Long Noncoding RNA MALAT1 Promotes Hepatocellular

Published OnlineFirst December 19, 2016; DOI: 10.1158/0008-5472.CAN-16-1508

Cancer Molecular and Cellular Pathobiology Research

Long Noncoding RNA MALAT1 Promotes Hepatocellular Carcinoma Development by SRSF1 Upregulation and mTOR Activation Pushkar Malakar1,AsafShilo1, Adi Mogilevsky1,IlanStein2,EliPikarsky2, Yuval Nevo3, Hadar Benyamini3, Sharona Elgavish3, Xinying Zong4, Kannanganattu V. Prasanth4, and Rotem Karni1

Abstract

Several long noncoding RNAs (lncRNA) are abrogated in way by modulating the alternative splicing of S6K1. Inhibition cancer but their precise contributions to oncogenesis are still of SRSF1 expression or mTOR activity abolishes the oncogenic emerging. Here we report that the lncRNA MALAT1 is upregu- properties of MALAT1, suggesting that SRSF1 induction and lated in hepatocellular carcinoma and acts as a proto-oncogene mTOR activation are essential for MALAT1-induced transfor- through Wnt pathway activation and induction of the onco- mation. Our results reveal a mechanism by which lncRNA genic splicing factor SRSF1. Induction of SRSF1 by MALAT1 MALAT1 acts as a proto-oncogene in hepatocellular carcinoma, modulates SRSF1 splicing targets, enhancing the production of modulating oncogenic alternative splicing through SRSF1 upre- antiapoptotic splicing isoforms and activating the mTOR path- gulation. Cancer Res; 77(5); 1155–67. 2016 AACR.

Introduction sequence is highly conserved among mammals (7). MALAT1 is assumed to play an important role in regulation of gene Recent estimations from transcriptome studies suggest that expression due to its residence in nuclear speckles (8). Local- more than 75% of the human genome is transcribed, generating ization of MALAT1 in nuclear speckles is dependent on active thousands of noncoding RNAs (ncRNA), which are not trans- transcription by RNA-polymerase II (9). lated into proteins (1). This large group of noncoding tran- MALAT1 was shown to modulate the positioning of a member scripts contains many small RNAs (2). However, the majority of of the SR family of pre-mRNA splicing factors to the transcription ncRNAs is longer than 200 nucleotides and is designated as site of an inducible transgene array (9). SR proteins are a family of long ncRNA (lncRNA; ref. 2). lncRNAs are involved in the RNA-binding proteins that regulate both general and alternative regulation of almost every step of gene expression, ranging splicing. SRSF1, a classic example of a SR protein family member, from chromatin remodeling, transcriptional control, regulation has been shown to regulate the alternative splicing of various of splicing, mRNA stability, mRNA translation, miRNA proces- oncogenes and tumor suppressor genes important for tumor sing, and protein stability (3). Expression and function of progression and maintenance (10). SRSF1 was shown to act as lncRNAs are deregulated in several human diseases, including an oncogene by activating the mTORC1 pathway (11). MALAT1 cancer (4). The ﬁrst lncRNA discovered with an established role was shown to bind active chromatin sites of many genes and to in cancer is metastasis-associated lung adenocarcinoma tran- bind several splicing factors, among them SRSF1. This binding script1(MALAT1),laterreferredtoasnuclear-enrichedabun- affects both its localization and phosphorylation by the kinase dant transcript 2 (NEAT2; ref. 5). The MALAT1 transcript is SRPK1, leading to changes in alternative splicing of its splicing greater than 6 kb in length and is highly abundant (6). MALAT1 targets (12, 13). The process of alternative splicing is widely deregulated in various cancers and many tumors express can- ﬁ 1Department of Biochemistry and Molecular Biology, Hebrew University-Hadas- cer-speci c splicing isoforms that are absent or are expressed at sah Medical School, Ein Karem, Jerusalem, Israel. 2Department of Immunology different levels in the corresponding normal tissues (14, 15). and Cancer Research, Hebrew University-Hadassah Medical School, Ein Karem, Many of these transcripts encode for oncogenes and tumor Jerusalem, Israel. 3Bioinformatics unit, the Institute for Medical Research Israel- suppressor genes (16–18). Canada, Hebrew University-Hadassah Medical School, Ein Karem, Jerusalem, In spite of its abundance, MALAT1 is dispensable for viabil- 4 Israel. Department of Cell and Developmental Biology, University of Illinois at ity, and MALAT1 knockout mice do not present any obvious Urbana-Champaign, Urbana, Illinois. abnormal phenotype (19, 20). One report using MALAT1 Note: Supplementary data for this article are available at Cancer Research knockout mice suggested that MALAT1 is not necessary for Online (http://cancerres.aacrjournals.org/). development but has the potential to regulate the expression of Corresponding Author: Rotem Karni, Hebrew University-Hadassah Medical nearby genes (21). In the past few years, studies have found that School, Ein Karem, Jerusalem 91120, Israel. Phone: 972-2675-8289; Fax: 972- MALAT1 is upregulated in several cancers, and its knockdown 2675-7379; E-mail: [email protected] inhibited tumorigenesis (22, 23). Several reports showed that doi: 10.1158/0008-5472.CAN-16-1508 MALAT1 regulates the Wnt-b-catenin pathway by enhancing 2016 American Association for Cancer Research. nuclear b-catenin levels and elevating c-Myc expression

