ASBEL–TCF3 Complex Is Required for the Tumorigenicity of Colorectal Cancer Cells

ASBEL–TCF3 complex is required for the tumorigenicity of colorectal cancer cells

Kenzui Taniuea,1, Akiko Kurimotoa,b,1, Yasuko Takedaa, Takeshi Nagashimac, Mariko Okada-Hatakeyamac, Yuki Katoud, Katsuhiko Shirahiged, and Tetsu Akiyamaa,2

aLaboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan; bOncology Research Laboratories, Daiichi Sankyo Co., Ltd, Shinagawa-ku, Tokyo 140-8710, Japan; cLaboratory for Integrated Cellular Systems, RIKEN Center for Integrative Medical Sciences, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan; and dLaboratory of Genome Structure and Function, Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan

Edited by Robert N. Eisenman, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved September 20, 2016 (received for review April 14, 2016) Wnt/β-catenin signaling plays a key role in the tumorigenicity of co- in the Abundant in neuroepithelium area (ANA)/B-cell transloca- lon cancer. Furthermore, it has been reported that lncRNAs are dys- tion gene 3 (BTG3) locus] and transcription factor 3 (TCF3) (20, regulated in several steps of cancer development. Here we show that 21). We previously identified ASBEL as an antisense transcript of β-catenin directly activates the transcription of the long noncoding the ANA/BTG3 gene, which encodes an antiproliferative protein, RNA (lncRNA) ASBEL [antisense ncRNA in the ANA (Abundant in neu- and showed that it suppresses the levels of ANA/BTG3 protein and roepithelium area)/BTG3 (B-cell translocation gene 3) locus] and tran- is required for the tumorigenicity of ovarian clear cell carcinoma scription factor 3 (TCF3), both of which are required for the survival ASBEL ASBEL (13). In this study, we show that is required for the tu- and tumorigenicity of colorectal cancer cells. interacts with morigenicity of colon cancer cells and that ASBEL and TCF3 form a and recruits TCF3 to the activating transcription factor 3 (ATF3)locus, complex to suppress the expression of activating transcription factor where it represses the expression of ATF3. Furthermore, we demon- ASBEL– – strate that ASBEL–TCF3–mediated down-regulation of ATF3 expres- 3 (ATF3). We further demonstrate that TCF3 mediated sion is required for the proliferation and tumorigenicity of colon down-regulation of ATF3 is critical for the tumorigenicity of colon tumor cells. ATF3, in turn, represses the expression of ASBEL.Our cancer cells. Finally, we show that ATF3 functions in a negative ASBEL results reveal a pathway involving an lncRNA and two transcription feedback loop that inhibits expression. CELL BIOLOGY factors that plays a key role in Wnt/β–catenin–mediated tumorigenesis. These results may provide insights into the variety of biological Results and pathological processes regulated by Wnt/β-catenin signaling. The lncRNA ASBEL Is a Target of β-Catenin. As a first step to identify genes that are the direct targets of β-catenin, we performed RNA- β-catenin | noncoding RNA | ASBEL | colorectal tumorigenesis | ATF3 sequencing (RNA-seq) analysis using DLD-1 cells. We found that knockdown of β-catenin led to the up-regulation of 2,072 genes, he canonical Wnt signaling pathway plays essential roles in the including 86 genes encoding lncRNAs, and to the down-regulation Tregulation of proliferation, cell fate, the self-renewal of stem of 1,512 genes, including 33 genes encoding lncRNAs (Fig. 1A and and progenitor cells, and tumorigenesis (1–4). In the absence of Fig. S1A and Datasets S1–S4). Functional pathway analyses using Wnt signaling, β-catenin is targeted for ubiquitin/proteasome- the Ingenuity Pathway Analysis (IPA) software revealed that genes mediated degradation by a destruction complex composed of the tumor suppressor adenomatous polyposis coli (APC), axin, glyco- Significance gen synthase kinase-3β, casein kinase I, and TAP/TAZ. Wnt signaling suppresses the function of this complex and induces the Wnt/β-catenin signaling plays crucial roles in the regulation of association of β-catenin with the TCF/LEF family of transcription proliferation, cell fate, the self-renewal of stem and progenitor factors, thereby activating the transcription of a variety of Wnt cells, and tumorigenesis. Long noncoding RNAs (lncRNAs), non– target genes. In the majority of colorectal cancers, β-catenin is protein-coding transcripts longer than 200 nt, also play important stabilized by mutations in APC or β-catenin, and thereby Wnt roles in a number of biological processes and in tumorigenesis. target genes involved in tumorigenesis, including c-Myc and cyclin We show that the lncRNA ASBEL [antisense ncRNA in the ANA D1, are constitutively activated. (Abundant in neuroepithelium area)/BTG3 (B-cell translocation Mammalian genomes encode numerous long noncoding RNAs gene 3) locus] and transcription factor 3 (TCF3) are directly trans- (lncRNAs), a class of non–protein-coding transcripts longer than activated by β-catenin and form a complex that downregulates the 200 nt (5–7). lncRNAs play important roles in a variety of biological expression of activating transcription factor 3 (ATF3). We further processes, including proliferation, differentiation, embryogenesis, demonstrate that ASBEL–TCF3–mediated downregulation of ATF3 neurogenesis, stem cell pluripotency, and tumorigenesis. Accumu- expression is required for the tumorigenicity of colon cancer cells. lating evidence indicates that a number of lncRNAs regulate gene Our results suggest that the β-catenin–ASBEL–TCF3–ATF3 pathway expression by interacting with epigenetic regulators and acting as may be a promising target for colon cancer therapy. scaffolds for the assembly of protein complexes (8–11). It has also been reported that many lncRNAs regulate transcription by mod- Author contributions: K.T., A.K., and T.A. designed research; K.T., A.K., Y.T., Y.K., and K.S. performed research; K.T., T.N., and M.O.