Cancer Research ID4 Imparts Chemoresistance and Cancer Sternness to Glioma Cells by Derepressing miR-9*-Mediated Suppression of SOX2 Hye-Min Jeon, Young-Woo Sohn, Se-Young Oh, et al. Cancer Res 2011 ;71:3410-3421. Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/71 /9/3410 Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2011/04/22/71.9.3410.DC1 .html Cited Articles This article cites by 44 articles, 6 of which you can access for free at: http://cancerres.aacrjournals.Org/content/71/9/3410.full.html#ref-list-1 Citing articles This article has been cited by 7 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.Org/content/71/9/3410.full.html#related-urls E-mail alerts Sign up to receive free email-alerts related to this article or journal. Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at pubs@aacr.org. Permissions To request permission to re-use all or part of this article, contact the AACR Publications Department at permissions@aacr.org. Downloaded from cancerres.aacrjournals.org on April 12, 2014. © 2011 American Association for Cancer Research. Tumor and Stern Cell Biology Cancer Research ID4 Imparts Chemoresistance and Cancer Sternness to Glioma Cells by Derepressing miR-9*-Mediated Suppression of SOX2 Hye-Min Jeon1, Young-Woo Sohn1, Se-Young Oh1, Sung-Hak Kim1, Samuel Beck1, Soonhag Kim2, and Hyunggee Kim1 Abstract Glioma stem cells (GSC) possess tumor-initiating potential and are relatively resistant to conventional chemotherapy and irradiation. Thus, they are considered to be major drivers for glioma initiation, progression, and recurrence. However, the precise mechanism governing acquisition of their drug resistance remains to be elucidated. Our previous study has shown that inhibitor of differentiation 4 (ID4) dedifferentiates Ink4a/Arf/~ mouse astrocytes and human glioma cells to glioma stem-like cells (induced GSCs or iGSCs). In this article, we report that ID4-driven iGSCs exhibit chemoresistant behavior to anticancer drugs through activation of ATP-binding cassette (ABC) transporters. We found that ID4 enhanced SOX2 protein expression by suppressing microRNA-9* (miR-9*), which can repress SOX2 by targeting its 3'-untranslated region. Consequently, ID4-mediated SOX2 induction enhanced ABCC3 and ABCC6 expression through direct transcriptional regulation, indicating that ID4 regulates the chemoresistance of iGSCs by promoting SOX2-mediated induction of ABC transporters. Furthermore, we found that short hairpin RNA-mediated knockdown of SOX2 in ID4-driven iGSCs resulted in loss of cancer sternness. Moreover, ectopic expression of SOX2 could dedifferentiate Ink4a/Arf~/~ astrocytes and glioma cells to iGSCs, indicating a crucial role of SOX2 in genesis and maintenance of GSCs. Finally, we found that the significance of the ID4-miR-9*-SOX2-ABCC3/ABCC6 regulatory pathway is recapitulated in GSCs derived from patients with glioma. Together, our results reveal a novel regulatory mechanism by which ID4-driven suppression of miR-9* induces SOX2, which imparts sternness potential and chemoresistance to glioma cells and GSCs. Cancer Res; 71(9); 3410-21. ©2011 AACR Introduction Glioblastoma multiforme (GBM), the most common and aggressive form of brain malignancies, remains mostly incurable and has about 1-year median survival postdiagnosis, despite considerable advances in diagnosis, therapies, and understanding of its molecular pathways (1-5). Recent studies have revealed that a cancer-cell subpopulation in brain tumors—glioma stem cells (GSC)—exhibits tumor-initiating ability, self-renewal, and aberrant differentiation and is responsible for glioma initiation and progression (6, 7). Interestingly, GSCs are remarkably resistant to chemotherapy and irradiation, which are first-line treatment options for patients with malignant gliomas (8, 9). Therefore, GSCs are considered major culprits for glioma