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(24, 25). It was shown recently that MALAT1 regulates the RT-PCR differentiation and metastasis of mammary tumors (26). How- Total RNA was extracted with TRI Reagent (Sigma) and 1 mgof ever, neither a direct causative role for MALAT1 in early steps of total RNA was reverse transcribed using M-MLV reverse transcrip- transformation and tumorigenesis, nor the mechanism by tase (Promega). PCR was performed on 1/10th volume (2 mL) of which MALAT1 causes cellular transformation has been shown the cDNA using PCR Mix (Kapa Biosystems). to date. In this study, we show that MALAT1 is upregulated in hepatocellular carcinoma, and acts as a proto-oncogene to induce qRT-PCR m transformation and tumorigenesis of liver progenitor cells by Total RNA was extracted with TRI Reagent (Sigma), and 1 gof Wnt pathway activation, SRSF1 upregulation, and mTORC1 total RNA was reverse transcribed using M-MLV reverse transcrip- activation. tase (Promega) after DNASE treatment (Promega). qPCR was performed on the cDNA using SYBR Green Mix (Roche) and CFX96 (Bio-Rad) real-time PCR machine. Primer list is supplied Materials and Methods in Supplementary Table S1. Animal care All animal experiments were performed in accordance with the Immunoblotting guidelines of the Hebrew University committee for the use of Cells were lysed in Laemmli buffer and analyzed for total animals for research and under the approval of the Hebrew protein concentration as described (10). Twenty micrograms of University Ethics committee. Veterinary care was provided to all total protein from each cell lysate was separated by SDS-PAGE animals by the Hebrew University animal care facility staff in and transferred on to a polyvinylidene difluoride (PVDF) mem- accord with AAALAC standard procedures and as approved by the brane. Primary antibodies used were TCF7L2 EP20334 Hebrew University Ethics committee. (1:10,000; Abcam), c-MYC Sc-40 (1:1,000; Santa Cruz Biotech- nology), SRSF1 (AK96 culture supernatant 1:300; ref. 29), Cell culture GAPDH (1:500; Santa Cruz Biotechnology), a-tubulin Liver progenitor cells from embryonic day 18 fetal livers from (1:1,000; Santa Cruz Biotechnology), b-catenin (1:2,000; TP53 / mice were isolated and immortalized with MSCV-based Sigma), b-actin (1:2,000; Santa Cruz Biotechnology), total retroviruses expressing MYC-IRES-GFP as previously described to S6K1-anti-p70 (1:1,000; BD Transduction Laboratories), phos- generate TP53 / hepatocytes overexpressing c-MYC (PHM-1) pho-4E-BP1 Thr70 (1:1,000; Cell Signaling Technology), 4E- cells (27, 28). PHM-1, FLC4, and BWTG-3 cells were grown in BP1 (1:1,000; Cell Signaling Technology). Secondary antibodies DMEM supplemented with 10% FCS, 0.1 mg/mL penicillin, and used were HRP-conjugated goat anti-mouse, goat anti-rabbit, 0.1 mg/mL streptomycin. All cell lines have been tested and donkey anti-goat IgG (HþL; 1:10,000; Jackson Laboratories). authenticated using STR loci (TH01, TPOX, vWA, CSF1PO, D16S539, D7S820, D13S317, and D5S818) plus Amelogenin for gender identification for human cell line authentication by the Colony formation assay biosynthesis DNA Identity Testing Centre. Cells were seeded in 6-well plates (1,000 cells/well) and grown for 10 days. After fixation with 2.5% glutaraldehyde, the plates were washed three times. Fixed cells were then stained with Stable cell lines methylene blue solution (1% methylene blue in 0.1 mol/L borate pCD513B1 empty (System Biosciences) and pCD513B1-hMA- buffer, pH 8.5) for 60 minutes at room temperature. Plates were LAT1 lentiviruses were prepared using the manufacturer's instruc- photographed after extensive washing and air drying. tions. These viruses were used to infect PHM-1 cells. Cells were selected by the addition of puromycin (2 mg/mL) for 72–96 hours. In the case of infection with MLP-puro-shRNA viruses, cells were Anisomycin-mediated cell death assay selected with puromycin (2 mg/mL) for 96 hours. Following transduction and selection, cells were seeded in 6-well plates (2 105 cells/well). Twenty-four hours later, cells Growth curve were incubated with 1 mmol/L anisomycin in DMEM containing PHM-1, FLC4, and BWTG-3 cells were either infected with 0.1% serum for 24 hours. Medium and PBS washes were collected the indicated lentiviruses or treated with siRNAs. After selection together with trypsinized cells from each well into 15-mL tubes or siRNA treatment, 500 (PHM-1) or 2,000 cells (FLC4 and and centrifuged at 1,500 rpm for 5 minutes. Cells were washed BWTG-3) were seeded in 96-well plates. Cells were stained with with PBS and resuspended in 50 mL of HEPES buffer. Ten micro- 1% methylene blue in 0.1 mol/L borate buffer and fixed. After liters of the cell suspension was mixed with 10 mL of 4% Trypan treatment with 0.1 N HCl, the absorbance (655 nm) of the acid blue solution and live/dead cells were counted using a Bio-Rad extracted stain was measured using a plate reader (Bio-Rad). TC-10 automated cell counter.

siRNA treatment Anchorage-independent growth Double-stranded siRNAs (Sigma) were used to deplete Colony formation in soft agar was assayed as described previ- MALAT1 from cells at specific concentrations. siRNAs against ously (10). After 14 to 21 days, colonies from 10 different fields in Luciferase (Dharmacon Thermo Scientific) or siRNA universal each of two wells were counted for each treatment and the average negative control (Sigma) was used as a control at specified con- number of colonies per well was calculated. The colonies were centrations. Lipofectamine 2000 reagent was used for transfection stained as described (10) and photographed under a light micro- as per the manufacturer's instructions (Invitrogen). scope at 10 magnification.

