RBM5-AS1 Is Critical for Self-Renewal of Colon Cancer

Published OnlineFirst August 12, 2016; DOI: 10.1158/0008-5472.CAN-15-1824

Cancer Priority Report Research

RBM5-AS1 Is Critical for Self-Renewal of Colon Cancer Stem-like Cells Serena Di Cecilia1,2,3,4, Fan Zhang5, Ana Sancho1,2,3, SiDe Li2, Francesca Aguilo1,2,3, Yifei Sun1,2,3, Madhumitha Rengasamy1,2,3, Weijia Zhang5, Luigi Del Vecchio6, Francesco Salvatore6, and Martin J. Walsh1,2,3

Abstract

Cancer-initiating cells (CIC) undergo asymmetric growth pat- characterized previously, and we determined it to be a nuclear- terns that increase phenotypic diversity and drive selection for retained transcript that selectively interacted with b-catenin. chemotherapeutic resistance and tumor relapse. WNT signaling is Mechanistic investigations showed that silencing or overexpres- a hallmark of colon CIC, often caused by APC mutations, which sion of RBM5-AS1 caused a respective loss or retention of b-cate- enable activation of b-catenin and MYC. Accumulating evidence nin from TCF4 complexes bound to the WNT target genes SGK1, indicates that long noncoding RNAs (lncRNA) contribute to the YAP1, and MYC. Our work suggests that RBM5-AS1 activity is stem-like character of colon cancer cells. In this study, we report critical for the functional enablement of colon cancer stem-like enrichment of the lncRNA RBM5-AS1/LUST during sphere for- cells. Furthermore, it deﬁnes the mechanism of action of RBM5- mation of colon CIC. Its silencing impaired WNT signaling, AS1 in the WNT pathway via physical interactions with b-catenin, whereas its overexpression enforced WNT signaling, cell growth, helping organize transcriptional complexes that sustain colon and survival in serum-free media. RBM5-AS1 has been little CIC function. Cancer Res; 76(19); 5615–27. 2016 AACR.

Introduction byaconstitutivelyactiveWingless (WNT)/b-catenin signaling pathway (4). Active WNT signaling is critical for intestinal stem Colorectal cancer represents one of the most common tumor cells, crypt cell proliferation, and turnover; however, "stem- types and the third leading cause of cancer mortalities in the like" characteristics result from the convergence of cell-intrinsic United States (1). The bulk of a tumor consists of rapidly pro- features, extracellular signals, and stochastic events that con- liferating, postmitotic, differentiated cells and a very small tinuously shape the self-renewing compartment (3). In partic- population of cancer-initiating cells (CIC) or cancer stem cell ular, the epigenetic programming of transcription involves (CSC). CICs/CSCs show a remarkable capacity for self-renewal long noncoding RNAs (lncRNA), in addition to chromatin and asymmetrical tumor cell growth and have been identiﬁed proteins and DNA. LncRNAs have various roles (for review, in several cancers, including human colon malignancies (2). see ref. 5) with previous reports demonstrating their direct Representative colon CICs (CCIC) surface markers and com- involvement in regulating, as well as maintaining, pluripotent bined phenotypes have been used to isolate presumed CCIC states at the chromatin level (6). Interestingly, the WNT path- populations, including the enrichment of CD133, CD44, þ þ way can be modulated by lncRNAs in several tumor types (7). CD24, and CD29 as cell surface antigens, and CD24 CD44 , þ þ þ þ Within the context of a population of CCICs, we hypothesize EphB2high, EpCAMhigh/CD44 /CD166 ,ALDH ,LGR5 ,and þ that lncRNAs coordinate the chromatin architecture to include CD44v6 as phenotypic marks of stem cells (3). From a speciﬁc lncRNAs that facilitate reprogramming of the epigen- molecular perspective, a hallmark of CCICs is represented ome, thereby enabling the emergence of stem–like progenitors in colon cancer.

1 We identify the RBM5 antisense (RBM5-AS1)transcriptor Department of Pharmacological Sciences, Icahn School of Medicine – fi at Mount Sinai, New York. 2Department of Pediatrics, Icahn School of LUST (Luca-15 speci ctranscript), herein, referenced as LUST as Medicine at Mount Sinai, New York. 3Department of Genetics and aspecific lncRNA elevated in CCICs population. The loss of Genomic Sciences, Icahn School of Medicine at Mount Sinai, New LUST lncRNA results in the progressive differentiation of York. 4SEMM, European School of Molecular Medicine, Naples, Italy. 5Department of Medicine, Division of Nephrology, Bioinformatics CCICs, whereas ectopic expression corresponds with resistance Laboratory, Icahn School of Medicine at Mount Sinai, New York. to cellular differentiation and the stable maintenance of CICCs 6CEINGE-Biotecnologie avanzate, Universita degli Studi di Napoli- population. LUST appears to selectively direct b-catenin trans- Federico II, Naples, Italy. activation via a TCF4 reporter system facilitating its capacity Note: Supplementary data for this article are available at Cancer Research to target gene expression. Moreover, we find that LUST rein- Online (http://cancerres.aacrjournals.org/). forces the chromatin association between b-catenin and TCF4 Corresponding Author: Martin J. Walsh, Departments of Pediatrics, Pharma- on specifictargetsCMYC, CCND1, SGK1, and YAP1 to provide cological Sciences, Genetics and Genomic Sciences, Icahn School of Medicine at a cell growth advantage, reflecting CCICs in vivo. These findings Mount Sinai, 1468 Madison Ave., Box 1656 New York, NY 10029. Phone: 212-241- demonstrate that the LUST transcript is a novel lncRNA involv- 9714; E-mail: [email protected] ing the recruitment and function of b-catenin in CCICs doi: 10.1158/0008-5472.CAN-15-1824 and regulates WNT pathwaybypromoting"stemness" 2016 American Association for Cancer Research. maintenance.

