Cancer-initiating cells (CIC) undergo asymmetric growth patterns that increase phenotypic diversity and drive selection for chemotherapeutic resistance and tumor relapse. WNT signaling is a hallmark of colon CIC, often caused by APC mutations, which enable activation of β-catenin and MYC. Accumulating evidence indicates that long noncoding RNAs (lncRNA) contribute to the stem-like character of colon cancer cells. In this study, we report enrichment of the lncRNA RBM5-AS1/LUST during sphere formation of colon CIC. Its silencing impaired WNT signaling, whereas its overexpression enforced WNT signaling, cell growth, and survival in serum-free media. RBM5-AS1 has been little characterized previously, and we determined it to be a nuclear-retained transcript that selectively interacted with β-catenin. Mechanistic investigations showed that silencing or overexpression of RBM5-AS1 caused a respective loss or retention of β-catenin from TCF4 complexes bound to the WNT target genes SGK1, YAP1, and MYC. Our work suggests that RBM5-AS1 activity is critical for the functional enablement of colon cancer stem-like cells. Furthermore, it defines the mechanism of action of RBM5-AS1 in the WNT pathway via physical interactions with β-catenin, helping organize transcriptional complexes that sustain colon CIC function. Cancer Res; 76(19); 5615–27. ©2016 AACR.
Colorectal cancer represents one of the most common tumor types and the third leading cause of cancer mortalities in the United States (1). The bulk of a tumor consists of rapidly proliferating, postmitotic, differentiated cells and a very small population of cancer-initiating cells (CIC) or cancer stem cell (CSC). CICs/CSCs show a remarkable capacity for self-renewal and asymmetrical tumor cell growth and have been identified in several cancers, including human colon malignancies (2). Representative colon CICs (CCIC) surface markers and combined phenotypes have been used to isolate presumed CCIC populations, including the enrichment of CD133, CD44, CD24, and CD29 as cell surface antigens, and CD24+CD44+, EphB2high, EpCAMhigh/CD44+/CD166+, ALDH+, LGR5+, and CD44v6+ as phenotypic marks of stem cells (3). From a molecular perspective, a hallmark of CCICs is represented by a constitutively active Wingless (WNT)/β-catenin signaling pathway (4). Active WNT signaling is critical for intestinal stem cells, crypt cell proliferation, and turnover; however, “stem-like” characteristics result from the convergence of cell-intrinsic features, extracellular signals, and stochastic events that continuously shape the self-renewing compartment (3). In particular, the epigenetic programming of transcription involves long noncoding RNAs (lncRNA), in addition to chromatin proteins and DNA. LncRNAs have various roles (for review, see ref. 5) with previous reports demonstrating their direct involvement in regulating, as well as maintaining, pluripotent states at the chromatin level (6). Interestingly, the WNT pathway can be modulated by lncRNAs in several tumor types (7). Within the context of a population of CCICs, we hypothesize that lncRNAs coordinate the chromatin architecture to include specific lncRNAs that facilitate reprogramming of the epigenome, thereby enabling the emergence of stem–like progenitors in colon cancer.
We identify the RBM5 antisense (RBM5-AS1) transcript or LUST (Luca-15–specific transcript), herein, referenced as LUST as a specific lncRNA elevated in CCICs population. The loss of LUST lncRNA results in the progressive differentiation of CCICs, whereas ectopic expression corresponds with resistance to cellular differentiation and the stable maintenance of CICCs population. LUST appears to selectively direct β-catenin transactivation via a TCF4 reporter system facilitating its capacity to target gene expression. Moreover, we find that LUST reinforces the chromatin association between β-catenin and TCF4 on specific targets CMYC, CCND1, SGK1, and YAP1 to provide a cell growth advantage, reflecting CCICs in vivo. These findings demonstrate that the LUST transcript is a novel lncRNA involving the recruitment and function of β-catenin in CCICs and regulates WNT pathway by promoting “stemness” maintenance.