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Tumorigenesis assays in nude mice were removed with cutadapt (version 1.11, http://cutadapt. PHM-1 cells overexpressing MALAT1 or an empty vector, with readthedocs.org/en/stable/), using a minimal overlap of 1 and without SRSF1 shRNA expression, were injected (3 106 (-O parameter), allowing for read wildcards, and filtering out cells/site in 200 mL of PBS) subcutaneously into each rear flank of reads that became shorter than 15 nt (-m parameter). The NOD-SCID mice using a 26-gauge needle. Tumor growth was remaining reads were further filtered to remove very low monitored twice a week. Tumor volume was calculated using the quality reads, using the fastq_quality_filter program of the formula, tumor volume ¼ (length width2)/2 (10). FASTX package (version 0.0.14, http://hannonlab.cshl.edu/ fastx_toolkit/), with a quality threshold of 20 at 90% or more Nuclear and cytoplasmic extract preparation of the read's positions. Cytoplasmic and nuclear extracts were prepared as described previously (30). After trypsinization, the cells were washed in cold Mapping and differential expression analysis PBS and spun down. The pellet was resuspended in CE buffer The processed fastq files were mapped to the mouse tran- (10 mmol/L HEPES pH 7.9, 1.5 mmol/L MgCl2, 10 mmol/L KCl- scriptome and genome using TopHat (v2.0.14). The genome containing protease inhibitor) and incubated on ice for 5 minutes. version was GRCm38, with the human MALAT1 gene added as An equal amount of CE buffer containing 0.2% NP40 was added an additional chromosome. Annotations were taken from to the cell suspension, incubated for 5 minutes on ice, and Ensembl release 84. Mapping allowed up to three mismatches centrifuged for 3 minutes at 6,500 rpm at 4C. The supernatant per read, a maximum gap of five bases, and a total edit distance is the cytoplasmic extract. The pellet was resuspended in NE buffer of 8 (full command: tophat -G genes.gtf -N 3–read-gap-length – – – (20 mmol/L HEPES pH 7.9, 1.5 mmol/L MgCl2, 0.42 mol/L NaCl, 5 read-edit-dist 8 segment-length 20 read-realign-edit-dist 0.2 mmol/L EDTA, 25% glycerol-containing protease inhibitors) 3–no-coverage-search genome processed.fastq). Quantification and vortexed at full speed for 1 minute. The nuclear extract was done using htseq-count (version 0.6.0, http://www-huber. suspension underwent three cycles of freeze (80C for 15 embl.de/users/anders/HTSeq/doc/count.html). Strand infor- minutes) and thaw (for 1 minute at 37C). Between each cycle mation was set to "no," and an annotation file that lacked of freeze–thaw, the suspension was vortexed for 1 minute at full information for genes of type IG, TR, Mt, rRNA, tRNA, miRNA, speed. The suspension was then spun at full speed for 15 minutes misc_RNA, scRNA, snRNA, snoRNA, sRNA, scaRNA, piRNA, at 4C. The supernatant is the nuclear extract. vaultRNA, ribozyme, artifact, and LRG_gene was used. Normal- ization and differential expression were done with the DESeq2 Oligonucleotide pull-down assay package (version 1.12.4). Genes with a sum of counts less than 2 Oligonucleotide pull-down assay was performed as over all samples were filtered out prior to normalization, then described previously (30). In brief, 30 mL of 1:1 bead slurry dispersion and size factors were calculated. Differential expres- was washed three times in 1 mL of GFB 100, blocked for 30 sion was calculated with default parameters. The significance P minutes at 4 C in 1 mL GFB 100–containing heparin, and then threshold was taken as adj < 0.1, testing for a log fold change washed in 1 mL of GFB 100. The binding reaction mixture with greater than 0.3 (lfcThreshold parameter to the results method). 5 mLof10pmol/mLbiotinylatedSRSF1ESEoligoderivedfrom Several quality control assays,suchascountsdistributions within the 30-UTR of SRSF1 transcript (see the sequence in and principal component analysis, as well as differential expres- Supplementary Table S1) and 20 mL of nuclear extract was sion results, were calculated and visualized in R (version 3.3.1, incubated at 30C for 30 minutes on a rotating shaker. The with packages "RColorBrewer_1.1-2," "pheatmap_1.0.8," and streptavidin bead suspension was added to the binding reaction "ggplot2_2.1.0"). Results were then combined with gene details mixture containing biotinylated RNA oligo and nuclear extracts (such as symbol, Entrez accession, etc.) taken from the results and incubated for 2 hours with rotation at 4C. After incuba- of a BioMart query (Ensembl, release 84) to produce the final tion, the coupled beads and binding reaction mixture were Excel file. washed four times in 500 mL of GFB100 containing 4 mmol/L MgCl2. The beads were then resuspended in 50 mLof2 SDS Gene set enrichment analysis (whole data) sample buffer and run on SDS-PAGE. Whole differential expression data were subjected to gene set enrichment analysis using GSEA (31) with the corresponding RNA-seq analysis human ortholog gene symbols. Best human orthologs were RNA from PHM-1 cells transduced with retroviruses encod- extracted from Ensembl. GSEA uses all differential expression ing for empty vector or MALAT1 was extracted and subjected data (cut-off independent) to determine whether a priori–defined to RNA-seq using the Ilumina Hi-seq sequencer. Approximately sets of genes show statistically significant, concordant differences 7 107 reads at the length of 50 bases was generated from each between two biological states. Gene sets of the MSigDB database sample. hallmark category were examined (v5.2, October 2016).

Trimming and filtering of raw reads Enrichment analysis (top changed genes only) Raw reads (fastq files) were inspected for quality issues A list of 880 statistically significant differentially expressed with FastQC (v0.11.2, http://www.bioinformatics.babraham. genes was subjected to pathway enrichment analysis using ac.uk/projects/fastqc/). According to the FastQC report, reads Qiagen's Ingenuity Pathway Analysis (IPA, Qiagen Redwood were quality-trimmed at both ends, using in-house Perl scripts, City; www.qiagen.com/ingenuity), GeneAnalytics (32), and with a quality threshold of 32. In short, the scripts use a sliding EnrichR (33) as well as functions/diseases enrichment analysis window of five bases from the read's end and trim one base at a using IPAA list of 880 genes whose expression shown the most time until the average quality of the window passes the given intensive change was subjected to enrichment analysis using threshold. Following quality-trimming, adapter sequences IPA, GeneAnalytics (32), and EnrichR (33).

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Network analysis overexpressing c-MYC (PHM-1 cells) with lentiviruses encoding To build and visualize a functional network of the enriched either hMALAT1 or an empty vector (Fig. 2A). We found that functions in differentially expressed genes, the ClueGO applica- overexpression of MALAT1 increased the proliferative capacity of tion for Cytoscape was applied to genes that were significantly the transduced PHM-1 cells as determined by staining cells with differentially expressed. ClueGO visualizes the nonredundant methylene blue and measuring the absorbance of the extracted dye biological terms for large clusters of genes in a functionally relative to that of the first day of the growth curve (Fig. 2B). grouped network. Enrichments of pathways (released November Overexpression of MALAT1 also increased survival of PHM-1 cells 2016) were analyzed. seeded sparsely in a clonogenic assay, suggesting that MALAT1 protects cells subjected to low-density stress conditions (Fig. 2C). To enumerate the possible role of MALAT1 in response to cellular Results stress, we treated transduced PHM-1 cells with anisomycin under lncRNA MALAT1 is upregulated in hepatocellular carcinoma low (0.1%) serum conditions. Overexpression of MALAT1 To examine whether MALAT1 plays a role in liver cancer decreased apoptotic cell death in response to anisomycin treat- development, we analyzed expression data from normal human ment as measured by Trypan blue exclusion and caspase-3 cleavage livers and liver cancer samples (hepatocellular carcinoma and (Fig. 2D). To investigate the role of MALAT1 in tumor mainte- liver cell dysplasia; https://www.oncomine.org/). Comparison of nance, we knocked down its expression using siRNAs in two normal liver samples to cancer liver samples showed elevated hepatocellular carcinoma cell lines (to eliminate cell type–specific levels of MALAT1 transcripts in hepatocellular carcinoma and effects): a human hepatocellular carcinoma cell line (FLC4), and liver cell dysplasia samples (Fig. 1A and B). Next, we compared mouse hepatocellular carcinoma cells (BWTG-3). MALAT1 knock- tumor liver samples and adjacent liver parenchyma from an down in FLC4 cells (Fig. 2E) resulted in reduced proliferative inflammation-induced liver cancer mouse model, Mdr2 / Mice capacity (Fig. 2F), reduced colony survival (Fig. 2G), and reduced (34, 35). We found elevated levels of MALAT1 RNA in most of the anchorage-independent growth (Fig. 2H). Knockdown of MALAT1 liver tumor samples compared with the adjacent inflamed liver also reduced proliferation capacity in the hepatocellular carcinoma parenchyma from Mdr2 / mice (Fig. 1C and D). cell line BWTG-3 and PHM-1 cells (Supplementary Fig. S1A–S1D).

