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EZH2 promotes progression of small cell lung cancer by suppressing the TGF-β-Smad-ASCL1 pathway

EZH2 promotes progression of small cell lung cancer by suppressing the TGF-β-Smad-ASCL1 pathway OPEN Citation: Cell Discovery (2015) 1, 15026; doi:10.1038/celldisc.2015.26 www.nature.com/celldisc ARTICLE EZH2 promotes progression of small cell lung cancer by suppressing the TGF-β-Smad-ASCL1 pathway 1 1 2 2 1 Fumihiko Murai , Daizo Koinuma , Aya Shinozaki-Ushiku , Masashi Fukayama , Kohei Miyaozono , Shogo Ehata Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan; Department of Pathology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan Transforming growth factor-β (TGF-β) induces apoptosis in many types of cancer cells and acts as a tumor suppressor. We performed a functional analysis of TGF-β signaling to identify a molecular mechanism that regulated survival in small cell lung cancer cells. Here, we found low expression of TGF-β type II receptor (TβRII) in most small cell lung cancer cells and tissues compared to normal lung epithelial cells and normal lung tissues, respectively. When wild-type TβRII was overexpressed in small cell lung cancer cells, TGF-β suppressed cell growth in vitro and tumor formation in vivo through induction of apoptosis. Components of polycomb repressive complex 2, including enhancer of zeste 2 (EZH2), were highly expressed in small cell lung cancer cells; this led to epigenetic silencing of TβRII expression and suppression of TGF-β- mediated apoptosis. Achaete-scute family bHLH transcription factor 1 (ASCL1; also known as ASH1), a Smad-dependent target of TGF-β, was found to induce survival in small cell lung cancer cells. Thus, EZH2 promoted small cell lung cancer progression by suppressing the TGF-β-Smad-ASCL1 pathway. Keywords: small cell lung cancer; epigenetics; apoptosis; EZH2; TGF-β; ASCL1; Smad Cell Discovery (2015) 1, 15026; doi:10.1038/celldisc.2015.26; published online 22 September 2015 Introduction Transforming growth factor (TGF)-β is a cytokine that exerts many biological functions. TGF-β binds to Lung cancer causes mortality more than any other two different types of serine-threonine kinase receptors, type of cancer [1]. Lung cancer is mainly classified as termed type II (TβRII) and type I receptors (TβRI is also known as activin receptor-like kinase 5, ALK-5) either small cell lung cancer (SCLC) or non-small cell expressed on the cell surface. Upon ligand binding, lung cancer (NSCLC), with incidences of ~ 15 and 84%, two TβRIIs and two TβRIs form a heterotetrameric respectively [2]. SCLC, high-grade neuroendocrine complex, and this activated complex phosphorylates tumors, has been reported to have the worst prognosis, the receptor-regulated Smads (R-Smads), Smad2 and with a 5-year survival rate of ~ 5% [3]. Those patients Smad3. The phosphorylated R-Smads form complexes are mostly treated with anti-cancer drugs and/or with their common-partner Smad (Co-Smad), Smad4, radiation. However, a primary clinical issue is the and the R-Smad/Co-Smad complex translocates to the acquisition of chemoresistance in SCLC cells [4]. Thus, nucleus. Then, the complexes associate with various it is essential to develop novel strategies for SCLC transcription factors and transcriptional co-activators therapy. For successful drug discovery, it is important or co-repressors, which in turn, regulate transcription to find molecular mechanism(s) that maintain survival of a wide spectrum of target genes [5–7]. in SCLC cells. TGF-β has been reported to have bi-directional roles in cancer progression [8]. TGF-β induces cell cycle arrest at G1 by regulating expression of cyclin- Correspondence: Kohei Miyazono dependent kinase inhibitor 1 A (CDKN1A, also Tel: +81 3 5841 3345; Fax: +81 3 5841 3354; known as p21), CDKN2B (also known as p15), E-mail: [email protected] Received 22 February 2015; accepted 3 August 2015 the v-myc avian myelocytomatosis viral oncogene EZH2 promotes SCLC by suppressing TGF-β signaling NSCLC SCLC SMAD7 2.5 *** ** ** A549 H146 H82 H209 H345 (-) -+ - + - + - + - + TGF-β 1.5 TGF-β pSmad2 Smad2/3 0.5 A549 H441 H146 H82 H209 H345 NSCLC SCLC TGFBR1 TGFBR2 1.2 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 NSCLC SCLC NSCLC SCLC SMAD2 SMAD4 SMAD3 2.5 1 3.5 2 0.8 2.5 1.5 0.6 1.5 1 0.4 0.2 0.5 0.5 0 0 0 NSCLC SCLC NSCLC SCLC NSCLC SCLC NSCLC cells SCLC cells Normal lung epithelial cells 2 SMAD4 SMAD2 TGFBR1 TGFBR2 SMAD3 -2 Figure 1 TGF-β signal transduction is attenuated in several SCLC cells due to decreased expression of TβRII. (a and b) SCLC and NSCLC cells were stimulated with TGF-β for 2 h. (a) Immunoblot of cell lysates probed with the indicated antibodies; (b) qRT-PCR analysis of SMAD7 expression. Data represent mean ± s.d. **Po0.01; ***Po0.001. (c) qRT-PCR analysis shows expression of TGF-β signaling components in SCLC and NSCLC cells. Data represent mean ± s.d. (d) Comprehensive gene expression analysis from the NCBI GEO database (GSE32036) shows expression profiles of TGF-β signaling components in normal lung epithelial cells (n = 59), SCLC cells (n = 29) and NSCLC cells (n = 119). Raw data were normalized by quantile algorithm. The color indicates the distance from the median of each row. GEO, gene expression omnibus; NCBI, National Center for Biotechnology Information; NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; TGF-β, transforming growth factor-β; qRT-PCR, quantitative real-time reverse transcription-PCR. Cell Discovery www.nature.com/celldisc Relative expression (/GAPDH) Relative expression (/GAPDH) H441 H441 A549 A549 H82 H82 H146 H146 H209 H209 H345 H345 Relative expression (/GAPDH) Relative expression (/GAPDH) H441 H441 A549 A549 H82 H82 H146 H146 H209 H209 H345 H345 Relative expression (/GAPDH) Relative expression (/GAPDH) H441 A549 H82 H146 H209 H345 Distance from median Fumihiko Murai et al. homolog (MYC), and cell division cycle protein 25A significant differences (Figure 1d, and Supplementary (CDC25A) [9]. TGF-β also induces apoptosis in several Figure S1). Since TGF-β signal is transduced even in types of cancer cells through multiple mechanisms the low expression levels of Smad3 if Smad2 is [9, 10]. In ~ 80% of NSCLC tissues, expression of expressed in H146 cells (Figure 1b), we assumed that TβRII was lower than that of normal lung tissues [11]. TGF-β signal transduction was attenuated in SCLC It was also shown that restoration of TβRII into cells through the decreased expression of TβRII, and NSCLC cells inhibited their growth in vitro and in vivo. therefore, we decided to focus on the roles of TβRII in Conversely, TGF-β also plays critical roles in cancer SCLC in the present study. metastasis via the epithelial-mesenchymal transition (EMT) [12]. Once EMT occurs in NSCLC cells, they TβRII suppresses SCLC tumor growth through acquire mesenchymal characteristics, which results in TGF-β-induced apoptosis invasion and metastasis [13]. In NSCLC cells, thyroid To examine the roles of TGF-β in SCLC progres- transcription factor-1 (TTF-1) suppresses TGF-β- sion, wild-type TβRII was introduced into H82 cells mediated EMT and inhibits cell migration and inva- (H82-TβRII cells) or H345 cells (H345-TβRII cells) sion [14]. Thus, the roles of TGF-β in the progression of with lentiviral vectors. Both phosphorylation of Smad2 NSCLC has been intensively studied. In contrast to and induction of SMAD7 by TGF-β were observed in NSCLC, the roles of TGF-β in the progression of TβRII-expressing cancer cells, but not in control SCLC SCLC have not been fully investigated. A few studies cells that expressed green fluorescent protein (GFP) have reported that expression of TGFBR2 (the gene that alone (H82-GFP cells and H345-GFP cells; Figure 2a encodes TβRII) was decreased in some SCLC cells, but and b). Thus, TGF-β signal transduction was success- the mechanisms were not detailed [15, 16]. Therefore, fully recovered by expressing TβRII. These cells were the present study aimed to clarify the roles of TGF-β in subcutaneously xenografted into nude mice to examine SCLC cells, to identify the mechanisms involved in the tumor growth in vivo. Tumor formation was decreased downregulation of TβRII, and to identify novel TGF-β in mice injected with H82-TβRII cells and H345-TβRII target genes in this type of cancer. cells, compared with mice xenografted with the control cells (Figure 2c). Although the expression of TGFBR2 Results mRNA was low (Figure 1c) and the TβRII protein was not detected by immunoblot analysis (data not shown), Downregulation of TβRII expression in SCLC cells Smad-dependent TGF-β signal was transduced in First, we investigated whether TGF-β signals were H146 cells (Figures 1a and b), suggesting that a low transduced in SCLC cells. Phosphorylation of Smad2 level of TβRII protein may be functioning in these cells. and induction of SMAD7, one of the direct targets of Thus, a GFP-tagged dominant-negative form of TβRII TGF-β, were examined in human SCLC cells (H146, (dnTβRII) was overexpressed in H146 cells (H146- H82, H209 and H345) and in NSCLC cells (A549 dnTβRII cells; Supplementary Figure S2a). Both phos- and H441) with immunoblotting and quantitative phorylation of Smad2 and induction of SMAD7 were real-time reverse transcription-PCR (qRT-PCR) inhibited by the introduction of dnTβRII (Supplemen- analyses. TGF-β-mediated phosphorylation of Smad2 tary Figures S2b and S2c). When these cells were sub- was observed in H146 cells as well as in A549 and cutaneously xenografted into mice, tumor formation H441 cells (Figure 1a and data not shown, see Isogaya was accelerated in mice injected with H146-dnTβRII et al [17]). Induction of SMAD7 by TGF-β was also cells compared with those injected with H146-GFP observed in H146, A549 and H441 cells (Figure 1b). cells (Supplementary Figure S2d). These results However, in the other SCLC cells, these responses suggested that TGF-β may act as a tumor suppressor were not induced by TGF-β. A qRT-PCR analysis in vivo. We assessed angiogenesis in these tumor tissues also showed that expression of TGFBR2 and SMAD3 by staining for CD31 expression (also known as was decreased in SCLC cells, but other TGF-β signal- platelet/endothelial cell adhesion molecule 1, ing components, including SMAD2, SMAD4 and PECAM1). However, there was no remarkable differ- TGFBR1 (the gene that encodes TβRI), were expressed ence in CD31 expression between H146-GFP and at normal levels in these cells (Figure 1c). These H146-dnTβRII cells xenografted tissues (Supplementary expression profiles were confirmed with comprehensive Figure S2e). This finding suggested that tumor gene expression analysis data from the gene expression suppression was mediated by the effect of TGF-β on omnibus (GEO) of the National Center for Bio- SCLC cells, not by its effect on angiogenesis in the technology Information (NCBI) with statistically tumor microenvironment. Cell Discovery www.nature.com/celldisc | EZH2 promotes SCLC by suppressing TGF-β signaling SMAD7 SMAD7 *** *** 3.5 0.6 0.5 -- + ++- - + --++ TGF-β 2.5 TGF-β 0.4 (-) 0.3 pSmad2 pSmad2 1.5 TGF-β 0.2 Smad2/3 Smad2/3 0.5 0.1 TβRII TβRII α-tubulin α-tubulin ** 700 50 *** 0.4 *** 0.4 400 0.3 0.3 (-) 0.2 0.2 TGF-β 0.1 10 0.1 0 0 0 0 TGF-β H345-GFP H345-TβRII sub-G0/G1 G0/G1 S G2/M () - -- + ++- - + TGF-β uncleaved PARP cleaved pRB pSmad2 Smad2/3 TGF-β () - ()++ () - () () + α-tubulin H345-GFP H345-TβRII PI intensity CDKN1A (p21) CDKN2B (p15) CDC25A MYC *** *** ** *** 3 4 2 2 3 1.5 1.5 (-) 2 1 1 TGF-β 1 0.5 0.5 0 0 0 0 Cell Discovery www.nature.com/celldisc Cell number Relative expression (/GAPDH) Tumor volume (mm ) HaCaT H82 H82-GFP H345-GFP H82- H82-TβRII H345-TβRII TβRII Tumor volume (mm ) H82- GFP Relative expression H345-GFP (/GAPDH) H345-TβRII HaCaT H345 H345-GFP H345- H345-TβRII GFP % of distribution H345- TβRII Relative expression (/GAPDH) Relative expression Absorbance (A450-A595) (/GAPDH) HaCaT H345-GFP H82 H82 H345-TβRII H82-GFP H82-GFP H82-TβRII H82-TβRII Relative expression (/GAPDH) Relative expression Absorbance (A450-A595) (/GAPDH) HaCaT HaCaT H345 H345-GFP H345 H345 H345- GFP H345-TβRII H345-GFP H345-GFP H345- TβRII H345-TβRII H345 -TβRII Fumihiko Murai et al. We postulated that TGF-β might suppress the pro- and SCLC tissues displayed higher expression of the liferative activity of SCLC cells. When we restored enhancer of zeste 2 (EZH2), SUZ12 polycomb TβRII expression in these cells, TGF-β significantly repressive complex 2 subunit (SUZ12), and embryonic suppressed the in vitro proliferation of H82 cells and ectoderm development (EED) than normal lung epi- H345 cells (Figure 2d). Moreover, dnTβRII expression thelial cells and normal lung tissues, respectively canceled TGF-β-mediated growth inhibition in H146 (Figure 3a, and