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Materials and Methods using Exiqon's GapmeR Design Algorithm: (http://www.exiqon. com/ls/Pages/GDTSequenceInput.aspx?SkipCheck¼true). Cells and reagents Human LS174T (ATCC #CL-188), SW480 (ATCC #CCL- TOPFlash dual luciferase assay 228), HT-29 (ATCC #HTB-38), CaCo-2 (ATCC #HTB-37), HT-29 cells were transiently transfected using Lipofectamine DLD-1 (ATCC #CCL-221), and HCT 116 (ATCC #CCL-247) (Invitrogen) according to the manufacturer's instructions, using colon cancer cell lines were purchased from the ATCC between 250 ng of the TOPFlash reporter gene construct (M50 Super 8x the years 2013 and 2014, propagated and passaged as adherent TOPFlash, Plasmid #12456, Addgene) and 500 ng of pcDNA3- cell cultures according to instructions provided by ATCC. For all LUST and/or 500 ng of pcDNA-b-catenin construct. Luciferase cell lines, the cells were received from ATCC as early passages reporter gene expression was measured according to the man- and guidelines for authentication were followed as described ufacturer's protocol (Dual-luciferase Reporter assay System, previously (8). However, no additional steps to authenticate Promega). The luciferase activity was normalized to Renilla were taken. All documentation related to the cell lines obtained luciferase activity from cotransfected internal control plasmid can be acquired through ATCC. Cells were maintained in pRL-CMV. adherent conditions, at 37Cinhumidified atmosphere containing 5% CO2. The medium was changed twice a week, cells Immunoblotting for proteins were passaged using 0.05% trypsin/EDTA (Corning) and pre- For western blot analysis, 30 mg of protein lysate was analyzed served at early passages. Mycoplasma detection was routinely by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) tested by qPCR methods (9). membranes (Bio-Rad), and blotted with indicated antibodies followed by ECL detection (Thermo Scientific). Western blot Flow cytometric analysis and cell sorting assays were performed using the fallowing commercially available HT-29, LS174T, and SW480 colon cancer cells were stained antibodies, at the indicated concentrations: anti-b-actin (Sigma, using fluorescein isothiocyanate (FITC)-conjugated CD24, phy- A5441, 1:1,000), anti-a-tubulin (Sigma, T5168, 1:1,000), anti- coerythrin (PE)-conjugated CD166, allophycocyanin (APC)-con- b-catenin (Bethyl Laboratories, A302-012A, 1:1,000), anti-active- jugated CD133 (BD Biosciences), and PE-Cy7–conjugated CD44 b-catenin (Millipore, 05-665, 1:1,000), anti-cyclin D1 (CCND1, (BioLegend). Samples were analyzed on a BD LSRII flow- Abcam, ab16663, 1:1,000), and anti-c-myc (Cell Signaling, 5605, cytometer (Becton Dickinson). Fluorescence-activated cell sorting 1:1,000). (FACS) of HT-29 cells was performed using BD FACSAria II (Becton Dickinson). Analysis of cytometric data was performed Cell transfections using FACSDiva software (Becton Dickinson; see Supplementary For lncRNA LUST knockdown, cells were transfected using 300 Information). pmole of LNA GapmeRs and Lipofectamine (Invitrogen) according to the manufacturer's protocol. For lncRNA LUST over- In vitro colonospheres formation assay expression, pcDNA3 and pcDNA3-LUST construct were Spheres formed with colon carcinoma cells (HT-29, LS174T, transiently transfected into HT-29 cells using 4 mg of DNA. SW480, DLD-1, HCT116) were obtained as previously described For colonospheres formation assay, HT-29, LS 174T, SW-480, (4) with minor modifications provided in Supplementary DLD -1, and HCT116 cells were transiently transfected with Information. pcDNA3 control or pcDNA-LUST andthenseededinalow- RNA extraction, qRT-PCR, and RNA-Seq attachment plate. Total RNA was extracted from HT-29, CaCo-2, LS174T, and SW480 cells, derived colonospheres using TRIzol and the Nuclear fractionation and FISH RNeasy Mini Kit (Qiagen) according to the manufacturer's Nuclear fractionation was performed as previously described protocol. Reverse transcription was performed using Prime- (12). For lncRNA LUST localization, RNA-FISH was performed Script RT Reagent Kit (Takara #6130). Quantitative PCR (qRT- using customized probes purchased from Exiqon, following the PCT) was performed using the GoTaq qPCR Master Mix manufacturer's instructions for use. (Promega). Hypoxanthine-guanine phosphoribosyltransfer- ase (HPRT) gene was used as housekeeping gene for normal- Crosslinked RNA immunoprecipitation, chromatin ization. Sequences of all the primers used for qRT-PCR are immunoprecipitation, photoactivatable ribonucleoside- listed in Supplementary Table S2. RNA-Seq datasets from enhanced crosslinking immunoprecipitation, and sequential Illumina HiSeq2500 sequencing approaches have been depos- chromatin immunoprecipitation ited in the NCBI GEO under the accession number GSE69236, Essentially all experiments performed in this section have m NCBI website: http://www.ncbi.nlm.nih.gov/geo/query/acc. been previously described (10, 13) and performed using 5 g b m cgi?token¼sbibkwgutdgzxyn&acc¼GSE69236. Gene ontology of anti- -catenin (Bethyl Laboratories, A302-012A) and 5 g analysis was performed as previously described (10). of rabbit control IgG-ChIP Grade (Abcam, ab46540). Chro- matin immunoprecipitation (ChIP) was performed as previ- lncRNA LUST knockdown and overexpression ously described (14) using 6 mgofanti-b-catenin (Bethyl lncRNA LUST knockdown was performed using LNA longRNA Laboratories, A302-012A) and equal amount of rabbit control GapmeR (Exiqon, #300600). Four different probes directed IgG-ChIP Grade (Abcam, ab46540). The sequential-ChIP against lncRNA LUST transcript and one unspecific negative (or Re-ChIP) studies were performed as described (15). control probe were used. The construct pcDNA3-LUST was used Photoactivatable ribonucleoside-enhanced crosslinking and and generated as previously described (11). The sequences of the immunoprecipitation (PAR-CLIP) was performed as described oligonucleotides and their LNATM spiking patterns were designed in (10, 16).