Materials and Methods
Cells and reagents
Human LS174T (ATCC #CL-188), SW480 (ATCC #CCL-228), HT-29 (ATCC #HTB-38), CaCo-2 (ATCC #HTB-37), DLD-1 (ATCC #CCL-221), and HCT 116 (ATCC #CCL-247) colon cancer cell lines were purchased from the ATCC between the years 2013 and 2014, propagated and passaged as adherent cell cultures according to instructions provided by ATCC. For all cell lines, the cells were received from ATCC as early passages and guidelines for authentication were followed as described previously (8). However, no additional steps to authenticate were taken. All documentation related to the cell lines obtained can be acquired through ATCC. Cells were maintained in adherent conditions, at 37°C in humidified atmosphere containing 5% CO2. The medium was changed twice a week, cells were passaged using 0.05% trypsin/EDTA (Corning) and preserved at early passages. Mycoplasma detection was routinely tested by qPCR methods (9).
Flow cytometric analysis and cell sorting
HT-29, LS174T, and SW480 colon cancer cells were stained using fluorescein isothiocyanate (FITC)-conjugated CD24, phycoerythrin (PE)-conjugated CD166, allophycocyanin (APC)-conjugated CD133 (BD Biosciences), and PE-Cy7–conjugated CD44 (BioLegend). Samples were analyzed on a BD LSRII flow-cytometer (Becton Dickinson). Fluorescence-activated cell sorting (FACS) of HT-29 cells was performed using BD FACSAria II (Becton Dickinson). Analysis of cytometric data was performed using FACSDiva software (Becton Dickinson; see Supplementary Information).
In vitro colonospheres formation assay
Spheres formed with colon carcinoma cells (HT-29, LS174T, SW480, DLD-1, HCT116) were obtained as previously described (4) with minor modifications provided in Supplementary Information.
RNA extraction, qRT-PCR, and RNA-Seq
Total RNA was extracted from HT-29, CaCo-2, LS174T, and SW480 cells, derived colonospheres using TRIzol and the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Reverse transcription was performed using PrimeScript RT Reagent Kit (Takara #6130). Quantitative PCR (qRT-PCT) was performed using the GoTaq qPCR Master Mix (Promega). Hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene was used as housekeeping gene for normalization. Sequences of all the primers used for qRT-PCR are listed in Supplementary Table S2. RNA-Seq datasets from Illumina HiSeq2500 sequencing approaches have been deposited in the NCBI GEO under the accession number GSE69236, NCBI website: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=sbibkwgutdgzxyn&acc=GSE69236. Gene ontology analysis was performed as previously described (10).
lncRNA LUST knockdown and overexpression
lncRNA LUST knockdown was performed using LNA longRNA GapmeR (Exiqon, #300600). Four different probes directed against lncRNA LUST transcript and one unspecific negative control probe were used. The construct pcDNA3-LUST was used and generated as previously described (11). The sequences of the oligonucleotides and their LNATM spiking patterns were designed using Exiqon's GapmeR Design Algorithm: (http://www.exiqon.com/ls/Pages/GDTSequenceInput.aspx?SkipCheck=true).
TOPFlash dual luciferase assay
HT-29 cells were transiently transfected using Lipofectamine (Invitrogen) according to the manufacturer's instructions, using 250 ng of the TOPFlash reporter gene construct (M50 Super 8x TOPFlash, Plasmid #12456, Addgene) and 500 ng of pcDNA3-LUST and/or 500 ng of pcDNA-β-catenin construct. Luciferase reporter gene expression was measured according to the manufacturer's protocol (Dual-luciferase Reporter assay System, Promega). The luciferase activity was normalized to Renilla luciferase activity from cotransfected internal control plasmid pRL-CMV.