Supplementary Figure S3). We also cells (Supplementary Figure S2f). Cell cycle analysis found that their expressions were increased in SCLC revealed that TGF-β increased the sub-G0/G1 popu- cells but not in NSCLC cells (Figure 3b and c). These lation in H345-TβRII cells compared with H345-GFP molecules associate to form the polycomb repressive cells (Figure 2e). TGF-β also induced the cleavage of complex 2 (PRC2), which inhibits gene transcription poly (ADP-ribose) polymerase (PARP) in H345-TβRII through methylation of lysine 27 in histone H3 cells (Figure 2f), which suggested that TGF-β (H3K27me3). High expression of H3K27me3 was decreased the number of SCLC cells by inducing observed in SCLC cells, which was attenuated by the apoptosis. TGF-β is also known to suppress prolifera- treatment with an EZH2 inhibitor, GSK343 tion of many types of cells by regulating CDK activa- (Figure 3c), suggesting that PRC2 is implicated in tors or inhibitors. We found that expression levels of transcriptional regulation of several genes in SCLC CDKN1A, CDKN2B, MYC or CDC25A in H345- cells. H146 cells showed different expression profiles of TβRII cells were not markedly altered by TGF-β the PRC2 complex from those in the other SCLC cells. (Figure 2g). However, in human keratinocyte The expression level of EZH2 protein was similar to HaCaT cells, TGF-β upregulated the expression of those in the other SCLC cells, while the expression CDKN1A and CDKN2B and downregulated the levels of EZH2, EED and SUZ12 mRNAs were lower expression of MYC and CDC25A. Moreover, expres- than those in the other SCLC cells (Figures 3b and c). sion of retinoblastoma protein (pRB) was not detected In order to directly examine whether EZH2 is in H345 cells (Figure 2f). These results suggested that involved in downregulation of TGFBR2, chromatin TGF-β suppressed proliferation of SCLC cells by immunoprecipitation (ChIP)-qRT-PCR analysis using inducing apoptosis, but not by regulating the cell cycle. anti-EZH2 antibody was performed (Figure 4a). EZH2 bound to the several loci in TGFBR2 in H345 cells. Moreover, transcription of TGFBR2 mRNA was Importance of EZH2-mediated silencing of TβRII for increased in GSK343-treated SCLC cells (Figure 4b). SCLC tumor formation When EZH2 expression was silenced in H345 cells with Histone methyltransferase mediates methylation on a short hairpin RNA (shRNA) (H345-shEZH2), the lysine or arginine residues of histones of the H3 and H4 knockdown of EZH2 led to an increase in TGFBR2 families to regulate transcription of various genes [18]. expression (Figure 4c); in turn, TGF-β induced Smad2 We postulated that, in SCLCs, the expression of TβRII phosphorylation and SMAD7 expression (Figures 4d might be epigenetically silenced through histone and e). These results suggested that EZH2 played a modification by histone methyltransferases. Therefore, we investigated histone methyltransferase expression in critical role in downregulating TβRII in SCLC cells. We also investigated whether EZH2 was important SCLC cells. Comprehensive gene expression analyses for TGF-β-mediated apoptosis and tumor formation in from the NCBI GEO data set revealed that SCLC cells Figure 2 TβRII suppresses SCLC tumor growth through TGF-β-induced apoptosis. (a and b) SCLC cells were infected with lentivirus vectors encoding GFP (H82-GFP and H345-GFP) or TβRII (H82-TβRII and H345-TβRII). Cells were stimulated with TGF-β for 2 h. (a) Immunoblots of cell lysates probed with the indicated antibodies. (b) qRT-PCR analysis of SMAD7 expression. Data represent mean ± s.d. ***Po0.001. (c) Mice received subcutaneous transplantations of H82-GFP (n = 15) and H82-TβRII cells (n = 12) or H345-GFP (n = 7) and H345-TβRII cells (n = 9), and tumor volumes were measured 16 days (H82) or 17 days (H345) after transplantation. Data represent mean ± s.e.m. *Po0.05; **Po0.01. (d) Cell proliferation assay. SCLC cells were stimulated with TGF-β for 6 days (H82) or 12 days (H345). Data represent mean ± s.d. ***Po0.001. (e) Cell cycle analysis. (left panels) H345-GFP and H345-TβRII cells were unstimulated (top panels) or stimulated (bottom panels) with TGF-β for 12 days; the number of cells in each cell cycle stage is shown (color coding shown in right panel). (right panel) Percentage of cells in each cell cycle stage. (f) Immunoblots of cell lysates probed with the indicated antibodies. SCLC and control HaCaT cells were stimulated with TGF-β for 48 h (HaCaT) or 12 days (H345). (g) qRT-PCR analysis shows cell cycle-related gene expression. H345-GFP, H345-TβRII and HaCaT cells were stimulated with TGF-β for 2 h (MYC)or24h (CDKN1A, CDKN2B and CDC25A). Data represent mean ± s.d. *Po0.05; **Po0.01; ***Po0.001. GFP, green fluorescent protein; SCLC, small cell lung cancer; TGF-β, transforming growth factor-β; qRT-PCR, quantitative real-time reverse transcription-PCR. Cell Discovery www.nature.com/celldisc | EZH2 promotes SCLC by suppressing TGF-β signaling NSCLC cells SCLC cells Normal lung epithelial cells EED EZH2 SUZ12 -2 EZH2 EED SUZ12 3.5 3.5 1.6 1.4 3 3 1.2 2.5 2.5 0.8 1.5 1.5 0.6 1 1 0.4 0.5 0.5 0.2 0 0 NSCLC SCLC NSCLC SCLC NSCLC SCLC EZH2 NSCLC SCLC A549 H441 H82 H146 H209 H345 GSK343 H3K27me3 H3S10p H3 (total) EZH2 α-tubulin NSCLC SCLC Figure 3 EZH2 is highly expressed in SCLC cells. (a) Expression profiles of PRC2 components in SCLC cells, based on the data in Figure 1d. The color indicates the distance from the median of each row. (b) qRT-PCR analysis shows expression of PRC2 components in SCLC and NSCLC cells. Data represent mean ± s.d. (c) Immunoblot of cell lysates probed with the indicated antibodies. (left panels) SCLC and NSCLC cells were treated with GSK343 in a wide concentration range (0, 0.4, 2 and 10 μM) for 3 days. (right panel) Relative expression of EZH2 protein in each cell without GSK343 was quantified. NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; qRT-PCR, quantitative real-time reverse transcription-PCR. H345 cells. Cell cycle analysis showed that TGF-β ChIP-sequencing (ChIP-seq) analysis was performed increased the sub-G0/G1 population in H345-shEZH2 with an anti-Smad2/3 antibody in H345-TβRII cells cells, but not in H345-shNTC cells (Figure 4f). to identify comprehensively Smad2/3-regulated genes. Moreover, the ability of H345-shEZH2 cells to form The characteristics of SCLC cells are thought to tumors was attenuated, compared to that of depend on the expression of several neuroendocrine- H345-shNTC cells (Figure 4g). related genes, including the achaete-scute family basic helix-loop-helix transcription factor 1 (ASCL1, also ASCL1 is negatively regulated by TGF-β in a known as ASH1), synaptophysin (SYP), neural cell Smad-dependent manner adhesion molecule (NCAM) and v-myc avian myelo- We next attempted to identify TGF-β target cytomatosis viral oncogene lung carcinoma derived genes that were involved in SCLC cell apoptosis. homolog (MYCL) [19]. Therefore, we focused on Cell Discovery www.nature.com/celldisc Relative expression (/GAPDH) H441 A549 H146 H82 H209 H345 Relative expression (/GAPDH) H441 A549 H146 H82 H209 H345 Relative expression (/GAPDH) H441 A549 Relative expression H146 H82 A549 H209 H441 H345 H82 H146 H209 Distance from median H345 Fumihiko Murai et al. Smad2/3 binding to these gene loci. ChIP-seq analysis locus or MYCL locus, in H345-TβRII cells (Figure 5a). showed that, in the presence of TGF-β, Smad2/3 sig- Among the binding regions in the NCAM1 locus, nificantly bound to two loci of the ASCL1 gene and Smad2/3 strongly bound to the first intron. Compre- several loci of the NCAM1 gene, but not to the SYP hensive gene expression analysis from the NCBI GEO TGFBR2 TGFBR2 TGFBR2 TGFBR2 HPRT1 (locus 1) (locus 2) (locus 3) (locus 4) TGFBR2 ** * * 6 0.01 0.015 0.015 0.008 0.008 0.008 0.006 0.006 4 ( ) 0.01 0.01 0.006 GSK343 0.004 0.004 0.004 0.005 0.005 0.002 0.002 0.002 H82 H146 H209 H345 0 0 0 0 0 EZH2 TGFBR2 *** *** SMAD7 *** *** 2.5 2.5 *** ** *** *** 2 2 *** 1.5 1.5 ( ) TGF- TGF- EZH2 0.5 0.5 pSmad2 0 0 0 Smad2/3 -tubulin TGF- H345-shNTC H345-shEZH2 #1 H345-shEZH2 #2 sub-G0/G1 G0/G1 S G2/M ( ) ( ) TGF- ( )( )( )( )( )( ) H345- H345- H345- shNTC shEZH2 #1 shEZH2 #2 PI intensity H345-shNTC H345-shEZH2 #1 100 H345-shNTC H345-shEZH2 #1 ** 123 4 Week after transplantation Cell Discovery www.nature.com/celldisc % of input Relative expression (/GAPDH) H345-shNTC IgG1 Cell number H345-shEZH2 #1 EZH2 H345-shEZH2 #2 % of input Relative expression (/GAPDH) IgG1 H345-shNTC EZH2 H345-shEZH2 #1 % of input H345-shEZH2 #2 IgG1 EZH2 % of input H345- GFP H345- 3 IgG1 Tumor volume (mm ) T RII EZH2 H345- % of input shNTC H345- shEZH2 #1 IgG1 H345- % of distribution shEZH2 #2 EZH2 Relative expression Relative expression (/GAPDH) (/GAPDH) H345-GFP H345-T RII H345-shNTC H345-shEZH2 #1 H345-shEZH2 #2 EZH2 promotes SCLC by suppressing TGF-β signaling data sets showed that the mRNA levels of each of these TGF-β could upregulate SMAD7 and downregulate neuroendocrine-related genes were elevated in SCLC ASCL1 expression (Figure 5f). This result suggested cells and SCLC tissues, except for SYP in the SCLC that, similar to SMAD7, the expression of ASCL1 was tissue (Supplementary Figures S4a and S4b). Next, directly regulated by TGF-β. TGF-β regulation of these genes was assessed by qRT- TGF-β can activate both Smad and non-Smad PCR analysis (Figure 5b and Supplementary Figures pathways. To determine which pathway played a S5a and S5b). In H345-TβRII cells, treatment with predominant role in regulating ASCL1 expression, we TGF-β caused ASCL1 expression to decrease within used a shRNA (shSmad4) to silence the expression of 1 h, and it reached a minimum at 4 h. TGF-β also Smad4 in H345-TβRII cells. The knockdown of Smad4 decreased ASCL1 expression within 2 h in H146 cells. attenuated the induction of SMAD7 by TGF-β However, MYCL, NCAM1 and SYP were not (Figures 5g and h). Moreover, TGF-β-mediated regulated by TGF-β in these cells. Moreover, TGF-β downregulation of ASCL1 expression was canceled in suppressed ASCL1 protein expression in SCLC cells H345-TβRII cells by shSmad4, but not in cells infected (Figure 5c). In accordance with a previous analysis with negative control shRNA (shNTC). These results (Supplementary Figure S4a), in NSCLC cells, ASCL1 suggested that TGF-β directly suppressed ASCL1 was only weakly expressed, and it was not regulated by expression in a Smad-dependent manner. TGF-β (A549 and H441) (Supplementary Figure S5c). These results suggested that ASCL1 was a TGF-β ASCL1 promotes survival of SCLC cells target, and regulation of ASCL1 by TGF-β was specific Next, we determined whether negative regulation for SCLC cells. of ASCL1 transcription was important for TGF-β- We then focused on the molecular mechanism mediated apoptosis of SCLC cells. When expression of underlying TGF-β-mediated transcriptional regulation ASCL1 was silenced in H345 cells with shRNA of ASCL1 in SCLC cells. ChIP-qRT-PCR analysis was (shASCL1) (Figure 6a), cell growth was inhibited performed to confirm whether Smad2/3 bound in by the induction of apoptosis (Figures 6b and c). the ASCL1 locus. We found that TGF-β-stimulated Conversely, when ASCL1 was exogenously introduced H345-TβRII cells showed a sixfold-enrichment of by transferring lentiviral vectors into H345-TβRII cells, Smad2/3 binding to these loci, compared with those to cell cycle analysis revealed that the TGF-β-mediated hemoglobin beta (HBB) locus (Figure 5d). In addition increase in the sub-G0/G1 population was attenuated to the recovery of TβRII expression and TGF-β signal (Figures 6d and e). These results suggested that ASCL1 transduction in H345-shEZH2 cells (see Figures 4d had an important role in enabling SCLC cells to escape and e), ASCL1 expression was decreased in these cells from TGF-β-induced apoptosis. (Figure 5e). Next, cycloheximide (CHX), a de novo Next, we performed subcutaneous transplantations protein synthesis inhibitor, was used to investigate in mice to determine whether knocking down ASCL1 whether ASCL1 expression was directly or indirectly expression would suppress tumor growth or formation. regulated by TGF-β. Even in the presence of CHX, We knocked down ASCL1 expression by treating cells Figure 4 EZH2-mediated silencing of TβRII is required for SCLC tumor formation. (a) qRT-PCR analysis post-immunoprecipitation with anti-EZH2 antibody shows EZH2 enrichment in the TGFBR2 locus of H345 cells. Hypoxanthine guanine phosphoribosyl transferase1 (HPRT1) was used as the negative control. Data represent mean ± s.d. TGFBR2 locus 1, chromosome 3: 30606416–30606492 bp; TGFBR2 locus 2, chromosome 3: 30605676–30605771 bp; TGFBR2 locus 3, chromosome 3: 30604616–30604714 bp; TGFBR2 locus 4, chromosome 3: 30603967–30604061 bp. *Po0.05; **Po0.01. (b) qRT-PCR analysis shows TGFBR2 expression. SCLC cells were treated with GSK343 (10 μM) for 7 days. Data represent mean ± s.d. (c) qRT-PCR analysis shows EZH2 and TGFBR2 expression. H345 cells were infected with lentivirus vectors with control shRNA (H345-shNTC) or shRNA that targeted EZH2 (H345-shEZH2). Data represent mean ± s.d. ***Po0.001. (d) Immunoblot of cell lysates from cells in (c) stimulated with TGF-β for 2 h and probed with the indicated antibodies. H345-GFP cells and H345-TβRII cells were negative and positive controls, respectively. (e) qRT-PCR analysis shows SMAD7 expression. Cells in (c) were stimulated with TGF-β for 4 h. Data represent mean ± s.d. H345-GFP cells and H345-TβRII cells were negative and positive controls, respectively. **Po0.01; ***Po0.001. (f) Cell cycle analysis of cells in (c) stimulated with TGF-β for 12 days. (left panels) The number of cells in each cell cycle stage is shown (color coding shown in right panel). (right panel) Percentage of cells in each cell cycle stage. (g) Mice received subcutaneous transplants of H345-shNTC cells (n = 7) or H345-shEZH2 #1 cells (n = 7). (left panels) Representative photographs 4 weeks after injection. Arrow heads indicate tumors. (right panel) Tumor volumes at the indicated time points. Data represent mean ± s.e.m. **Po0.01. SCLC, small cell lung cancer; qRT-PCR, quantitative real-time reverse transcription-PCR. Cell Discovery www.nature.com/celldisc | Fumihiko Murai et al. with small interfering RNA (siRNA) that targeted seven mice, but siASCL1-treated H345 cells formed ASCL1 (siASCL1). Control cells were treated with tumors in only one out of seven mice (Figure 6g). These negative control siRNA (siNTC) (Figure 6f). The results suggested that ASCL1 was involved in SCLC siNTC treated-H345 cells formed tumors in six out of tumor formation. 