recurrence after therapy (10). Authors' Affiliations: 1School of Life Sciences and Biotechnology, Korea University; and department of Applied Bioscience, CHA Stem Cell Institute, CHA University, Seoul, Republic of Korea Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Hyunggee Kim, School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea. Phone: 82-2-3290-3059; Fax: 82-2-953-0737; E-mail: hg-kim@korea.ac.kr doi: 10.1158/0008-5472.CAN-10-3340 ©2011 American Association for Cancer Research. Although resistance to temozolomide is associated with the expression of 06-methylguanine-DNA-methyltransferase, which is abundantly detected in CD133-positive GBMs (11), a major cause of chemoresistance in GSCs is the activation of multiple drug resistance genes, such as those encoding ATP-binding cassette (ABC) transporters (8). ABC transporters can cause efflux of molecules across the cell membrane by using ATP and are highly expressed in normal and cancer stem cells compared with their differentiated counterpart cells (12,13). A recent study has shown that PI3K/Akt signaling, which is activated by PTEN loss, confers chemoresistance to GSCs by enhancing ABCG2 activity (14). However, the mechanism underlying the expression of various ABC transporters involved in the chemoresistance of GSCs remains poorly understood. MicroRNAs (miRNA or miR) are an evolutionarily conserved group of small noncoding RNAs that play pivotal roles in regulating most biological processes of normal development and various diseases, including cancers, by suppressing mRNA stability and/or translation (15). Recently, the biological consequences of miRNAs have been revealed in many studies showing that miRNAs specifically regulate maintenance and differentiation of embryonic stem cells (ESC) and induce pluripotent stem cells (iPSC; ref. 16). For example, miR-145, which is highly upregulated during differentiation, can 3410 Cancer Res; 71 (9) May 1, 2011 J^^^Ammcan Association [or CamcerResearch Downloaded from cancerres.aacrjournals.org on April 12, 2014. © 2011 American Association for Cancer Research. ID4-miR-9*-SOX2 Pathway in Glioma Stem Cell Traits repress pluripotency in human ESCs by suppressing many reprogramming factors, including OCT4, SOX2, and KLF4 (17). Conversely, miR-291-3p, miR-294, and miR-295, which are specifically expressed in ESCs, can increase the efficiency of iPSC genesis by inducing OCT4, SOX2, and KLF4 (18). These reprogramming factors are upregulated in various human malignancies, and some are encoded by classical oncogenes (19-21). In addition, SOX2 is highly expressed in patients with glioma and plays a crucial role in GSC maintenance (22). However, the precise roles, such as tumor initiation, self-renewal, aberrant differentiation, and chemo/radioresistance. of these reprogramming factors in GSC nature remain to be elucidated. Previously, we have shown that inhibitor of differentiation 4 (ID4) induces dedifferentiation of Ink4a/Arj~/~ mouse astrocytes, generating GSC-like cells (iGSCs) through activation of cyclin E and Notch signaling (23). However, the molecular mechanism governing the chemoresistance of ID4-driven iGSCs has not been elucidated. In this article, our data show that ID4 confers chemoresistance to GSCs by inducing the expression of 2 SOX2-mediated ABC transporters—ABCC3 and ABCC6—through direct suppression of miRNA-9* (miR-9*), a SOX2-targeting miRNA. Materials and Methods Cell culture and conditions Human glioma cell lines (Al72, A1207, LN18, and LN229) were purchased from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium/high glucose supplemented with 10% FBS (Hyclone), 1% penicillin and streptomycin (Life Technologies), and 2 mmol/L L-gluta-mine (Life Technologies). Human glioma stem cells (hGSC-1. hGSC-2, and hGSC-3; ref. 24) and glioma cells were grown in suspension culture with neurobasal