Immunoblotting for proteins
For western blot analysis, 30 μg of protein lysate was analyzed by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad), and blotted with indicated antibodies followed by ECL detection (Thermo Scientific). Western blot assays were performed using the fallowing commercially available antibodies, at the indicated concentrations: anti-β-actin (Sigma, A5441, 1:1,000), anti-α-tubulin (Sigma, T5168, 1:1,000), anti-β-catenin (Bethyl Laboratories, A302-012A, 1:1,000), anti-active-β-catenin (Millipore, 05-665, 1:1,000), anti-cyclin D1 (CCND1, Abcam, ab16663, 1:1,000), and anti-c-myc (Cell Signaling, 5605, 1:1,000).
For lncRNA LUST knockdown, cells were transfected using 300 pmole of LNA GapmeRs and Lipofectamine (Invitrogen) according to the manufacturer's protocol. For lncRNA LUST overexpression, pcDNA3 and pcDNA3-LUST construct were transiently transfected into HT-29 cells using 4 μg of DNA. For colonospheres formation assay, HT-29, LS 174T, SW-480, DLD -1, and HCT116 cells were transiently transfected with pcDNA3 control or pcDNA-LUST and then seeded in a low-attachment plate.
Nuclear fractionation and FISH
Nuclear fractionation was performed as previously described (12). For lncRNA LUST localization, RNA-FISH was performed using customized probes purchased from Exiqon, following the manufacturer's instructions for use.
Crosslinked RNA immunoprecipitation, chromatin immunoprecipitation, photoactivatable ribonucleoside-enhanced crosslinking immunoprecipitation, and sequential chromatin immunoprecipitation
Essentially all experiments performed in this section have been previously described (10, 13) and performed using 5 μg of anti-β-catenin (Bethyl Laboratories, A302-012A) and 5 μg of rabbit control IgG-ChIP Grade (Abcam, ab46540). Chromatin immunoprecipitation (ChIP) was performed as previously described (14) using 6 μg of anti-β-catenin (Bethyl Laboratories, A302-012A) and equal amount of rabbit control IgG-ChIP Grade (Abcam, ab46540). The sequential-ChIP (or Re-ChIP) studies were performed as described (15). Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) was performed as described in (10, 16).
Mouse xenograft studies
A total of 2 × 106 HT-29–transfected cells were recovered, placed in 1× PBS, and inoculated by subcutaneous engraftment in the hind rump of male immunodeficient CB17-SCID mice (Charles River Laboratories) between 8 and 10 weeks of age. Tumor cell growth was established directly from HT-29 cells manipulated either by transfection with the expression vector for the LUST RNA transcript or by depleting LUST using locked nucleic RNAs (purchased through Exiqon). The growth of the tumors was followed by means of caliper measurements. Monitoring of tumor growth was performed at least twice per week using calipers. Details on analyses of tumor volume and survival are described within Supplementary Information.
Chromatin isolation by RNA purification
Chromatin isolation by RNA purification (ChIRP) experiments were performed as previously described (17, 18). Twenty-five probes were generated against the RBM5-AS1 (LUST), RBM5-S1 (sense), and HOTAIR transcripts. Bound chromatin was detected by qPCR from HOXD3, MYC 5′, MYC 3′, CCND1, SGK1 promoter-specific primers. Sequences of RBM5-AS1 and RBM5 and HOTAIR RNA probes are listed in Supplementary Table S2.
All experiments were performed in triplicate at least 3 times. All values were expressed as mean ± SEM. Statistical analysis was performed by the unpaired Student t test. A probability value of P ≤ 0.05 was considered statistically significant.