30 30 ASCL1 MYCL 20 20 10 10 0 0 -+ - + TGF-β 30 30 SYP ASCL1 NCAM1 pSmad2 10 10 Smad2/3 0 0 -tubulin SMAD7 ASCL1 12 1.5 1.2 0.9 0.6 H345-GFP 0.3 H345-TβRII 0 0 0 20406080 0 20406080 TGF- treatment time (h) TGF- treatment time (h) ASCL1 ASCL1 (locus 1) ASCL1 (locus 2) SMAD7 HBB *** 0.02 * 0.02 ** 0.02 0.3 *** (-) *** 0.25 0.015 0.015 0.015 0.2 TGF-β 1.5 0.01 0.01 0.01 0.15 0.1 0.005 0.005 0.005 0.05 0.5 0 0 IP IgG1 Smad2/3 IP IgG1 Smad2/3 IgG1 Smad2/3 IP IP IgG1 Smad2/3 SMAD7 ASCL1 SMAD4 SMAD7 ASCL1 *** *** * *** * *** *** *** *** *** *** *** *** 7 1.4 3 1.5 6 1.2 2.5 5 1 1.5 (-) 2 1 4 0.8 (-) 3 TGF-β 0.6 1.5 1 TGF-β 2 0.4 1 0.5 1 0.2 0.5 0.5 0 0 0 0 DMSO CHX DMSO CHX shNTC shSmad4 shSmad4 shNTC shSmad4 shNTC Cell Discovery www.nature.com/celldisc %of input Relative expression (/GAPDH) H345-GFP Mapped tag number H345-TβRII H345-GFP H345-GFP H345-TβRII H345-TβRII H345-GFP Fold change Relative expression (/GAPDH) H345-TβRII (/GAPDH/non-treatment) % of input H345-GFP H345-TβRII H345-GFP H345-GFP H345-TβRII H345-TβRII H345-GFP Relative expression (/GAPDH) H345-TβRII % of input H345-GFP H345-TβRII H345-GFP H345-GFP Fold change H345-TβRII (/GAPDH/non-treatment) H345-TβRII H345-GFP H345-TβRII Relative expression (/GAPDH) % of input H345-GFP H345-TβRII H345-GFP H345-GFP H345-TβRII H345-TβRII H345-GFP Relative expression (/GAPDH) H345-TβRII Relative expression (/GAPDH) H345-GFP H345- H345-TβRII GFP H345-shNTC H345-GFP H345- H345-shEZH2 #1 TβRII H345-TβRII H345-shEZH2 #2 EZH2 promotes SCLC by suppressing TGF-β signaling EZH2-mediated silencing of TβRII in SCLC tissues suppressed, which resulted in high ASCL1 levels and Finally, we compared the expression of EZH2, progression of SCLC. These results suggested new TβRII and ASCL1 in SCLC tissues and NSCLC tissues therapeutic strategies for targeting EZH2 and ASCL1 to that in normal lung tissues (Supplementary Table S1). in SCLC therapy. Immunohistochemical analysis showed that EZH2 was We demonstrated that TGF-β inhibited prolifera- strongly expressed in the nuclei of all SCLC cells tion of SCLC cells in vivo and in vitro. The SCLC cells (Figure 7a). However, its expression was not observed used in this study carried mutations in the DNA in those of normal lung epithelial cells. In contrast, binding region of TP53, and they did not express pro- TβRII was weakly expressed in SCLC cells, while it apoptotic pRB, except for the H209 cells (Figure 2f, was expressed on the surface of normal lung epithelial data not shown) [20]. Thus, TGF-β-mediated apoptosis cells (Figure 7a). Although ASCL1 was also detected in in SCLC cells may occur independently of p53 and nuclei of some SCLC cells, it was not observed in most pRB. TGF-β causes cell cycle arrest at the G1 phase or of normal lung epithelial cells and other lung cancer apoptosis in many types of cancer cells [9]. For cell cells (Figure 7a and Supplementary Figure S6). The cycle arrest at the G1 phase, TGF-β regulates p21, p15, expression profiles of EZH2, TβRII and ASCL1 are c-Myc and CDC25A in various types of cells, but it did shown in Figure 7b. The number of positive samples, not regulate these genes in SCLC cells. TGF-β was also grouped according to expression frequency, is shown in reported to induce apoptosis by inducing expression of Supplementary Table S2. When all lung tissues in BCL2-like 11 (BCL2L11, also known as Bim), growth Figure 7 were considered, there was a negative corre- arrest and DNA-damage-inducible beta (GADD45B) lation between EZH2 and TβRII and a positive cor- and inositol polyphosphate-5-phosphatase 145 kDa relation between EZH2 and ASCL1 (Supplementary (INPP5D, also known as SHIP) [9]. In contrast, our Table S3). These results supported the notion that comprehensive gene expression analysis and ChIP-seq EZH2 had a role in epigenetic silencing of TβRII in analysis showed that the TGF-β-mediated apoptosis in human SCLC tissues, and that the loss of TβRII SCLC cells appeared to be independent of those genes upregulated the expression of ASCL1. (data not shown). Based on these observations, we attempted to identify novel target(s) for TGF-β, which could regulate survival in SCLC cells. Discussion In many types of cancers, TβRII is dysfunctional through either genetic mutation or transcriptional The present study clarified the tumor suppressive role of TGF-β in SCLCs. We showed that TβRII was repression [21–23]. The TGFBR2 locus was shown to be mutated in the 10-adenine (A10) tract of exon 3 and expressed in normal lung epithelial cells, and that it inhibited abnormal cell growth by downregulating serine-threonine kinase domain in some cancers [21–24]. Previous studies demonstrated that genetic ASCL1 in a Smad-dependent manner (Figures 8a and b). However, EZH2 was highly expressed in mutations in the TGFBR2 A10 tract or expression of a truncated TβRII were not common in SCLC [15, 25]. SCLC cells, which epigenetically attenuated the expression of TβRII; thus, TGF-β signaling was Moreover, loss of heterozygosity in chromosome 3, Figure 5 ASCL1 is negatively regulated by TGF-β in a Smad-dependent manner. (a) ChIP-seq analysis using anti-Smad2/3 antibody. H345-TβRII cells were stimulated with TGF-β for 1.5 h. Arrows indicate transcription start sites and direction. (b) qRT-PCR analysis shows SMAD7 and ASCL1 expression in H345-GFP and H345-TβRII cells after TGF-β stimulation for the indicated times. Data represent mean ± s.d. (c) Immunoblot of cell lysates probed with the indicated antibodies. H345-GFP and H345-TβRII cells were stimulated with TGF-β for 12 days. (d) qRT-PCR analysis post-immunoprecipitation with anti-Smad2/3 antibody shows Smad2/3 enrichment. H345-GFP and H345-TβRII cells were stimulated with TGF-β for 1.5 h. Hemoglobin beta (HBB) was used as a negative control. Data represent mean ± s.d. ASCL1 locus 1, chromosome 12: 103351326–103352062 bp; ASCL1 locus 2, chromosome 12: 103351326–103352062 bp. *Po0.05; **Po0.01. (e) qRT-PCR analysis shows ASCL1 expression in indicated cells. Data represent mean ± s.d. ***Po0.001. (f) qRT-PCR analysis shows SMAD7 and ASCL1 expression. H345-GFP and H345-TβRII cells were stimulated with TGF-β for 4 h after pre-treatment with CHX (3 μM) for 24 h. Data represent mean ± s.d. ***Po0.001. (g) qRT-PCR analysis shows SMAD4 expression. H345-GFP and H345-TβRII cells were infected with lentivirus vector with control shRNA (shNTC) or shRNA that targeted Smad4 (shSmad4). Data represent mean ± s.d. (h) qRT-PCR analysis shows SMAD7 and ASCL1 expression. Cells in (g) were stimulated with TGF-β for 4 h. Data represent mean ± s.d. *Po0.05; ***Po0.001. ChIP, chromatin immunoprecipitation; TGF-β, transforming growth factor-β; qRT-PCR, quantitative real-time reverse transcription-PCR. Cell Discovery www.nature.com/celldisc | Fumihiko Murai et al. including in the TGFBR2 locus, was not observed in of TβRII in SCLC. In contrast, transcriptional SCLC [25]. Those studies indicated that genetic muta- repression of TGFBR2 was reported in retinoblastoma tion was not a common mechanism for the dysfunction and hematopoietic malignancies [23, 26]. ASCL1 * *** *** 2 0.4 *** *** *** *** *** 1.5 0.3 1 0.2 0.5 0.1 0 0 (-)TGF-β (-)TGF-β H345-TβΡΙΙ shNTC shASCL1 shASCL1 shASCL1 H345-TβRII shNTC shASCL1 shASCL1 shASCL1 #1 #2 #3 #1 #2 #3 sub-G0/G1 G0/G1 S G2/M H345 shNTC shASCL1 #1 shASCL1 #2 shASCL1 #3 shNTC shASCL1 shASCL1 shASCL1 #1 #2 #3 PI intensity GFP ASCL1 ASCL1 siNTC siASCL1 1.6 *** 1.4 1.2 -- + + --++ TGF-β 0.8 ASCL1 ASCL1 0.6 pSmad2 0.4 0.2 10 Smad2/3 α-tubulin GFP ASCL1 sub-G0/G1 G0/G1 S G2/M TGF-β H345-GFP H345-TβRII H345-GFP H345-TβRII (-) (+) TGF-β (-)(+)(-)(+) (-) (+) (-) (+) H345- H345- H345- H345- - GFP TβRII GFP TβRII GFP ASCL1 PI intensity Cell Discovery www.nature.com/celldisc Cell number Cell number Relative expression (/GAPDH) H345- GFP H345- TβRII H345- GFP H345- TβRII Relative expression (/GAPDH) siNTC siASCL1 Absorbance (A450-A595) % of distrubution % of distribution % of tumor formation siNTC siASCL1 EZH2 promotes SCLC by suppressing TGF-β signaling In this study, we showed that PRC2 components report was the first to reveal the mechanism underlying were highly expressed in most of the SCLC cells, and TGF-β regulation of ASCL1 expression. ASCL1, a that increased EZH2 expression caused silencing of member of the basic helix-loop-helix family transcrip- TβRII expression in SCLC cells. Sato et al [27]. tion factors, has a crucial role in the differentiation of reported that many kinds of genes, such as JUB, neural stem cells into neuronal lineages [40]. ASCL1 is PTRF, DMKN, AXL and EPHB4, were identified as also expressed in neuroendocrine tumors, especially in targets for EZH2 in SCLC cells by ChIP-seq analysis. cases with poor prognoses [41–48]. In addition, several They also found that introduction of JUB inhibited genes were identified as targets for ASCL1 [49]. Among cellular growth, suggesting that suppression of these them, miRNA-375 (miR-375) was supposed to inacti- genes by EZH2 other than TGFBR2 might be involved vate Yes-associated protein (YAP)1 in SCLC [50]. In in the growth of SCLC. Genetic alternations in TP53 H345-TβRII cells, TGF-β exhibited a downgulation of and RB1 are commonly observed in patients with ASCL1, followed by upregulation of primary-miR 375 SCLC; consequently, these were considered as early (pri-miR-375) (data not shown). Thus, our present events that triggered SCLC development [28–30]. findings suggested that the TGF-β-Smad-ASCL1 EZH2 expression was upregulated in Rb1 knockout pathway is important in SCLC progression, and that it MEF cells [31]. Based on those studies, our finding that may also have an important role in other neuroendo- EZH2 was highly expressed in SCLC suggests that crine tumors. EZH2 is an oncogenic factor. EZH2 was also reported to be highly expressed in breast cancer and prostate Materials and Methods cancer [32, 33]; moreover, EZH2 inhibitors have been considered a promising therapy for certain types of Cell culture and reagents tumors [34, 35]. A specific EZH2 inhibitor induced Human SCLC H82, H146, H209, H345 cells and human TGFBR2 expression (Figure 4b); therefore, EZH2 NSCLC A549 and H441 cells were purchased from American inhibitors may also effectively eradicate SCLC cells Type Culture Collection (ATCC, Manassas, VA, USA) and by restoring TβRII expression, and thus, enabling cultured as recommended. Human skin keratinocyte HaCaT cells were previously described [51]. TGF-β3(R&D TGF-β-mediated apoptosis. Systems, Minneapolis, MN, USA) was reconstituted in 4 mM TGF-β-target genes have been comprehensively HCl and 0.1% bovine serum albumin (BSA, Sigma-Aldrich, identified in many kinds of tumors, including liver − 1 St Louis, MO, USA) and used at a concentration of 1 ng ml . cancer, pancreatic cancer, NSCLC and breast cancer GSK343 (Sigma-Aldrich) and CHX (Sigma-Aldrich) were [17, 36–38], but rarely in neuroendocrine tumors. reconstituted in dimethyl sulfoxide. See also Supplementary Identification of novel target(s) may