medium (NBE; Invitro-gen) supplemented with modified N2, B27, epidermal growth factor (EGF; 20 ng/mL; R&D Systems), and basic fibroblast growth factor (bFGF; 20 ng/mL; R&D Systems). For neuro-sphere-formation assays, cells were seeded at a density of 2 cells/mm2 in 12-well plates and then grown in suspension with NBE supplemented with N2, B27, EGF, and bFGF (all 20 ng/ mL), as described previously (23). EGF and bFGF were replaced every 3 days, and the neurosphere numbers were determined after 14 days. Luciferase reporter gene assay, RNA analysis, plasmid and gene transduction, Western blot analysis, immunofluorescence assay, bromodeoxyuridine (BrdU) incorporation assay, fluorescence activated cell-sorting (FACS) assay, in vivo tumori-genicity assay, apoptosis analysis, side population cell analysis, and statistical analysis were done. Details of experimental procedures are provided in the Supplementary Information. Results ID4 primes anticancer drug resistance through induction of ABC transporters To investigate whether ID4 confers chemoresistance to cells, we established ID4-overexpressing glioma cells (A172- ID4 and A1207-ID4) by transduction of retroviral vector-encoding ID4 genes, as well as ID4-depleted cells (LN229-shID4) using short hairpin RNA interference (shRNAi). Compared with their controls, cell death evaluated on flow cytometric analysis revealed that A172-ID4 and A1207-ID4 were significantly resistant whereas LN229-shID4 was sensitive to l,3-bis(2-chloroethyl)-l-nitrosourea (BCNU)-induced apoptosis (Supplementary Fig. SI). Our previous study has shown that ID4 allows Ink4a/Ar]~/~ astrocytes and glioma cells to acquire GSC properties through activation of cyclin E and Jaggedl-Notchl signaling (23). Thus, we examined whether these pathways were required for ID4-mediated antiapoptosis by treating cells with BCNU, in combination with purvalanol A (a cyclin E inhibitor; ref. 25) and/or DAPT (a Notch inhibitor), and found that cyclin E and Notch signaling were not involved in ID4-mediated chemoresistance of A172, A1207, and LN229 (Supplementary Fig. SI). A recent study had shown that GSCs acquire anticancer drug resistance through PI3K/Akt-mediated ABCG2 activation (14) and, therefore, we investigated whether ID4-driven chemoresistance was associated with ABC transporter activity. First, we cotreated cells with BCNU and the pan-ABC transporter inhibitor verapamil (14) and found that verapamil suppressed BCNU-induced apoptosis in A172-ID4 (Fig. 1A) but not in LN229-shID4 (Fig. 1A), indicating that ID4 causes cells to retain chemoresistance, at least in part, through ABC transporter activity. Next, we carried out semiquantitative real-time PCR (RT-PCR) analysis to examine which ABC transporters are differentially regulated by ID4 and found that the levels of ABCC3. ABCC6, and ABCA2 were significantly elevated in A172-ID4 (Supplementary Fig. S2A) and the levels of ABCC3, ABCC4. ABCC6, and ABCG2 were increased in Ink4a/Arj~/~ astrocyte-ID4 (Supplementary Fig. S2B). On the contrary, the levels of ABCC2, ABCC3, ABCC6, and ABCG2 were markedly reduced in LN229-shID4 (Supplementary Fig. S2C). In addition, we examined the expression of ABC transporters in 2 human GSCs (hGSC) derived from GBM patients (24) and found that the levels of ABCC3 and ABCC6 were diminished in ID4-depleted hGSCl (Supplementary Fig. S2D) and the levels of ABCC3. ABCC6, and ABCG2 were decreased in ID4-depleted hGSC2 (Supplementary Fig. S2E). Thus, to further investigate a possible role of ABC transporters in ID4-driven chemoresistance. we focused on ABCC3 and ABCC6. We depleted ABCC3 and ABCC6 in ID4-overexpressing cells by using shRNAi and confirmed their depletion in A172-ID4 and A1207-ID4 by quantitative RT-PCR (qRT-PCR) and Western blot assays (Fig. IB; Supplementary Fig. S3A and B). The depletion of ABCC3 and ABCC6 in ID4-overexpressing