Published OnlineFirst August 27, 2014; DOI: 10.1158/1078-0432.CCR-14-0706 Clinical Cancer Biology of Human Tumors Research Identification of Differentially Expressed Long Noncoding RNAs in Bladder Cancer Stefan Peter1, Edyta Borkowska1, Ross M Drayton1, Callum P. Rakhit1, Aidan Noon1,2, Wei Chen3, and James WF Catto1 Abstract Purpose: Loss of epigenetic gene regulation through altered long noncoding RNA (lncRNA) expression seems important in human cancer. LncRNAs have diagnostic and therapeutic potential, and offer insights into the biology disease, but little is known of their expression in urothelial cancer. Here, we identify differentially expressed lncRNAs with potential regulatory functions in urothelial cancer. Experimental Design: The expression of 17,112 lncRNAs and 22,074 mRNAs was determined using microarrays in 83 normal and malignant urothelial (discovery) samples and selected RNAs with qPCR in 138 samples for validation. Significantly differentially expressed RNAs were identified and stratified according to tumor phenotype. siRNA knockdown, functional assays, and whole-genome transcriptomic profiling were used to identify potential roles of selected lncRNAs. Results: We observed upregulation of many lncRNAs in urothelial cancer that was distinct to corre- sponding, more balanced changes for mRNAs. In general, lncRNA expression reflected disease phenotype. We identified 32 lncRNAs with potential roles in disease progression. Focusing upon a promising candidate, we implicate upregulation of AB074278 in apoptosis avoidance and the maintenance of a proproliferative state in cancer through a potential interaction with EMP1, a tumor suppressor and a negative regulator of cell proliferation. Conclusions: We report differential expression profiles for numerous lncRNA in urothelial cancer. We identify phenotype-specific expression and a potential mechanistic target to explain this observation. Further studies are required to validate lncRNAs as prognostic biomarkers in this disease. Clin Cancer Res; 1–11. Ó2014 AACR. Introduction resection (4). In contrast, high-grade BCs are aggressive Bladder cancer (BC) is the fourth commonest male tumors that may present before or after the onset of malignancy and one of the most expensive human can- muscle invasion (5). These poorly differentiated urothe- cers to manage (1, 2). The majority of tumors are urothe- lial cancers have widespread chromosomal instability, lial carcinoma in histologic type. Clinicopathological multiple mutations, and are best characterized by defi- data suggest that urothelial cancers are best stratified into ciency of p53-mediated pathways (3). High-grade tumors two distinct phenotypes characterized by low- and high- share many molecular alterations regardless of stage and grade cellular differentiation. Low-grade urothelial can- respond poorly to chemotherapy (6). cers frequently have mutations of FGFR3 and STAG2, Although many reports detail genetic events in urothe- partial deletion of chromosome 9 (3), and rarely progress lial cancer, alterations of epigenetic gene regulation are to muscle invasion or metastases following endoscopic also important in this disease (7). In general, epigenetic alterations reflect urothelial cancer disease phenotypes and associated genetic events (8, 9). For example, Wolff 1Academic Urology Unit and Unit of Molecular Oncology, University of and colleagues reported that high-grade invasive urothe- Sheffield, Sheffield, United Kingdom. 2Department of Urology, University of Toronto, Toronto, Canada. 3Max Delbruck€ Center for Molecular Medicine, lial cancers had widespread aberrant hypermethylation, Berlin, Germany. whereas low-grade noninvasive tumors had regional Note: Supplementary data for this article are available at Clinical Cancer hypomethylation (10). In general, epigenetic events Research Online (http://clincancerres.aacrjournals.org/). reflect chromosomal changes with the disease and can S. Peter and E. Borkowska share first authorship. be used as predictor of disease progression (11). With regard to miRNA, low-grade urothelial cancers are char- Corresponding Author: James Catto, Department of Oncology, G Floor, The Medical School, University of Sheffield, Beech Hill Road, Sheffield S10 acterized by loss of expression of many species, including 2RX, United Kingdom. Phone: 44-114-226-1229; Fax: 44-114-271-2268; miRs99a/100 that target FGFR3 (12, 13). In contrast, E-mail: j.catto@sheffield.ac.uk high-grade tumors have upregulation of many miRs, doi: 10.1158/1078-0432.CCR-14-0706 including miR21 that targets the p53 pathway (14) and Ó2014 American Association for Cancer Research. miR129 (15). www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst August 27, 