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Colon Cancer Stemness Is Regulated through LUST

Mouse xenograft studies transcripts as a prominent signature characteristic of some cancer A total of 2 106 HT-29–transfected cells were recovered, stem cells (25), we performed RNA-Seq on CD24bright/CD44bright placed in 1 PBS, and inoculated by subcutaneous engraftment in (CCICs) and CD24dim/CD44dim (a differentiated comparison) the hind rump of male immunodeficient CB17-SCID mice sorted subpopulations from HT-29 colon cancer cell line, and (Charles River Laboratories) between 8 and 10 weeks of age. spheres derived from the CD24bright/CD44bright subpopulation, Tumor cell growth was established directly from HT-29 cells to explore pathways that are dysregulated. Approximately, 3,000 manipulated either by transfection with the expression vector for genes were elevated in CCICs, when compared with the differ- the LUST RNA transcript or by depleting LUST using locked entiated population, indicated with a fold change > 1.5 (Fig. 1D, nucleic RNAs (purchased through Exiqon). The growth of the top left). Analysis of the elevated genes identifies them among tumors was followed by means of caliper measurements. Mon- regulation of cell proliferation, cell migration, and other biolog- itoring of tumor growth was performed at least twice per week ical processes characteristic of cancer cell phenotype (Fig. 1D, using calipers. Details on analyses of tumor volume and survival bottom left). Approximately, 2,000 genes resulted to be repressed are described within Supplementary Information. between CD24bright/CD44bright and CD24dim/CD44dim subpopulations (Fig. 1D, top right), involved as well in regulation of cell fi Chromatin isolation by RNA puri cation proliferation or cellular metabolism (Fig. 1D, bottom right). We fi Chromatin isolation by RNA puri cation (ChIRP) experi- then analyzed the expression of genes defining colonic mucosa ments were performed as previously described (17, 18). Twen- cells differentiation, such as MUCIN 2 (MUC2) or keratin B20 fi ty- ve probes were generated against the RBM5-AS1 (LUST), (KB20/KRT80) that resulted downregulated, or colon cancer stem RBM5-S1 (sense), and HOTAIR transcripts. Bound chromatin 0 0 cells markers, in particular CD24, CD44, CD166, and ALDH1A1 was detected by qPCR from HOXD3, MYC 5 , MYC 3 , CCND1, that were upregulated (Fig. 1E, shown as the green and red bars, fi SGK1 promoter-speci c primers. Sequences of RBM5-AS1 and respectively). Members of the canonical WNT-mediated signaling RBM5 and HOTAIR RNA probes are listed in Supplementary pathway, such as ASCL2, IGFBP2, LGR4, DKK1, MYCL1, FGFR2, Table S2. SP5, MMP7 and the receptor EPHB3 were elevated in the CCICs compared with the more differentiated counterpart, with a fold Statistical analysis increase > 1.5 (Fig. 1E and F, shown as the blue bars), as shown All experiments were performed in triplicate at least 3 times. All form the IGV tracks (Supplementary Fig. S1). RNA-Seq, per- values were expressed as mean SEM. Statistical analysis was formed on spheres derived from CCICs, was used to compare performed by the unpaired Student t test. A probability value of the expression of WNT-target genes and stem-like markers with P 0.05 was considered statistically significant. the subpopulation (CD24bright/CD44bright) they originated from. The expression of ASCL2, MYCL1, LGR4, EPHB3, CLDN1, CD44, Results CD24, ALDH1A1, and AKR1B10 was strongly increased during b-Catenin target genes are stimulated within HT-29 CCICs spheres formation. Genes involved in differentiation, such as compared with their more differentiated counterparts Kruppel-like factor 9 (KLF9), KB20, and the b-catenin–regulated Here, we confirmed the expression of surface markers (2, 19) on gene peptidyl arginine deiminase, type 1 (PADI1), were the most 3 different colon cancer cell lines chosen for their differentiated suppressed (fold change > 1.5) transcripts in both spheres and status: SW480 (poorly differentiated), HT-29 (moderately differ- the parental subpopulation, as well as the retinoic acid receptor entiated), and LS174T (well-differentiated). Within the HT-29 cell responder protein 1 (RARRES1), known to have a tumor line, we identified 2 subpopulations coexpressing all 4 surface suppressor role (26). Intriguingly, 2 lncRNAs, KCNQ1OT1 and markers with signal intensity corresponding respectively as LUST, were increased in this process, and the overexpression of "bright" and "dim" (CD24, CD44, CD133, and CD166, LUST seemed to be more sphere specific (Fig. 1G). Furthermore, Fig. 1A). For LS174T and SW480 colon cancer cell lines, 2 different when cells were induced to differentiate, the mRNA levels of subpopulations could be discriminated as well, on the basis of the CCDN1, LGR4, and CMYC were reduced (Fig. 1H), showing signal intensity of CD24 and CD44 coexpression (bright and that WNT signaling is highly and selectively active during HT- dim). However, the intensity of CD133 and CD166 antigens was 29 cells sphere formation and that the activity is reduced upon lower than that of the HT-29 subpopulation (Fig. 1A). To assess FBS-induced differentiation, confirming its role in maintenance the ability of self-renewal of undifferentiated (CD24bright/ of colon cancer cell spheres (27). CD44bright) versus differentiated (CD24dim/CD44dim) HT-29 cell subpopulations, we performed in vitro colon carcinoma spheres The lncRNA LUST transcript expression corresponds strongly assay as previously described (2, 19, 20). Colon cancer progenitor with the stem-like capacity of CCICs cells can grow as spherical aggregates that, in the presence of serum Recent data have shown that dysregulation of coding as well or extracellular matrix, differentiate upon growth factor removal as noncoding RNAs contribute to CCIC generation, reviewed in (21–23). Toward this aim, we first isolated HT-29 CD24bright/ ref. 28 and recently has been demonstrated that in cancer the CD44bright (CCICs) and CD24dim/CD44dim (the differentiated WNT signaling can be additionally regulated by lncRNAs counterpart) by FACS (Fig. 1B) and then grew the sorted cells in through cell-autonomous mechanisms (29). To investigate the suspension, in serum-free media, to induce spheres formation involvement of lncRNAs in CCIC maintenance and their role in (19). After 3 weeks of culture, we obtained spheres formed as the WNT signaling regulation, we performed an lncRNA pro- aggregates of exponentially growing undifferentiated cells filer array analysis on CCICs and the more differentiated CD24bright/CD44bright (CCICs) that were larger in terms of size counterpart isolated from HT-29 colon cancer cells. Several and number than the ones obtained from the CD24dim/CD44dim lncRNAs were dramatically elevated in CCICs compared with colon cancer cells, as expected and previously reported their differentiated counterpart (Table S1), with an increase (Fig. 1C; ref. 24). On the basis of WNT/b-catenin signaling higher than 2.5-fold.