β-Catenin target genes are stimulated within HT-29 CCICs compared with their more differentiated counterparts
Here, we confirmed the expression of surface markers (2, 19) on 3 different colon cancer cell lines chosen for their differentiated status: SW480 (poorly differentiated), HT-29 (moderately differentiated), and LS174T (well-differentiated). Within the HT-29 cell line, we identified 2 subpopulations coexpressing all 4 surface markers with signal intensity corresponding respectively as “bright” and “dim” (CD24, CD44, CD133, and CD166, Fig. 1A). For LS174T and SW480 colon cancer cell lines, 2 different subpopulations could be discriminated as well, on the basis of the signal intensity of CD24 and CD44 coexpression (bright and dim). However, the intensity of CD133 and CD166 antigens was lower than that of the HT-29 subpopulation (Fig. 1A). To assess the ability of self-renewal of undifferentiated (CD24bright/CD44bright) versus differentiated (CD24dim/CD44dim) HT-29 cell subpopulations, we performed in vitro colon carcinoma spheres assay as previously described (2, 19, 20). Colon cancer progenitor cells can grow as spherical aggregates that, in the presence of serum or extracellular matrix, differentiate upon growth factor removal (21–23). Toward this aim, we first isolated HT-29 CD24bright/CD44bright (CCICs) and CD24dim/CD44dim (the differentiated counterpart) by FACS (Fig. 1B) and then grew the sorted cells in suspension, in serum-free media, to induce spheres formation (19). After 3 weeks of culture, we obtained spheres formed as aggregates of exponentially growing undifferentiated cells CD24bright/CD44bright (CCICs) that were larger in terms of size and number than the ones obtained from the CD24dim/CD44dim colon cancer cells, as expected and previously reported (Fig. 1C; ref. 24). On the basis of WNT/β-catenin signaling transcripts as a prominent signature characteristic of some cancer stem cells (25), we performed RNA-Seq on CD24bright/CD44bright (CCICs) and CD24dim/CD44dim (a differentiated comparison) sorted subpopulations from HT-29 colon cancer cell line, and spheres derived from the CD24bright/CD44bright subpopulation, to explore pathways that are dysregulated. Approximately, 3,000 genes were elevated in CCICs, when compared with the differentiated population, indicated with a fold change > 1.5 (Fig. 1D, top left). Analysis of the elevated genes identifies them among regulation of cell proliferation, cell migration, and other biological processes characteristic of cancer cell phenotype (Fig. 1D, bottom left). Approximately, 2,000 genes resulted to be repressed between CD24bright/CD44bright and CD24dim/CD44dim subpopulations (Fig. 1D, top right), involved as well in regulation of cell proliferation or cellular metabolism (Fig. 1D, bottom right). We then analyzed the expression of genes defining colonic mucosa cells differentiation, such as MUCIN 2 (MUC2) or keratin B20 (KB20/KRT80) that resulted downregulated, or colon cancer stem cells markers, in particular CD24, CD44, CD166, and ALDH1A1 that were upregulated (Fig. 1E, shown as the green and red bars, respectively). Members of the canonical WNT-mediated signaling pathway, such as ASCL2, IGFBP2, LGR4, DKK1, MYCL1, FGFR2, SP5, MMP7 and the receptor EPHB3 were elevated in the CCICs compared with the more differentiated counterpart, with a fold increase > 1.5 (Fig. 1E and F, shown as the blue bars), as shown form the IGV tracks (Supplementary Fig. S1). RNA-Seq, performed on spheres derived from CCICs, was used to compare the expression of WNT-target genes and stem-like markers with the subpopulation (CD24bright/CD44bright) they originated from. The expression of ASCL2, MYCL1, LGR4, EPHB3, CLDN1, CD44, CD24, ALDH1A1, and AKR1B10 was strongly increased during spheres formation. Genes involved in differentiation, such as Kruppel-like factor 9 (KLF9), KB20, and the β-catenin–regulated gene peptidyl arginine deiminase, type 1 (PADI1), were the most suppressed (fold change > 1.5) transcripts in both spheres and the parental subpopulation, as well as the retinoic acid receptor responder protein 1 (RARRES1), known to have a tumor suppressor role (26). Intriguingly, 2 lncRNAs, KCNQ1OT1 and LUST, were increased in this process, and the overexpression of LUST seemed to be more sphere specific (Fig. 1G). Furthermore, when cells were induced to differentiate, the mRNA levels of CCDN1, LGR4, and CMYC were reduced (Fig. 1H), showing that WNT signaling is highly and selectively active during HT-29 cells sphere formation and that the activity is reduced upon FBS-induced differentiation, confirming its role in maintenance of colon cancer cell spheres (27).