cells resulted in a marked increase in apoptosis (Fig. 1C), suggesting that ID4-mediated chemoresistance to BCNU is partially attributed to increased ABCC3 and ABCC6 expression. ID4 upregulates SOX2 by suppressing miR-9* expression The preceding data raise the question pertaining to how ID4 upregulates ABCC3 and ABCC6 mRNA expression, because ID4 is a helix-loop-helix (HLH) protein that lacks a DNA-binding domain, and it can suppress transcription by acting as www.aacrjournals.org Cancer Res; 71(9) May 1, 2011 3411 Downloaded from cancerres.aacrjournals.org on April 12, 2014. © 2011 American Association for Cancer Research. Jeon et al. DMSO V50 B100 V+B T3 CD 70- ■ LN229-shSr 60 - □l_N229-shlD4 50 - 40 - 30 - 20 " 10 " 0 - DMSO V50 B100 V+B B A1207 A1207 25 g> 20 03 < z 15 10 co O O m < ConScM 2 200 i CD g 150 < z | 100 CO O < 0 ID4 A172 Con Scr 1 ID4 A172 5 i s 4 03 z 3 cc co O m 1 -< 0 co O o m < 30 J2 25 S * 20 < Con Scr 1 2 15 10 CO O O m < ID4 ID4 50 40 30 20 10 0 40 35 2 30 S 25 § 20 15 10 5 0 A1207 ■ Control □ ID4-shScr ■ ID4-shABCC3-1 ■ ID4-shABCC3-2 ■ ID4-shABCC6-1 DMSO BCNU A172 ■ Control □ ID4-sriScr ■ ID4-shABCC3-1 B ID4-shABCC3-2 ■ ID4-shABCC6-1 ■ ID4-shABCC6-2 DMSO BCNU Figure 1. ID4 induces anticancer drug resistance in glioma cells by inducing ABCC3 and ABCC6 transporters. A, percentage of dead cells in A172-ID4, LN229-shlD4, and their controls treated with BCNU (100 jig/mL; B100) in the presence or absence of verapamil (50 jimol/L; V50) was determined by AnnexinV/ propidium iodide (Pl)-mediated FACS analysis. Verapamil + BCNU (V + B). B, ABCC3 and ABCC6 mRNA levels in A1207-ID4 and A172-ID4, and in ABCC3- or ABCC6-depleted A1207-ID4 and A172-ID4, together with their controls, were examined by qRT-PCR. The A1207-ID4-shABCC6-1 cell line was used in the qRT-PCR analysis because we failed to establish additional A1207-ID4-shABCC6 cell lines. C, apoptosis rate of cells described in (B) treated with or without BCNU (100 ng/mL); *, P < 0.05; **, P < 0.01. a dominant-negative transcriptional repressor (26). During the investigation of regulatory mechanisms pertaining to ID4-driven ABCC3 and ABCC6 expression, we found that the ID4 level correlated with SOX2 protein expression—that is. increased SOX2 protein levels were observed in A1207-ID4 and A172-ID4, whereas decreased SOX2 protein levels were observed in LN229-shID4 (Fig. 2A; Supplementary Fig. S4; ref. 23). In addition, SOX2 protein levels correlated with ABCC3 and ABCC6 protein levels in these cells. Interestingly, qRT-PCR analysis revealed that ID4-induced SOX2 protein levels were not correlated with its mRNA levels (Fig. 2A), indicating that ID4 might regulate SOX2 expression at the posttranscriptional level. Emerging evidence suggests that various biological processes are regulated by miRNAs that act as negative regulators of gene expression at the posttranscriptional level (15). Therefore, we attempted to explore miRNAs based on 3 criteria: (i) miRNAs that can target the 3'-untranslated region (UTR) of SOX2 (examined using bioinformatics programs such as Pic-Tar, TargetScan, miRBase, and Miranda); (ii) miRNAs that are enriched in brain tissues and are involved in differentiation of neural stem cells (NSC) or progenitors; and (iii) miRNAs that possess E-boxes—ID4-responsive elements—in their promoters. Consequently, we found that one such candidate is miR-9*, known to be specifically expressed during brain neurogenesis (27, 28). To validate our bioinformatics criteria, we initially carried out a TaqMan microRNA detection assay and found that miR-9* expression was significantly decreased in A1207-ID4 and A172-ID4 but enhanced in LN229-shID4 (Fig. 2B). The luciferase activity of the reporter construct containing 3 copies of the miR-9*-binding motif (27) was dose-dependently elevated in 293T cells cotransfected with ID4 (Fig. 2B) and, in