Integrative Genomics Identified RFC3 As an Amplified Candidate Oncogene in Esophageal Adenocarcinoma

Published OnlineFirst February 10, 2012; DOI: 10.1158/1078-0432.CCR-11-1431

Clinical Cancer Human Cancer Biology Research

Integrative Genomics Identiﬁed RFC3 As an Ampliﬁed Candidate Oncogene in Esophageal Adenocarcinoma

William W. Lockwood1, Kelsie L. Thu1, Lin Lin2, Larissa A. Pikor1, Raj Chari1, Wan L. Lam1, and David G. Beer2

Abstract Purpose: Esophageal adenocarcinoma (EAC) is a lethal malignancy that can develop from the premalignant condition, Barrett’s esophagus (BE). Currently, there are no validated simple methods to predict which patients will progress to EAC. A better understanding of the genetic mechanisms driving EAC tumorigenesis is needed to identify new therapeutic targets and develop biomarkers capable of identifying high-risk patients that would benefit from aggressive neoadjuvant therapy. We employed an integrative genomics approach to identify novel genes involved in EAC biology that may serve as useful clinical markers. Experimental Design: Whole genome tiling-path array comparative genomic hybridization was used to identify significant regions of copy number alteration in 20 EACs and 10 matching BE tissues. Copy number and gene expression data were integrated to identify candidate oncogenes within regions of amplification and multiple additional sample cohorts were assessed to validate candidate genes. Results: We identified RFC3 as a novel, candidate oncogene activated by amplification in approximately 25% of EAC samples. RFC3 was also amplified in BE from a patient whose EAC harbored amplification and was differentially expressed between nonmalignant and EAC tissues. Copy number gains were detected in other cancer types and RFC3 knockdown inhibited proliferation and anchorage-independent growth of cancer cells with increased copy number but had little effect on those without. Moreover, high RFC3 expression was associated with poor patient outcome in multiple cancer types. Conclusions: RFC3 is a candidate oncogene amplified in EAC. RFC3 DNA amplification is also prevalent in other epithelial cancer types and RFC3 expression could serve as a prognostic marker. Clin Cancer Res; 18(7); 1936–46. 2012 AACR.

Introduction esophagus (BE) is a premalignant condition that can give Over the past 3 decades there has been a dramatic rise in rise to EAC. Histologically, BE is characterized by the the incidence of esophageal adenocarcinoma (EAC) espe- replacement of the stratified squamous epithelium of lower cially in Western countries (1, 2). However, progress in early esophagus with a metaplastic columnar epithelium (4). detection and treatment strategies have been unable to Larger length of the Barrett’s segment may be associated improve the poor 5-year survival rate of 16% (3). Barrett’s with higher risk of progression to EAC, and there is prelim- inary evidence to support that genomic instability is a marker for BE progression (4, 5). Improved knowledge of EAC cancer biology and the identification of markers of Authors' Affiliations: 1Integrative Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada; and 2Department progression in BE could significantly improve patient prog- of Surgery, University of Michigan, Ann Arbor, Michigan nosis by invoking early interventions. Note: Supplementary data for this article are available at Clinical Cancer To discover genes involved in cancer progression, we Research Online (http://clincancerres.aacrjournals.org/). identified recurrent genetic alterations in EAC tumors and

Current address for W.W. Lockwood: National Human Genome Research assessed their presence in BE. Analysis of more than 80 Institute–NIH, Bethesda, MD. Current address for R. Chari: Department of EAC tumors revealed RFC3 (Replication Factor C-3 at Genetics, Harvard Medical School, Boston, MA. chromosome 13q13), a gene involved in DNA replication W.W. Lockwood and K.L. Thu contributed equally to this work. and cell proliferation, to be frequently ampliﬁed in EAC tumors. RFC3 ampliﬁcation was also detected in the BE of W.L. Lam and D.G. Beer contributed equally to this work as principal investigators. a patient, suggesting it could be an early event in tumorigenesis. Furthermore, we showed that RFC3 gain is Corresponding Author: William W. Lockwood, Cancer Genetics Branch, RFC3 National Human Genome Research Institute, NIH, 50 South Drive, prevalent in several types of cancer, knockdown of Bethesda, MD 20892. Phone: 301-594-9773; Fax: 301-443-9139; E-mail: has an antiproliferative effect, and that RFC3 expression [email protected] is associated with poor prognosis in multiple patient doi: 10.1158/1078-0432.CCR-11-1431 cohorts, which collectively suggests an oncogenic role 2012 American Association for Cancer Research. for RFC3.