The lncRNA LUST transcript expression corresponds strongly with the stem-like capacity of CCICs
Recent data have shown that dysregulation of coding as well as noncoding RNAs contribute to CCIC generation, reviewed in ref. 28 and recently has been demonstrated that in cancer the WNT signaling can be additionally regulated by lncRNAs through cell-autonomous mechanisms (29). To investigate the involvement of lncRNAs in CCIC maintenance and their role in the WNT signaling regulation, we performed an lncRNA profiler array analysis on CCICs and the more differentiated counterpart isolated from HT-29 colon cancer cells. Several lncRNAs were dramatically elevated in CCICs compared with their differentiated counterpart (Table S1), with an increase higher than 2.5-fold.
Among these lncRNAs, LUST and KCNQ1OT1 were overexpressed, as confirmed by RNA-Seq also in spheres derived from HT29 cells (Fig. 1G and I). To address the functional role of LUST in cancer stem cell maintenance, we analyzed by qRT-PCR the lncRNA and stem-like markers expression in spheres derived from CCICs and in FBS-induced differentiation cells. During the sphere formation process, the lncRNA LUST expression results strongly increased (Fig. 2A, left). Of note, the increase appears to be significant already after 14 days in culture, when the stem-like markers CD24 and CD44 mRNA levels (Fig. 2A, middle and right) are not elevated yet, and reaches a 10-fold change increase when cells are grown for 5 weeks in the same conditions (Fig. 2A, left). This result suggests that LUST upregulation could be an early event in the sphere formation process. As expected, when differentiation of the spheres is induced by adding FBS for additional 48 hours (20), the mRNA expression levels of conventional cancer stem cell markers, CD24 and CD44, are abrogated (Fig. 2A, middle and right), confirming the loss of the stem-like potential. Interestingly, the expression level of the lncRNA LUST drastically decreased (Fig. 2A, left), and the loss of markers CD24 and CD44, shown at the protein level (Fig. 2B, blue population), strongly correlates with the switch from spheroid toward more adherent cell morphology (Fig. 2C, top and bottom, respectively).
WNT signaling activation is impaired upon LUST knockdown
We performed loss-of-function assays in HT-29 cells by using two distinct locked nucleic acid (LNA) RNA GapmeRs that knocked down the expression of LUST to about 60% and 50%, respectively, compared with the control unspecific probe (Fig. 3A) and analyzed the expression of WNT signaling target genes by RT-qPCR. While control cells retained the expression of WNT target genes, a strong reduction of mRNA transcripts such as AXIN2, CCND1, CD44, and TCF4 as well as CD24 mRNA levels occurs upon LUST knockdown (Fig. 3B). A reduction at the protein level was seen for CCND1 and C-MYC (Fig. 3C) and, of note, LUST knockdown caused also a reduction of active β-catenin (Fig. 3C).