addition, significantly elevated in A1207-ID4 and A172-ID4 (Fig. 2B), indicating that miR-9* activity was lower in ID4-overexpressing cells. A 6-bp sequence in the 5'-ends of miR-9* was perfectly complementary to SOX2-3'-UTR (Supplementary Fig. S5A). To determine whether this predicted binding site was responsible for SOX2 repression, we created luciferase reporter constructs containing either wild-type (WT) or miR-9*-binding site-mutated SOX2-3'-UTR (Supplementary Fig. S5A; ref. 17). WT, but not mutated SOX2-3'-UTR repressed its reporter activity in LN229-shID4 but increased in A1207-ID4 (Fig. 2C), indicating that ID4 enhances SOX2 expression, in part, by suppressing miR-9*. The miR-9* transcripts are located in 3 loci of human chromosomes: chromosomes 3412 Cancer Res; 71 (9) May 1, 2011 Cancer Research Downloaded from cancerres.aacrjournals.org on April 12, 2014. © 2011 American Association for Cancer Research. ID4-miR-9*-SOX2 Pathway in Glioma Stem Cell Traits LN229 A1207 A172 A1207 A172 -------------------------------------------1------ C ! D Control shlD4 Control ID4 Csh|D4 C ID4 C ID4 CshlD4 C ID4 C ID4 LN229 A1207 LN229 A1207 A172 LN229 A1207 A172 Figure 2. ID4 upregulates SOX2 protein by inhibiting miR-9* expression. A, Western blot analysis showed that SOX2, ABCC3, and ABCC6 protein levels were decreased in LN229-shlD4 compared with control LN229-shScrambled and increased in A1207-ID4 and A172-ID4as compared with their controls. qRT-PCR was carried out to examine SOX2 mRNA levels in these cells. B, miR-9* levels increased in A1207-ID4 and A172-ID4 and decreased in LN229-shlD4. The luciferase activity of the reporter gene containing 3 copies of the miR-9*-binding motif was dose-dependently elevated in 293T cells after transfecting different concentrations of ID4. Furthermore, this activity was significantly increased in ID4-overexpressing A1207 and A172 compared with their controls. C, the luciferase activity of the reporter gene containing WT SOX2-3'-UTR, but not miR-9*-binding site-mutated SOX2-3'-UTR, in A1207-ID4, LN229-shlD4, and their controls. D, qRT-PCR analysis revealed that expression of the dORF61 gene encoding miR-9* was decreased in ID4-overexpressing A1207 and A172 and increased in ID4-depleted LN229. Luciferase reporter assays revealed that the transcriptional activity of the reporter gene containing the dORF61 promoter was elevated in LN229-shlD4 but decreased in A1207-ID4 and A172-ID4; *, P < 0.05; **, P < 0.01. 1 (hsa-miR-9-1), 5 (hsa-miR-9-2), and 15 (hsa-miR-9-3). In particular, the hsa-miR-9-1 transcript is located in the intron region of the Clorf61 gene whereas hsa-miR-9-2 and hsa-miR-9-3 transcripts are located in the intergenic region. Because the Clorf61 gene has 3 conserved "E-box" elements in its promoter (Supplementary Fig. S5B), we carried out qRT-PCR to measure Clorf61 mRNA expression and found that its mRNA level was markedly diminished in A1207-ID4 and A172-ID4 but significantly increased in LN229-shID4 (Fig. 2D). Furthermore, ID4 overexpression decreased the luciferase activity of the reporter gene containing the Clorf61 gene promoter and ID4 knockdown enhanced Clorf61 promoter-luciferase activity (Fig. 2D), indicating that ID4 suppresses miR-9* expression through transcriptional repression of the Clorf61 gene. SOX2 promotes anticancer drug resistance through ABCC3 and ABCC6 expression We wondered whether SOX2 was associated with ID4-mediated chemoresistance to anticancer drugs. Therefore, we established several SOX2-overexpressing glioma cells (A1207-SOX2, A172-SOX2, LN18-SOX2, and LN229-SOX2) by transducing the retroviral vector-encoding S0X2 gene. In addition, we employed shRNAi to downregulate SOX2 in LN229 (LN229-shSOX2), which expresses a high level of endogenous SOX2. All SOX2-overexpressing glioma cells showed a marked decrease in apoptosis when treated with BCNU (Fig. 3A). On