MALAT1 expression enhances proliferation and survival of MALAT1 expression transforms hepatocytes and is essential hepatocytes for hepatocellular carcinoma tumor maintenance To examine the oncogenic potential of MALAT1 in hepatocytes, To determine whether MALAT1 is required for tumor mainte- we transduced TP53 / mouse embryonic progenitor hepatocytes nance, we examined the effect of knockdown of MALAT1 on

A B 4.0 5.0

3.5 4.5 4.0 3.0 3.5 2.5 Figure 1. 3.0 Elevated MALAT1 expression in 2.0 2.5 hepatocellular carcinoma, liver cell 1.5 2.0 dysplasia, and mouse liver tumors. A, 1.5 1.0 Box plot representation of MALAT1 RNA 1.0 levels in normal liver (n ¼ 10) and 0.5 0.5 n ¼ Log2 median-centered intensity Log2 median-centered intensity hepatocellular carcinoma ( 35) 0.0 0.0 1 2 1 2 samples. P ¼ 1.38 E6; t test ¼ 6.279; Normal liver HCC Normal liver Liver cell dysplasia fold change ¼ 3.233. B, Box blot representation of MALAT1 RNA levels in normal liver (n ¼ 10) and liver cell C D dysplasia (n ¼ 17) samples. P ¼ 2.59 60 8 E6; t test ¼ 6.284; fold change ¼ 3.170. ** Analysis is based on RNA-Seq data from MALAT1 7 50 Oncomine database (https://www. oncomine.org). C, qRT-PCR of MALAT1 6 expression using RNA isolated from ﬁve 40 ﬂ 5 adjacent in amed liver parenchyma and 20 tumor liver samples from Mdr2/ 30 4 mice. All samples were normalized to tubulin mRNA levels. Error bars, SD of 3 three technical repeats. Number 20 indicates sample number. D, Average

Relative expression Relative 2

Relative expression Relative value of MALAT1 expression level from 10 C. Tubulin was used for normalization 1 (, P ¼ 0.0084). 0 0 N1 N3 N5 T2 T4 T6 T8 T10 T12 T14 T16 T18 T20 Normal HCC livers Tumors Liver samples

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A E 1.2 300 1 250 0.8 200 0.6 150 100 0.4 50 0.2 Relave expression Relave

Relative expression Relative 0 0 Empty MALAT1 siLuc siMALAT1 Figure 2. Modulation of MALAT1 expression B 60 F 9 affects proliferation, survival, and anchorage-independent growth. A, 50 Empty 8 siLuc qRT-PCR of MALAT1 expression using 7 RNA from PHM-1 cells transduced with 40 MALAT 1 6 siMALAT1 lentiviruses encoding either full-length 30 5 human MALAT1 or an empty vector. 4 Relative expression was normalized to 20 3 actin. B, Proliferation assay of cells

Relative Abs. Abs. Relative 2 described in A. Error bars, SD from six 10 1 repeats. C, Colony survival assay of 0

Relave absorbance absorbance Relave 0 cells described in A. D, Trypan blue 050100 exclusion assay of cells described in A. 0 24487296 Bottom, Western blot analysis of C Time (hrs) G Time (hrs) cleaved caspase-3 in lysates from cells # of Cells described in A, as a marker for apoptosis. E, qRT-PCR of MALAT1 seeded: 100 500 1,000 RNA levels after knockdown of siRNA: MALAT1 in FLC4 cells by siRNA MALAT1 (siMALAT1). siRNA against luciferase was used as control. Relative Empty expression was normalized to tubulin. F, Proliferation assay of cells described in E. G, Colony survival assay of cells 80 described in E. H, Growth in soft agar D assay on cells described in E. Graph 60 represents the average and SD of number of colonies/well (n ¼ 3). 40 H 1,000 20 750

% Cell death death Cell % 0 Empty MALAT1 500

Cl. Casp3 Colonies/well 250

0 β-Catenin siLuc siMALAT1

transformation of hepatocellular carcinoma cells. We found that can act as an oncogenic driver in hepatocellular carcinoma MALAT1 knockdown by siRNA inhibited colony formation in soft development. agar of mouse hepatocellular carcinoma cells (BWTG-3; Fig. 3A and B) and PHM-1 cells (Supplementary Fig. S1A and Fig. 3C). MALAT1 regulates the expression of the oncogenic splicing Overexpression of MALAT1 was able to transform mouse PHM-1 factor SRSF1 cells enabling them to form colonies on soft agar (Figs. 2A One of the proteins known to bind MALAT1 is the splicing and 3D). To further investigate whether MALAT1 overexpression factor SRSF1. Splicing factor SRSF1 is upregulated in various types can render cells tumorigenic in vivo, we injected PHM-1 cells of cancer, acts as a proto-oncogene in hepatocellular carcinoma expressing either MALAT1 or the empty vector subcutaneously (36), and transforms liver progenitor cells when upregulated (28). into NOD-SCID mice. We found that PHM-1 cells overexpressing Overexpression of MALAT1 in PHM-1 cells upregulated the MALAT1 formed large tumors in mice when compared with expression of SRSF1. SRSF1 upregulation occurred at both the cells expressing empty vector (Fig. 3E and F). These results suggest mRNA and protein level (Fig. 4A–C). Knockdown of MALAT1, in that MALAT1 is a cellular proto-oncogene in hepatocytes and PHM-1 cells, resulted in the reduction of SRSF1 protein (Fig. 4D

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A 1.2 B

1 1.2 1 0.8 ell 0.8 0.6

expression 0.6 e Figure 3. i v 0.4 0.4 MALAT1 overexpression induced Colonies/w transformation and tumorigenesis in PHM- Relat 0.2 0.2 1 cells. A, qRT-PCR of MALAT1 expression 0 0 using RNA from mouse hepatocellular siLuc siMALAT1-1 siMALAT1-2 siLuc siMALAT1-1 siMALAT1-2 carcinoma cells and BWTG-3 cells after knockdown of MALAT1 by siRNAs (siMALAT1-1,2). siRNA against luciferase C D 1,000 wasusedascontrol.B, Colony 1.2 formation in soft agar assay of cells 800 described in A.Graphshowsthe l

l 1 ll e e average number of colonies/well and

/w 0.8 600 SD. n ¼ 3. C, Growth in soft agar assay using PHM-1 cells transfected with siRNAs 0.6 400 speciﬁc for mouse MALAT1 (siMALAT1-1, 2) 0.4 or a control siRNA. Graph represents the Colonies Colonies/w average and SD of number of colonies/ 0.2 200 well. n ¼ 3. D, Growth in soft agar assay using PHM-1 cells transduced with the 0 0 lentiviruses encoding either MALAT1 siCont. siMALAT1-1siMALAT1-2 Empty MALAT1 or an empty vector. Graph represents the E F average and SD of number of colonies/ well. n ¼ 3. E, Growth of tumors after 1,400 subcutaneous injection of cells (3 106/ Empty * )

3 1,200 site) described in D into NOD-SCID mice MALAT1 (n ¼ 8). Tumor volume was measured and 1,000 ** MALAT1 calculated as described in Materials and 800 Methods. F, Pictures of representative tumors described in E.