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Colon Cancer Stemness Is Regulated through LUST

Among these lncRNAs, LUST and KCNQ1OT1 were overex- and induce luciferase activity. Interestingly, the cotransfection of pressed, as confirmed by RNA-Seq also in spheres derived from pcDNA3-LUST and pcDNA3-b-catenin induced a significant strong HT29 cells (Fig. 1G and I). To address the functional role of LUST increase in luciferase activity compared with pcDNA3-LUST and in cancer stem cell maintenance, we analyzed by qRT-PCR the pcDNA3-b-catenin alone. These results demonstrated that the lncRNA and stem-like markers expression in spheres derived from lncRNA LUST and b-catenin synergistically activate WNT/b-catenin CCICs and in FBS-induced differentiation cells. During the sphere signaling (Fig. 3D). To further evaluate the LUST-mediated regu- formation process, the lncRNA LUST expression results strongly lation of WNT signaling in colon cancer, we analyzed the mRNA increased (Fig. 2A, left). Of note, the increase appears to be levels of WNT signaling target genes upon LUST overexpression. significant already after 14 days in culture, when the stem-like pcDNA3 and pcDNA3-LUST constructs were transiently trans- markers CD24 and CD44 mRNA levels (Fig. 2A, middle and right) fected into HT-29 cells, and RNA samples were analyzed through are not elevated yet, and reaches a 10-fold change increase when qRT-PCR. The expression of AXIN2, CCND1, MYC, TCF4 and of the cells are grown for 5 weeks in the same conditions (Fig. 2A, left). stemness markers CD24 and CD44 was significantly elevated under This result suggests that LUST upregulation could be an early event these conditions (Fig. 3E). Although total b-catenin protein level in the sphere formation process. As expected, when differentiation remains stable, we verified a strong increase of active b-catenin, of the spheres is induced by adding FBS for additional 48 hours MYC, and CCND1 expression, (Fig. 3F), showing that the increase (20), the mRNA expression levels of conventional cancer stem cell is induced at both mRNA and protein levels and therefore dem- markers, CD24 and CD44, are abrogated (Fig. 2A, middle and onstrating that LUST enhances WNT signaling activation. More- right), confirming the loss of the stem-like potential. Interestingly, over, transient overexpression of LUST in HT29 cells corresponds the expression level of the lncRNA LUST drastically decreased (Fig. with more profound colony formation when evaluated by crystal 2A, left), and the loss of markers CD24 and CD44, shown at the violet on soft agar (Fig. 3G). To investigate a key role of LUST in protein level (Fig. 2B, blue population), strongly correlates with tumor initiation, we performed in vitro sphere formation assay the switch from spheroid toward more adherent cell morphology from HT-29 cells and showed that LUST overexpression promotes (Fig. 2C, top and bottom, respectively). an earlier onset of spheroids compared with thecontrol. In fact, HT- 29 LUST–overexpressing cells are able to form spheres within 7 WNT LUST signaling activation is impaired upon knockdown days in serum-free media culture conditions and the full spherical We performed loss-of-function assays in HT-29 cells by using morphology is reached within 14 days. Instead, HT-29 cells trans- two distinct locked nucleic acid (LNA) RNA GapmeRs that fected with empty vector required a longer timeframe correspond- knocked down the expression of LUST to about 60% and ing to 21 days (Fig. 3H). fi 50%, respectively, compared with the control unspeci cprobe We extended our study using 4 additional human colon cancer (Fig. 3A) and analyzed the expression of WNT signaling target cell lines to further validate the role of LUST as a colon cancer stem genes by RT-qPCR. While control cells retained the expression cell regulator. Exogenous LUST overexpression in LS174T and SW- of WNT target genes, a strong reduction of mRNA transcripts 480 (both APCwild-type/CTNNB1mutant) and in DLD-1 and HT116 such as AXIN2, CCND1, CD44, and TCF4 as well as CD24 (both APCmutant/CTNNB1wild-type) confirmed the mRNA level mRNA levels occurs upon LUST knockdown (Fig. 3B). A reduc- induction of CCND1, CD44, C-MYC, and TCF4, as well as the tion at the protein level was seen for CCND1 and C-MYC (Fig. protein level increases of c-MYC, CCND1, and active b-catenin 3C) and, of note, LUST knockdown caused also a reduction of (Fig. 4A and B). Even in this case, we could notice an earlier onset b active -catenin (Fig. 3C). of spheroid formation after only 14 days in culture (Fig. 4C–G). These results collectively demonstrate that LUST is an lncRNA LUST overexpression potentiates WNT/b-catenin signaling transcript that we consider as a key component in regulating CCIC cascade and accelerates spheroid formation across multiple spheroid formation. colon carcinoma cell lines We then performed dual luciferase assay using TOPFlash promoter (a reporter plasmid containing multiple copies of wild-type LUST is a nuclear-retained lncRNA that directly interacts with Tcf4-binding sites), transiently integrated in HT-29 cells (TOP- b-catenin and enhances occupation of b-catenin at promoters Flash-HT-29 cells). Relative luciferase activity was measured in associated with WNT signaling target genes TOPFlash-HT-29 cells transfected with pcDNA3-LUST,pcDNA3- LUST lncRNA localization resulted to be mostly nuclear b-catenin, or pcDNA3-LUST and pcDNA3-b-catenin. The lncRNA (Fig. 5A), and RNA-FISH performed in HT-29 cells transiently LUST alone as well as b-catenin were able to activate TCF reporter transfected with pcDNA3 or pcDNA3-LUST confirmed the nuclear

Figure 1. RNA-Seq analysis of CCICs and the more differentiated counterpart in the HT-29 cell line reveals deregulation of WNT signaling and noncoding RNAs. A, FACS analysis of HT-29, LS174T, and SW480 colon cancer cells. Plots are gated on CD24bright/CD44bright (CCICs) and CD24dim/CD44dim cells (more differentiated cells). Histograms show CD133 and CD166 surface antigen expression in CCICs (green) and more differentiated cell (blue) subpopulations for the above cells lines. B, sorting of CCICs and more differentiated cells from HT-29 colon cell line; plots are gated on CD24bright/CD44bright and CD24dim/CD44dim cells; percentage of sorted populations is shown. C, spheres formed with colon carcinoma cells obtained from CD24bright/CD44bright and CD24dim/CD44dim sorted cells. D, top, number of upregulated (left) and downregulated (right) genes with a >1.5-fold change between CD24bright/CD44bright and CD24dim/CD44dim subpopulations. Bottom, Gene Ontology analysis of upregulated (left) and downregulated genes (right). E, differential expression of genes between CD24bright/CD44bright and CD24dim/ CD44dim subpopulations. Fold change > 1.5; P < 0.01. F, qRT-PCR validation of WNT target genes. G, heatmap showing elevated (red) and repressed genes (blue) in CD24bright/CD44bright compared with CD24dim/CD44dim subpopulations and in spheres formed with colon carcinoma cells compared with the CD24dim/CD44dim subpopulation. Fold change > 1.5; P < 0.01. H, qRT-PCR validation of WNT target genes differentially expressed in spheres formed with colon carcinoma cells and FBS-induced differentiation cells. For qRT-PCR, HPRT was used as housekeeping control gene for normalization. Error bars, SEM. , P < 0.05. I, Venn diagram showing overlapping elevated lncRNAs between CCICs and HT-29–derived spheres.