improve our Information. understanding of SCLC cell characteristics. In the present study, our comprehensive gene expression Cell proliferation assay analysis and ChIP-seq analysis demonstrated that the 4 4 H82 cells (1 × 10 cells), H146 cells (3 × 10 cells) and H345 TGF-β-induced apoptosis in SCLC cells could be cells (3 × 10 cells) were seeded on 12-well plates, and then attributed to negative regulation of ASCL1 by TGF-β. stimulated with TGF-β. Cell proliferation was evaluated with It was previously shown that ASCL1 expression was Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan). inhibited by Notch signaling in SCLC cells [39]. This Absorbance at 450 nm was measured with a Model 680 Figure 6 Downregulation of ASCL1 is important for TGF-β-mediated apoptosis in SCLC cells. (a) qRT-PCR analysis shows ASCL1 expression. H345 cells were infected with lentivirus vectors encoding control shRNA (shNTC) or shRNA that targeted ASCL1 (shASCL1). H345-TβRII cells stimulated with TGF-β for 48 h served as control. Data represent mean ± s.d. *Po0.05; ***Po0.001. (b) Cell proliferation assay. Cells in (a) were incubated for 12 days. Data represent mean ± s.d. ***Po0.001. (c) Cell cycle analysis of cells in (b). (left panels) The number of cells in each cell cycle stage is shown (color coding shown in right panel). (right panel) Percentage of cells in each cell cycle stage. (d) Immunoblot of cell lysates probed with the indicated antibodies. H345-GFP and H345-TβRII cells were infected with lentivirus vectors encoding GFP alone or ASCL1, and then stimulated with TGF-β for 12 days. (e) Cell cycle analysis of cells in (d). (left panels) The number of cells in each cell cycle stage is shown (color coding shown in right panel). (right panel) Percentage of each cell cycle stage in indicated cells is shown. (f) qRT-PCR analysis shows ASCL1 expression in H345 cells transfected with control siRNA (siNTC) or siRNA that targeted ASCL1 (siASCL1). ASCL1 expression was determined 72 h post-transfection. Data represent mean ± s.d. ***Po0.001. (g) Mice received subcutaneous transplants of cells in (f) (siNTC, n = 7, siASCL1, n = 7). (Left panels) Representative photographs; (right panel) incidence of tumor formation 2 weeks after injection. Arrow heads indicate tumors. GFP, green fluorescent protein; SCLC, small cell lung cancer; TGF-β, transforming growth factor-β; qRT-PCR, quantitative real-time reverse transcription-PCR. Cell Discovery www.nature.com/celldisc | Fumihiko Murai et al. HE EZH2 TβRII ASCL1 EZH2 TβRII ASCL1 5 5 5 4 4 3 3 3 2 2 2 1 1 1 0 0 0 -1 -1 -1 Normal SCLC Ad Sq LCNEC Normal SCLC Ad Sq LCNEC Normal SCLC Ad Sq LCNEC *** *** *** *** * *** *** * *** *** Figure 7 Expression profiles of EZH2, TβRII and ASCL1 in human normal lung tissues and human lung cancer tissues. (a) Lung tissues were stained with HE, anti-EZH2 antibody, anti-TβRII antibody and anti-ASCL1 antibody. Representative images show normal tissues (top and third rows) and SCLC tissues (second and bottom rows) from two patients, as indicated. (Insets) ASCL1 staining in boxed region is shown at high magnification. Scale bars are 30 μm. (b) Expression profiles of samples in (a) and Supplementary Figure S6 were analyzed by defining scores (s) that corresponded to the frequency of positive cells (f) in each sample, as follows: s = 4 for 80⩽ f⩽ 100; s = 3 for 50⩽ fo80; s = 2 for 20⩽ fo50; s = 1 for 0ofo20; s = 0 for f = 0. Data represent means. *Po0.05; ***Po0.001. HE, hematoxylin-eosin; LCNEC, large cell neuroendocrine carcinoma; normal, normal lung; SCLC, small cell carcinoma; Ad, adenocarcinoma; Sq, squamous cell carcinoma. Cell Discovery www.nature.com/celldisc Patient #2 Patient #1 Normal lung tissues Normal lung tissues SCLC tissues SCLC tissues Average score (s) Average score (s) Average score (s) EZH2 promotes SCLC by suppressing TGF-β signaling TGF-β TGF-β Normal lung epithelial cells SCLC cells P P TβRI TβRI PRC2 PRC2 P P (ALK-5) (ALK-5) TβRII TβRII SUZ12 SUZ12 EED EED Smad2/3 Smad2/3 EZH2 EZH2 Smad2/3 Smad2/3 Smad4 Smad4 open chromatin open chromatin ASCL1 ASCL1 TGFBR2 TGFBR2 apoptosis apoptosis Figure 8 Disruption of TGF-β-mediated tumor suppression in SCLC cells. (a) In normal lung epithelial cells, TGF-β induces apoptosis through the suppression of ASCL1 expression in a Smad-dependent manner. (b) In SCLC cells, high EZH2 expression attenuates TβRII expression through histone H3K27 tri-methylation. Disruption of TGF-β signaling elevates ASCL1 expression, which in turn protects SCLC cells from apoptosis. SCLC, small cell lung cancer; TGF-β, transforming growth factor-β. Microplate Reader (Bio-Rad, Melville, NY, USA), followed by gel electrophoresis, and transferred to Fluoro Trans W mem- subtraction of absorbance at 595 nm. brane (Pall, East Hills, NY, USA). Chemiluminescence images were captured on ImageQuant LAS4000 (Fujifilm, Tokyo, In vivo tumor growth assay Japan). Image J software (NIH) was used to quantify blot band In vivo experiments were performed as previously described intensities in Figure 3c. See also Supplementary Information. [52]. The protocols were approved by the Animal Ethics Committee of The University of Tokyo (approval number: Lentiviral vector construction and lentivirus production 2186). BALB/c nu/nu mice (4 weeks, male) were purchased from A lentiviral vector system (provided by Dr Hiroyuki Charles River Laboratories (Yokohama, Japan). Cells were Miyoshi, RIKEN) was used to induce specific gene introduction resuspended in culture media supplemented with 50% BD and knockdown. For gene introduction, we inserted com- Matrigel (BD Bioscience, San Jose, CA, USA), and then sub- plementary DNAs encoding the human wild-type TGFBR2, cutaneously injected into mice (3 × 10 cells in 100 μl per mouse). TGFBR2 with a truncated intracellular domain and a carboxy- For xenograft transplantations, H345 cells were treated with terminal GFP tag (dnTβRII), and human wild-type ASCL1, siRNA against ASCL1 for 72 h in vitro, followed by sub- into the entry vector, pENTR201 [55]. Then, pENTR201 cutaneous transplantation of ASCL1-silenced cells into mice. vectors were inserted into the lentiviral destination vector, pCSII-EF-RfA or pCSII-CMV-RfA, as previously described [56]. Vectors encoding GFP were also generated as controls. Gene expression analysis Total RNA was extracted with the RNeasy Mini Kit Similarly, shRNAs designed to knockdown a specified gene (Qiagen, Valencia, CA, USA). Complementary DNA was syn- were inserted into the entry vector pENTR4-H1. Then, thesized with the random hexamer protocol described in the pENTR4-H1 vectors that carried shRNAs specific for human PrimeScript II 1st strand complementary DNA Synthesis Kit ASCL1 or EZH2 were inserted into the lentiviral destination (Takara, Otsu, Japan). For qRT-PCR analysis, gene expression vector, pCS-RfA-EG. The shRNA target sequences for was quantified with the StepOne Plus Real time-PCR System gene knockdowns were obtained from Dharmacon siDESIGN (Life Technologies, Tokyo, Japan) and the Fast SYBR Green Center (GE Healthcare, Piscataway, NJ, USA; Supplementary Master Mix (Life Technologies). The expression level of each Table S5). Lentiviral vectors were produced as described pre- gene was normalized to that of glyceraldehyde-3-phosphate viously [53]. Culture supernatant was concentrated with Lenti-X dehydrogenase (GAPDH). Primer sequences are shown in Concentrator (Clontech, Palo Alto, CA, USA), then used for Supplementary Table S4. lentiviral vector infections. siRNA Immunoblotting An Accell-siRNA SMARTpool specific for human ASCL1 Immunoblotting was previously described [53, 54]. Cells was purchased from Dharmacon (GE Healthcare), and recon- were lysed in radio-immunoprecipitation assay buffer (50 mM stituted in 1 × siRNA buffer (100 μM, GE Healthcare). The Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, siRNA target sequences are shown in Supplementary Table S6. and 0.5% sodium deoxycholate) with the Complete Protease Cells were treated with siRNA at a final concentration of 1 μM. Inhibitor Cocktail (Roche Diagnostics, Tokyo, Japan) and an EDTA-free phosphatase inhibitor cocktail (Nacalai Tesque). Protein concentrations were quantified with the BCA Protein Cell cycle analysis Assay (ThermoFisher Scientific, Yokohama, Japan). Equal After washing with phosphate-buffered saline, cells were amounts of total protein were applied to SDS-polyaclylamide fixed with ice-cold 70% EtOH in phosphate-buffered saline Cell Discovery www.nature.com/celldisc | Fumihiko Murai et al. and stored for more than 16 h at − 20 °C. The fixed cells were performed with the analysis of variance; one-way analysis were resuspended in phosphate-buffered saline containing of variance (Tukey’s method) was applied to comprehensive − 1 0.25 mg ml RNase A, and then incubated at 37 °C for 1 h. gene expression analyses and immunohistochemical analyses. − 1 Cells were labeled with 50 μgml propidium iodide (PI, Life The repeated measure analysis of variance was applied to in vivo Technologies) for 30 min at 4 °C; then, cell cycle analysis was experiments. Significant differences were defined as Po0.05. performed with a Gallios Flow Cytometer (Beckman Coulter, Miami, FL, USA). The distribution of each cell cycle stage was Conflict of Interest analyzed with the FlowJo (Tomy Digital Biology, Tokyo, Japan) Watson Pragmatic cell cycle analysis program. The authors declare no conflict of interest. ChIP-qRT-PCR analysis and ChIP-seq analysis Acknowledgements ChIP was previously described [51, 57]. See also Supplementary Information. ChIP-qRT-PCR analyses were We thank Yasuyuki Morishita and Kei Sakuma (The performed with the StepOne Plus Real time-PCR System and University of Tokyo) for technical assistance, and Hiroyuki FastStart Universal SYBR Green Master (Rox) (Roche). Primer Miyoshi (RIKEN) for providing the lentiviral vectors. This sequences for each gene locus are shown in Supplementary work was supported by a KAKENHI Grant-in-Aid for scientific Table S4. research on Innovative Area (Integrative Research on Cancer For ChIP-seq analysis, total amounts of double stranded Microenvironment Network; grant number 22112002) from the DNA were quantified with Qubit dsDNA HS Assay Kits (Life Ministry of Education, Culture, Sports, Science and Technology Technologies). Libraries were prepared with IonXpress Plus (MEXT) of Japan, a KAKENHI Grant-in-Aid for Young Fragment Library Kit (Life Technologies). Libraries were Scientists (B) (grant number 22700967) from the Japan Society quantified with Ion Library Quantification Kit (Life Technolo- for the Promotion of Science (JSPS) (SE), the Strategic Basic gies). Emulsion PCR and product purification were performed Research Program from Japan Science and Technology Agency with Ion PGM Template OT2 400 Kit (Life Technologies). The (KM), and a Specific Research Grant from The Cell Science amplified samples were sequenced with Ion PGM Sequencer Research Foundation (SE). This study was performed in part as (Life Technologies) with Ion PGM Hi-Q Sequencing Kit. The a research program for the Project for Development of Inno- acquired read tags were mapped onto the NCBI hg19 human vative Research on Cancer Therapeutics (P-Direct), MEXT. genome assembly. Analyses of ChIP-seq data were previously described [58, 59]. The significant Smad2/3 binding region was Author contributions calculated using CisGenome version 2 using default parameters except for window size (400 bp), cut-off counts (⩾10 reads (P = 0.023516)), and step size (25 bp). Raw ChIP-seq and peak FM, SE and KM designed the study, analyzed the data and call data are available at GEO (GSE63871). wrote the manuscript. FM and SE performed experiments. DK assisted with and performed the ChIP-seq analysis. 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This work is licensed under a Creative Commons 51 Koinuma D, Tsutsumi S, Kamimura N et al. Chromatin Attribution 4.0 International License. The images or immunoprecipitation on microarray analysis of Smad2/3 other third party material in this article are included in the article’s binding sites reveals roles of ETS1 and TFAP2A in trans- Creative Commons license, unless indicated otherwise in the forming growth factor β signaling. Mol Cell Biol 2009; 29: credit line; if the material is not included under the Creative 172–186. Commons license, users will need to obtain permission from the 52 Shirai YT, Ehata S, Yashiro M, Yanagihara K, Hirakawa license holder to reproduce the material. To view a copy of this K, Miyazono K. Bone morphogenetic protein-2 and -4 play license, visit http://creativecommons.org/licenses/by/4.0/ Cell Discovery www.nature.com/celldisc http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Discovery Springer Journals