o lume (mm 600 400 ** Empty

u mor v 200

T * 0 0 8 16 24 32 40 Days after injection

and E). To examine the functional activity of SRSF1 in MALAT1- roles in transformation and apoptosis. We focused on the splicing overexpressing cells, we measured binding of nuclear SRSF1 to an pattern of three previously reported SRSF1 target genes: pro- RNA oligonucleotide containing the consensus ESE motif taken apoptotic Bcl-2 family member BIM, tumor suppressor BIN1, from the SRSF1 30UTR region (37), through direct RNA affinity and the transcription factor TEAD-1 (TEF-1; refs. 38–40). We purification (Fig. 4F). Cytoplasmic and nuclear extracts were found that PHM-1 cells overexpressing MALAT1 increased expres- prepared from PHM-1 empty and PHM-1 MALAT1 cells over- sion of the ES (extra short) isoform of BIM (Fig. 5A; Supplemen- expressing SRSF1. The purity of the extracts was examined with tary Fig. S2A), which lacks exon 4, the BH3 domain. The ES known cytoplasmic and nuclear protein (Supplementary Fig. isoform of BIM has been shown to behave in a similar fashion to S1E). Nuclear extracts of PHM-1 cells overexpressing MALAT1 that of the g1 isoform, as an antiapoptotic protein (28). Transient and transfected with T7-SRSF1 had more binding of SRSF1 as MALAT1 knockdown by siRNAs in PHM-1 MALAT1 cells resulted compared with PHM-1 cells with empty vector transfected with in reduced expression of the ES isoform of BIM (Fig. 5B; Supple- T7-SRSF1 (Fig. 4G). This result suggests that there are higher mentary Fig. S2A). The BIN1 protein interacts with c-MYC and amounts of active SRSF1 in the nucleus of PHM-1 cells over- suppresses its oncogenic activity (39). Inclusion of exon 12A in expressing MALAT1. the BIN1 transcript abolishes its tumor suppressor activity while inclusion of exon 13 is required for the tumor suppressor activity MALAT1 upregulation induces oncogenic alternative splicing of BIN1 (39). It was shown previously that SRSF1 overexpression events regulated by SRSF1 results in increased inclusion of exon12A of BIN1 in human, To establish whether SRSF1 upregulation induced by MALAT1 mouse, and rat cells (10, 28). In agreement with these findings, we overexpression affects alternative splicing events known to be found that in PHM-1 cells overexpressing MALAT1, inclusion of regulated by SRSF1, we examined splicing patterns of genes with exon 12A was increased compared with cells containing empty

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A 5 pre-mRNA of SRSF1 B C 3 4 2.5 Figure 4. MALAT1 regulates the expression and 3 SRSF1 2 function of SRSF1. A, Detection of SRSF1 pre-mRNA by qRT-PCR in PHM-1 cells 2 β-Catenin 1.5 transduced with lentiviruses encoding 1 either MALAT1 or an empty vector. PCR 1 PHM-1

primers were designed to amplify an intron Relative intensity

Relative expression Relative 0.5 and neighboring exon sequences. B, DetectionofSRSF1proteinbyWestern 0 0 blotting in cells described in A.Total Empty MALAT1 Empty MALAT1 b-catenin was used as a loading control. C, Quantiﬁcation of the D E 1.2 average SRSF1 protein levels upon MALAT1 overexpression (B)fromthree 1 experiments. Empty vector was set as 1. D, 0.8 DetectionofSRSF1proteininPHM-1cells SRSF1 0.6 transfected with either siLuc or siMALAT1 (1, 2) by Western blot analysis. Total 0.4 b-catenin and b-tubulin were used as β-Tubulin 0.2 loading controls. E, Quantiﬁcation of the Relative intensityRelative 0 average SRSF1 protein levels upon MALAT1 β-Catenin knockdown (D) from three experiments. Empty vector was set as 1. F, Scheme PHM-1 SiLuc illustrating pull-down assay using either ESE or SCR biotinylated RNA siMALAT1-1siMALAT1-2 oligonucleotides. G, Top, input of nuclear F Input extracts of PHM-1 cells transduced with G lentiviruses encoding either empty vector Nuc. extract: Empty MALAT1 Empty MALAT1 or MALAT1 transiently transfected with either pCDNA3 or T7 tagged SRSF1. SRSF1 expression in nuclear extracts was detected by Western blot analysis SRSF1 using anti-T7 antibodies; bottom, Western +Oligo T7 blot analysis of proteins pulled down using ESE/SCR Biotin a biotinylated RNA oligo containing an ESE Pull down motif (described in Materials and Methods) Avidin SCR from nuclear extracts of cells described Pull down Oligo: ESE in F. Biotinylated SCR oligo was used as a control. N.E: Immunoblot T7-SRSF1

vector control (Fig. 5A; Supplementary Fig. S2A). Alternatively, (mTORC1; refs. 10, 41). However, SRSF1 can activate mTOR and transient MALAT1 knockdown by siRNA in PHM-1 MALAT1 cells protein translation also by alternative splicing-independent induced skipping of exon 12A (Fig. 5B; Supplementary Fig. S2A). mechanisms (42, 43). We therefore examined the splicing pattern Finally, we examined the alternative splicing of TEAD1. SRSF1 of S6K1 in MALAT1-overexpressing cells. PHM-1 cells overexpres- affects the alternative splicing of the pre-mRNA of the transcrip- sing MALAT1 showed increased Iso-2/Iso-1 ratios at the mRNA tion factor TEAD1 by promoting the inclusion of exon 5, resulting level (Fig. 5C) and elevated levels of the Iso-2 isoform at the in more proliferative activity (10, 28). Consistent with this, we protein level (Supplementary Fig. S2B). Transient knockdown found increased inclusion of exon 5 of TEAD1 in PHM-1 cells of MALAT1 by siRNA in PHM-1– and PHM-1–overexpressing overexpressing MALAT1 (Fig. 5A and Supplementary Fig. S2A). In MALAT1 cells resulted in a decreased ratio of Iso2/Iso1 at the contrast, transient MALAT1 knockdown by siRNA in PHM-1 cells mRNA and protein levels (Fig. 5D; Supplementary Fig. S2B). The overexpressing MALAT1 induced skipping of exon 5 of TEAD1 S6K1 short isoform has been shown to bind and activate (Fig. 5B; Supplementary Fig. S2A). mTORC1, resulting in enhanced 4E-BP1 phosphorylation (41). We therefore examined the phosphorylation status of 4E-BP1 in MALAT1 upregulation activates the mTOR pathway PHM-1 cells overexpressing MALAT1. These cells had increased We have shown previously that SRSF1 regulates alternative phosphorylation of 4E-BP1 (Fig. 5E). Knockdown of MALAT1 splicing of RPS6KB1, increasing the expression of a shorter spliced resulted in decreased phosphorylation of 4E-BP1 in PHM-1– variant of S6K1, called Iso-2 (10). Iso-2 possesses oncogenic overexpressing MALAT1 cells (Fig. 5F). Cumulatively, these ﬁnd- activity, binds mTOR, and activates the mTOR complex 1 ings suggest that MALAT1 can activate the mTORC1 pathway.