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Quantitative real-time PCR validation of 13q13 genes Translational Relevance Genomic DNA for the 85 EAC tumors acquired at the Esophageal adenocarcinoma (EAC) is a lethal cancer University of Michigan was analyzed using Quantitative that can develop from the premalignant condition, real-time PCR (qPCR) to validate DNA copy number status Barrett’s esophagus. Little progress has been made in of 2 genes situated in the 13q13 amplicon identified by improving the survival rate for EAC patients; thus, an GISTIC (STARD13 and RFC3). Primer sequences and anal- improved understanding of the genetic alterations driv- ysis details are provided in Supplementary Methods and ing EAC development is essential to improve patient Supplementary Table S2. RFC3 copy number status was also prognosis. assessed by genomic DNA qPCR for the esophageal cell lines We employed an integrative genomics approach to OE33, Flo-1, and Het-1a. identify novel genes involved in EAC tumorigenesis that may serve as clinical markers. We identified RFC3 as a Gene expression profiling candidate oncogene activated by DNA amplification in Gene expression profiles for 11 of the 20 discovery set approximately 25% of EAC patients. Knockdown of this EAC tumors were generated using Affymetrix U133A expres- gene inhibited cancer cell lines with amplification but sion arrays as previously described (Supplementary Tables had little effect on cancer cell lines without the alteration S1 and S3; ref. 10). Expression arrays were carried out by the or nonmalignant cells. Furthermore, high RFC3 expres- University of Michigan Microarray Core. The probe display- sion was associated with poor patient outcome in mul- ing the maximum average intensity across the 11 samples tiple epithelial cancer types. Our findings suggest that was used to assess gene expression when multiple probes for activation of RFC3 may play a role in EAC development the same gene were present as previously described (10). and that its expression could serve as a prognostic marker. Externally generated SNP, CGH, and expression data Additional array data were accessed from publically available sources to investigate RFC3 in external cohorts (Sup- plementary Fig. S1 and Supplementary Table S3). Data Materials and Methods analyses done on these datasets are described in Supple- Discovery set (University of Michigan) tissue accrual mentary Methods. and processing The discovery set includes 20 EACs, 10 matched BE tissue, Integration of DNA copy number and gene expression and 6 nonmalignant esophagus tissue samples correspond- data ing to the 10 BEs (Supplementary Table S1). An additional Spearman’s correlation and Mann–Whitney U tests con- set of 65 EAC tumor specimens was collected for validation ducted using MATLAB software were used to investigate (Supplementary Table S1). All samples were collected (fresh whether increased gene dosage influenced expression for the frozen in liquid nitrogen and stored at 80C) with written 4 13q13 amplicon genes. A correlation coefficient more than patient consent and according to the ethics guidelines of the 0.6 and P values less than 0.05 were considered significant. University of Michigan Institutional Review Board from radiation and chemotherapy-naive patients who underwent Cell culture and shRNA-mediated RFC3 knockdown esophagectomy for adenocarcinoma at the University of EAC cell lines, OE33 (Sigma-Aldrich 96070808-1VL) and Michigan Health System between 1991 and 2004. DNA and OE19 (Sigma-Aldrich 96071721-1VL), and breast ductal RNA were isolated from microdissected tissue that con- adenocarcinoma cell line, HCC1395 (ATCC CRL-2324), tained at least 70% tumor cell or Barrett’s metaplasia con- were cultured in RPMI-1640 media supplemented with tent (for more details, see Supplementary Methods). 10% FBS and 0.1% Penicillin–Streptomycin (Invitrogen). Flo-1, also an EAC line, and Het-1a, a nonmalignant esoph- Array comparative genomic hybridization and GISTIC ageal cell line, were cultured in Dulbecco’s modified Eagle’s analysis for altered regions medium supplemented with 10% FBS and 10 Antibiotic– A total of 20 EACs, 10 matched BE cases, and 6 Antimycotic (Invitrogen 15240-096). DNA was isolated nonmalignant esophageal tissues were profiled as previ- using standard phenol:chloroform extractions and RNA ously described (6, 7). The EAC profiles were analyzed was extracted using TRIzol reagent (Invitrogen). PLKO using the GISTIC (Genomic identification of significant plasmid constructs containing short hairpin RNAs (shRNA) targets in cancer) algorithm for copy number alteration targeting RFC3 were purchased from Open Biosystems frequency and amplitude, using the following para- (Catalog RHS4533-NM_181558). Lentiviral production meters: q-value threshold of 0.10, refgene file Hg18, and infections were carried out as previously described amplification threshold 0.1, deletion threshold 0.1, join (11). RFC3 knockdown was quantified by quantitative segment size ¼ 2 (8). CNAs were filtered for natural copy reverse transcriptase PCR (qRT-PCR) using TaqMan gene number variants within the GISTIC analysis (9). High- expression assays for RFC3 (Hs00161357_m1) with 18S level copy number amplifications and deletions were rRNA (Hs99999901_s1) as an endogenous control (11). defined as DNA segments with log2 ratios more than Four shRNAs designed to target RFC3 were tested and 0.8 or log2 ratios less than 1.3, respectively. the one giving the greatest RFC3 mRNA knockdown