EZH2 promotes progression of small cell lung cancer by suppressing the TGF-β-Smad-ASCL1 pathway

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Life Sciences; Life Sciences, general; Cell Biology; Stem Cells; Cell Culture; Cell Cycle Analysis; Cell Physiology
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OPEN Citation: Cell Discovery (2015) 1, 15026; doi:10.1038/celldisc.2015.26 www.nature.com/celldisc ARTICLE EZH2 promotes progression of small cell lung cancer by suppressing the TGF-β-Smad-ASCL1 pathway 1 1 2 2 1 Fumihiko Murai , Daizo Koinuma , Aya Shinozaki-Ushiku , Masashi Fukayama , Kohei Miyaozono , Shogo Ehata Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan; Department of Pathology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan Transforming growth factor-β (TGF-β) induces apoptosis in many types of cancer cells and acts as a tumor suppressor. We performed a functional analysis of TGF-β signaling to identify a molecular mechanism that regulated survival in small cell lung cancer cells. Here, we found low expression of TGF-β type II receptor (TβRII) in most small cell lung cancer cells and tissues compared to normal lung epithelial cells and normal lung tissues, respectively. When wild-type TβRII was overexpressed in small cell lung cancer cells, TGF-β suppressed cell growth in vitro and tumor formation in vivo through induction of apoptosis. Components of polycomb repressive complex 2, including enhancer of zeste 2 (EZH2), were highly expressed in small cell lung cancer cells; this led to epigenetic silencing of TβRII expression and suppression of TGF-β- mediated apoptosis. Achaete-scute family bHLH transcription factor 1 (ASCL1; also known as ASH1), a Smad-dependent target of TGF-β, was found to induce survival in small cell lung cancer cells. Thus, EZH2 promoted small cell lung cancer progression by suppressing the TGF-β-Smad-ASCL1 pathway. Keywords: small cell lung cancer; epigenetics; apoptosis; EZH2; TGF-β; ASCL1; Smad Cell Discovery (2015) 1, 15026; doi:10.1038/celldisc.2015.26; published online 22 September 2015 Introduction Transforming growth factor (TGF)-β is a cytokine that exerts many biological functions. TGF-β binds to Lung cancer causes mortality more than any other two different types of serine-threonine kinase receptors, type of cancer [1]. Lung cancer is mainly classified as termed type II (TβRII) and type I receptors (TβRI is also known as activin receptor-like kinase 5, ALK-5) either small cell lung cancer (SCLC) or non-small cell expressed on the cell surface. Upon ligand binding, lung cancer (NSCLC), with incidences of ~ 15 and 84%, two TβRIIs and two TβRIs form a heterotetrameric respectively [2]. SCLC, high-grade neuroendocrine complex, and this activated complex phosphorylates tumors, has been reported to have the worst prognosis, the receptor-regulated Smads (R-Smads), Smad2 and with a 5-year survival rate of ~ 5% [3]. Those patients Smad3. The phosphorylated R-Smads form complexes are mostly treated with anti-cancer drugs and/or with their common-partner Smad (Co-Smad), Smad4, radiation. However, a primary clinical issue is the and the R-Smad/Co-Smad complex translocates to the acquisition of chemoresistance in SCLC cells [4]. Thus, nucleus. Then, the complexes associate with various it is essential to develop novel strategies for SCLC transcription factors and transcriptional co-activators therapy. For successful drug discovery, it is important or co-repressors, which in turn, regulate transcription to find molecular mechanism(s) that maintain survival of a wide spectrum of target genes [5–7]. in SCLC cells. TGF-β has been reported to have bi-directional roles in cancer progression [8]. TGF-β induces cell cycle arrest at G1 by regulating expression of cyclin- Correspondence: Kohei Miyazono dependent kinase inhibitor 1 A (CDKN1A, also Tel: +81 3 5841 3345; Fax: +81 3 5841 3354; known as p21), CDKN2B (also known as p15), E-mail: [email protected] Received 22 February 2015; accepted 3 August 2015 the v-myc avian myelocytomatosis viral oncogene EZH2 promotes SCLC by suppressing TGF-β signaling NSCLC SCLC SMAD7 2.5 *** ** ** A549 H146 H82 H209 H345 (-) -+ - + - + - + - + TGF-β 1.5 TGF-β pSmad2 Smad2/3 0.5 A549 H441 H146 H82 H209 H345 NSCLC SCLC TGFBR1 TGFBR2 1.2 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 NSCLC SCLC NSCLC SCLC SMAD2 SMAD4 SMAD3 2.5 1 3.5 2 0.8 2.5 1.5 0.6 1.5 1 0.4 0.2 0.5 0.5 0 0 0 NSCLC SCLC NSCLC SCLC NSCLC SCLC NSCLC cells SCLC cells Normal lung epithelial cells 2 SMAD4 SMAD2 TGFBR1 TGFBR2 SMAD3 -2 Figure 1 TGF-β signal transduction is attenuated in several SCLC cells due to decreased expression of TβRII. (a and b) SCLC and NSCLC cells were stimulated with TGF-β for 2 h. (a) Immunoblot of cell lysates probed with the indicated antibodies; (b) qRT-PCR analysis of SMAD7 expression. Data represent mean ± s.d. **Po0.01; ***Po0.001. (c) qRT-PCR analysis shows expression of TGF-β signaling components in SCLC and NSCLC cells. Data represent mean ± s.d. (d) Comprehensive gene expression analysis from the NCBI GEO database (GSE32036) shows expression profiles of TGF-β signaling components in normal lung epithelial cells (n = 59), SCLC cells (n = 29) and NSCLC cells (n = 119). Raw data were normalized by quantile algorithm. The color indicates the distance from the median of each row. GEO, gene expression omnibus; NCBI, National Center for Biotechnology Information; NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; TGF-β, transforming growth factor-β; qRT-PCR, quantitative real-time reverse transcription-PCR. Cell Discovery www.nature.com/celldisc Relative expression (/GAPDH) Relative expression (/GAPDH) H441 H441 A549 A549 H82 H82 H146 H146 H209 H209 H345 H345 Relative expression (/GAPDH) Relative expression (/GAPDH) H441 H441 A549 A549 H82 H82 H146 H146 H209 H209 H345 H345 Relative expression (/GAPDH) Relative expression (/GAPDH) H441 A549 H82 H146 H209 H345 Distance from median Fumihiko Murai et al. homolog (MYC), and cell division cycle protein 25A significant differences (Figure 1d, and Supplementary (CDC25A) [9]. TGF-β also induces apoptosis in several Figure S1). Since TGF-β signal is transduced even in types of cancer cells through multiple mechanisms the low expression levels of Smad3 if Smad2 is [9, 10]. In ~ 80% of NSCLC tissues, expression of expressed in H146 cells (Figure 1b), we assumed that TβRII was lower than that of normal lung tissues [11]. TGF-β signal transduction was attenuated in SCLC It was also shown that restoration of TβRII into cells through the decreased expression of TβRII, and NSCLC cells inhibited their growth in vitro and in vivo. therefore, we decided to focus on the roles of TβRII in Conversely, TGF-β also plays critical roles in cancer SCLC in the present study. metastasis via the epithelial-mesenchymal transition (EMT) [12]. Once EMT occurs in NSCLC cells, they TβRII suppresses SCLC tumor growth through acquire mesenchymal characteristics, which results in TGF-β-induced apoptosis invasion and metastasis [13]. In NSCLC cells, thyroid To examine the roles of TGF-β in SCLC progres- transcription factor-1 (TTF-1) suppresses TGF-β- sion, wild-type TβRII was introduced into H82 cells mediated EMT and inhibits cell migration and inva- (H82-TβRII cells) or H345 cells (H345-TβRII cells) sion [14]. Thus, the roles of TGF-β in the progression of with lentiviral vectors. Both phosphorylation of Smad2 NSCLC has been intensively studied. In contrast to and induction of SMAD7 by TGF-β were observed in NSCLC, the roles of TGF-β in the progression of TβRII-expressing cancer cells, but not in control SCLC SCLC have not been fully investigated. A few studies cells that expressed green fluorescent protein (GFP) have reported that expression of TGFBR2 (the gene that alone (H82-GFP cells and H345-GFP cells; Figure 2a encodes TβRII) was decreased in some SCLC cells, but and b). Thus, TGF-β signal transduction was success- the mechanisms were not detailed [15, 16]. Therefore, fully recovered by expressing TβRII. These cells were the present study aimed to clarify the roles of TGF-β in subcutaneously xenografted into nude mice to examine SCLC cells, to identify the mechanisms involved in the tumor growth in vivo. Tumor formation was decreased downregulation of TβRII, and to identify novel TGF-β in mice injected with H82-TβRII cells and H345-TβRII target genes in this type of cancer. cells, compared with mice xenografted with the control cells (Figure 2c). Although the expression of TGFBR2 Results mRNA was low (Figure 1c) and the TβRII protein was not detected by immunoblot analysis (data not shown), Downregulation of TβRII expression in SCLC cells Smad-dependent TGF-β signal was transduced in First, we investigated whether TGF-β signals were H146 cells (Figures 1a and b), suggesting that a low transduced in SCLC cells. Phosphorylation of Smad2 level of TβRII protein may be functioning in these cells. and induction of SMAD7, one of the direct targets of Thus, a GFP-tagged dominant-negative form of TβRII TGF-β, were examined in human SCLC cells (H146, (dnTβRII) was overexpressed in H146 cells (H146- H82, H209 and H345) and in NSCLC cells (A549 dnTβRII cells; Supplementary Figure S2a). Both phos- and H441) with immunoblotting and quantitative phorylation of Smad2 and induction of SMAD7 were real-time reverse transcription-PCR (qRT-PCR) inhibited by the introduction of dnTβRII (Supplemen- analyses. TGF-β-mediated phosphorylation of Smad2 tary Figures S2b and S2c). When these cells were sub- was observed in H146 cells as well as in A549 and cutaneously xenografted into mice, tumor formation H441 cells (Figure 1a and data not shown, see Isogaya was accelerated in mice injected with H146-dnTβRII et al [17]). Induction of SMAD7 by TGF-β was also cells compared with those injected with H146-GFP observed in H146, A549 and H441 cells (Figure 1b). cells (Supplementary Figure S2d). These results However, in the other SCLC cells, these responses suggested that TGF-β may act as a tumor suppressor were not induced by TGF-β. A qRT-PCR analysis in vivo. We assessed angiogenesis in these tumor tissues also showed that expression of TGFBR2 and SMAD3 by staining for CD31 expression (also known as was decreased in SCLC cells, but other TGF-β signal- platelet/endothelial cell adhesion molecule 1, ing components, including SMAD2, SMAD4 and PECAM1). However, there was no remarkable differ- TGFBR1 (the gene that encodes TβRI), were expressed ence in CD31 expression between H146-GFP and at normal levels in these cells (Figure 1c). These H146-dnTβRII cells xenografted tissues (Supplementary expression profiles were confirmed with comprehensive Figure S2e). This finding suggested that tumor gene expression analysis data from the gene expression suppression was mediated by the effect of TGF-β on omnibus (GEO) of the National Center for Bio- SCLC cells, not by its effect on angiogenesis in the technology Information (NCBI) with statistically tumor microenvironment. Cell Discovery www.nature.com/celldisc | EZH2 promotes SCLC by suppressing TGF-β signaling SMAD7 SMAD7 *** *** 3.5 0.6 0.5 -- + ++- - + --++ TGF-β 2.5 TGF-β 0.4 (-) 0.3 pSmad2 pSmad2 1.5 TGF-β 0.2 Smad2/3 Smad2/3 0.5 0.1 TβRII TβRII α-tubulin α-tubulin ** 700 50 *** 0.4 *** 0.4 400 0.3 0.3 (-) 0.2 0.2 TGF-β 0.1 10 0.1 0 0 0 0 TGF-β H345-GFP H345-TβRII sub-G0/G1 G0/G1 S G2/M () - -- + ++- - + TGF-β uncleaved PARP cleaved pRB pSmad2 Smad2/3 TGF-β () - ()++ () - () () + α-tubulin H345-GFP H345-TβRII PI intensity CDKN1A (p21) CDKN2B (p15) CDC25A MYC *** *** ** *** 3 4 2 2 3 1.5 1.5 (-) 2 1 1 TGF-β 1 0.5 0.5 0 0 0 0 Cell Discovery www.nature.com/celldisc Cell number Relative expression (/GAPDH) Tumor volume (mm ) HaCaT H82 H82-GFP H345-GFP H82- H82-TβRII H345-TβRII TβRII Tumor volume (mm ) H82- GFP Relative expression H345-GFP (/GAPDH) H345-TβRII HaCaT H345 H345-GFP H345- H345-TβRII GFP % of distribution H345- TβRII Relative expression (/GAPDH) Relative expression Absorbance (A450-A595) (/GAPDH) HaCaT H345-GFP H82 H82 H345-TβRII H82-GFP H82-GFP H82-TβRII H82-TβRII Relative expression (/GAPDH) Relative expression Absorbance (A450-A595) (/GAPDH) HaCaT HaCaT H345 H345-GFP H345 H345 H345- GFP H345-TβRII H345-GFP H345-GFP H345- TβRII H345-TβRII H345 -TβRII Fumihiko Murai et al. We postulated that TGF-β might suppress the pro- and SCLC tissues displayed higher expression of the liferative activity of SCLC cells. When we restored enhancer of zeste 2 (EZH2), SUZ12 polycomb TβRII expression in these cells, TGF-β significantly repressive complex 2 subunit (SUZ12), and embryonic suppressed the in vitro proliferation of H82 cells and ectoderm development (EED) than normal lung epi- H345 cells (Figure 2d). Moreover, dnTβRII expression thelial cells and normal lung tissues, respectively canceled