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direct transcriptional target of c-MYC (44). c-MYC is the direct A B transcriptional target of the Wnt-b-catenin pathway. To better understand the transcriptional program induced by MALAT1 in hepatocytes, we performed RNA-seq analysis on PHM-1 EL EL BIM L cells overexpressing MALAT1 compared with cells with empty S L vector (Supplementary Fig. S3; Supplementary Table S2). We ES BIM S identified, by two different bioinformatic tools (IPA and +12A+13A ES GeneAnalytics; refs. 31–33), activation of the Wnt pathway BIN1 +12A (Supplementary Figs. S4–S6; Supplementary Table S3). We -12A-13 +12A+13A identified upregulation of several activators upstream of the +12A Wnt pathway (e.g., both ligands Wnt2 and Wnt10a, as well as TEAD1 +E5 BIN1 +13 -E5 the receptor Frizzled, which activate the pathway; Supplemen- -12A-13 tary Figs. S4–S6). In addition, several suppressors of the GAPDH TEAD1 +E5 pathway were downregulated (e.g., DKK, TGFb,TGFbR; Sup- PHM-1 -E5 plementary Fig. S4B). Both c-Myc and Cyclin D1 are known Tubulin transcriptional targets of the Wnt pathway and contribute to proliferation and oncogenesis. Moreover, c-Myc is a transcrip- PHM-1 MALAT1 tional activator of SRSF1 (44). Thus, we examined the expres- 2 RPS6KB1 CD1.2 sion of c-MYC and Cyclin D1 upon MALAT1 overexpression. RPS6KB1 In PHM-1 cells, overexpression of MALAT1 resulted in 1 1.5 increased expression of c-MYC and cyclin D1, at both the 0.8 mRNA and protein levels while MALAT1 knockdown reduced 1 0.6 their levels (Fig. 6A and B; Supplementary Fig. S7). A hallmark of Wnt pathway activation is translocation of b-catenin into Short/Long 0.4 0.5 the nucleus (45, 46). Overexpression of MALAT1 was shown Short/Long 0.2 to increase the nuclear localization of b-catenin in LoVo cells 0 0 (24). We therefore examined b-catenin levels in cytoplasmic Empty MALAT1 and nuclear extracts upon MALAT1 overexpression. Overex- pression of MALAT1 in PHM-1 cells resulted in increased nuclear b-catenin (Fig. 6C). These results suggest that TCF/ E F LEF/b-catenin transcriptional activity is enhanced in these cells. Knockdown of MALAT1 by siRNA, in PHM-1, BWTG- p-4EBP1 p-4EBP1 3, FLC4, and PHM-1 MALAT1 cells resulted in reduced expression of c-MYC protein (Supplementary Fig. S7A) as well as c- Total 4EBP1 Total 4EBP1 MYC and cyclin D1 expression (Fig. 6D and E). Because SRSF1 is a transcriptional target of c-Myc, these results can explain GAPDH Tubulin SRSF1 upregulation in cells overexpressing MALAT1. Another transcriptional target of the Wnt pathway is the receptor Figure 5. tyrosine kinase ERBB3 (47). Indeed, the RNA-seq analysis Effect of MALAT1 expression on alternative splicing of endogenous targets of showed increased expression of ERBB3 in cells expressing SRSF1. A, RT-PCR of SRSF1 target genes (BIM, BIN-1,andTEAD-1)fromRNA MALAT1, which was also validated by qRT-PCR (Supplemen- extracted from PHM-1 cells transduced with lentiviruses encoding either tary Figs. S4B, S5B, and S6A). In addition, ERBB4, another fi MALAT1 or an empty vector. Isoform-speci c primers were used for member of the EGFR family was induced by MALAT1 (Sup- RT-PCR. GAPDH was used as a control. B, RT-PCR of SRSF1 target genes – from RNA extracted from MALAT1-overexpressing PHM-1 cells transfected plementary Figs. S4B and S5B), suggesting that the ERBB3 with either siLUC or siMALAT1. Splicing patterns were determined using ERBB4 signaling pathway is activated by MALAT1. Cells over- isoform-specific primers. Tubulin was used as control. C, qRT-PCR of expressing MALAT1 also showed hallmarks of mTOR pathway RPSKB1 isoforms in RNA extracted from cells described in A.Resultsare activation based on gene expression patterns (Supplementary expressed as a ratio of Iso2/Iso1 isoform. Expression was normalized to Fig. S6C). actin. D, qRT-PCR of RPS6KB1 isoforms in RNA extracted from cells described in B. Results are expressed as a ratio of Iso2/Iso1 isoform. Actin was used for normalization. E, Western blot analysis of SRSF1 upregulation and mTOR activation are essential for phosphorylated and total 4E-BP1 protein in extracts from cells described in MALAT1 transformation and tumorigenesis A. GAPDH was used as a loading control. F, Western blot analysis of To assess the importance of mTOR activation for MALAT1- phosphorylated and total 4E-BP1 protein in extracts from cells described mediated transformation, we used the mTOR inhibitor rapa- in B. Tubulin was used as a loading control. mycin. Rapamycin blocks mTORC1 activity (48). The oncogenic properties of MALAT1-expressing PHM-1 cells were abolished in the presence of rapamycin as seen by decreased survival in MALAT1 activates a transcriptional program, resulting in colony survival assay (Fig. 7A), reduced formation of colonies activation of the Wnt and ERBB3-4 signaling pathways and on soft agar (Fig. 7B), and reduced proliferative capacity (Fig. increased expression of c-MYC and cyclin D1 7C). These results demonstrate that cells overexpressing The results presented above suggest that MALAT1 regulates MALAT1 are highly sensitive to rapamycin and mTOR activa- the expression and activity of SRSF1. SRSF1 was shown to be a tion is essential for MALAT1-induced transformation.

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A 2.5 D 1.2 c-MYC 2 1 1.5 0.8 c-MYC

expression 1

e 0.6 expression e i v

0.5 i v 0.4

Relat 0 0.2 Relat Empty MALAT1 0 10 Cyclin D1 7.5 Figure 6. MALAT1 increases nuclear b-catenin 1.2 expression 5 and upregulates the expression of e 1 c-MYC and cyclin D1. A, qRT-PCR of i v 2.5 Cyclin D1 c-MYC and cyclin D1 mRNA levels in 0.8

PHM-1 cells transduced with Relat 0.6 lentiviruses encoding either MALAT1 0 expression

or an empty vector. B, Western blot Empty MALAT1 e 0.4 analysis of c-MYC and cyclin D1 in cells i v 0.2 described in A. b-Catenin was used as a loading control. C, Western blot 0 Relat analysis of b-catenin in cytoplasmic B and nuclear fractions isolated from cells described in A. Sam68 was used Cyclin D1 as nuclear-speciﬁc marker, tubulin as a cytoplasmic-speciﬁc marker, and GAPDH as a loading control. D, c-MYC qRT-PCR of c-MYC and cyclin D1 mRNA levels in BWTG-3 cells E following MALAT1 knockdown by siRNAs. E, Western blot analysis of β-Catenin c-MYC c-MYC and cyclin D1 in cells described PHM-1 Cells in D. Tubulin and GAPDH were used as loading controls. Cyclin D1 Cytoplasm Nucleus Tubulin