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(R2–TRCN0000072649) was used for cell model experi- Investigation of amplified regions without known ments. Knockdowns were also measured by Western blot- oncogenes ting, as described in Supplementary Methods. Although both amplifications and deletions are known to be important in cancer development, the gain-of-function MTT cell proliferation assay effect of gene amplifications makes them ideal targets for the OE33, OE19, HCC1395, Flo-1, and Het-1a cell lines were development of biomarkers and strategies for therapeutic stably transduced with shRNA targeting RFC3 and were intervention. Thus, we chose to focus our study on DNA investigated for differences in cell proliferation rates using amplifications, as they are a prominent mechanism of the MTT assay (Trevigen) as previously described (11). oncogene activation (7). In total, 9 of the 11 regions Vector-only lines (OE33-PLKO, OE19-PLKO, HCC1395- in Fig. 1 can be attributed to known oncogenes in EAC or MYC ERBB2 EGFR. PLKO, Flo-1-PLKO, and Het-1a-PLKO) served as controls other tumor types, such as , , and Driver 7q21.11 13q13.1 to assess proliferation in the knockdown lines (OE33-R2, oncogenes for the and regions remain OE19-R2, HCC1395-R2, Flo-1-R2, and Het-1a-R2). Assay elusive despite frequent reporting of DNA amplifications 7q 13q details are further described in Supplementary Methods. on and in EAC copy number studies. In our discovery set, high-level amplifications of 13q13 were more prevalent Colony formation assay than amplifications of 7q21; thus, we focused on the 13q13 Anchorage-independent growth was assessed in stably amplicon which contained only 4 genes: PDS5B, KL, transfected OE33, OE19, HCC1395, Flo-1 and Het-1a PLKO STARD13, and RFC3 (Fig. 2). and RFC3 knockdown cell lines by the soft agar method as previously described (11). Cells were plated at a density of 1,000 cells per well in supplemented media containing Copy number and gene expression integration for 0.3% low melting point agarose. Each cell line was seeded 13q13 genes in triplicate and cultured for 2 (OE33 and Het-1a) or 3 Next, we sought to identify the driver gene of 13q13 weeks (HCC1395, OE19, and Flo-1) at 37 Cin5%CO2. amplification in EAC. Matched copy number and gene Colonies were stained with MTT, counted, and the mean expression data for 11 of the 20 discovery set EAC tumors SEM were normalized to the average of control (PLKO) cells. were integrated to determine whether PDS5B, KL, STARD13,orRFC3 exhibited elevated gene expression as Survival analysis a consequence of DNA amplification. Correlation analysis Gene expression and patient survival data for 6 indepen- revealed a positive correlation between DNA copy number dent tumor datasets were obtained including 1 EAC, 1 (using the moving average of log2 ratios) and gene expres- breast cancer, and 4 lung cancer datasets (Supplementary sion for STARD13 (r ¼ 0.65) and RFC3 (r ¼ 0.69). Expres- Table S3). For each dataset, patients were grouped into sion of both genes was significantly higher in tumors with tertiles based on expression levels (i.e., low, middle, and DNA copy number gain as opposed to those without (P ¼ high) and survival times of patients with expression values 0.01), providing further evidence that amplification of in the lowest tertile were compared with those of patients STARD13 and RFC3 is a mechanism of gene deregulation whose expression ranked in the highest tertile. Survival in EAC tumors (Fig. 3A). To determine whether any of the 4 associations were tested for RFC3, PDS5B, KL, and candidate genes were differentially expressed between EAC STARD13. Log-rank Mantel–Cox tests were done and and nonmalignant tissue, we interrogated expression levels Kaplan–Meier survival curves were generated using Graph- of PDS5B, KL, STARD13, and RFC3 throughout the histo- Pad Prism 5 software to assess RFC3 expression associations logic progression of EAC in a publically available gene with patient survival. A P value less than 0.05 was consid- expression dataset consisting of 8 sets of nonmalignant, BE ered significant. Multivariate analyses were carried out using and EAC cases, each originating from the same patient (Fig. the robust likelihood–based survival modeling package in R 3B–E; ref. 22). RFC3 was the only gene differentially (rbsurv; ref. 12) as described in Supplementary Methods. expressed between nonmalignant and EAC tumor tissues (P < 0.05). Analyses of ours and externally generated data Results revealed RFC3 as the only 13q13-amplified gene that exhib- Identification of significant regions of copy number ited a strong correlation between gene dosage and expres- alteration in EAC sion and differential expression in tumors relative to non- We generated tiling resolution comparative genomic malignant tissue. Thus, we concluded that RFC3 is the driver hybridization (CGH) profiles for a discovery set of 20 EAC gene of the 13q13 amplicon and a novel candidate onco- tumors. A total of 11 significant regions of amplification gene in EAC development. (Fig. 1) and 8 significantly deleted regions (Supplementary Fig. S2) were detected. The characteristics of each region are RFC3 amplification is a prevalent feature of EAC provided in Fig. 1 and Supplementary Table S4. We iden- tumors tified several frequently reported EAC alterations, including To validate our array CGH data, we conducted qPCR on gains of 7p, 8q, and 17q as well as losses of 3p, 5q, and 17p, genomic DNA for each EAC tumor in the discovery set. A showing our tumors are consistent with the EAC genomic strong correlation between qPCR gene dosage and moving landscape (5, 13–21). average array CGH log2 ratios was observed for RFC3