TGF-β-mediated growth inhibition in H146 (Figure 3a, and Supplementary Figure S3). We also cells (Supplementary Figure S2f). Cell cycle analysis found that their expressions were increased in SCLC revealed that TGF-β increased the sub-G0/G1 popu- cells but not in NSCLC cells (Figure 3b and c). These lation in H345-TβRII cells compared with H345-GFP molecules associate to form the polycomb repressive cells (Figure 2e). TGF-β also induced the cleavage of complex 2 (PRC2), which inhibits gene transcription poly (ADP-ribose) polymerase (PARP) in H345-TβRII through methylation of lysine 27 in histone H3 cells (Figure 2f), which suggested that TGF-β (H3K27me3). High expression of H3K27me3 was decreased the number of SCLC cells by inducing observed in SCLC cells, which was attenuated by the apoptosis. TGF-β is also known to suppress prolifera- treatment with an EZH2 inhibitor, GSK343 tion of many types of cells by regulating CDK activa- (Figure 3c), suggesting that PRC2 is implicated in tors or inhibitors. We found that expression levels of transcriptional regulation of several genes in SCLC CDKN1A, CDKN2B, MYC or CDC25A in H345- cells. H146 cells showed different expression profiles of TβRII cells were not markedly altered by TGF-β the PRC2 complex from those in the other SCLC cells. (Figure 2g). However, in human keratinocyte The expression level of EZH2 protein was similar to HaCaT cells, TGF-β upregulated the expression of those in the other SCLC cells, while the expression CDKN1A and CDKN2B and downregulated the levels of EZH2, EED and SUZ12 mRNAs were lower expression of MYC and CDC25A. Moreover, expres- than those in the other SCLC cells (Figures 3b and c). sion of retinoblastoma protein (pRB) was not detected In order to directly examine whether EZH2 is in H345 cells (Figure 2f). These results suggested that involved in downregulation of TGFBR2, chromatin TGF-β suppressed proliferation of SCLC cells by immunoprecipitation (ChIP)-qRT-PCR analysis using inducing apoptosis, but not by regulating the cell cycle. anti-EZH2 antibody was performed (Figure 4a). EZH2 bound to the several loci in TGFBR2 in H345 cells. Moreover, transcription of TGFBR2 mRNA was Importance of EZH2-mediated silencing of TβRII for increased in GSK343-treated SCLC cells (Figure 4b). SCLC tumor formation When EZH2 expression was silenced in H345 cells with Histone methyltransferase mediates methylation on a short hairpin RNA (shRNA) (H345-shEZH2), the lysine or arginine residues of histones of the H3 and H4 knockdown of EZH2 led to an increase in TGFBR2 families to regulate transcription of various genes [18]. expression (Figure 4c); in turn, TGF-β induced Smad2 We postulated that, in SCLCs, the expression of TβRII phosphorylation and SMAD7 expression (Figures 4d might be epigenetically silenced through histone and e). These results suggested that EZH2 played a modification by histone methyltransferases. Therefore, we investigated histone methyltransferase expression in critical role in downregulating TβRII in SCLC cells. We also investigated whether EZH2 was important SCLC cells. Comprehensive gene expression analyses for TGF-β-mediated apoptosis and tumor formation in from the NCBI GEO data set revealed that SCLC cells Figure 2 TβRII suppresses SCLC tumor growth through TGF-β-induced apoptosis. (a and b) SCLC cells were infected with lentivirus vectors encoding GFP (H82-GFP and H345-GFP) or TβRII (H82-TβRII and H345-TβRII). Cells were stimulated with TGF-β for 2 h. (a) Immunoblots of cell lysates probed with the indicated antibodies. (b) qRT-PCR analysis of SMAD7 expression. Data represent mean ± s.d. ***Po0.001. (c) Mice received subcutaneous transplantations of H82-GFP (n = 15) and H82-TβRII cells (n = 12) or H345-GFP (n = 7) and H345-TβRII cells (n = 9), and tumor volumes were measured 16 days (H82) or 17 days (H345) after transplantation. Data represent mean ± s.e.m. *Po0.05; **Po0.01. (d) Cell proliferation assay. SCLC cells were stimulated with TGF-β for 6 days (H82) or 12 days (H345). Data represent mean ± s.d. ***Po0.001. (e) Cell cycle analysis. (left panels) H345-GFP and H345-TβRII cells were unstimulated (top panels) or stimulated (bottom panels) with TGF-β for 12 days; the number of cells in each cell cycle stage is shown (color coding shown in right panel). (right panel) Percentage of cells in each cell cycle stage. (f) Immunoblots of cell lysates probed with the indicated antibodies. SCLC and control HaCaT cells were stimulated with TGF-β for 48 h (HaCaT) or 12 days (H345). (g) qRT-PCR analysis shows cell cycle-related gene expression. H345-GFP, H345-TβRII and HaCaT cells were stimulated with TGF-β for 2 h (MYC)or24h (CDKN1A, CDKN2B and CDC25A). Data represent mean ± s.d. *Po0.05; **Po0.01; ***Po0.001. GFP, green fluorescent protein; SCLC, small cell lung cancer; TGF-β, transforming growth factor-β; qRT-PCR, quantitative real-time reverse transcription-PCR. Cell Discovery www.nature.com/celldisc | EZH2 promotes SCLC by suppressing TGF-β signaling NSCLC cells SCLC cells Normal lung epithelial cells EED EZH2 SUZ12 -2 EZH2 EED SUZ12 3.5 3.5 1.6 1.4 3 3 1.2 2.5 2.5 0.8 1.5 1.5 0.6 1 1 0.4 0.5 0.5 0.2 0 0 NSCLC SCLC NSCLC SCLC NSCLC SCLC EZH2 NSCLC SCLC A549 H441 H82 H146 H209 H345 GSK343 H3K27me3 H3S10p H3 (total) EZH2 α-tubulin NSCLC SCLC Figure 3 EZH2 is highly expressed in SCLC cells. (a) Expression profiles of PRC2 components in SCLC cells, based on the data in Figure 1d. The color indicates the distance from the median of each row. (b) qRT-PCR analysis shows expression of PRC2 components in SCLC and NSCLC cells. Data represent mean ± s.d. (c) Immunoblot of cell lysates probed with the indicated antibodies. (left panels) SCLC and NSCLC cells were treated with GSK343 in a wide concentration range (0, 0.4, 2 and 10 μM) for 3 days. (right panel) Relative expression of EZH2 protein in each cell without GSK343 was quantified. NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; qRT-PCR, quantitative real-time reverse transcription-PCR. H345 cells. Cell cycle analysis showed that TGF-β ChIP-sequencing (ChIP-seq) analysis was performed increased the sub-G0/G1 population in H345-shEZH2 with an anti-Smad2/3 antibody in H345-TβRII cells cells, but not in H345-shNTC cells (Figure 4f). to identify comprehensively Smad2/3-regulated genes. Moreover, the ability of H345-shEZH2 cells to form The characteristics of SCLC cells are thought to tumors was attenuated, compared to that of depend on the expression of several neuroendocrine- H345-shNTC cells (Figure 4g). related genes, including the achaete-scute family basic helix-loop-helix transcription factor 1 (ASCL1, also ASCL1 is negatively regulated by TGF-β in a known as ASH1), synaptophysin (SYP), neural cell Smad-dependent manner adhesion molecule (NCAM) and v-myc avian myelo- We next attempted to identify TGF-β target cytomatosis viral oncogene lung carcinoma derived genes that were involved in SCLC cell apoptosis. homolog (MYCL) [19]. Therefore, we focused on Cell Discovery www.nature.com/celldisc Relative expression (/GAPDH) H441 A549 H146 H82 H209 H345 Relative expression (/GAPDH) H441 A549 H146 H82 H209 H345 Relative expression (/GAPDH) H441 A549 Relative expression H146 H82 A549 H209 H441 H345 H82 H146 H209 Distance from median H345 Fumihiko Murai et al. Smad2/3 binding to these gene loci. ChIP-seq analysis locus or MYCL locus, in H345-TβRII cells (Figure 5a). showed that, in the presence of TGF-β, Smad2/3 sig- Among the binding regions in the NCAM1 locus, nificantly bound to two loci of the ASCL1 gene and Smad2/3 strongly bound to the first intron. Compre- several loci of the NCAM1 gene, but not to the SYP hensive gene expression analysis from the NCBI GEO TGFBR2 TGFBR2 TGFBR2 TGFBR2 HPRT1 (locus 1) (locus 2) (locus 3) (locus 4) TGFBR2 ** * * 6 0.01 0.015 0.015 0.008 0.008 0.008 0.006 0.006 4 ( ) 0.01 0.01 0.006 GSK343 0.004 0.004 0.004 0.005 0.005 0.002 0.002 0.002 H82 H146 H209 H345 0 0 0 0 0 EZH2 TGFBR2 *** *** SMAD7 *** *** 2.5 2.5 *** ** *** *** 2 2 *** 1.5 1.5 ( ) TGF- TGF- EZH2 0.5 0.5 pSmad2 0 0 0 Smad2/3 -tubulin TGF- H345-shNTC H345-shEZH2 #1 H345-shEZH2 #2 sub-G0/G1 G0/G1 S G2/M ( ) ( ) TGF- ( )( )( )( )( )( ) H345- H345- H345- shNTC shEZH2 #1 shEZH2 #2 PI intensity H345-shNTC H345-shEZH2 #1 100 H345-shNTC H345-shEZH2 #1 ** 123 4 Week after transplantation Cell Discovery www.nature.com/celldisc % of input Relative expression (/GAPDH) H345-shNTC IgG1 Cell number H345-shEZH2 #1 EZH2 H345-shEZH2 #2 % of input Relative expression (/GAPDH) IgG1 H345-shNTC EZH2 H345-shEZH2 #1 % of input H345-shEZH2 #2 IgG1 EZH2 % of input H345- GFP H345- 3 IgG1 Tumor volume (mm ) T RII EZH2 H345- % of input shNTC H345- shEZH2 #1 IgG1 H345- % of distribution shEZH2 #2 EZH2 Relative expression Relative expression (/GAPDH) (/GAPDH) H345-GFP H345-T RII H345-shNTC H345-shEZH2 #1 H345-shEZH2 #2 EZH2 promotes SCLC by suppressing TGF-β signaling data sets showed that the mRNA levels of each of these TGF-β could upregulate SMAD7 and downregulate neuroendocrine-related genes were elevated in SCLC ASCL1 expression (Figure 5f). This result suggested cells and SCLC tissues, except for SYP in the SCLC that, similar to SMAD7, the expression of ASCL1 was tissue (Supplementary Figures S4a and S4b). Next, directly regulated by TGF-β. TGF-β regulation of these genes was assessed by qRT- TGF-β can activate both Smad and non-Smad PCR analysis (Figure 5b and Supplementary Figures pathways. To determine which pathway played a S5a and S5b). In H345-TβRII cells, treatment with predominant role in regulating ASCL1 expression, we TGF-β caused ASCL1 expression to decrease within used a shRNA (shSmad4) to silence the expression of 1 h, and it reached a minimum at 4 h. TGF-β also Smad4 in H345-TβRII cells. The knockdown of Smad4 decreased ASCL1 expression within 2 h in H146 cells. attenuated the induction of SMAD7 by TGF-β However, MYCL, NCAM1 and SYP were not (Figures 5g and h). Moreover, TGF-β-mediated regulated by TGF-β in these cells. Moreover, TGF-β downregulation of ASCL1 expression was canceled in suppressed ASCL1 protein expression in SCLC cells H345-TβRII cells by shSmad4, but not in cells infected (Figure 5c). In accordance with a previous analysis with negative control shRNA (shNTC). These results (Supplementary Figure S4a), in NSCLC cells, ASCL1 suggested that TGF-β directly suppressed ASCL1 was only weakly expressed, and it was not regulated by expression in a Smad-dependent manner. TGF-β (A549 and H441) (Supplementary Figure S5c). These results suggested that ASCL1 was a TGF-β ASCL1 promotes survival of SCLC cells target, and regulation of ASCL1 by TGF-β was specific Next, we determined whether negative regulation for SCLC cells. of ASCL1 transcription was important for TGF-β- We then focused on the molecular mechanism mediated apoptosis of SCLC cells. When expression of underlying TGF-β-mediated transcriptional regulation ASCL1 was silenced in H345 cells with shRNA of ASCL1 in SCLC cells. ChIP-qRT-PCR analysis was (shASCL1) (Figure 6a), cell growth was inhibited performed to confirm whether Smad2/3 bound in by the induction of apoptosis (Figures 6b and c). the ASCL1 locus. We found that TGF-β-stimulated Conversely, when ASCL1 was exogenously introduced H345-TβRII cells showed a sixfold-enrichment of by transferring lentiviral vectors into H345-TβRII cells, Smad2/3 binding to these loci, compared with those to cell cycle analysis revealed that the TGF-β-mediated hemoglobin beta (HBB) locus (Figure 5d). In addition increase in the sub-G0/G1 population was attenuated to the recovery of TβRII expression and TGF-β signal (Figures 6d and e). These results suggested that ASCL1 transduction in H345-shEZH2 cells (see Figures 4d had an important role in enabling SCLC cells to escape and e), ASCL1 expression was decreased in these cells from TGF-β-induced apoptosis. (Figure 5e). Next, cycloheximide (CHX), a de novo Next, we performed subcutaneous transplantations protein synthesis inhibitor, was used to investigate in mice to determine whether knocking down ASCL1 whether ASCL1 expression was directly or indirectly expression would suppress tumor growth or formation. regulated by TGF-β. Even in the presence of CHX, We knocked down ASCL1 expression by treating cells Figure 4 EZH2-mediated silencing of TβRII is required for SCLC tumor formation. (a) qRT-PCR analysis post-immunoprecipitation with anti-EZH2 antibody shows EZH2 enrichment in the TGFBR2 locus of H345 cells. Hypoxanthine guanine phosphoribosyl transferase1 (HPRT1) was used as the negative control. Data represent mean ± s.d. TGFBR2 locus 1, chromosome 3: 30606416–30606492 bp; TGFBR2 locus 2, chromosome 3: 30605676–30605771 bp; TGFBR2 locus 3, chromosome 3: 30604616–30604714 bp; TGFBR2 locus 4, chromosome 3: 30603967–30604061 bp. *Po0.05; **Po0.01. (b) qRT-PCR analysis shows TGFBR2 expression. SCLC cells were treated with GSK343 (10 μM) for 7 days. Data represent mean ± s.d. (c) qRT-PCR analysis shows EZH2 and TGFBR2 expression. H345 cells were infected with lentivirus vectors with control shRNA (H345-shNTC) or shRNA that targeted EZH2 (H345-shEZH2). Data represent mean ± s.d. ***Po0.001. (d) Immunoblot of cell lysates from cells in (c) stimulated with TGF-β for 2 h and probed with the indicated antibodies. H345-GFP cells and H345-TβRII cells were negative and positive controls, respectively. (e) qRT-PCR analysis shows SMAD7 expression. Cells in (c) were stimulated with TGF-β for 4 h. Data represent mean ± s.d. H345-GFP cells and H345-TβRII cells were negative and positive controls, respectively. **Po0.01; ***Po0.001. (f) Cell cycle analysis of cells in (c) stimulated with TGF-β for 12 days. (left panels) The number of cells in each cell cycle stage is shown (color coding shown in right panel). (right panel) Percentage of cells in each cell cycle stage. (g) Mice received subcutaneous transplants of H345-shNTC cells (n = 7) or H345-shEZH2 #1 cells (n = 7). (left panels) Representative photographs 4 weeks after injection. Arrow heads indicate tumors. (right panel) Tumor volumes at the indicated time points. Data represent mean ± s.e.m. **Po0.01. SCLC, small cell lung cancer; qRT-PCR, quantitative real-time reverse transcription-PCR. Cell Discovery www.nature.com/celldisc | Fumihiko Murai et al. with small interfering RNA (siRNA) that targeted seven mice, but siASCL1-treated H345 cells formed ASCL1 (siASCL1). Control cells were treated with tumors in only one out of seven mice (Figure 6g). These negative control siRNA (siNTC) (Figure 6f). The results suggested that ASCL1 was involved in SCLC siNTC treated-H345 cells formed tumors in six out of tumor formation. 