C GAPDH

Sam68

β-Catenin

Tubulin

GAPDH

To examine whether SRSF1 upregulation mediates MALAT1- observations suggest that SRSF1 is required for the tumorigenic induced transformation, we knocked down the expression effects induced by MALAT1 and functions downstream to of SRSF1 in PHM-1 cells overexpressing human MALAT1 MALAT1. To examine whether MALAT1 oncogenic activity is (Fig. 7D). Stable knockdown of SRSF1 in these cells resulted speciﬁcally sensitive to SRSF1 downregulation, we transformed in reduced proliferative capacity (Fig. 7E) and decreased sur- PHM-1 cells by oncogenic Ras and silenced SRSF1. We found vival when sparsely seeded for colony survival (Supplementary that although SRSF1 knockdown did not affect colony survival Fig. S8A). To test whether MALAT1-induced tumorigenesis can and proliferation (Supplementary Fig. S8B), it did inhibit, to be inhibited by SRSF1 knockdown in vivo,weinjectedNOD- some extent, colony formation in soft agar (Supplementary SCID mice with PHM-1 cells overexpressing MALAT1 with or Fig. S8C and S8D). These results suggest that MALAT1 onco- without SRSF1 knockdown. Knockdown of SRSF1 greatly genic activity is highly sensitive to inhibition of SRSF1, more so inhibited tumor growth in vivo (Fig.7FandG).Theabove than other oncogenes (such as Ras), and that SRSF1 is essential

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ABC3 30 2.5 25 Empty Empty MALAT1 +DMSO 2 20 MALAT1+ Rapa 1.5 15 Figure 7. MALAT1 1 +DMSO 10 mTOR activity and SRSF1 are required for

0.5 Abs. Relative 5 MALAT1 transformation activity. A, Colony survival assay of PHM-1 cells transduced MALAT1 0 0 +Rapamycin with lentiviruses encoding either MALAT1

Relative # of of colonies # Relative 0244872 or an empty vector in the presence or Time (hrs) absence (DMSO) of rapamycin. B, Quantiﬁcation of growth in soft agar assay of cells described in A.Resultsare 30 presented as relative to PHM-1 cells DEFVector 25 transduced with empty vector. C, SRSF1 Sh1 Proliferation assay on cells described in A. 20 Vector SRSF1 SRSF1 Sh2 Error bars, SD from six experimental 15 repeats. D, Western blot analysis of PHM-1 β-Actin 10 SRSF1 sh1 cells transduced with MALAT1 lentivirus

Relative Abs. Abs. Relative 5 SRSF1 sh2 cotransduced with lentiviruses containing PHM-1 MALAT1 0 shRNAs against SRSF1 or an empty vector. 0244872 b-Actin was used as loading control. E, Time (hrs) Proliferation assay of cells described in D. F, Cells described in D were injected G H MALAT1 (3 106 cells/site) subcutaneously into NOD-SCID mice (n ¼ 10). Pictures of

) 2,000 3 Vector representative tumors. G, Tumor volume Wnt Pathway c-MYC 1,500 SRSF1Sh1 * was measured and calculated as described activation SRSF1Sh2 ERBB 3-4 SRSF1 in Materials and Methods. H, Scheme 1,000 Signaling, summarizing the role of MALAT1 as a * other potential oncogene in hepatocellular Cyclin D1 Splicing carcinoma. Red lines represent changes or 500 targets * changes targets through which MALAT1 acts as an oncogene in hepatocellular carcinoma in Tumor volume (mm volume Tumor 0 our experimental studies. Black lines 02040 RPS6KB1 Apoptosis represent alternative pathways or targets Days after injection TEAD1 (BIM, BIN1) through which MALAT1 can function as an mTORC1 oncogene.

Proliferation, survival, tumorigenesis

to some of the oncogenic properties of MALAT1 (e.g., proliferative splicing, leading to activation of a pro-oncogenic signal tion/survival as determined by the clonogenic assay) whereas transduction pathway. other properties (motility/invasiveness) might also be mediated by additional targets. To determine whether SRSF1 regulation of MALAT1 is upregulated and acts as a proto-oncogene in alternative splicing also functions downstream to MALAT1, we hepatocellular carcinoma knocked down MALAT1 by siRNA in PHM-1 cells overexpressing Here, we show that not only is MALAT1 mRNA expression SRSF1 (Supplementary Fig. S8E). We did not observe any signif- high in different forms of human liver cancer, but also in liver icant change in the splicing patterns of BIM, BIN-1,orTEAD-1, tumors from a mouse model of hepatic carcinogenesis (Fig. 1). suggesting that SRSF1 regulates splicing downstream to MALAT1 We further show that overexpression of MALAT1 in PHM-1 cells and not vice versa (Supplementary Fig. S8F). resulted in transformation of these cells in vitro and in vivo (Figs. 2A–Dand3D–F). In contrast, knockdown of MALAT1 in Discussion cells resulted in reduced proliferative capacity and decreased capacity to form colonies on soft agar (Figs. 2E–Hand3A–C; In this study, we show that upregulation of the lncRNA Supplementary Fig. S1). Our results suggest that MALAT1 acts MALAT1 can act as an oncogenic driver in hepatocellular as an oncogene. Indeed, recent results show that the MALAT1 carcinoma, and that this activity is mediated by the induction gene is mutated in liver and breast cancers, and functions as an of the oncogenic splicing factor SRSF1. SRSF1 is known to affect oncogene in breast cancer as well (26, 49, 50). alternative splicing of genes involved in transformation and apoptosis and to activate the mTOR pathway. Here, we present MALAT1 induces SRSF1 upregulation activating a pro- evidence that SRSF1 upregulation and mTOR activation are oncogenic alternative splicing program essential for MALAT1-induced transformation and tumorigen- Our results suggest that the oncogenic potential of MALAT1 esis, suggesting a mechanism by which lncRNA alters alterna- could be in part attributed to increased activity of the oncogenic

