Biosystems, Foster City, CA). Gene expression levels were quantified using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Comparative RT-PCR was performed in triplicate, including no-template controls. The fold difference for each sample was obtained using the equation 2dCt; Ct is the threshold cycle, the cycle number at which the fluorescence generated within a reaction crosses the threshold; dCt ¼ (Ct average sample gene) – (Ct average RNU6B). Total RNAs from 15 N, 15 BM, 15 LG, 15 HG and 15 BAc biopsy samples were used in the qRT-PCR analysis.
treated with Proteinase K (DakoCytomation) for 30 min at room temperature, rinsed several times with dH2O and immersed in 95% ethanol for 10 sec before air drying. Slides were prehybridized at 49–56 C for 1 hr with mRNA ISH buffer (Ambion) before incubation overnight at 49–56 C in buffer containing the 50 -biotin labeled miR-203, miR-205, let-7c miRCURYTM LNA detection probe (Exiqon, Woburn, MA) or the scrambled negative control probe (U6, Exiqon) at 200 nM final concentration. Slides were washed in both TBST washing buffer and GenPoint stringent wash solution (54 C for 30 min). Slides were then exposed to H2O2 blocking solution (DakoCytomation) for 20 min and further blocked in a blocking buffer (DakoCytomation) for 30 min before being exposed to primary Streptavidin-HRP antibody, biotinyl tyramide, secondary Streptavidin-HRP antibody and DAB chromogen solutions following the manufacturer’s protocol. Slides were then briefly counterstained in hematoxylin and rinsed with both TBST and water before mounting. For the ISH study, samples obtained from 5 BAc esophagectomies were considered.
In situ hybridization
ISH was performed using the GenPointTM catalyzed signal amplification system (DakoCytomation) following the manufacturer’s protocol. Briefly, slides were incubated at 60 C for 30 min and deparaffinized as described.22 Sections were C 2010 UICC Int. J. Cancer: 129, 1661–1670 (2011) V
cDNA microarray analysis
The Oncomine database and gene microarray analysis tool, a repository for published cDNA microarray data (www.oncomine.org),23,24 was explored (15th December 2009) for
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(green, black and red, respectively). The scale represents the intensity of gene expression (log2 scale ranges between 3 and 3).
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Table 1. Differently expressed miRNAs in Barrett’s carcinogenesis and between diseased and normal esophageal tissues
miRNA
Correlation coefficient
p
FDR-adjusted p
Fold change
p
FDR-adjusted P
Fold change
p
FDR-adjusted p
62.77