-H. analyzed data; Y.K. and K.S. performed se- ulating the activity of transcription factors or posttranscriptional quence analysis; and K.T. and T.A. wrote the paper. processes, including splicing, transport, translation, and degrada- The authors declare no conflict of interest. – tion of mRNA (12 17).Furthermore,ithasbeenshownthat This article is a PNAS Direct Submission. lncRNAs are frequently dysregulated in many types of cancer, in- Data deposition: The data reported in this paper have been deposited in the DNA Data cluding colorectal cancer, and can have oncogenic or antioncogenic Bank of Japan Sequence Read Archive (DRA) database, trace.ddbj.nig.ac.jp/dra/index.html functions (12, 18, 19). (accession no. DRA004515). In the present study, to gain insights into Wnt/β-catenin–mediated 1K.T. and A.K. contributed equally to this work. tumorigenesis, we attempted to identify lncRNAs that are directly 2To whom correspondence should be addressed. Email: [email protected]. β β transactivated by -catenin. We show that -catenin directly en- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. hances the transcription of the lncRNA ASBEL [antisense ncRNA 1073/pnas.1605938113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1605938113 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 involved in cell survival, movement, and proliferation were over- of DLD-1 cells and found that knockdown of ASBEL caused represented among the affected genes (Fig. S1B and Dataset S5). stronger growth inhibition than knockdown of AK092875 (Figs. S1 We next subjected DLD-1 cells to ChIP-sequencing (ChIP-seq) E and F and Fig. S2C). In addition, we found that knockdown of analysis to examine the genome-wide localization of β-catenin in AK092875, but not of ASBEL, inhibited the growth of normal gene promoter regions, i.e., from approximately −2,000 to ap- keratinocyte HaCaT cells (Fig. S1F). proximately +2,000 bp from the transcription start sites (TSSs) We therefore decided to focus our analysis on ASBEL.Wefirst (Fig. 1A). We found that β-catenin bound to the promoter regions confirmed that knockdown of β-catenin resulted in decreased of 813 genes, including 105 genes encoding lncRNAs (Fig. S1A and expression of ASBEL, as well as AXIN2, a known β-catenin target Datasets S6 and S7). Comparison of the RNA-seq and ChIP-seq gene, in both DLD-1 and SW480 cells (Fig. 1B and Fig. S1G). We data suggested that β-catenin directly down-regulated 74 genes, also observed that knockdown of APC caused an increase in the including one encoding an lncRNA, and up-regulated 66 genes, expression of ASBEL and AXIN2 in 293FT cells (Fig. 1C and Fig. including two encoding lncRNAs (Fig. 1A and Fig. S1A and S1H). Moreover, ChIP analyses with anti–β-catenin antibody Datasets S8 and S9). Consistent with previous reports (22), Gene revealed that β-catenin bound to the TSSs but not to the upstream Set Enrichment Analysis (GSEA) using the C3 gene set revealed (−3,500 bp) regions of the ASBEL and AXIN2 promoters in DLD-1 that the β-catenin target genes were enriched for LEF1 motifs in and SW480 cells (Fig. 1D and Fig. S1I). In addition, we found their promoter regions (Fig. S1C and Dataset S10). In addition, that β-catenin is recruited to the TSSs but not to the upstream GSEA pathway analysis showed that β-catenin regulates the ex- (−3,500 bp) regions of the ASBEL and AXIN2 promoters in pression of known APC target and ES cell-related genes (Fig. S1D 293FT cells in which APC had been knocked down (Fig. 1E). This and Datasets S11 and S12). recruitment was not observed in control 293FT cells (Fig. 1E). Among the genes identified in the above experiments, we fo- These results suggest that β-catenin transactivates ASBEL directly cused on two lncRNAs that are directly up-regulated by β-catenin, by binding to its TSS region in colon tumor cells. AK092875 and ASBEL (BC028229), the latter of which is known We next examined whether β-catenin could up-regulate a region to be required for the tumorigenicity of ovarian cancer (13). We of the ASBEL promoter (from approximately −1,000 to approxi- then examined the effects of these lncRNAs on the proliferation mately +1 bp) inserted into a reporter construct driving the luciferase gene (WT-ASBEL). We also generated variants of this reporter construct containing a mutated TCF-binding element (TBE) in the ABSEL promoter region (MT1–3-ASBEL; Fig. S1J). A siβ-catenin#1 siCont siβ-catenin#2 C ASBEL 5 APC * When transfected into DLD-1 cells, the activity of WT- was ASBEL * – ASBEL 4 TCF3 significantly higher than in mock or MT1 3- reporter RNA-seq 3 plasmids (Fig. S1J). Moreover, we found that cotransfection of WT- * 2 * ASBEL ASBEL β-catenin-regulatedd genesgeness , but not of mock or MT3- , into HeLa cells together 1 β β β IPA -catenin * * with a constitutively active mutant of -catenin, -cateninS33Y, Relative expression 0 J β-catenin target genes ChIP-seq resulted in a significant increase in reporter activity (Fig. S1 and GSEA TF K ASBEL siCont siCont siCont ). Quantitative RT-PC (qRT-PCR) analysis revealed that GSEA pathway ASBEL, TCF3 siAPC#1 siAPC#2 siAPC#1 siAPC#2 siAPC#1 siAPC#2 1.2 β D 0.06% Anti-β-catenin expression was up-regulated in the colon cancer cell lines HCT116, B -catenin Axin2 ASBEL TCF3 * Anti-IgG * DLD-1, and Caco2, in which Wnt signaling is activated, compared 0.8 * L M 0.03% with the normal keratinocyte cell line HaCaT (Fig. S1 and ). * * 0.4 * * * * These results suggest that β-catenin up-regulates ASBEL expres- % of Input * * sion by binding to the TBEs in the ASBEL promoter region.