LUST overexpression potentiates WNT/β-catenin signaling cascade and accelerates spheroid formation across multiple colon carcinoma cell lines
We then performed dual luciferase assay using TOPFlash promoter (a reporter plasmid containing multiple copies of wild-type Tcf4-binding sites), transiently integrated in HT-29 cells (TOPFlash-HT-29 cells). Relative luciferase activity was measured in TOPFlash-HT-29 cells transfected with pcDNA3-LUST, pcDNA3-β-catenin, or pcDNA3-LUST and pcDNA3-β-catenin. The lncRNA LUST alone as well as β-catenin were able to activate TCF reporter and induce luciferase activity. Interestingly, the cotransfection of pcDNA3-LUST and pcDNA3-β-catenin induced a significant strong increase in luciferase activity compared with pcDNA3-LUST and pcDNA3-β-catenin alone. These results demonstrated that the lncRNA LUST and β-catenin synergistically activate WNT/β-catenin signaling (Fig. 3D). To further evaluate the LUST-mediated regulation of WNT signaling in colon cancer, we analyzed the mRNA levels of WNT signaling target genes upon LUST overexpression. pcDNA3 and pcDNA3-LUST constructs were transiently transfected into HT-29 cells, and RNA samples were analyzed through qRT-PCR. The expression of AXIN2, CCND1, MYC, TCF4 and of the stemness markers CD24 and CD44 was significantly elevated under these conditions (Fig. 3E). Although total β-catenin protein level remains stable, we verified a strong increase of active β-catenin, MYC, and CCND1 expression, (Fig. 3F), showing that the increase is induced at both mRNA and protein levels and therefore demonstrating that LUST enhances WNT signaling activation. Moreover, transient overexpression of LUST in HT29 cells corresponds with more profound colony formation when evaluated by crystal violet on soft agar (Fig. 3G). To investigate a key role of LUST in tumor initiation, we performed in vitro sphere formation assay from HT-29 cells and showed that LUST overexpression promotes an earlier onset of spheroids compared with the control. In fact, HT-29 LUST–overexpressing cells are able to form spheres within 7 days in serum-free media culture conditions and the full spherical morphology is reached within 14 days. Instead, HT-29 cells transfected with empty vector required a longer timeframe corresponding to 21 days (Fig. 3H).
We extended our study using 4 additional human colon cancer cell lines to further validate the role of LUST as a colon cancer stem cell regulator. Exogenous LUST overexpression in LS174T and SW-480 (both APCwild-type/CTNNB1mutant) and in DLD-1 and HT116 (both APCmutant/CTNNB1wild-type) confirmed the mRNA level induction of CCND1, CD44, C-MYC, and TCF4, as well as the protein level increases of c-MYC, CCND1, and active β-catenin (Fig. 4A and B). Even in this case, we could notice an earlier onset of spheroid formation after only 14 days in culture (Fig. 4C–G). These results collectively demonstrate that LUST is an lncRNA transcript that we consider as a key component in regulating CCIC spheroid formation.
LUST is a nuclear-retained lncRNA that directly interacts with β-catenin and enhances occupation of β-catenin at promoters associated with WNT signaling target genes
LUST lncRNA localization resulted to be mostly nuclear (Fig. 5A), and RNA-FISH performed in HT-29 cells transiently transfected with pcDNA3 or pcDNA3-LUST confirmed the nuclear expression of the transcript (magenta color; Fig. 5B). A role for lncRNAs with WNT/β-catenin signaling in cancer has been indicated (29, 30). Therefore, we analyzed the role played by LUST in the regulation of β-catenin activity in CICs, by performing RNA immunoprecipitation (RIP). Results demonstrated that the lncRNA LUST strongly binds to β-catenin (Fig. 5C and Supplementary Fig. S2). To determine the activity of the lncRNA as a transcriptional regulator of WNT signaling, we performed β-catenin ChIP experiments in HT-29 LUST–overexpressing and control cells. Analysis of ChIP-qPCR indicates that LUST induces the enrichment of β-catenin at the promoters of WNT-signaling target genes as MYC, CXXC, YAP1, HDAC4, and SGK1, previously shown to be β-catenin targets (Fig. 5D; ref. 31). For additional evidence where the binding of LUST with β-catenin could be detected, we performed RIP assay (32) in CaCo-2 cells (Fig. 5E and F) that harbor both APC and β-catenin mutations (33). PAR-CLIP assays, performed in CaCo-2 cells, validated that the binding of β-catenin to the lncRNA is selective when the enrichment is compared