the contrary, LN229-shSOX2 was significantly sensitive to anticancer-drug-induced apoptosis when treated with BCNU, etoposide, and staurosporine (Fig. 3A, far right graph). SOX2, a member of the SRY-related www.aacrjournals.org Cancer Res; 71(9) May 1, 2011 3413 Downloaded from cancerres.aacrjournals.org on April 12, 2014. © 2011 American Association for Cancer Research. Jeon et al. ■ A1207 Control ■ A172 Control ■ LN18 Control □ LN18SOX2 ■ LN229 Control □ LN229 SOX2 70 ■ LN229 shScr □ LN229 shSox2 I BCNU Etoposide STS B i Control SOX2 2.5 2 1.5 1 0.5 0 Hi 2.5 2 1.5 Control SOX2 Control SOX2 2.5 » 2 a a < 1.5 z X I 1 o o 2] < 0.5 0 - 2 - 25 n T 2 - 1.5 - - 1 - ■ 1.5 - 1 0.5 - 1 - 1 0.5 - 1 0 - 0 ■ Control SOX2 Control SOX2 Control SOX2 i Con Scr 1 SOX2 A172 Con Scr shABCC6 i SOX2 A172 i ■ I Con Scr 1 Figure 3. SOX2 induces anticancer drug resistance by upregulating ABCC3 and ABCC6 transporters. A, percentage of dead cells in SOX2-overexpressing glioma cells (A1207, A172, LN18, and LN229) and their controls treated with BCNU (80-100 ng/mL), and percentage of dead cells in control LN229-shScrambled and LN229-shSOX2 treated with BCNU (80 p:g/ml_), etoposide (180 p:mol/ L),andstaurosporine(100 nmol/L; STS). B, qRT-PCR analysis revealed that ABCC3 and ABCC6 mRNA levels increased in all SOX2-overexpressing glioma cell lines that were tested. C, ABCC3 and ABCC6 mRNA levels were markedly reduced in ABCC3- or ABCC6-depleted A1207-SOX2 and A172-SOX2 compared with their controls. Percentage of dead cells in these cells treated with BCNU (100 ng/mL) was determined by AnnexinV/PI-mediated FACS analysis. D, cotransfection of luciferase reporter genes containing ABCC3 (C3-1 and C3-2) or ABCC6 promoters (C6-1 and C6-2) at different concentrations of SOX2 in 293T cells revealed that SOX2 increases the transcription of ABCC3 (C3-1) and ABCC6 (C6-1 and C6-2) in a dose-dependent manner; *, P < 0.05; **, P < 0.01. high mobility group (HMG) box family, regulates expression of various target genes that contain a consensus SOX2-binding motif (A/TA/TCAAA/TG) in their promoters (29) and is upregulated in several human malignancies, including GBM (22, 30); however, its role in tumorigenesis is poorly understood. Initially, qRT-PCR was conducted to examine the expression of ABCC3 and ABCC6 and showed that their expression levels were relatively increased in all SOX2-over-expressing cells (Fig. 3B). To determine whether SOX2 exerted an antiapoptotic effect through induction of ABCC3 and ABCC6, we used shRNAi to knock down ABCC3 and ABCC6 expression in SOX2-overexpressing cells 3414 Cancer Res; 71 (9) May 1, 2011 Cancer Research Downloaded from cancerres.aacrjournals.org on April 12, 2014. © 2011 American Association for Cancer Research. ID4-miR-9*-SOX2 Pathway in Glioma Stem Cell Traits Figure 4. SOX2 regulates ABCC3/ ABCC6-dependent chemoresistance in ID4-overexpressing glioma cells. A, SOX2 mRNA levels were reduced in A1207-ID4 and A172-ID4 when transduced with 2 SOX2-specific shRNAi constructs. The SOX2 mRNA level was increased by reconstitution of a miR-9*-resistant version of SOX2 in LN229-shlD4 (LN229-shlD4-SOX2), and Western blot analysis revealed induction of SOX2 protein level in these cells. B, ABCC3 and ABCC6 mRNA levels were diminished in A1207-ID4 and A172-ID4 when transduced with 2 SOX2-specific shRNAi constructs but increased in LN229-shlD4-SOX2. C, percentage of dead cells in 2 A1207-ID4-shSOX2 cell lines, 2 A172-ID4-shSOX2 cell lines, and LN229-shlD4-SOX2, and their controls treated with or without BCNU (100 ng/mL); *, P < 0.05; " P < 0.01. A1207 A172 LN229 3 2.5 2 1.5 ' 1 0.5 0 shSOX2 Con Scr 1 2 ID4 LN229 Con. shlD4 shlD4 + SOX2 shSOX2 SOX2 ID4 ß-Actin Con shlD4 shlD4 + SOX2 B A1207 shSOX2 Con Scr 1 2 ID4 LN229 A1207 7 « 6 < 4 e Ü Ü shSOX2 \MMl Con Scr 1 2 shSOX2 Con Scr 1 2 ID4 LN229 shlD4 shlD4+SOX2 Con shlD4 shlD4+SOX2 A1207 DMSO BCNU 60 -I "Control □ ID4-shScr 50 J«ID4-shSOX2-1 m |alD4-shSOX2-2 ö 40 ■ A172 DMSO BCNU LN229 DMSO BCNU (A1207-SOX2-shC3 and A1207-SOX2-shC6; A172-SOX2-shC3 and A172-SOX2-shC6) and found that complete knockdown occurred (Fig. 3C). The depletion of these mRNAs in A1207-SOX2 and A172-SOX2 resulted in significant resistance to BCNU (Fig. 3C), indicating that SOX2 causes anticancer drug resistance by inducing ABC transporters. Among several putative binding sites for SOX2 in their promoter regions. 