2014; DOI: 10.1158/1078-0432.CCR-14-0706 Peter et al. cle invasive (NMI), high-grade NMI, and invasive urothelial Translational Relevance cancer. All tissues were fresh frozen in liquid nitrogen. Bladder cancer is a common disease whose biology is Histologic confirmation was obtained before use. We also poorly understood. Here, we show that many long analyzed the human telomerase reverse transcriptase noncoding RNAs have altered expression in the disease (hTERT) immortalized normal human urothelial (NHU) and may play key roles in proliferation and disease cells maintained in keratinocyte serum-free medium con- progression. We identify one long noncoding RNA that taining bovine pituitary extract, EGF (Invitrogen), and appears a strong candidate as a prognostic biomarker cholera toxin. The hTERT NHU cells were obtained as a and show that it affects mRNA expression, cell prolifer- gift directly from Prof. M.A. Knowles, University of Leeds ation, and death, and may have potential as a therapeutic (Leeds, United Kingdom), and tested by this laboratory target. using comparative genomic hybridization and mutational analysis of CDKN2A and TP53 as described (25). The cells were not authenticated in Sheffield before use. Epigenetic gene regulation may occur directly or indirec- Long noncoding RNA expression profiling  m tly through noncoding RNA (ncRNA) species. These are From each sample, 10 10 m thick sections were classified according to size, function, and gene location microdissected to extract normal and malignant urothelial > (16). Most reports of ncRNA in cancer have focused upon cells ( 90% content). Total RNA was extracted using the miRNAs, which modulate gene expression through target- mirVana kit (Ambion) according to manufacturer’s proto- ing complementary seed sequences within mRNA 30 UTRs col (26), and measured using a 2100 Bioanalyzer (Agilent). (17). The role of miRNAs in disease varies with cell type, The expression of long ncRNAs and protein coding mRNAs biologic stress, species conservation, and processing (9, 18). was determined using microarrays (NCode Human Non- To date, few data have reported the role of long noncoding coding RNA Microarrays, Invitrogen). Each sample was RNA (lncRNA) in cancer and little is known of their func- prepared according to Agilent’s one-color microarray pro- tion (16, 19). Exceptions include lncRNAs clearly implicat- tocol. Briefly, 200 ng RNA samples were mixed with spike ed in carcinogenesis (such as MALAT-1 or HOTAIR, in control RNA (Agilent), labeled with cyanine 3-CTP (Low reviewed in Spizzo and colleagues; ref. 20). LncRNAs are Input Quick Amp Labeling Kit, one-color, Agilent), and annotated according to gene location (intergenic, exonic, hybridized to the microarray (Gene Expression Hybridiza- intronic, anti-sense etc.) and size (21). The GENCODE tion Kit, Agilent) according to the manufacturer’s protocol. consortium recently annotated 9,277 long ncRNA genes, After washing (Gene Expression Wash Buffer 1 and 2, corresponding to 14,880 transcripts (22). In contrast with Agilent), the microarray slides were scanned using the protein coding genes, the majority of reported lncRNAs had Agilent Microarray Scanner platform (High Resolution few gene exons, were located in the nucleus and chromatin, Microarray Scanner C) and raw probe fluorescence and many were not conserved between species. As multiple extracted. The microarray data are deposited with Gene reports suggest that lncRNAs are important in human cancer Expression Omnibus (GSE55433). The NCode microarray (16, 19, 23) and preliminary data suggest this is true for contains duplicate probes to 17,112 ncRNAs and 22,074 urothelial cancer (24), we hypothesized that they play a role mRNAs. The lncRNAs were identified by various strategies in urothelial carcinogenesis. To test this hypothesis, we without annotation (27). To annotate the array, we con- profiled the expression of 9,351 lncRNAs in normal and verted all probes to hg 19.0 loci (using LiftOver, UCSC malignant urothelial samples. We identified global changes genome browser), matched to target gene, structure and that reflect the disease phenotype and differ to patterns of the Gencode v7.0 annotation (22), and the nearest CpG mRNA alteration. We performed explorative analysis of island/differentially methylated region (28). For microar- selected species and identified those with potential prog- ray validation, individual lncRNA expression was mea- nostic roles. sured in triplicate using qRT-PCR. Total RNA was tran- scribed using random hexamer primers (Applied Biosys- Materials and Methods tems) and the High Capacity Reverse Transcription Kit Patient samples and cell lines (Applied Biosystems) before diluting 10-fold in nucle-