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Figure 1. Significant regions of DNA amplification in EAC. A, GISTIC analysis revealed 11 significantly amplified regions in the discovery set of 20 EAC tumors. Chromosomes are displayed vertically with boundaries indicated by alternating white and black segments. Red bars represent DNA amplification peaks identified by GISTIC. The green vertical line marks the q value threshold, 0.10. Amplification peaks extending past the green threshold line are considered significant. B, genomic coordinates (March 2006, hg18 genome build), size, frequency, and candidate driver genes for the 11 significant regions of DNA amplification identified by GISTIC in the discovery set of EAC tumors (n ¼ 20).

(r ¼ 0.81, P < 0.05) validating our array CGH findings ing it may play a role in the progression of BE to EAC (Supplementary Table S5). To investigate the prevalence (Fig. 4). These findings showed RFC3 DNA amplification of RFC3 amplification, we carried out genomic qPCR to is prevalent in a broad spectrum of EAC tumors and is not assess gene copy number in an additional set of 65 EAC limited to our study. tumors. DNA copy number gain, defined as greater than 1.6-fold increase in gene dosage relative to normal (dip- RFC3 is a broad-spectrum oncogene amplified in loid) tissue, was observed in 22 of 85 (26%) samples. cancers from tissues of diverse origin High-level amplification of RFC3,definedasgreaterthan Because a number of established oncogenes are acti- 2-fold increase in gene dosage relative to normal (dip- vated in a broad spectrum of cancer tissues, we investi- loid) tissue, was observed in the 19% of tumors. We also gated whether RFC3 was altered in cancers other than assessed RFC3 copy number status in a publically avail- EAC. To address this question we analyzed copy number able dataset (GSE22524), which showed RFC3 was gained data for more than 700 cancer cell lines spanning 31 or amplified in 2 of 7 of EACs. Lastly, we looked for RFC3 different tissue types. RFC3 copy number gain (defined as amplification in BE samples obtained from the same more than 2 copies) was detected in 14% of all cases in 23 patients in the discovery set, whose tumors we profiled tissue types, whereas 6% of all malignancies harbored with array CGH (Supplementary Table S1). In the 10 EAC gains of more than 3 copies. A variety of cancer tissues, cases that had corresponding BE samples examined, one most of which were epithelial in origin, displayed elevat- (10%) harbored RFC3 amplification. RFC3 was amplified ed RFC3 gene dosage (Supplementary Fig. S3A). Interest- in both the tumor and corresponding BE tissue, indicat- ingly, RFC3 gains were most prevalent in cancers of the

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Figure 2. DNA amplification of 13q13 in EAC tumors. 13q13 DNA amplification is a recurrent event in the discovery set of EAC tumors. Array CGH copy number profiles for 6 different tumors harboring the 13q13 amplicon are shown (A). Orange shading marks the 13q13 region boundaries identified by GISTIC. Each blue dot is an individual array element, and those shifted to the right (toward þ1) of the yellow neutral line (0) exemplify copy number gains whereas those shifted to the left (toward 1) exemplify copy number loss. The numbers 1, 0, and þ1 indicate the log2 ratio scale. The 13q13 amplicon is shown in the UCSC genome browser using the March 2006 hg18 build (C). The focal region of amplification is approximately 1.3 Mb in size and encompasses 4 RefSeq genes: PDS5B, KL, STARD13, and RFC3.