Relative expression 0 0.00%

siCont siCont siCont siCont TSS TSS ASBEL Axin2 -3500 -3500 Is Required for the Tumorigenicity of Colon Tumor Cells. To β-catenin#1β-catenin#2β-catenin#1β-catenin#2β-catenin#1β-catenin#2β-catenin#1β-catenin#2 ASBEL TCF3 si si si si si si si si investigate the importance of ASBEL in colorectal tumorigenesis, E 0.10% Anti-β-catenin ** Anti-IgG we infected DLD-1, HCT116, and HT29 cells with a lentivirus expressing an shRNA targeting ASBEL (shASBEL) and examined 0.05% ** their tumorigenicity. When these cells were transplanted into nude

% of Input ** mice, the cells infected with shASBEL showed significantly retarded 0.00% cell growth compared with those infectedwithacontrollentivirus (Fig. 2A and Fig. S2 A and B). In addition, Cell Titer-Glo assays siCont siCont siCont siCont siCont revealed that knockdown of ASBEL by siRNA (siASBEL)causeda siAPC#1 siAPC#2 siAPC#1 siAPC#2 siAPC#1 siAPC#2 siAPC#1 siAPC#2 siAPC#1 siAPC#2 TSS -3500 TSS -3500 TSS Axin2 ASBEL TCF3 significant reduction in the growthofDLD-1,HCT116,andCaco2 cells but not of normal keratinocyte HaCaT cells in vitro (Fig. 2B Fig. 1. Transactivation of ASBEL and TCF3 by β-catenin is required for and Fig. S2C). Annexin V assays also showed that knockdown of β-catenin–mediated proliferation of colon cancer cells. (A) Depiction of a ASBEL ledtoamarkedincreaseinapoptoticcelldeathofHCT116 screen to identify β-catenin target genes. (B) qRT-PCR analysis of the ex- cells but not of HaCaT cells (Fig. S2 C and D). Moreover, we found pression of the indicated genes in DLD-1 cells transfected with an siRNA that knockdown of ASBEL reduced the invasiveness of DLD-1 and targeting β-catenin or a control siRNA (siCont). Two distinct siRNAs targeting C C β β HCT116 cells (Fig. 2 and Fig. S2 ). -catenin (si -catenin #1 and #2) were used. Results are expressed as the ASBEL mean ± SD (n = 3). *P < 0.05. (C) qRT-PCR analysis of the expression of the We next examined expression in human colorectal tu- indicated genes in 293FT cells transfected with an siRNA targeting APC. mors and adjacent noncancerous tissues by qRT-PCR analysis. We Results are expressed as the mean ± SD (n = 3). *P < 0.05. (D) ChIP assays found that ASBEL expression was higher in stage I–III colon cancer were performed with DLD-1 cells using anti–β-catenin antibody. The pro- than in normal tissues (Fig. S2E). Taken together, these results moter region of AXIN2 was amplified as a positive control. The regions suggest that ASBEL is required for the proliferation, survival, and around −3,500 bp of ASBEL and −3,500 bp of TCF3 were amplified as neg- tumorigenicity of colon cancer cells. ± = < ative controls. Results are expressed as the mean SD (n 3). *P 0.05. Knockdown of ASBEL in HCT116 cells resulted in the up- (E) ChIP assays were performed with 293FT cells transfected with an siRNA targeting APC (siAPC) using anti–β-catenin antibody. The promoter region of regulation of 375 genes and in the down-regulation of 233 genes, as AXIN2 was amplified as a positive control. The regions around −3,500 bp of determined by RNA-seq analysis. Consistent with the above results, ASBEL and −3,500 bp of TCF3 were amplified as negative controls. Results this gene set was enriched with genes involved in cell proliferation, are expressed as the mean ± SD (n = 3). *P < 0.05. death, and movement (Fig. 2D and Datasets S13 and S14).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1605938113 Taniue et al. Downloaded by guest on October 1, 2021 A 2000 B 1.5 CE1.2 HCT116 D -log(pvalue)

) HCT116 DLD-1 Caco2 HaCaT DLD1 ) 3 0 5 10 15 20 -2 10 * shCont Cellular Growth * + 1 0.8 ** and Proliferation **

shCont Cellular TCF3 -3 1000 ** ** 0.4 Development shASBEL#A 0.5 ** ** Cell Death and * + Cell viability Survival Relative invasion

shATF3 Relative 0 0 Cellular * Movement expression (Log -4 N I II III IV shASBEL#A IPA Molecular and Cellular Functions Tumor volume (mm + 0 1.5 * siCont siCont * siCont siCont siCont shCont siCont * 012345Weeks J siASBEL#1 siASBEL#2 siASBEL#1 siASBEL#2 siASBEL#1 siASBEL#2 siASBEL#1 siASBEL#2 siASBEL#1 siASBEL#2 siASBEL#1 siASBEL#2 1 F 1.2 G 2500 H 50