with other known RNA–protein interactions of EZH2 and CBX7 (Supplementary Fig. S2). To assess RNA-binding affinities, we examined the binding of nuclear proteins isolated from CaCo-2 cells to biotinylated RNA probe representing the LUST transcript and show from the shifted band that the RNA–nuclear protein complex involves β-catenin and TCF4 (Supplementary Fig. S3A). Moreover, we show that affinity-purified β-catenin protein selectively binds the LUST transcript and as a control fails to bind the sense RBM5 transcript (Supplementary Fig. S3B). These results suggest that the lncRNA LUST plays a role in CICC maintenance by regulating the WNT signaling pathway, hence we sought to investigate an additional role as a prognostic marker in colorectal cancer. We compared survival from 187 patients with a graded stage III or greater of colorectal cancer progression following resection, obtained from The Cancer Genome Atlas (TCGA) database (34). We indicate that LUST and AKR1B10 transcript abundance corresponds with a poorer survival outcome (35) in patients following surgical resection, as the expression of AKR1B10, corresponds with mortality in patients with colon adenocarcinoma. Weaker outcomes in survival were shown when the expression of either LUST or AKR1B10 transcripts was above the median from all patients from TCGA datasets, recorded following surgical resection (Supplementary Fig. S4) using algorithms previously described (34, 36). Therefore, to validate the role of LUST in directing tumor cell fate, we generated a xenograft mouse model using HT-29 cells that were depleted of LUST (LNA_RMB5-AS1), HT-29 cells that were transiently overexpressing (xEx_RBM5-AS1), and relative control (LNA_Control). We find a strong link of RBM5-AS1 expression with HT-29 cell tumor growth from xenograft implants into immunocompromised mice in both volume and survival outcomes. This suggests that in vivo, LUST expression in HT-29 cells leads to increased tumor cell growth, whereas depletion corresponds with tumor growth loss and better survival rate (Fig. 5G).
LUST targets CMYC, CCND1, and SGK1 by reinforcing TCF4:β-catenin interactions
Finally, to assess the mechanism by which LUST transcript can target chromatin, we generated biotin-tagged oligonucleotide probes that were tiled across the RBM5-AS1 transcript and performed ChIRP as previously described (17, 18). Subsequent quantification using qPCR determined that the LUST transcript is capable of selectively target the MYC promoter at the 3′ end, as well as CCND1 and SGK1 promoters. As expected, RBM5, as a sense transcript, showed no enrichment at the mentioned regions. HOXD3-4 genomic site, known to be HOTAIR target site (37), was used as negative control (Fig. 6A). Furthermore, we verified the selectivity of our ChIRP assay by testing the enrichment of RNA-bound chromatin for β-catenin/TCF4 target loci through qPCR. Specifically, we examined CCND1, SGK1, and YAP1 loci, on the basis of the ChIP evidence, we provided from the CCIC population previously characterized in Figs. 1 and 2. Results now demonstrate how the LUST transcript targets and modulates the chromatin context surrounding specific β-catenin targets such as CMYC, CCND1, YAP1, and SGK1 (Fig. 6B).
In this study, we isolated by FACS the CD24brightCD44bright and the CD24dimCD44dim subpopulations from HT-29 colon cancer cell line and identified them respectively as CCICs and more differentiated counterpart. Through RNA sequencing analysis, we identified several coding and lncRNA transcripts differentially expressed and confirmed a dysregulation of WNT signaling in CCICs (4). LncRNAs can regulate gene expression by diverse mechanisms (38) and their involvement in colorectal cancer has been demonstrated (39). We defined LUST, an lncRNA, among the most highly expressed transcript in CCICs, as responsible of promoting CCICs self-renewal. Here, we show a function of LUST as a co-transcriptional activator of the WNT signaling by facilitating β-catenin binding to the TCF4 transcription factor. The interaction between β-catenin and LUST amplifies the signaling to maintain self-renewal of CCICs. Constitutive activation of WNT signaling is a hallmark of CCICs as indicated previously (4). Therefore, we now define LUST as a transcriptional regulator of WNT signaling during the process of spheroids formation. During this process, in fact, the mRNA expression levels of CD24 and CD44 increase, as evidence of cancer stem cell–like potential and