large intestine and esophagus, further supporting our Four separate shRNAs designed to target RFC3 were findings. Similar to EAC tumors, cell lines harboring tested, and the one that consistently showed the greatest RFC3 copy number gains showed higher expression than knockdown (R2) was used for all subsequent experiments those without gain (P < 0.0001; Supplementary Fig. S3B). (Methods, Supplementary Fig. S6). Indeed, we observed a significant reduction in cell proliferation and anchorage- Knockdown of RFC3 inhibits proliferation and independent growth in both OE33 and OE19 R2-knock- anchorage-independent growth down lines relative to the vector-only control lines To assess the functional role of RFC3 in esophageal (Fig. 5A–D, Supplementary Fig. S7 and S8) and minimal tumorigenesis, we conducted shRNA-mediated RFC3 to no effect in Flo-1 EAC and nonmalignant Het-1a cells knockdown. Array CGH for the commonly used EAC model (Fig. 5A, B, F, and G, Supplementary Fig. S7 and S8). Growth cell line, OE33, revealed a 13q13 DNA amplification inhibition in OE33 was also seen using the second best encompassing RFC3 (Supplementary Fig. S4A). We con- hairpin (R3)—albeit to a lesser degree than R2, consistent firmed the presence of RFC3 DNA amplification in OE33 by with the lower level of knockdown—reducing the proba- analyzing additional copy number profiles independently bility of off-target effects (Supplementary Fig. S9). To deter- generated using single-nucleotide polymorphism (SNP) mine whether RFC3 functions as a putative oncogene in arrays by the Wellcome Trust Sanger Institute and GlaxoS- other cancer types, we also inhibited RFC3 in the breast mithKline (Supplementary Fig. S4A), and by genomic DNA cancer cell line, HCC1395, which harbored RFC3 DNA copy qPCR (data not shown). Knockdowns were also done in number gain (Supplementary Fig. S4B). Knockdown of additional EAC cell lines, OE19 (RFC3 gain, data not RFC3 resulted in similar reductions in expression, cell shown) and Flo-1 (no gain of RFC3, data not shown), as proliferation, and colony formation in HCC1395 (Figs. well as Het-1a, a nonmalignant esophageal line (no gain of 5A, B, and E, Supplementary Fig. S8). These cell experiments RFC3, data not shown). Consistent with the copy number supported our hypothesis, showing a significant biologic status, cell lines with increased gene dosage also displayed effect of RFC3 knockdown in cancer cells, consistent with an higher expression of RFC3 as determined by qPCR (Sup- oncogenic role for RFC3 in cancer. plementary Fig. S5). We hypothesized that if RFC3 plays an oncogenic role, OE33 and OE19 (lines harboring RFC3 Overexpression of RFC3 is associated with increased gains) would be dependent on RFC3 expression, and that cell growth and proliferation pathways knockdown would result in a reduction in cell proliferation In an attempt to elucidate gene networks and biologic and anchorage-independent growth, whereas Flo-1 and pathways to which RFC3 belongs, we conducted a signif- Het-1a, which lack RFC3 copy number gains, would not icance analysis of microarrays analysis on a panel of EAC be affected. tumors (Supplementary Methods and Supplementary

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Figure 3. Integration of DNA copy number and gene expression identifies RFC3 as the driver oncogene of the 13q13 amplicon in EACs. Gene expression of the 4 genes located within the 13q13 amplicon (PDS5B, KL, STARD13, and RFC3) was compared in EAC tumors with 13q13 DNA amplification or gain (green) versus tumors without amplification or gain of 13q13 (blue; A). Only 2 of the genes in the 13q13 amplicon, STARD13 and RFC3, had significantly higher expression in tumors with amplification and gain (U test, P < 0.05), suggesting increased dosage of these genes drives their overexpression. Gene expression values are scaled across samples from 0 to 100. Gene expression levels for the 4 genes were assessed in a publically available data set of 8 groups of matched normal, BE and EAC tumor samples from the same patient (B–E). Only RFC3 is differentially expressed between tumor and normal samples (U test, P < 0.05).

Table S3). Expression of 167 genes was positively or neg- survival in publically available expression data for 48 EAC atively correlated with that of RFC3 (Supplementary Table tumors (23). Because RFC3 was gained/amplified in S6). These genes were investigated using Ingenuity Pathway approximately 25% to 35% of tumors (approximately Analysis software to determine whether they were enriched 25% from qPCR of all 85 samples, approximately 35% for any annotated cell pathways or functions. Applying this from CGH in the initial discovery set), and this subset of strategy, we revealed these genes were enriched in cell tumors also showed the highest expression of the gene, we growth and proliferation and DNA replication, recombina- segregated the samples by RFC3 expression into tertiles tion and repair networks (Supplementary Table S7). More- based on the assumption that the highest expressers would over, these genes were enriched for involvement in the likely represent the cases with RFC3 gain/amplification. We Wntb-catenin signaling pathway. These findings validated then compared survival between the top and bottom patient the known functional involvement of RFC3 in DNA repli- tertiles (see Methods). Although the association did not cation and implicated RFC3 in cancer promoting pathways reach statistical significance, a trend toward poorer survival and networks, providing additional evidence to support our in patients with higher RFC3 expression was evident (P ¼ hypothesis that RFC3 is an important oncogene in cancer. 0.1565; Fig. 6A). We also assessed associations between STARD13, KL, and PDS5B expression with patient survival High RFC3 mRNA levels are associated with poor in this EAC cohort. Consistent with our data that RFC3 is the patient survival driver of 13q13 amplification, RFC3 expression had the As a final assessment of the importance of increased RFC3 strongest association with patient survival in EAC. Because copy number in EAC biology, we sought to determine the small sample size of EAC tumors may have prevented a whether RFC3 gene expression was associated with patient significant association with patient survival in EAC and we