HCT116 DLD-1 Caco2 HaCaT )

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2000 -cat/siCont) 0.5 β ASBEL Input + GAPDH 0.8 shCont 30 Cell viability TCF3 (si ** 1500 0 ** ** shTCF3 20 0.4 + α-tubulin Cell viability 1000 * 10 * shATF3 0 RNA siRNA siCont siCont siCont siCont 500 shTCF3 0 -catenin#1 -catenin#1 -catenin#1 -catenin#1 Tumor volume (mm

+ U1 U6 shCont β β β β si si si si

siCont siCont siCont siCont 0 ASBEL HPRT1 MALAT Plasmid Mock ASBEL TCF3 ASBEL GAPDH

012345Weeks Relative enrichment to IgG siTCF3#1 siTCF3#2 siTCF3#1 siTCF3#2 siTCF3#1 siTCF3#2 siTCF3#1 siTCF3#2 TCF3

Fig. 2. TCF3 is associated with ASBEL and is required for the proliferation of colon cancer cells. (A) HCT116 cells infected with a lentivirus expressing an shRNA targeting ASBEL and/or ATF3 were injected into nude mice. Results are expressed as the mean ± SD (n = 6). *P < 0.05. shCont, control. (B) The viability of HCT116, DLD-1, Caco2, and HaCaT cells transfected with an siRNA targeting ASBEL was assessed by Cell Titer-Glo assays. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (C) Invasion assays were performed with HCT116 and DLD-1 cells transfected with siASBEL followed by Cell Titer-Glo assays to detect invading cells. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (D)IPApathwayanalysisofASBEL-regulated genes. (E) qRT-PCR analysis of TCF3 expression in human colon cancerous and noncancerous tissues. TCF3 expression was quantitated as the percentage relative to β-Actin mRNA (N, stage 0, n = 8; I, stage I, n = 5; II, stage II, n = 9; III, stage III, n = 16; IV, stage IV, n = 10). *P < 0.05. (F) Viability of HCT116, DLD-1, Caco2, and HaCaT cells transfected with siTCF3. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (G) HCT116 cells infected with a lentivirus expressing an shRNA targeting TCF3 and/or ATF3 were injected into nude mice. Results are expressed as the mean ± SD (n = 6). *P < 0.05. (H) Lysates from HCT116 cells were subjected to immunoprecipitation with anti-TCF3 antibody or anti-rabbit IgG antibody followed by qRT-PCR analysis to detect ASBEL. GAPDH, HPRT1, U1, U6,andMALAT were used as negative controls. Results are expressed as the mean ± SD (n = 2). (I) In vitro-

transcribed ASBEL interacts with TCF3. Lysates from HCT116 cells were incubated with biotinylated ASBEL or GAPDH generated in vitro, and bound proteins were CELL BIOLOGY precipitated with streptavidin beads and subjected to immunoblotting analysis with anti-TCF3 or anti–α-tubulin antibody. α-Tubulin was used as a negative control. (J) The viability of HCT116 cells transfected with an siRNA targeting β-catenin along with the indicated plasmids was assessed by Cell Titer-Glo assays. Results are expressed as the mean ± SD (n = 4). *P < 0.05. HPRT1, hypoxanthine phosphoribosyltransferase 1; MALAT, metastasis associated lung adenocarcinoma transcript; U1, small nuclear RNA U1; U6, small nuclear RNA U6.