30 30 ASCL1 MYCL 20 20 10 10 0 0 -+ - + TGF-β 30 30 SYP ASCL1 NCAM1 pSmad2 10 10 Smad2/3 0 0 -tubulin SMAD7 ASCL1 12 1.5 1.2 0.9 0.6 H345-GFP 0.3 H345-TβRII 0 0 0 20406080 0 20406080 TGF- treatment time (h) TGF- treatment time (h) ASCL1 ASCL1 (locus 1) ASCL1 (locus 2) SMAD7 HBB *** 0.02 * 0.02 ** 0.02 0.3 *** (-) *** 0.25 0.015 0.015 0.015 0.2 TGF-β 1.5 0.01 0.01 0.01 0.15 0.1 0.005 0.005 0.005 0.05 0.5 0 0 IP IgG1 Smad2/3 IP IgG1 Smad2/3 IgG1 Smad2/3 IP IP IgG1 Smad2/3 SMAD7 ASCL1 SMAD4 SMAD7 ASCL1 *** *** * *** * *** *** *** *** *** *** *** *** 7 1.4 3 1.5 6 1.2 2.5 5 1 1.5 (-) 2 1 4 0.8 (-) 3 TGF-β 0.6 1.5 1 TGF-β 2 0.4 1 0.5 1 0.2 0.5 0.5 0 0 0 0 DMSO CHX DMSO CHX shNTC shSmad4 shSmad4 shNTC shSmad4 shNTC Cell Discovery www.nature.com/celldisc %of input Relative expression (/GAPDH) H345-GFP Mapped tag number H345-TβRII H345-GFP H345-GFP H345-TβRII H345-TβRII H345-GFP Fold change Relative expression (/GAPDH) H345-TβRII (/GAPDH/non-treatment) % of input H345-GFP H345-TβRII H345-GFP H345-GFP H345-TβRII H345-TβRII H345-GFP Relative expression (/GAPDH) H345-TβRII % of input H345-GFP H345-TβRII H345-GFP H345-GFP Fold change H345-TβRII (/GAPDH/non-treatment) H345-TβRII H345-GFP H345-TβRII Relative expression (/GAPDH) % of input H345-GFP H345-TβRII H345-GFP H345-GFP H345-TβRII H345-TβRII H345-GFP Relative expression (/GAPDH) H345-TβRII Relative expression (/GAPDH) H345-GFP H345- H345-TβRII GFP H345-shNTC H345-GFP H345- H345-shEZH2 #1 TβRII H345-TβRII H345-shEZH2 #2 EZH2 promotes SCLC by suppressing TGF-β signaling EZH2-mediated silencing of TβRII in SCLC tissues suppressed, which resulted in high ASCL1 levels and Finally, we compared the expression of EZH2, progression of SCLC. These results suggested new TβRII and ASCL1 in SCLC tissues and NSCLC tissues therapeutic strategies for targeting EZH2 and ASCL1 to that in normal lung tissues (Supplementary Table S1). in SCLC therapy. Immunohistochemical analysis showed that EZH2 was We demonstrated that TGF-β inhibited prolifera- strongly expressed in the nuclei of all SCLC cells tion of SCLC cells in vivo and in vitro. The SCLC cells (Figure 7a). However, its expression was not observed used in this study carried mutations in the DNA in those of normal lung epithelial cells. In contrast, binding region of TP53, and they did not express pro- TβRII was weakly expressed in SCLC cells, while it apoptotic pRB, except for the H209 cells (Figure 2f, was expressed on the surface of normal lung epithelial data not shown) [20]. Thus, TGF-β-mediated apoptosis cells (Figure 7a). Although ASCL1 was also detected in in SCLC cells may occur independently of p53 and nuclei of some SCLC cells, it was not observed in most pRB. TGF-β causes cell cycle arrest at the G1 phase or of normal lung epithelial cells and other lung cancer apoptosis in many types of cancer cells [9]. For cell cells (Figure 7a and Supplementary Figure S6). The cycle arrest at the G1 phase, TGF-β regulates p21, p15, expression profiles of EZH2, TβRII and ASCL1 are c-Myc and CDC25A in various types of cells, but it did shown in Figure 7b. The number of positive samples, not regulate these genes in SCLC cells. TGF-β was also grouped according to expression frequency, is shown in reported to induce apoptosis by inducing expression of Supplementary Table S2. When all lung tissues in BCL2-like 11 (BCL2L11, also known as Bim), growth Figure 7 were considered, there was a negative corre- arrest and DNA-damage-inducible beta (GADD45B) lation between EZH2 and TβRII and a positive cor- and inositol polyphosphate-5-phosphatase 145 kDa relation between EZH2 and ASCL1 (Supplementary (INPP5D, also known as SHIP) [9]. In contrast, our Table S3). These results supported the notion that comprehensive gene expression analysis and ChIP-seq EZH2 had a role in epigenetic silencing of TβRII in analysis showed that the TGF-β-mediated apoptosis in human SCLC tissues, and that the loss of TβRII SCLC cells appeared to be independent of those genes upregulated the expression of ASCL1. (data not shown). Based on these observations, we attempted to identify novel target(s) for TGF-β, which could regulate survival in SCLC cells. Discussion In many types of cancers, TβRII is dysfunctional through either genetic mutation or transcriptional The present study clarified the tumor suppressive role of TGF-β in SCLCs. We showed that TβRII was repression [21–23]. The TGFBR2 locus was shown to be mutated in the 10-adenine (A10) tract of exon 3 and expressed in normal lung epithelial cells, and that it inhibited abnormal cell growth by downregulating serine-threonine kinase domain in some cancers [21–24]. Previous studies demonstrated that genetic ASCL1 in a Smad-dependent manner (Figures 8a and b). However, EZH2 was highly expressed in mutations in the TGFBR2 A10 tract or expression of a truncated TβRII were not common in SCLC [15, 25]. SCLC cells, which epigenetically attenuated the expression of TβRII; thus, TGF-β signaling was Moreover, loss of heterozygosity in chromosome 3, Figure 5 ASCL1 is negatively regulated by TGF-β in a Smad-dependent manner. (a) ChIP-seq analysis using anti-Smad2/3 antibody. H345-TβRII cells were stimulated with TGF-β for 1.5 h. Arrows indicate transcription start sites and direction. (b) qRT-PCR analysis shows SMAD7 and ASCL1 expression in H345-GFP and H345-TβRII cells after TGF-β stimulation for the indicated times. Data represent mean ± s.d. (c) Immunoblot of cell lysates probed with the indicated antibodies. H345-GFP and H345-TβRII cells were stimulated with TGF-β for 12 days. (d) qRT-PCR analysis post-immunoprecipitation with anti-Smad2/3 antibody shows Smad2/3 enrichment. H345-GFP and H345-TβRII cells were stimulated with TGF-β for 1.5 h. Hemoglobin beta (HBB) was used as a negative control. Data represent mean ± s.d. ASCL1 locus 1, chromosome 12: 103351326–103352062 bp; ASCL1 locus 2, chromosome 12: 103351326–103352062 bp. *Po0.05; **Po0.01. (e) qRT-PCR analysis shows ASCL1 expression in indicated cells. Data represent mean ± s.d. ***Po0.001. (f) qRT-PCR analysis shows SMAD7 and ASCL1 expression. H345-GFP and H345-TβRII cells were stimulated with TGF-β for 4 h after pre-treatment with CHX (3 μM) for 24 h. Data represent mean ± s.d. ***Po0.001. (g) qRT-PCR analysis shows SMAD4 expression. H345-GFP and H345-TβRII cells were infected with lentivirus vector with control shRNA (shNTC) or shRNA that targeted Smad4 (shSmad4). Data represent mean ± s.d. (h) qRT-PCR analysis shows SMAD7 and ASCL1 expression. Cells in (g) were stimulated with TGF-β for 4 h. Data represent mean ± s.d. *Po0.05; ***Po0.001. ChIP, chromatin immunoprecipitation; TGF-β, transforming growth factor-β; qRT-PCR, quantitative real-time reverse transcription-PCR. Cell Discovery www.nature.com/celldisc | Fumihiko Murai et al. including in the TGFBR2 locus, was not observed in of TβRII in SCLC. In contrast, transcriptional SCLC [25]. Those studies indicated that genetic muta- repression of TGFBR2 was reported in retinoblastoma tion was not a common mechanism for the dysfunction and hematopoietic malignancies [23, 26]. ASCL1 * *** *** 2 0.4 *** *** *** *** *** 1.5 0.3 1 0.2 0.5 0.1 0 0 (-)TGF-β (-)TGF-β H345-TβΡΙΙ shNTC shASCL1 shASCL1 shASCL1 H345-TβRII shNTC shASCL1 shASCL1 shASCL1 #1 #2 #3 #1 #2 #3 sub-G0/G1 G0/G1 S G2/M H345 shNTC shASCL1 #1 shASCL1 #2 shASCL1 #3 shNTC shASCL1 shASCL1 shASCL1 #1 #2 #3 PI intensity GFP ASCL1 ASCL1 siNTC siASCL1 1.6 *** 1.4 1.2 -- + + --++ TGF-β 0.8 ASCL1 ASCL1 0.6 pSmad2 0.4 0.2 10 Smad2/3 α-tubulin GFP ASCL1 sub-G0/G1 G0/G1 S G2/M TGF-β H345-GFP H345-TβRII H345-GFP H345-TβRII (-) (+) TGF-β (-)(+)(-)(+) (-) (+) (-) (+) H345- H345- H345- H345- - GFP TβRII GFP TβRII GFP ASCL1 PI intensity Cell Discovery www.nature.com/celldisc Cell number Cell number Relative expression (/GAPDH) H345- GFP H345- TβRII H345- GFP H345- TβRII Relative expression (/GAPDH) siNTC siASCL1 Absorbance (A450-A595) % of distrubution % of distribution % of tumor formation siNTC siASCL1 EZH2 promotes SCLC by suppressing TGF-β signaling In this study, we showed that PRC2 components report was the first to reveal the mechanism underlying were highly expressed in most of the SCLC cells, and TGF-β regulation of ASCL1 expression. ASCL1, a that increased EZH2 expression caused silencing of member of the basic helix-loop-helix family transcrip- TβRII expression in SCLC cells. Sato et al [27]. tion factors, has a crucial role in the differentiation of reported that many kinds of genes, such as JUB, neural stem cells into neuronal lineages [40]. ASCL1 is PTRF, DMKN, AXL and EPHB4, were identified as also expressed in neuroendocrine tumors, especially in targets for EZH2 in SCLC cells by ChIP-seq analysis. cases with poor prognoses [41–48]. In addition, several They also found that introduction of JUB inhibited genes were identified as targets for ASCL1 [49]. Among cellular growth, suggesting that suppression of these them, miRNA-375 (miR-375) was supposed to inacti- genes by EZH2 other than TGFBR2 might be involved vate Yes-associated protein (YAP)1 in SCLC [50]. In in the growth of SCLC. Genetic alternations in TP53 H345-TβRII cells, TGF-β exhibited a downgulation of and RB1 are commonly observed in patients with ASCL1, followed by upregulation of primary-miR 375 SCLC; consequently, these were considered as early (pri-miR-375) (data not shown). Thus, our present events that triggered SCLC development [28–30]. findings suggested that the TGF-β-Smad-ASCL1 EZH2 expression was upregulated in Rb1 knockout pathway is important in SCLC progression, and that it MEF cells [31]. Based on those studies, our finding that may also have an important role in other neuroendo- EZH2 was highly expressed in SCLC suggests that crine tumors. EZH2 is an oncogenic factor. EZH2 was also reported to be highly expressed in breast cancer and prostate Materials and Methods cancer [32, 33]; moreover, EZH2 inhibitors have been considered a promising therapy for certain types of Cell culture and reagents tumors [34, 35]. A specific EZH2 inhibitor induced Human SCLC H82, H146, H209, H345 cells and human TGFBR2 expression (Figure 4b); therefore, EZH2 NSCLC A549 and H441 cells were purchased from American inhibitors may also effectively eradicate SCLC cells Type Culture Collection (ATCC, Manassas, VA, USA) and by restoring TβRII expression, and thus, enabling cultured as recommended. Human skin keratinocyte HaCaT cells were previously described [51]. TGF-β3(R&D TGF-β-mediated apoptosis. Systems, Minneapolis, MN, USA) was reconstituted in 4 mM TGF-β-target genes have been comprehensively HCl and 0.1% bovine serum albumin (BSA, Sigma-Aldrich, identified in many kinds of tumors, including liver − 1 St Louis, MO, USA) and used at a concentration of 1 ng ml . cancer, pancreatic cancer, NSCLC and breast cancer GSK343 (Sigma-Aldrich) and CHX (Sigma-Aldrich) were [17, 36–38], but rarely in neuroendocrine tumors. reconstituted in dimethyl sulfoxide. See also