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Discussion DNA CNAs can play significant roles in tumorigenesis and provide insight into gene deregulation and cancer biology, as exemplified by DNA amplifications of the oncogenes HER-2 and EGFR (24, 25). CNAs in EAC tumors have been studied using a variety of detection methods with various resolutions including conventional CGH, microsat- ellite mapping, array CGH, and SNP arrays (4). The goal of these studies has generally been to identify the targeted driver genes of CNAs, as they are postulated to have causal roles in EAC tumorigenesis. Findings from copy number profiling studies in EAC have resulted in the compilation of a well-established genomic landscape of EAC; recurrent genomic events include gains of 7p, 8q, and 17q and losses Figure 4. RFC3 DNA amplification may be an early event in EAC of 3p, 4p, 5q, and 17p (26–31). Most recurrently altered tumorigenesis. Comparison of copy number profiles generated from nonmalignant BE and associated EAC tissues, each set derived from regions in EAC contain candidate or proven oncogenes or individual patients in the Michigan discovery set, revealed 13q13 tumor suppressors such as MYC, EGFR, CDKN2A, and amplification was present in a BE and retained in the corresponding TP53, however, the driver genes for some frequently altered EAC tumor. regions have yet to be discovered. In this study, we identified and investigated a frequently observed RFC3 amplification in other cancer types includ- amplified region of DNA located on chromosome 13q13. ing lung and breast, we interrogated 5 additional datasets to Recurrent DNA amplification of this region was initially determine whether RFC3 expression was associated with identified in a discovery set of 20 EAC tumors and was patient survival. Comparison of survival times in patients validated in the discovery set and an additional 65 EAC with high versus low expression in 295 breast cancers and tumors. 13q copy number gains are frequently reported in 4 independent sets of lung adenocarcinoma (n ¼ 58, n ¼ 82, EACs, including several recent high-resolution studies, n ¼ 107, and n ¼ 92) revealed survival was significantly although no driver gene has been conclusively identified worse in patients with high RFC3 expression in the breast (5, 21, 26–28, 30, 32). This could reflect the fact that 13q cancer (P ¼ 0.0027) and 2 of 4 lung adenocarcinoma sets DNA gains are often large, segmental alterations encom- (P ¼ 0.0079 and P ¼ 0.0102; Fig. 6B–D). The same passing hundreds of genes, making the elucidation of target trend was evident in the 2 other lung datasets (P ¼ genes difficult. Our analysis detected a discrete, minimal 0.0603 and P ¼ 0.0653). Of the 4 genes we identified in common region of DNA amplification in an EAC tumor and the 13q13 amplicon, RFC3 had the strongest association matched BE sample encompassing only 4 genes. Integration with patient survival in each cohort assessed, except for one of copy number and gene expression data revealed RFC3 as lung adenocarcinoma dataset, in which STARD13 had the the candidate oncogene responsible for driving recurrent strongest association with patient survival (P ¼ 0.0136). 13q13 amplification. Finally, we aimed to determine the prognostic perfor- RFC3, replication factor C 3, is one of 5 replication factor mance of RFC3 in relation to other known oncogenes subunits that form of a multiprotein complex known as targeted by amplification in EAC. For this purpose, we activator 1, which is required for DNA replication and repair conducted robust likelihood-based survival modeling by the DNA polymerases e and d. Collectively, the RFC (ref. 12; see Supplementary Methods) using RFC3 along complex acts as a clamp loader, enabling binding of the with the known driver genes of 9 other significantly ampli- proliferating cell nuclear antigen (PCNA) clamp onto fied regions identified in the EAC samples (MYB, EGFR, primed DNA (33). PCNA is required for processive elon- MET, MYC, CCND1, KRAS, ERBB2, GATA6, and CCNE1). gation during DNA synthesis, emphasizing the importance This method selects survival-associated genes based on the of RFC3 in cell proliferation because of its role in DNA partial likelihood of the Cox model and can discover replication (33). Human replication factor C genes are multiple sets of genes by iterative forward selection. When highly homologous to RFC genes in yeast, and RFC3 is applied to the same 5 datasets described above, we found essential for growth in fission yeast (34, 35). In human cells, that RFC3 waspartofaselectedmodelfor3ofthese RFC3 protein was found associated with the mitogenic datasets (Supplementary Table S8). RFC3 and MYC were transcription factor, MYC, which suggests RFC3 could have the amplified oncogenes that most consistently contrib- an integral role in driving cell proliferation in human cells as uted to a prognostic model across the datasets, suggesting well and could explain the antiproliferative knockdown that RFC3 may indeed be useful from a prognostic stand- effect we observed in cancer cells (36). point. Together, the association of RFC3 expression with Interestingly, RFC3 expression is regulated by several survival in multiple datasets and cancer types further transcription factors encoded by genes with established implies the significance of this gene and suggests RFC3 roles in cancer. MYCN and SHH, two proteins that could potentially be developed as a pan-cancer marker for promote proliferation in cancer cells, increase RFC3 patient prognosis.