ASBEL Is Associated with the Transcription Factor TCF3. We pre- Based on the above findings, we hypothesized that ASBEL may viously identified ASBEL as an antisense transcript of the ANA/ cooperate with TCF3 and promote its transcriptional activity in BTG3 gene and found that ASBEL promotes the tumorigenicity of colorectal cancer cells. To test this hypothesis, we performed RNA ovarian clear cell carcinoma by inhibiting the transportation of immunoprecipitation (RIP) assays with anti-TCF3 antibody using ANA/BTG3 mRNA, thereby suppressing its protein levels (13). lysates from HCT116 cells. qRT-PCR analysis of the immunopre- Consistent with this finding, we found that knockdown of ASBEL cipitates revealed that TCF3 was associated with endogenous resulted in increased levels of ANA/BTG3 protein but had no ef- ASBEL but not with GAPDH, HPRT1, U1, U6,orMALAT (Fig. fect on ANA/BTG3 mRNA in HCT116 and DLD-1 cells (Fig. S2 F 2H). In addition, we carried out RNA pull-down assays and found and G). However, knockdown of ANA/BTG3 did not restore the that TCF3 precipitated with ASBEL generated in vitro but not with viability of HCT116 or DLD-1 cells in which ASBEL had been GAPDH (Fig. 2I). Consistent with these results, subcellular frac- knocked down (Fig. S2 H and I). Thus, ASBEL-mediated sup- tionation followed by qRT-PCR analysis of ASBEL and immuno- pression of the ANA/BTG3 protein levels may not be critical for blotting analysis of TCF3 revealed that ASBEL and TCF3 were the proliferation of colon cancer cells. localized to the nucleus in colon cancer cells (Fig. S2O). In situ To clarify other potential functions of ASBEL in colon cancer cells, hybridization analysis also showed that ASBEL was localized to the we analyzed ASBEL-regulated genes by RNA-seq analysis and IPA nucleus in colon cancer specimens (Fig. S2P). Anti-TCF3 antibody software (Fig. S2J) (23). We found that the gene-expression signature suitable for immunostaining analysis was not available. observed in ASBEL-knockdown cells overlapped those regulated by the transcription factor TCF3 (Fig. S2K and Dataset S15). TCF3/ The ASBEL–TCF3 Complex Is Important for Wnt/β–Catenin Signaling. E2A belongs to the basic helix–loop–helix (bHLH) family of tran- We noticed that TCF3 is contained in the list of β-catenin target scription factors, which bind to E-box (CANNTG) sites present in the genes in Dataset S8. We confirmed that knockdown of β-catenin promoters or enhancer regions of their target genes (20, 21). The reduced the expression of TCF3 in DLD-1 and SW480 cells (Fig. bHLH family member E12/E47 participates in the repression of 1B and Fig. S1G), whereas knockdown of APC led to increased E-cadherin expression and induction of the epithelial-to-mesenchymal expression of TCF3 in 293FT cells (Fig. 1C). We also observed that transition, leading to the acquisition of invasive properties (24, 25). β-catenin binds to the TSS region but not to the upstream Moreover, TCF3/E2A is overexpressed in colon cancer (26, 27), (−3,500 bp) region of the TCF3 promoter in DLD-1, SW480, and prostate cancer (28, 29), and renal cancer (30) and plays a critical role 293FTcellsinwhichAPChadbeenknockeddown(Fig.1D and E in the proliferation and survival of tumor cells (29). Consistent with and Fig. S1I). Luciferase assays showed that the activity of a re- these reports, we found that TCF3 was highly expressed in colon porter containing the promoter and the 5′ UTR region of TCF3 cancer tissues compared with the noncancerous tissues (Fig. 2E)and (from approximately −1,000 to approximately +1,000 bp), but not was required for the viability of DLD-1, HCT116, and Caco2 cells but of reporters containing a mutated TBE, was higher than that of the not of HaCaT cells (Fig. 2F and Fig. S2 L and M). Moreover, when control reporter (Fig. S2Q). Furthermore, this activity was further s.c. injected into nude mice, HCT116 cells infected with a lentivirus enhanced by cotransfection of β-cateninS33Y (Fig. S2 Q and R). expressing an shRNA targeting TCF3 showed reduced tumorigenicity qRT-PCR analysis revealed that TCF3 expression was up-regulated compared with control HCT116 cells (Fig. 2G and Fig. S2N). in the colon cancer cell lines HCT116, DLD-1, and Caco2, in which

Taniue et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 Wnt signaling is activated, compared with the normal keratinocyte 1.2 -1

A B ) M S * cell line HaCaT (Fig. S1 and Fig. S2 ). These results suggest that 10 **** β 0.8 -catenin directly enhances the transcription of TCF3. * -2 To investigate the significance of the ASBEL–TCF3 complex in Wnt/β-catenin signaling, we examined the effect of overexpression 0.4 Cell viability -3 of ASBEL and TCF3 on the viability of HCT116 cells in which 0 Relative ATF3 β-catenin had been knocked down by siRNA. Although knockdown expression (Log siCont + + - - - - N I II III IV of β-catenin caused a reduction in the viability of HCT116 cells, siASBEL#1 - - ++- - -4 siASBEL#2 ----++ overexpression of ASBEL together with TCF3, but not of ASBEL siCont + - + - + - or TCF3 alone, partially restored their viability (Fig. 2J). These siATF3#1 - + - + - + 8 12 results suggest that the ASBEL–TCF3 complex plays an important C * D * β – 6 role in Wnt/ -catenin mediated proliferation of colon cancer cells. * 8 4 siCont siTCF3#1 * siTCF3#2 ASBEL/TCF3-Mediated Down-Regulation of ATF3 Is Required for the 4 siCont siASBEL#1 2 siASBEL#2 ATF3 Tumorigenicity of Colon Cancer Cells. To identify the target genes of ATF3 ATF3 expression the ASBEL–TCF3 complex that are involved in the proliferation of 0 ATF3 expression 0 TCF3 α-tubulin colon cancer cells, we used the IPA software platform to analyze α-tubulin siCont genes whose expression levels are altered by knockdown of ASBEL. siCont Relative Relative siTCF3#1 siTCF3#2