enrichment of CCICs (27), whereas the increased expression of LUST, also confirmed by RNA-seq analysis, is further induced within only 14 days in culture, when the so-called “stemness” markers levels are not increased yet. This expression reaches a 10-fold change increase when cells are grown for 5 weeks in the same conditions, suggesting that LUST expression is an early event in the sphere formation process. The differentiation process induced in spheres derived from colon carcinoma cells (19, 20) show that both mRNA and protein expression levels of the “stem-like” markers CD24 and CD44 is abrogated, confirming reduced CCIC-like potential. Interestingly, LUST expression strongly decreases upon induced differentiation, showing as evidence of its involvement with stem-like characteristics. Our loss-of-function assays demonstrate that LUST inhibition 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 reduction of CCND1 and MYC gene product levels may indicate a reduction of cell proliferation (40, 41). LUST knockdown reduces active β-catenin, confirming the decrease of WNT signaling activation. The dual luciferase assay performed on LUST-overexpressing cells showed that the lncRNA alone as well as β-catenin are able to activate the TCF-4 reporter mini-gene. The increased luciferase activity in cells co-transfected with pcDNA3-LUST and pcDNA3-β-catenin, instead, demonstrates that the LUST transcript and β-catenin coordinately regulate WNT/β-catenin signaling. Ectopic expression of the transcript in HT-29 cells induces WNT signaling target genes and stem-like markers transcriptional activation, and the increase is also reflected at the protein level. Moreover, LUST overexpression induces enrichment of β-catenin at the promoters of WNT signaling target genes and facilitates an earlier sphere formation process in several colorectal cancer cell lines, further demonstrating the important role of LUST in CICC maintenance by modulating the pathway activation. Nuclear/cytoplasmic RNA fractionation and RNA-FISH experiments revealed that LUST is a nuclear lncRNA to conceptualize a transcriptional or chromatin-based role.
HT-29 colon cancer cells harbor an APC inactivating mutation (as a deletion of the carboxyl terminus at residue 1555; ref. 42), inducing the cells to be inert to WNT ligands but carry constitutively active β-catenin/TCF4 transcription (43). We anticipated that lncRNAs have a direct role during the spheres formation process in CCICs maintenance by regulating the WNT-mediated signaling. Previous studies reported that β-catenin could selectively bind RNA (44, 45). The strong enrichment of LUST binding to β-catenin suggests that LUST could bind to a mutated isoform of the same protein. CaCo-2 cells harbor both APC and β-catenin mutations (33), and we show that even in this case LUST binds to β-catenin. We demonstrated that LUST enforces the binding of β-catenin and TCF4 facilitating oncogenic transcription. Furthermore, xenograft experiments and survival data from human patients suggest a reliable role of this transcript in tumor initiation and growth. This is the first detailed characterization of LUST localization and role in CCICs self-renewal and established a coactivating regulatory model whereby LUST function is critical for the transcriptional activation of the WNT signaling targets (Fig. 6C) essential to insure CCIC maintenance.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: S. Di Cecilia, M.J. Walsh
Development of methodology: S. Di Cecilia, F. Zhang, A. Sancho-Medina, S. Li, F. Aguiló, M. Rengasamy, M.J. Walsh
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Di Cecilia, M.J. Walsh
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Di Cecilia, F. Zhang, W. Zhang, L. Del Vecchio, M.J. Walsh
Writing, review, and/or revision of the manuscript: S. Di Cecilia, A. Sancho-Medina, F. Aguiló, F. Salvatore, M.J. Walsh
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): F. Zhang, A. Sancho-Medina, Y. Sun
Study supervision: L. Del Vecchio, F. Salvatore, M.J. Walsh
We thank Dr. A. Alonso of the Weill-Cornell College of Medicine's Epigenomic 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 illustration.
The study was supported by Senior Scholar Award in Aging (AG-SS-2482-10) to M.J. Walsh from the Ellison Medical Foundation and awards 5RO1 CA154903 and 5RO1 HL103967 from the NIH to M.J. Walsh.
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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.