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splicing factor SRSF1 (Fig. 4). The role of SRSF1 as an alter- by MALAT1 (Supplementary Figs. S4B, S5B, and S6), suggesting native splicing factor strongly suggests that MALAT1 oncogenic that MALAT1 oncogenic activity might also be attributed to activity might be mediated by SRSF1-dependent alternative enhanced EGFR family signaling. Consistent with this, ERBB3 splicing changes. We expect that it is not a single splicing event, expression is upregulated in a subset of hepatocellular carci- but rather the cumulative effect of a set of isoforms, which noma tumors correlating with poor prognosis (54). drives the transformation process. We therefore investigated the effects of MALAT1 overexpression or knockdown on alternative splicing of some of the well-established SRSF1 targets SRSF1 upregulation and activation of the mTOR pathway is that are known to be involved in transformation and apopto- essential for MALAT1-mediated transformation sis. Our results suggest that MALAT1 activates an alternative To examine the importance of mTOR activation for MALAT1- splicing program that enhances the antiapoptotic isoforms of mediated transformation, we blocked mTOR activity using fi genes, such as BIM, as well as the oncogenic isoforms of genes, the mTOR-speci c inhibitor rapamycin. Indeed, we found that such as S6K1 and TEAD-1, which contribute to transformation rapamycin fully inhibited the oncogenic properties of MALAT1- – (Fig. 5A-D). overexpressing cells (Fig. 7A C).ThisresultsuggeststhatmTOR The Ras–MAPK and PI3K–mTOR pathways are deregulated activation is essential for MALAT1-mediated transformation. in many cancers, contributing to the establishment and main- Activation of the mTOR pathway in various tumors has been tenance of the transformed phenotype (51, 52). We investi- reported in many studies (51). Rapamycin and its analogues are gated whether MALAT1 can modulate the mTOR-signaling extremely selective for mTOR and are already in clinical use pathway. We found that alternative splicing of S6K1, a down- for treating cancers and have been shown to have antitumor stream component of this pathway, is deregulated upon activity (48). MALAT1-transformed cells proved to be extreme- MALAT1 overexpression, leading to more expression of the ly sensitive to mTOR inhibition, raising the possibility that short S6K1 isoform, Iso2 (Fig. 5C; Supplementary Fig. S2B; clinical tumors with MALAT1 upregulation can be targeted fi ref. 41). It was also shown that the short S6K1 isoform binds with mTOR inhibitors. This nding may pave the way for and activates mTORC1, resulting in increased phosphorylation better diagnosis and treatment of cancers with MALAT1 of 4E-BP1 (11, 41). SRSF1 was shown to be present in a overexpression. complex with mTOR and to increase 4E-BP1 phosphorylation in translational extracts (42). SRSF1 also affects RNA proces- Conclusion sing steps, other than splicing, that can attribute to its oncogenic activity. It enhances mRNA transport (53), and also Taken together, our data suggest that MALAT1 acts as an directly affects translation of approximately 1,500 transcripts, oncogene in the context of hepatocellular carcinoma. Our pro- some of which belong to tumor suppressors and oncogenes posed model of how MALAT1 acts as an oncogene is presented in a regulating cell cycle and correct chromosomal segregation scheme (Fig. 7H). Overexpression of MALAT1 activates the Wnt (43). We observed more phosphorylation of 4EBP1 in pathway, inducing c-myc and cyclin D1. c-Myc activates the MALAT1-overexpressing cells, suggesting activation of the transcription of SRSF1, resulting in the modulation of alternative mTOR pathway (Fig. 5E). Moreover, the RNA-seq analysis we splicing of key target genes known to play an important role in performed showed a gene expression pattern, which suggests cancer progression and maintenance. Modulation of the alterna- mTOR pathway activation (Supplementary Fig. S5C). Taken tive splicing of S6K1 generates the oncogenic splicing isoform together, these results point to the deregulation of the mTOR Iso2, promotes activation of the mTOR pathway, leading to pathway, as a result of increased SRSF1 activity, to be one of enhanced 4E-BP1 phosphorylation. Alternatively, SRSF1 directly the major mechanisms by which MALAT1 exerts its oncogenic activates mTOR. Our data suggest that the mTOR pathway is properties. necessary for the oncogenic potential of MALAT1 with SRSF1 being an important mediator. These results suggest that down- MALAT1 activates SRSF1 and mTOR through a transcriptional regulation of MALAT1 levels or inhibition of mTOR, in tumors program, leading to Wnt and ERBB3-4 pathway activation where MALAT1 is upregulated, should be considered as a new SRSF1 was shown to be a direct transcriptional target of strategy for hepatocellular carcinoma therapy. c-MYC (44). c-MYC is the direct transcriptional target of the Wnt-b-catenin pathway. To better understand the transcription- Disclosure of Potential Conflicts of Interest al program induced by MALAT1 in hepatocytes, we performed No potential conflicts of interest were disclosed. RNA-seq analysis on PHM-1 cells overexpressing MALAT1. Our analysis revealed that MALAT1 induced upstream activators of the Wnt pathway (e.g., both ligands Wnt2 and Wnt10a, as well Authors' Contributions as the receptor, Frizzled, which activates the pathway). In Conception and design: P. Malakar, A. Shilo, R. Karni Development of methodology: P. Malakar, A. Shilo, S. Elgavish, X. Zong, addition, several suppressors of the pathway were downregu- K.V. Prasanth lated (e.g., DKK, TGFb, TGFbR; Supplementary Figs. S4–S6). Acquisition of data (provided animals, acquired and managed patients, These findings can explain why b-catenin translocated to the provided facilities, etc.): P. Malakar, I. Stein, E. Pikarsky, K.V. Prasanth nucleus and activated c-myc and cyclin D1 upon MALAT1 Analysis and interpretation of data (e.g., statistical analysis, biostatistics, overexpression (Fig. 6). The increased expression of c-MYC by computational analysis): P. Malakar, Y. Nevo, H. Benyamini, R. Karni MALAT1-overexpressing PHM-1 cells is in agreement with Writing, review, and/or revision of the manuscript: P. Malakar, I. Stein, E. Pikarsky, K.V. Prasanth, R. Karni earlier studies (24, 25). Interestingly, one of the transcriptional Administrative, technical, or material support (i.e., reporting or organizing targets of the Wnt pathway is the tyrosine kinase receptor data, constructing databases): A. Mogilevsky, S. Elgavish, X. Zong ERBB3 (47). Both ERBB3 and ERBB4 expression was induced Study supervision: R. Karni

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Acknowledgments ratory is supported by grants from American Cancer Society (RSG-11-174- The authors wish to acknowledge Drs. Zahava Kluger and Rami Aqeilan 01-RMC) and NIH (GM088252). for comments on the manuscript. 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 Grant Support this fact. This work was supported by the Israeli Science Foundation (ISF Grant No. 1290/12 to R. Karni). P. Malakar was funded by a postdoctoral fellowship of Received June 1, 2016; revised November 26, 2016; accepted December 6, the Israel Higher Education Committee. Research in K.V. Prasanth's labo- 2016; published OnlineFirst December 19, 2016.

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www.aacrjournals.org Cancer Res; 77(5) March 1, 2017 1167

Long Noncoding RNA MALAT1 Promotes Hepatocellular Carcinoma Development by SRSF1 Upregulation and mTOR Activation

Pushkar Malakar, Asaf Shilo, Adi Mogilevsky, et al.

Cancer Res 2017;77:1155-1167. Published OnlineFirst December 19, 2016.

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