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Figure 2. The lncRNA LUST overexpression is an early event in the spheres formation process. A, LUST, CD24, and CD44 mRNA levels measured by qRT-PCR in HT-29– derived spheres formed with colon carcinoma cells, respectively, after 14 and 35 days cultured in ultra-low-attachment conditions and in FBS-induced differentiation cells. HPRT transcript was used as housekeeping control gene for normalization. Error bars, SEM. , P 0.05. B, FACS analysis of spheres formed with colon adenocarcinoma cell lines (top) and FBS-induced differentiated cells (bottom) showing corresponding loss of expression of stem-like markers (blue population). Histograms report the mean ﬂuorescence intensity (range, 0–10,000) of CD24 (FITC) and CD44 (PE-Cy7). C, representative images of HT-29–derived spheres formed with colon adenocarcinoma cells with characteristic spheroid morphology (top) and differentiated spheres showing adherent morphology upon FBS-induced differentiation (bottom).

expression of the transcript (magenta color; Fig. 5B). A role for performed RIP assay (32) in CaCo-2 cells (Fig. 5E and F) that lncRNAs with WNT/b-catenin signaling in cancer has been indi- harbor both APC and b-catenin mutations (33). PAR-CLIP assays, cated (29, 30). Therefore, we analyzed the role played by LUST in performed in CaCo-2 cells, validated that the binding of b-catenin the regulation of b-catenin activity in CICs, by performing RNA to the lncRNA is selective when the enrichment is compared with immunoprecipitation (RIP). Results demonstrated that the other known RNA–protein interactions of EZH2 and CBX7 (Sup- lncRNA LUST strongly binds to b-catenin (Fig. 5C and Supple- plementary Fig. S2). To assess RNA-binding affinities, we exam- mentary Fig. S2). To determine the activity of the lncRNA as a ined the binding of nuclear proteins isolated from CaCo-2 cells to transcriptional regulator of WNT signaling, we performed b-cate- biotinylated RNA probe representing the LUST transcript and nin ChIP experiments in HT-29 LUST–overexpressing and control show from the shifted band that the RNA–nuclear protein com- cells. Analysis of ChIP-qPCR indicates that LUST induces the plex involves b-catenin and TCF4 (Supplementary Fig. S3A). enrichment of b-catenin at the promoters of WNT-signaling target Moreover, we show that affinity-purified b-catenin protein selec- genes as MYC, CXXC, YAP1, HDAC4, and SGK1, previously shown tively binds the LUST transcript and as a control fails to bind the to be b-catenin targets (Fig. 5D; ref. 31). For additional evidence sense RBM5 transcript (Supplementary Fig. S3B). These results where the binding of LUST with b-catenin could be detected, we suggest that the lncRNA LUST plays a role in CICC maintenance

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Figure 3. The lncRNA LUST is required for WNT signaling target genes activation. A, knockdown of the lncRNA LUST using two different antisense oligo probes (LNA RNAs). An unspeciﬁc probe was used as negative control (LNA RNA control). B, relative mRNA levels of WNT target genes upon LUST knockdown, analyzed by qRT-PCR. C, Western blot analysis (right) and relative protein abundance (left) of total b-catenin, active b-catenin, c-Myc, and cyclin D1 in control cells and in LUST-knockdown cells; a-tubulin was used as a loading control. D, relative luciferase activity measured in TOPFlash HT-29 cells transiently transfected with pcDNA3-LUST, pcDNA3-b-catenin, or pcDNA3-LUST and pcDNA3-b-catenin. E, relative mRNA levels of WNT target genes upon LUST overexpression analyzed by qRT-PCR. F, Western blot analysis (left) and relative protein abundance (right) of total b-catenin, active b-catenin, c-Myc, and cyclin D1 in pcDNA3 control cells and in LUST-overexpressing cells. b-Actin was used as a loading control. For qRT-PCR experiments, HPRT was used as housekeeping control gene for normalization. Error bars, SEM. ,P 0.05. G, soft agar colony formation assay for HT-29 pcDNA3 control and LUST-overexpressing cells. H, representative images of spheres formed with colon adenocarcinoma cells derived from pcDNA3 and pcDNA3-LUST–transfected cells. Cells were cultured in ultra-low-attachment conditions, in serum-free media for 7, 14, and 21 days.

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Figure 4. LUST expression promotes spheroid formation across multiple human colon adenocarcinoma cell lines. A, Western blot analysis of total cellular b-catenin, active b-catenin, c-Myc, and cyclin D1 in LS174T control cells and LUST-overexpressing cells, a-Tubulin was used as a loading control. B, qRT-PCR validation of WNT target genes expression upon LUST overexpression. C, representative images of colonospheres formed with colon cancer cells derived from pcDNA3 and pcDNA3-LUST transfected cells. D, relative mRNA levels of WNT target genes upon LUST overexpression in SW-480 colon cancer cells, analyzed by qRT-PCR. E–G, representative images of spheres formed respectively with SW-480, DLD-1, and HT116 colon cancer cells derived from pcDNA3 and pcDNA3-LUST transfected cells. Cells were cultured in "ultra-low" attachment conditions, in serum-free media for 7, 14, and 21 days. For qRT-PCR experiments, HPRT was used as housekeeping control gene for normalization. Error bars, SEM. , P 0.05.

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Colon Cancer Stemness Is Regulated through LUST