Supplementary Identification of novel target(s) may improve our Information. understanding of SCLC cell characteristics. In the present study, our comprehensive gene expression Cell proliferation assay analysis and ChIP-seq analysis demonstrated that the 4 4 H82 cells (1 × 10 cells), H146 cells (3 × 10 cells) and H345 TGF-β-induced apoptosis in SCLC cells could be cells (3 × 10 cells) were seeded on 12-well plates, and then attributed to negative regulation of ASCL1 by TGF-β. stimulated with TGF-β. Cell proliferation was evaluated with It was previously shown that ASCL1 expression was Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan). inhibited by Notch signaling in SCLC cells [39]. This Absorbance at 450 nm was measured with a Model 680 Figure 6 Downregulation of ASCL1 is important for TGF-β-mediated apoptosis in SCLC cells. (a) qRT-PCR analysis shows ASCL1 expression. H345 cells were infected with lentivirus vectors encoding control shRNA (shNTC) or shRNA that targeted ASCL1 (shASCL1). H345-TβRII cells stimulated with TGF-β for 48 h served as control. Data represent mean ± s.d. *Po0.05; ***Po0.001. (b) Cell proliferation assay. Cells in (a) were incubated for 12 days. Data represent mean ± s.d. ***Po0.001. (c) Cell cycle analysis of cells in (b). (left panels) The number of cells in each cell cycle stage is shown (color coding shown in right panel). (right panel) Percentage of cells in each cell cycle stage. (d) Immunoblot of cell lysates probed with the indicated antibodies. H345-GFP and H345-TβRII cells were infected with lentivirus vectors encoding GFP alone or ASCL1, and then stimulated with TGF-β for 12 days. (e) Cell cycle analysis of cells in (d). (left panels) The number of cells in each cell cycle stage is shown (color coding shown in right panel). (right panel) Percentage of each cell cycle stage in indicated cells is shown. (f) qRT-PCR analysis shows ASCL1 expression in H345 cells transfected with control siRNA (siNTC) or siRNA that targeted ASCL1 (siASCL1). ASCL1 expression was determined 72 h post-transfection. Data represent mean ± s.d. ***Po0.001. (g) Mice received subcutaneous transplants of cells in (f) (siNTC, n = 7, siASCL1, n = 7). (Left panels) Representative photographs; (right panel) incidence of tumor formation 2 weeks after injection. Arrow heads indicate tumors. GFP, green fluorescent protein; SCLC, small cell lung cancer; TGF-β, transforming growth factor-β; qRT-PCR, quantitative real-time reverse transcription-PCR. Cell Discovery www.nature.com/celldisc | Fumihiko Murai et al. HE EZH2 TβRII ASCL1 EZH2 TβRII ASCL1 5 5 5 4 4 3 3 3 2 2 2 1 1 1 0 0 0 -1 -1 -1 Normal SCLC Ad Sq LCNEC Normal SCLC Ad Sq LCNEC Normal SCLC Ad Sq LCNEC *** *** *** *** * *** *** * *** *** Figure 7 Expression profiles of EZH2, TβRII and ASCL1 in human normal lung tissues and human lung cancer tissues. (a) Lung tissues were stained with HE, anti-EZH2 antibody, anti-TβRII antibody and anti-ASCL1 antibody. Representative images show normal tissues (top and third rows) and SCLC tissues (second and bottom rows) from two patients, as indicated. (Insets) ASCL1 staining in boxed region is shown at high magnification. Scale bars are 30 μm. (b) Expression profiles of samples in (a) and Supplementary Figure S6 were analyzed by defining scores (s) that corresponded to the frequency of positive cells (f) in each sample, as follows: s = 4 for 80⩽ f⩽ 100; s = 3 for 50⩽ fo80; s = 2 for 20⩽ fo50; s = 1 for 0ofo20; s = 0 for f = 0. Data represent means. *Po0.05; ***Po0.001. HE, hematoxylin-eosin; LCNEC, large cell neuroendocrine carcinoma; normal, normal lung; SCLC, small cell carcinoma; Ad, adenocarcinoma; Sq, squamous cell carcinoma. Cell Discovery www.nature.com/celldisc Patient #2 Patient #1 Normal lung tissues Normal lung tissues SCLC tissues SCLC tissues Average score (s) Average score (s) Average score (s) EZH2 promotes SCLC by suppressing TGF-β signaling TGF-β TGF-β Normal lung epithelial cells SCLC cells P P TβRI TβRI PRC2 PRC2 P P (ALK-5) (ALK-5) TβRII TβRII SUZ12 SUZ12 EED EED Smad2/3 Smad2/3 EZH2 EZH2 Smad2/3 Smad2/3 Smad4 Smad4 open chromatin open chromatin ASCL1 ASCL1 TGFBR2 TGFBR2 apoptosis apoptosis Figure 8 Disruption of TGF-β-mediated tumor suppression in SCLC cells. (a) In normal lung epithelial cells, TGF-β induces apoptosis through the suppression of ASCL1 expression in a Smad-dependent manner. (b) In SCLC cells, high EZH2 expression attenuates TβRII expression through histone H3K27 tri-methylation. Disruption of TGF-β signaling elevates ASCL1 expression, which in turn protects SCLC cells from apoptosis. SCLC, small cell lung cancer; TGF-β, transforming growth factor-β. Microplate Reader (Bio-Rad, Melville, NY, USA), followed by gel electrophoresis, and transferred to Fluoro Trans W mem- subtraction of absorbance at 595 nm. brane (Pall, East Hills, NY, USA). Chemiluminescence images were captured on ImageQuant LAS4000 (Fujifilm, Tokyo, In vivo tumor growth assay Japan). Image J software (NIH) was used to quantify blot band In vivo experiments were performed as previously described intensities in Figure 3c. See also Supplementary Information. [52]. The protocols were approved by the Animal Ethics Committee of The University of Tokyo (approval number: Lentiviral vector construction and lentivirus production 2186). BALB/c nu/nu mice (4 weeks, male) were purchased from A lentiviral vector system (provided by Dr Hiroyuki Charles River Laboratories (Yokohama, Japan). Cells were Miyoshi, RIKEN) was used to induce specific gene introduction resuspended in culture media supplemented with 50% BD and knockdown. For gene introduction, we inserted com- Matrigel (BD Bioscience, San Jose, CA, USA), and then sub- plementary DNAs encoding the human wild-type TGFBR2, cutaneously injected into mice (3 × 10 cells in 100 μl per mouse). TGFBR2 with a truncated intracellular domain and a carboxy- For xenograft transplantations, H345 cells were treated with terminal GFP tag (dnTβRII), and human wild-type ASCL1, siRNA against ASCL1 for 72 h in vitro, followed by sub- into the entry vector, pENTR201 [55]. Then, pENTR201 cutaneous transplantation of ASCL1-silenced cells into mice. vectors were inserted into the lentiviral destination vector, pCSII-EF-RfA or pCSII-CMV-RfA, as previously described [56]. Vectors encoding GFP were also generated as controls. Gene expression analysis Total RNA was extracted with the RNeasy Mini Kit Similarly, shRNAs designed to knockdown a specified gene (Qiagen, Valencia, CA, USA). Complementary DNA was syn- were inserted into the entry vector pENTR4-H1. Then, thesized with the random hexamer protocol described in the pENTR4-H1 vectors that carried shRNAs specific for human PrimeScript II 1st strand complementary DNA Synthesis Kit ASCL1 or EZH2 were inserted into the lentiviral destination (Takara, Otsu, Japan). For qRT-PCR analysis, gene expression vector, pCS-RfA-EG. The shRNA target sequences for was quantified with the StepOne Plus Real time-PCR System gene knockdowns were obtained from Dharmacon siDESIGN (Life Technologies, Tokyo, Japan) and the Fast SYBR Green Center (GE Healthcare, Piscataway, NJ, USA; Supplementary Master Mix (Life Technologies). The expression level of each Table S5). Lentiviral vectors were produced as described pre- gene was normalized to that of glyceraldehyde-3-phosphate viously [53]. Culture supernatant was concentrated with Lenti-X dehydrogenase (GAPDH). Primer sequences are shown in Concentrator (Clontech, Palo Alto, CA, USA), then used for Supplementary Table S4. lentiviral vector infections. siRNA Immunoblotting An Accell-siRNA SMARTpool specific for human ASCL1 Immunoblotting was previously described [53, 54]. Cells was purchased from Dharmacon (GE Healthcare), and recon- were lysed in radio-immunoprecipitation assay buffer (50 mM stituted in 1 × siRNA buffer (100 μM, GE Healthcare). The Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, siRNA target sequences are shown in Supplementary Table S6. and 0.5% sodium deoxycholate) with the Complete Protease Cells were treated with siRNA at a final concentration of 1 μM. Inhibitor Cocktail (Roche Diagnostics, Tokyo, Japan) and an EDTA-free phosphatase inhibitor cocktail (Nacalai Tesque). Protein concentrations were quantified with the BCA Protein Cell cycle analysis Assay (ThermoFisher Scientific, Yokohama, Japan). Equal After washing with phosphate-buffered saline, cells were amounts of total protein were applied to SDS-polyaclylamide fixed with ice-cold 70% EtOH in phosphate-buffered saline Cell Discovery www.nature.com/celldisc | Fumihiko Murai et al. and stored for more than 16 h at − 20 °C. The fixed cells were performed with the analysis of variance; one-way analysis were resuspended in phosphate-buffered saline containing of variance (Tukey’s method) was applied to comprehensive − 1 0.25 mg ml RNase A, and then incubated at 37 °C for 1 h. gene expression analyses and immunohistochemical analyses. − 1 Cells were labeled with 50 μgml propidium iodide (PI, Life The repeated measure analysis of variance was applied to in vivo Technologies) for 30 min at 4 °C; then, cell cycle analysis was experiments. Significant differences were defined as Po0.05. performed with a Gallios Flow Cytometer (Beckman Coulter, Miami, FL, USA). The distribution of each cell cycle stage was Conflict of Interest analyzed with the FlowJo (Tomy Digital Biology, Tokyo, Japan) Watson Pragmatic cell cycle analysis program. The authors declare no conflict of interest. ChIP-qRT-PCR analysis and ChIP-seq analysis Acknowledgements ChIP was previously described [51, 57]. See also Supplementary Information. ChIP-qRT-PCR analyses were We thank Yasuyuki Morishita and Kei Sakuma (The performed with the StepOne Plus Real time-PCR System and University of Tokyo) for technical assistance, and Hiroyuki FastStart Universal SYBR Green Master (Rox) (Roche). Primer Miyoshi (RIKEN) for providing the lentiviral vectors. This sequences for each gene locus are shown in Supplementary work was supported by a KAKENHI Grant-in-Aid for scientific Table S4. research on Innovative Area (Integrative Research on Cancer For ChIP-seq analysis, total amounts of double stranded Microenvironment Network; grant number 22112002) from the DNA were quantified with Qubit dsDNA HS Assay Kits (Life Ministry of Education, Culture, Sports, Science and Technology Technologies). Libraries were prepared with IonXpress Plus (MEXT) of Japan, a KAKENHI Grant-in-Aid for Young Fragment Library Kit (Life Technologies). Libraries were Scientists (B) (grant number 22700967) from the Japan Society quantified with Ion Library Quantification Kit (Life Technolo- for the Promotion of Science (JSPS) (SE), the Strategic Basic gies). Emulsion PCR and product purification were performed Research Program from Japan Science and Technology Agency with Ion PGM Template OT2 400 Kit (Life Technologies). The (KM), and a Specific Research Grant from The Cell Science amplified samples were sequenced with Ion PGM Sequencer Research Foundation (SE). This study was performed in part as (Life Technologies) with Ion PGM Hi-Q Sequencing Kit. The a research program for the Project for Development of Inno- acquired read tags were mapped onto the NCBI hg19 human vative Research on Cancer Therapeutics (P-Direct), MEXT. genome assembly. Analyses of ChIP-seq data were previously described [58, 59]. The significant Smad2/3 binding region was Author contributions calculated using CisGenome version 2 using default parameters except for window size (400 bp), cut-off counts (⩾10 reads (P = 0.023516)), and step size (25 bp). Raw ChIP-seq and peak FM, SE and KM designed the study, analyzed the data and call data are available at GEO (GSE63871). wrote the manuscript. FM and SE performed experiments. DK assisted with and performed the ChIP-seq analysis. 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This work is licensed under a Creative Commons 51 Koinuma D, Tsutsumi S, Kamimura N et al. Chromatin Attribution 4.0 International License. The images or immunoprecipitation on microarray analysis of Smad2/3 other third party material in this article are included in the article’s binding sites reveals roles of ETS1 and TFAP2A in trans- Creative Commons license, unless indicated otherwise in the forming growth factor β signaling. Mol Cell Biol 2009; 29: credit line; if the material is not included under the Creative 172–186. Commons license, users will need to obtain permission from the 52 Shirai YT, Ehata S, Yashiro M, Yanagihara K, Hirakawa license holder to reproduce the material. To view a copy of this K, Miyazono K. Bone morphogenetic protein-2 and -4 play license, visit http://creativecommons.org/licenses/by/4.0/ Cell Discovery www.nature.com/celldisc

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