We found that the ASBEL–TCF3 complex up-regulated five genes siASBEL#1 siASBEL#2 J anddown-regulated16genes(Fig. S2 and Dataset S16). To clarify Fig. 3. The ASBEL–TCF3 complex represses the expression of ATF3 in colon the significance of the up-regulated genes, we transfected siRNAs cancer cells. (A) The viability of HCT116 cells transfected with an siRNA tar- targeting these genes into HCT116 cells and examined the viability geting ASBEL along with a control siRNA (siCont) or siATF3 was assessed by ofthesecells.Weobservedthat knockdown of CD79B resulted in Cell Titer-Glo assays. Results are expressed as the mean ± SD (n = 3). *P < decreased cell viability (Fig. S3 A and B). We also examined the 0.05. (B) qRT-PCR analysis of ATF3 expression in human colon cancerous and viability of cells that had been transfected with siRNAs targeting noncancerous tissues. ATF3 expression was quantitated as the percentage the down-regulated genes together with siASBEL. We found that relative to β-Actin mRNA (N, stage 0, n = 8; I, stage I, n = 5; II, stage II, n = 9; = = < knockdown of ATF3, CCNB2, CDH1, or GADD45B partially re- III, stage III, n 16; IV, stage IV, n 10). *P 0.05. (C and D, Left) qRT-PCR ASBEL A analysis of ATF3 mRNA in HCT116 cells transfected with an siRNA targeting stored the si -induced decrease in cell viability (Fig. 3 and ± = < C D ASBEL ASBEL (C) or TCF3 (D). Results are expressed as the mean SD (n 3). *P Fig. S3 and ). Moreover, knockdown of resulted in the 0.05. (Right) Cell lysates were subjected to immunoblotting analysis with up-regulation of these genes in HCT116 cells but not in HaCaT anti-ATF3 or anti–α-tubulin antibody. α-Tubulin was used as a loading con- cells (Fig. S3E). By contrast, AK092875 knockdown did not lead to trol. The asterisk indicates an irrelevant background band. the up-regulation of these genes in HCT116 cells (Fig. S3F). Of these genes, knockdown of ATF3 had the greatest effect; we hereafter focused our analysis on ATF3. sites that may bind TCF3. Data from the ENCODE project (https:// ATF3 is a member of the ATF/CREB family of transcription genome.ucsc.edu/ENCODE/) indicated an association of TCF3 factors whose expression is induced rapidly by a wide range of with a region 1,641–2,220 bp downstream (the TCF-binding site, cellular stresses, including DNA damage, cellular injury, and oxi- TBS) of the ATF3 gene TSS, as determined in the lymphoblastoid dative stress (31). ATF3 has been shown to suppress tumor growth cell line GM12878 (Fig. S4B). Consistent with these data, our ChIP and metastasis in many cancer types, including glioblastoma, colon, analysis with anti-TCF3 antibody confirmed that TCF3 was indeed bladder, and lung cancer (32–35). We examined the expression of associated with a TBS ∼2,000 bp downstream of the ATF3 gene but ATF3 in human colorectal tumor tissues and found that ATF3 was not with two other regions located upstream and downstream of the localizedtothenucleus(Fig. S2P). Subcellular fractionation fol- gene (Fig. 4 C and D). Furthermore, knockdown of ASBEL resulted lowed by immunoblotting analysis also revealed that ATF3, as well in a significant decrease in TCF3 binding to the TBS in the ATF3 as ASBEL and TCF3, was localized to the nucleus of HCT116 and gene (Fig. 4 C and D). In addition, we found by qRT-PCR and DLD-1 cells (Fig. S2O). qRT-PCR analysis revealed that ATF3 immunoblotting analyses that ASBEL did not affect the expression mRNA was expressed at lower levels in colorectal tumors than in levels of TCF3 in HCT116 cells (Fig. S4C), nor did it affect TCF3 noncancerous tissues (Fig. 3B). Moreover, we found that knock- dimer formation, which is critical for the binding of TCF3 to TBSs down of ASBEL or TCF3 resulted in increased expression of the (Fig. S4 D and E) (20, 21). We next examined whether ASBEL ATF3 mRNA and protein (Fig. 3 C and D and Fig. S3 G and H). interacts physically with the TBS at the ATF3 locus. We transfected Furthermore, we found that knockdown of either ASBEL or TCF3 HCT116 cells with biotin-labeled ASBEL RNA and precipitated restored the expression of ATF3 in 293FT cells in which APC had ASBEL-associated DNA using streptavidin-coated beads. We found been knocked down (Fig. S3I). Consistent with these results, in situ that ASBEL, but not antisense ASBEL, precipitated ATF3 sequences hybridization analysis of ASBEL and immunohistochemical analysis containing the TBS but did not precipitate the upstream or down- of ATF3 revealed that an inverse correlation between ASBEL and stream region (Fig. 4 C and E). ATF3 is also found in colorectal tissues (Fig. S3J). These results We next investigated the cis-activating potential of the DNA raised the possibility that the growth inhibition caused by siASBEL region containing this TBS. The ATF3 TBS region was inserted or siTCF3 may be an indirect consequence of increased ATF3 downstream of a reporter cassette consisting of the SV40 promoter protein expression. In line with this notion, knockdown of ATF3 and luciferase gene (Fig. 4F). We found that knockdown of ASBEL could partially rescue HCT116 cells from the reduction in cell vi- or TCF3 resulted in a significant increase in luciferase reporter ability caused by either ASBEL knockdown or TCF3 knockdown activity (Fig. 4 G and H). By contrast, overexpression of both (Figs. 3A and 4A). In addition, knockdown of ATF3 had similar ASBEL and TCF3, but not of either alone, resulted in the in- effects on the growth of cells in which β-catenin had been knocked hibition of reporter activity (Fig. 4I). These results raise the pos- down (Fig. 4B). Furthermore, we found that ATF3 knockdown sibility that ASBEL recruits TCF3 to the ATF3 locus and thereby could restore the tumorigenicity of HCT116 cells infected with a represses ATF3 expression in colon cancer cells. lentivirus targeting ASBEL or TCF3 (Fig. 2A and G and Figs. S2 A and N and S4A). Negative Feedback Regulation of the Expression of ASBEL by ATF3 in As a possible mechanism underlying ASBEL-mediated down- Colon Cancer Cells. The GSEA transcription factor analysis in Fig. regulation of ATF3 expression, we searched the ATF3 locus for 1A revealed that ATF/CREB (TGACGTCA) motifs are enriched