Figure 5. The nuclear lncRNA LUST recruits b-catenin promoting WNT signaling activation. A, relative subcellular abundance of LUST transcript determined by qRT-PCR; MALAT-1 was used as positive control for nuclear localization; HPRT was used as housekeeping control gene for normalization. B, single-molecule localization of LUST in HT-29 pcDNA3 (left) or pcDNA3-LUST (right) cells. Nuclei were stained with DAPI (blue); lncRNA LUST was detected using LNA double-DIG mRNA probe (red). C, RIP assay showing the physical association between LUST and b-catenin in HT-29 cells. b-Catenin was immunoprecipitated from nuclear extracts of formaldehyde-crosslinked HT-29 cells and associated RNA was detected by qRT-PCR. Enrichment of LUST binding to b-catenin is shown as % input. RIP-Western blot showing b-catenin immunoprecipitation. D, ChIP-RT-qPCR analysis of b-catenin binding at the MYC, CXXC, YAP1, HDAC4, and SGK1 loci. Enrichment of b-catenin is shown as % input. E, RIP assay showing the physical association between LUST and b-catenin in CaCo-2 cells. Enrichment of LUST binding to b-catenin is shown as % input. RIP-Western blot showing b-catenin immunoprecipitation. C–E, IgG was used as negative control. Error bars, SEM. ,P< 0.05. F, IGV tracks of LUST lncRNA bound to b-catenin recovered after immunoprecipitation. Peaks respectively correspond to b-catenin and input in CaCo-2 and HT-29 colon cancer cell lines. G, tumor progression and sizes (left) were evaluated following the orthotopically introduced HT-29 cells, upon either LUST overexpression or by LNA depletion, into male CB17-SCID mice. Mice were followed over a period of 35 days (n ¼ 12 for each group) for tumor volume and for 55 days for overall survival. Tumor volume was monitored by using calipers during tumor progression and measured from the mice three times weekly.

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Di Cecilia et al.

by regulating the WNT signaling pathway, hence we sought to signaling by facilitating b-catenin binding to the TCF4 tran- investigate an additional role as a prognostic marker in colorectal scription factor. The interaction between b-catenin and LUST cancer. We compared survival from 187 patients with a graded amplifies the signaling to maintain self-renewal of CCICs. stage III or greater of colorectal cancer progression following Constitutive activation of WNT signaling is a hallmark of resection, obtained from The Cancer Genome Atlas (TCGA) CCICs as indicated previously (4). Therefore, we now define database (34). We indicate that LUST and AKR1B10 transcript LUST as a transcriptional regulator of WNT signaling during the abundance corresponds with a poorer survival outcome (35) in process of spheroids formation. During this process, in fact, the patients following surgical resection, as the expression of mRNA expression levels of CD24 and CD44 increase, as evi- AKR1B10, corresponds with mortality in patients with colon dence of cancer stem cell–like potential and enrichment of adenocarcinoma. Weaker outcomes in survival were shown when CCICs (27), whereas the increased expression of LUST, also the expression of either LUST or AKR1B10 transcripts was above confirmed by RNA-seq analysis, is further induced within only the median from all patients from TCGA datasets, recorded 14 days in culture, when the so-called "stemness" markers following surgical resection (Supplementary Fig. S4) using algo- levels are not increased yet. This expression reaches a 10-fold rithms previously described (34, 36). Therefore, to validate the change increase when cells are grown for 5 weeks in the same role of LUST in directing tumor cell fate, we generated a xenograft conditions, suggesting that LUST expression is an early event in mouse model using HT-29 cells that were depleted of LUST the sphere formation process. The differentiation process (LNA_RMB5-AS1), HT-29 cells that were transiently overexpres- induced in spheres derived from colon carcinoma cells (19, sing (xEx_RBM5-AS1), and relative control (LNA_Control). We 20) show that both mRNA and protein expression levels of the find a strong link of RBM5-AS1 expression with HT-29 cell tumor "stem-like" markers CD24 and CD44 is abrogated, confirming growth from xenograft implants into immunocompromised mice reduced CCIC-like potential. Interestingly, LUST expression in both volume and survival outcomes. This suggests that in vivo, strongly decreases upon induced differentiation, showing