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Figure 5. RFC3 confers a growth advantage to cancer cells, suggesting it may be a broad-spectrum oncogene. shRNA-mediated knockdown of RFC3 was done in the EAC cell lines, OE33, OE19 and the breast ductal carcinoma cell line, HCC1395, all of which harbor RFC3 DNA amplifications; knockdown was also done in the EAC line Flo-1 and nonmalignant esophageal line Het-1a which do not harbor RFC3 gains. RFC3 knockdowns were verified by qRT-PCR and relative expression in the control (PLKO) and knockdown lines (R2) are shown (A). Knockdown of RFC3 caused a prominent reduction in anchorage-independent growth (as measured by colony formation in soft agar) in cancer cell lines with RFC3 amplification compared with those without RFC3 amplification (B). Knockdown ofRFC3 in all cancer cell lines with RFC3 gain caused a decrease in cell proliferation, showing the oncogenic effect ofRFC3 activation (C–E), whereas knockdown in EAC cells and nonmalignant esophageal cells lacking RFC3 gain had no significant effect on proliferation (F and G). Asterisks indicate significant differences in proliferation and colony formation measurements between PLKO and knockdown lines (Student t test, P < 0.05).

expression levels whereas tumor suppressors, TP53 and esize RFC3 functions as an oncogene by driving prolif- CDKN2A, suppress RFC3 expression (37–40). Expres- eration and enabling continuous expansion of cancer sion analysis in a cohort of 52 EACs corroborated these cells. findings, as RFC3 expression was negatively correlated Other replication factor C genes have also been impli- with P53 expression and positively correlated with SHH cated in tumorigenesis. RFC4 upregulation was discovered expression, although the fold change in P53 and SHH in hepatocellular carcinoma (HCC) tissues relative to non- expression between high and low expressing RFC3 malignant liver tissue, and knockdown of endogenous tumors did not reach our 2-fold criteria for pathway RFC4 decreased cellular proliferation, increased apoptosis, analysis. The transcription factors E2F1 and E2F4, which and enhanced the chemosensitivity of the HCC cell line induce transition of cells from G1 to S-phase in the cell HepG2 (42). Moreover, RFC4 seems to be overexpressed in cycle, thereby promoting DNA replication, are also cancer cells from the lung, prostate, colon, stomach, and knowntobindtheRFC3 gene promoter, suggesting they skin relative to tissue-matched nonmalignant cells (43). could positively regulate RFC3 (41). Pathway analysis Importantly, RFC4 was one of several genes whose expres- on genes whose expression was associated with RFC3 sion was upregulated in response to SV40 immortalization expression revealed an enrichment of genes in the Wnt/ in lung fibroblasts, implicating the replication factor C b-catenin signaling pathway, which stimulates the tran- complex in one of the earliest and essential steps in malig- scription of proliferation stimulating genes, as well as nant transformation (43). RFC2 is overexpressed in naso- gene networks involved in cellular growth and prolifer- pharyngeal carcinoma and was also found associated with ation and DNA replication, recombination and repair. the oncogenic protein c-MYC, whereas RFC5 is upregulated Induction by oncogenes and negative regulation by in human papillomavirus–positive squamous cell carcino- tumor suppressors, in addition to the involvement of mas of the head and neck (36, 44, 45). Thus, there is RFC3 in cell pathways and networks with roles in cell abundant evidence implicating replication factor C genes proliferation are consistent with a cancer-promoting in tumorigenesis and our study has identified a novel role for RFC3. Considering these findings, we hypoth- cancer-promoting role for factor C3 in EAC. Interestingly,

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Figure 6. High RFC3 expression is associated with poor patient survival in multiple cancer types. A Mantel–Cox test was used to assess the association between RFC3 gene expression and patient survival time in publically available datasets: Peters and colleagues EAC (n ¼ 48; A), Van DeVijver and colleagues breast cancer (n ¼ 295; B), Bild and colleagues lung adenocarcinoma (n ¼ 58; C), and Director's Challenge Consortium (DCC) lung adenocarcinoma (n ¼ 92; D). Survival time among patients having RFC3 expression in the top tertile (blue curves) was compared with that in patients with expression in the bottom tertile (red curves). High RFC3 expression was signiﬁcantly associated with poor patient survival in the breast and lung cancers (B and D). A trend toward poorer survival in EAC patients with high RFC3 expression was also observed, although it was nonsigniﬁcant (A).