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0.4 0.4 Activity ATF3

Cell viability 1 Cell viability

0 0 Relative Luciferase 0 ASBEASBELL siCont + + + - + - Luc-Mock + - + - + - + - β-catenin TCF3 β-ca-catenin siCont +-+- AAAAAAAAA siTCF3#1 - - + + - - siβ-catenin#1-+ - + Luc-ATF3-Enh - + - + - + - + TCF3 - - + + - - + + siTCF3#2 - - - - + + siCont ++-- siATF3#1-- + + ASBEL - - - - + + + + siCont + - + - + - Luc-Mock SV40 Luc siATF3#1 - + - + - + F Intron 1 Luc-ATF3-Enh SV40 Luc ATF3 +1641 +2240 ASBEL C-5500 +2000 +4000 * * TCF3 G 24 * * H 16 * * AAA * * UP TBS DO Luc-Mock Luc-Mock * 1.6% 16 0.1% Luc-ATF3-Enh Luc-ATF3-Enh D E * * SuppressionSu of 8 ASBEL TCF3 Tumorigenicity 8 AAA 0.8% 0 0 % of Input % of Input siCont siCont 0.0% 0.0% siCont siCont Relative Luciferase Activity UP TBS DO UP TBS DO Relative Luciferase Activity siTCF3#1 siTCF3#2 siTCF3#1 siTCF3#2 siASBEL#1 siASBEL#1 siCont siASBEL#1 NO ASBEL AntiASBEL siASBEL#2 siASBEL#2

Fig. 4. Down-regulation of ATF3 is required for the tumorigenicity of colon cancer cells. (A) The viability of HCT116 cells transfected with an siRNA targeting TCF3 along with siCont or siATF3 was assessed by Cell Titer-Glo assays. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (B) The viability of HCT116 cells transfected with an siRNA targeting β-catenin and/or ATF3 was assessed by Cell Titer-Glo assays. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (C) Schematic representation of the human ATF3 promoter region. Amplified regions are indicated by black bars. The regions around −5,500 bp (UP) and +4,000 bp (DO) were amplified as negative controls. (D) ChIP assays were performed with HCT116 cells that had been transfected with siASBEL using anti-TCF3 antibody. The regions around UP (−5,500) and DO (+4,000) were amplified as negative controls. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (E) RNA ChIP assays were performed with HCT116 cells that had been transfected with biotinylated sense or antisense ASBEL RNA and subjected to precipitation using streptavidin beads. The regions around UP (−5,500 bp) and DO (+4,000 bp) were amplified as negative controls. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (F) Schematic representation of the Luc-Mock and Luc-ATF3-Enh (Intron 1; ATF3 enhancer) reporter constructs for enhancer assays. (G and H) HCT116 cells that had

been transfected with an siRNA targeting ASBEL (G)orTCF3(H) or control siRNA were transfected with the ATF3 enhancer reporter construct and subjected to CELL BIOLOGY luciferase assays. pRL-SV40 was used as an internal control. Results are expressed as the mean ± SD (n = 3). *P < 0.05. (I) HeLa cells were transfected with a mock, ASBEL, and/or TCF3 expression vector along with an enhancer reporter construct containing ATF3 intron 1 sequence (Luc-ATF3-Enh) and were subjected to luciferase assays. pRL-TK was used as an internal control. Results are expressed as the mean ± SD (n = 4). *P < 0.05. (J)TheASBEL–TCF3 complex is required for the tumorigenicity of colon cancer cells. β-Catenin directly transactivates ASBEL and TCF3 in colon cancer. ASBEL is associated with TCF3 and represses the expression of ATF3. The ASBEL–TCF3–mediated down-regulation of ATF3 is required for the tumorigenicity of colon cancer cells. DO, downstream; UP, upstream.