as LUST expression in HT-29 cells leads to increased tumor cell evidence of its involvement with stem-like characteristics. Our growth, whereas depletion corresponds with tumor growth loss loss-of-function assays demonstrate that LUST inhibition and better survival rate (Fig. 5G). impairs the transcriptional activation of WNT signaling target genes AXIN2, CCND1, MYC and of stemness markers CD24 and CD44, suggesting the loss of stem-like character; moreover, the LUST targets CMYC, CCND1, and SGK1 by reinforcing reduction of CCND1 and MYC gene product levels may indicate TCF4:b-catenin interactions a reduction of cell proliferation (40, 41). LUST knockdown Finally, to assess the mechanism by which LUST transcript reduces active b-catenin, confirming the decrease of WNT can target chromatin, we generated biotin-tagged oligonucle- signaling activation. The dual luciferase assay performed on otideprobesthatweretiledacrosstheRBM5-AS1 transcript and LUST-overexpressing cells showed that the lncRNA alone as performed ChIRP as previously described (17, 18). Subsequent well as b-catenin are able to activate the TCF-4 reporter mini- quantification using qPCR determined that the LUST transcript 0 gene. The increased luciferase activity in cells co-transfected is capable of selectively target the MYC promoter at the 3 end, with pcDNA3-LUST and pcDNA3-b-catenin, instead, demon- as well as CCND1 and SGK1 promoters. As expected, RBM5, as strates that the LUST transcript and b-catenin coordinately a sense transcript, showed no enrichment at the mentioned regulate WNT/b-catenin signaling. Ectopic expression of the regions. HOXD3-4 genomic site, known to be HOTAIR target transcript in HT-29 cells induces WNT signaling target genes site (37), was used as negative control (Fig. 6A). Furthermore, and stem-like markers transcriptional activation, and the we verified the selectivity of our ChIRP assay by testing the increase is also reflected at the protein level. Moreover, LUST enrichment of RNA-bound chromatin for b-catenin/TCF4 target overexpression induces enrichment of b-catenin at the promo- loci through qPCR. Specifically, we examined CCND1, SGK1, ters of WNT signaling target genes and facilitates an earlier and YAP1 loci, on the basis of the ChIP evidence, we provided sphere formation process in several colorectal cancer cell lines, from the CCIC population previously characterized in Figs. 1 further demonstrating the important role of LUST in CICC and 2. Results now demonstrate how the LUST transcript targets maintenance by modulating the pathway activation. Nuclear/ and modulates the chromatin context surrounding specific cytoplasmic RNA fractionation and RNA-FISH experiments b-catenin targets such as CMYC, CCND1, YAP1,andSGK1 revealed that LUST is a nuclear lncRNA to conceptualize a (Fig. 6B). transcriptional or chromatin-based role. HT-29 colon cancer cells harbor an APC inactivating muta- Discussion tion (as a deletion of the carboxyl terminus at residue 1555; In this study, we isolated by FACS the CD24brightCD44bright ref. 42), inducing the cells to be inert to WNT ligands but carry and the CD24dimCD44dim subpopulations from HT-29 colon constitutively active b-catenin/TCF4 transcription (43). We cancer cell line and identified them respectively as CCICs and anticipated that lncRNAs have a direct role during the spheres more differentiated counterpart. Through RNA sequencing formation process in CCICs maintenance by regulating the analysis, we identified several coding and lncRNA transcripts WNT-mediated signaling. Previous studies reported that b-cate- differentially expressed and confirmed a dysregulation of WNT nin could selectively bind RNA (44, 45). The strong enrichment signaling in CCICs (4). LncRNAs can regulate gene expression of LUST binding to b-catenin suggests that LUST couldbindtoa by diverse mechanisms (38) and their involvement in colorec- mutated isoform of the same protein. CaCo-2 cells harbor both tal cancer has been demonstrated (39). We defined LUST,an APC and b-catenin mutations (33), and we show that even in lncRNA, among the most highly expressed transcript in CCICs, this case LUST binds to b-catenin. We demonstrated that LUST as responsible of promoting CCICs self-renewal. Here, we show enforces the binding of b-catenin and TCF4 facilitating onco- afunctionofLUST as a co-transcriptional activator of the WNT genic transcription. Furthermore, xenograft experiments and