a recent study reported frequent loss of function mutations and we cannot confirm whether the only area showing and downregulation of RFC3 in gastric and colorectal increased copy number were from dysplastic or nondys- cancers (46). The findings of this study and ours suggest plastic cells in this specimen. Therefore, we interpret this RFC3 may act in a tissue-specific manner in the context of finding with caution and conclude that further investiga- cancer. tion in BE samples is required to confirm whether or not We validated the biologic effect of RFC3 upregulation RFC3 DNA amplification is an early event in EAC tumor- in cell model experiments by showing that RFC3 sup- igenesis. Although genomic data on premalignant BE pression impedes cell proliferation and anchorage-inde- samples are rare, a segmental copy number gain of 13q pendent growth exclusively in cancer cell lines with copy was also identified in a BE case in the study by Akagi and gain, an observation consistent with our oncogene colleagues (21), showing the 13q amplicon we observed hypothesis. Although knockdown of RFC3 waslesspro- may be a recurrent event not only in EAC but also in its BE nounced in the nonmalignant esophageal line Het-1a as precursors. The observation of RFC3 amplification and compared with all other lines (50% vs. 75%–90%), 13q copy number gain in BE samples of our own and from even a 50% reduction in mRNA expression would be external datasets points to the significance of this alter- expected to cause a phenotypic effect. We are therefore ation. These findings suggest that RFC3 amplification may confident in our observation that knockdown of RFC3 have a role early in the development of EAC and that has no significant effect on the growth of nonmalignant amplification could potentially be a marker of BE pro- esophageal cells. gression to EAC; however, we emphasize that a large We were also interested in when RFC3 becomes prospective cohort is needed to investigate the signifi- involved during EAC development. Our unique dataset cance of RFC3 amplification in BE samples. allowed us to screen preneoplastic, BE tissues from indi- Finally, we showed that RFC3 DNA copy number vidualswhoprogressedtodevelopEAC.Weaskedwheth- alterations are not restricted to EAC but are also preva- er RFC3 DNA amplification was present in any of the BE lent in other cancers (Supplementary Fig. S3A). More- tissues of patients who developed EAC, based on the over, high RFC3 expression was associated with poor hypothesis that if present in preneoplastic tissue, RFC3 patient survival in multiple lung and breast adenocarci- amplification could be an early, causal event in EAC noma datasets, as well as in EACs. Taken together, our development. Intriguingly, we observed a high-level, data provide multifaceted evidence that RFC3 is a can- focal DNA amplification encompassing RFC3 in one of didate oncogene with potential predictive and prognostic the BE samples whose corresponding tumor also har- implications in addition to its biologic significance in bored the DNA alteration (Fig. 4). However, areas of EAC development, and perhaps, in other malignancies as dysplasia are present in many BE from patients with EAC, well.

1944 Clin Cancer Res; 18(7) April 1, 2012 Clinical Cancer Research

RFC3 Activation in Esophageal Cancer

Transcript Profiling collection of the data bear no responsibility for the further analysis or interpretation of it by the authors. The authors thank Jennifer Kennett and GEO Accession Numbers: GSE22524, GSE1420, Ivy Tsui for technical assistance. GSE19417, GSE2845, GSE3141. caArray: jacob-00182. caBIG (https://cabig.nci.nih.gov/tools/caArray_GSKdata). Grant Support This work was supported by the Canadian Institutes of Health Research Wellcome Trust Sanger Institute CGP Data Archive (W.L. Lam), Canadian Cancer Society (W.L. Lam), and Canadian Institutes of (www.sanger.ac.uk/genetics/CGP/Archive/). Health Research Vanier Canada Graduate Scholarships (K.L. Thu and L.A. Pikor), and CIHR Jean-Francois Saint Denis Fellowship in Cancer Research fl (W.W. Lockwood). Disclosure of Potential Con icts of Interest The costs of publication of this article were defrayed in part by the No potential conflicts of interest were disclosed. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Acknowledgments this fact. SNP 6.0 cell line data were downloaded from the Wellcome Trust Sanger Institute CGP Data Archive (www.sanger.ac.uk/genetics/CGP/Archive/). Received June 3, 2011; revised January 1, 2012; accepted January 30, 2012; Authors declare that those who carried out the original analysis and published OnlineFirst February 10, 2012.

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1946 Clin Cancer Res; 18(7) April 1, 2012 Clinical Cancer Research

Integrative Genomics Identified RFC3 As an Amplified Candidate Oncogene in Esophageal Adenocarcinoma

William W. Lockwood, Kelsie L. Thu, Lin Lin, et al.

Clin Cancer Res 2012;18:1936-1946. Published OnlineFirst February 10, 2012.

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