in genes targeted by β-catenin (Fig. S1C and Dataset S10). We that TCF3 is up-regulated by hypomethylation of its promoter and therefore examined whether ATF3 regulates the expression of that this up-regulation is correlated with recurrence in stage II and ASBEL in colon cancer cells. We found that knockdown of ATF3 III colorectal cancers (27). Thus, TCF3 is up-regulated by multiple indeed resulted in increased expression of ASBEL in HCT116 mechanisms in colon cancer, including aberrant activation of Wnt/ cells (Fig. S5 A and B). ChIP analyses with anti-ATF3 antibody β-catenin signaling and hypomethylation of its promoter. revealed that ATF3 was associated with the TSS but not with the We previously identified ASBEL as an antisense transcript of the upstream (−3,500 bp) region of the ASBEL promoter or the antiproliferative ANA/BTG3 gene and found that it is required for CCND1 promoter (Fig. S5C). Furthermore, we found that knock- the proliferation and tumorigenicity of ovarian clear cell carcinoma down of ATF3 resulted in increased activity of a reporter plasmid (13). We found that ASBEL forms complexes with ANA/BTG3 containing the ASBEL promoter (Fig. S5D). Taken together, these mRNA in the nucleus and suppresses its cytoplasmic transportation, results suggest that ATF3 functions as a component in the negative thereby suppressing the levels of ANA/BTG3 protein. Furthermore, feedback loop that inhibits ASBEL expression. we reported that knockdown of ANA/BTG3 rescues the growth inhibition caused by ASBEL knockdown. Thus, ASBEL may pro- Discussion mote the tumorigenicity of ovarian clear cell carcinoma by inhibiting In this study, we attempted to identify lncRNAs that are critical for its cytoplasmic transportation of the sense gene, thereby suppressing Wnt/β-catenin–mediated tumorigenesis. We performed RNA-seq its translation. Similarly, we observed that ASBEL suppresses the and ChIP-seq analyses of colorectal cancer cells and found that expression of ANA/BTG3 protein in colon cancer cells. However, we β-catenin directly activates the transcription of the lncRNA ASBEL. found that knockdown of ANA/BTG3 does not restore the reduced We observed that ASBEL is required for the proliferation and tu- viability of HCT116 or DLD-1 cells caused by ASBEL knockdown. morigenicity of colon cancer cells. Furthermore, analysis of ASBEL- Thus, ASBEL-mediated suppression of the levels of ANA/BTG3 regulated genes using IPA software revealed that ASBEL cooperates protein may not be critical for the growth of colon cancer cells. Our with the transcription factor TCF3, which is also directly trans- results suggest that ASBEL is a multifunctional lncRNA and that the activated by β-catenin. ASBEL forms a complex with TCF3, and this significance of each function varies depending on cell type. complex, but not either individual molecule, plays an important role We identified ATF3 as a target of the ASBEL–TCF3 complex in Wnt/β-catenin signaling in colon cancer cells. Interestingly, over- and showed that ASBEL- and TCF3-mediated repression of ATF3 expression of ASBEL together with TCF3 restores the viability of expression is required for the tumorigenicity of colon cancer cells. colon cancer cells in which β-catenin has been knocked down, Our results suggest that repression of ATF3 is critical for Wnt/ whereas overexpression of ASBEL or TCF3 alone has no effect. β-catenin–mediated tumorigenesis. We also found that ATF3 ex- In agreement with previous reports (26, 27), we found that pression is down-regulated in colon cancer cells compared with TCF3 is overexpressed in colon cancer tissues compared with ad- noncancerous tissues. These results appear to be consistent with a jacent noncancerous tissues. It has also been reported that TCF3 is previous report showing that ATF3 suppresses tumor growth in highly expressed in various cancers, including prostate, gastric, and colon cancer and many other types of cancer, including glioblas- renal cancers (28–30). Furthermore, it has been reported recently toma and bladder and lung cancer (32–35).

Taniue et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 Our ChIP analysis with anti-TCF3 antibody showed that TCF3 is it is possible that ATF3 also represses β-catenin target genes other associated with the TBS at the ATF locus in an ASBEL-dependent than ASBEL. These negative feedback loops may play important manner. Consistent with this result, we confirmed that ASBEL roles in the regulation of Wnt/β-catenin signaling in both tumor and interacts physically with the ATF3 locusTBS.Furthermore,we normal cells. showed that both ASBEL and TCF3 are required for the repression In conclusion, we found that ASBEL and TCF3 are directly of ATF3 expression. These results suggest that the ASBEL–TCF3 transactivated by β-catenin and form a complex that down-regulates complex binds to the ATF3 locus and thereby represses ATF3 the expression of ATF3. We further showed that the ASBEL–TCF3 expression in colon cancer cells. The function of TCF3 is known to complex–mediated down-regulation of ATF3 is required for the be regulated at the level of dimer formation. TCF3 needs to form a tumorigenicity of colon cancer cells (Fig. 4J). The pathway iden- homodimer or heterodimer via its HLH domain to bind to the TBS tified in this study may play important roles in Wnt/β-catenin– in the promoter or enhancer regions of its target genes (20, 21). In mediated tumorigenesis as well as in various other biological addition, the DNA-binding activity of TCF3 is inhibited by heter- processes. These findings may provide insights into the devel- odimer formation with ID1–3 (inhibitor of DNA binding 1–3, opment of cancer treatments. dominant-negative helix–loop–helix proteins) (20, 21, 36). How- ever, we found that ASBEL does not affect TCF3 homodimer Materials and Methods formation. We also observed that ASBEL does not affect the levels Further details are provided in SI Materials and Methods. All animal experi- of TCF3 protein or mRNA. We therefore speculate that ASBEL mental protocols were performed in accordance with the guidelines of the may facilitate the recruitment of TCF3 to the ATF3 locus. In- Animal Ethics Committee of the University of Tokyo. Primer sequences and terestingly, it has been reported recently that the lncRNA MEG3 shRNA sequences are listed in Dataset S17. Cell lines are described in Dataset regulates TGF-β pathway genes by binding to chromatin through S18. Cell viability was determined by measuring the intracellular levels of ATP formation of RNA–DNA triplex structures (37). We also speculate using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega). Lumi- nescence was measured using a Mithras LB 940 (Berthold). that the ASBEL–TCF3 complex may recruit corepressor factors that participate in repressing ATF3 expression. ACKNOWLEDGMENTS. This work was supported by the Innovative Technol- It has been reported that AP-1 motifs (TGANTCA) are enriched ogy Research Program of Innovative Cell Biology (Integrated Systems Analysis in genes targeted by β-catenin/TCF/LEF (38–40). We found that in of Cellular Oncogenic Signaling Networks), Grants-in-Aid for Scientific Re- addition to AP-1 motifs, ATF/CREB motifs (TGAYRTCA) are search on Innovative Areas “Integrative Research on Cancer Microenvironment β Network” and “Non-Coding RNA Neo-Taxonomy,” Grants-in-Aid for Scientific also enriched in genes targeted by -catenin. Furthermore, we ob- Research (C), and the Project for the Development of Innovative Research on served that ATF3 binds to the ATF motif in the promoter region of Cancer Therapeutics, Ministry of Education, Culture, Sports, Science, and Tech- ASBEL and represses its expression in colon cancer cells. Therefore nology, Japan, and Takeda Science Foundation.

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