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Colon Cancer Stemness Is Regulated through LUST

Figure 6. The RBM5-AS1 (LUST) transcript reinforces chromatin-associated b-catenin to TCF4 in HT-29 colon carcinoma cells. A, RBM5-AS1, RBM5-S1, and HOTAIR ChIRP-qPCR in HT-29 cells. HOXD3-4 and MYC 50 were used as negative controls. B, sequential ChIP (Re-ChIP) was performed to independently and sequentially select for interactions between TCF4 and b-catenin on the chromatin targets indicated. Comparisons were made between HT-29 cells transfected with pcDNA3 control, pcDNA3-LUST, or LNA RNAs against RBM5-AS1 (LUST). Na€ve mouse and rabbit IgG were used as negative controls. C, model schematic depicting role of the LUST lncRNA to facilitate expression of WNT signal–induced transcription. Copyright 2016 Jill Gregory and the Icahn School of Medicine at Mount Sinai.

survival data from human patients suggest a reliable role of this Authors' Contributions transcript in tumor initiation and growth. This is the first Conception and design: S. Di Cecilia, M.J. Walsh detailed characterization of LUST localization and role in Development of methodology: S. Di Cecilia, F. Zhang, A. Sancho-Medina, CCICs self-renewal and established a coactivating regulatory S. Li, F. Aguilo, M. Rengasamy, M.J. Walsh Acquisition of data (provided animals, acquired and managed patients, model whereby LUST function is critical for the transcriptional provided facilities, etc.): S. Di Cecilia, M.J. Walsh activation of the WNT signaling targets (Fig. 6C) essential to Analysis and interpretation of data (e.g., statistical analysis, biostatistics, insure CCIC maintenance. computational analysis): S. Di Cecilia, F. Zhang, W. Zhang, L. Del Vecchio, M.J. Walsh Disclosure of Potential Conflicts of Interest Writing, review, and/or revision of the manuscript: S. Di Cecilia, A. Sancho- No potential conflicts of interest were disclosed. Medina, F. Aguilo, F. Salvatore, M.J. Walsh

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Administrative, technical, or material support (i.e., reporting or organizing Grant Support data, constructing databases): F. Zhang, A. Sancho-Medina, Y. Sun The study was supported by Senior Scholar Award in Aging (AG-SS-2482-10) Study supervision: L. Del Vecchio, F. Salvatore, M.J. Walsh to M.J. Walsh from the Ellison Medical Foundation and awards 5RO1 CA154903 and 5RO1 HL103967 from the NIH to M.J. Walsh. Acknowledgments The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in We thank Dr. A. Alonso of the Weill-Cornell College of Medicine's Epige- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. nomic Sequencing Core for support for RNA-Seq and RIP-Seq studies. We acknowledge expertise from Stephen Hearn from the CSHL for help conducting the RNA-FISH studies. We thank Jill Gregory for providing the model Received July 7, 2015; revised June 20, 2016; accepted July 11, 2016; illustration. published OnlineFirst August 12, 2016.

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www.aacrjournals.org Cancer Res; 76(19) October 1, 2016 5627

RBM5-AS1 Is Critical for Self-Renewal of Colon Cancer Stem-like Cells

Serena Di Cecilia, Fan Zhang, Ana Sancho, et al.

Cancer Res 2016;76:5615-5627. Published OnlineFirst August 12, 2016.

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