2 sites in each ABCC3 and ABCC6 promoter were highly conserved and contained a predicted SOX2-binding consensus motif (Supplementary Fig. S5C). We created luciferase reporter genes containing the promoter regions of ABCC3 [C3-1 (-7,489 to -7,114) and C3-2 (-1,376 to -881); upstream from the transcription start site] and ABCC6 [C6-1 (-6,482 to -5,986) and C6-2 (-4,246 to -3,792)], with each harboring a predicted SOX2-binding motif, and conducted promoter-luciferase reporter assays to examine the transcriptional activity of S0X2. Transfection of different concentrations of the S0X2 gene in 293T cells resulted in a dose-dependent increase in all reporter gene activities tested (Fig. 3D). Together, our results suggest that SOX2 promotes anticancer drug resistance through direct activation of ABCC3 and ABCC6 expression. www.aacrjournals.org Cancer Res; 71(9) May 1, 2011 3415 Downloaded from cancerres.aacrjournals.org on April 12, 2014. © 2011 American Association for Cancer Research. Jeon et al. ID4 regulates anticancer drug resistance via SOX2 activation To investigate whether ID4 endows chemoresistance to glioma cells by SOX2-mediated induction of ABCC3 and ABCC6, we stably depleted SOX2 in ID4-overexpressing cells (A1207-ID4-shSOX2 and A172-ID4-shSOX2), using shRNAi, or reconstituted SOX2 in ID4-depleted cells (LN229-shID4-SOX2) by transduction of a retroviral vector-encoding S0X2 gene that lacks its own 3'-UTR (to avoid a possible miR-9*-driven repression) and confirmed SOX2 expression in these cells using qRT-PCR (Fig. 4A). Notably, Western blot and immunofluorescence analyses revealed that SOX2 expression in LN229-shID4-SOX2 was similar to that in control LN229 (Fig. 4A; Supplementary Fig. S6). Next, we conducted qRT-PCR to determine expression levels of ABCC3 and ABCC6 in A1207-ID4-shSOX2 and A172-ID4-shSOX2 and found that, as expected, ABCC3 and ABCC6 in these cells were diminished to levels similar to those of their parental cells (Fig. 4B). Furthermore, reconstitution of SOX2 in LN229-shID4 resulted in increases in ABCC3 and ABCC6 expression at levels similar to those of control cells (Fig. 4B). We then examined the chemoresistance of A1207-ID4-shSOX2 and A172-ID4-shSOX2 to BCNU and found that SOX2 knockdown in these cells led to a marked increase in apoptosis (Fig. 4C). Conversely, reconstitution of SOX2 in LN229-shID4 rescued the chemoresistance to BCNU at a level similar to that of parental LN229 (Fig. 4C). In summary, these data indicate that SOX2-mediated induction of ABCC3 and ABCC6 plays a crucial role in the acquisition of anticancer drug resistance of ID4-overexpres-sing cells. SOX2 promotes tumorigenicity and self-renewal of glioma cells SOX2 plays pivotal roles in maintaining sternness properties of NSCs and GSCs (22, 31, 32). Furthermore, ectopic over-expression of SOX2, along with other reprogramming factors, including OCT4, KLF4, and c-MYC, reprograms somatic cells into iPSCs (16). However, the functional role of SOX2 in gliomagenesis, particularly in GSC biology, has not been studied. Therefore, we subcutaneously inoculated SOX2-over-expressing glioma cells in immunocompromised mice and compared their tumorigenic potentials with control cells. We found that A1207-SOX2 and A172-SOX2 displayed a marked increase in tumor growth (Fig. 5A and B). Moreover, SOX2 overexpression caused Ink4a/Arf~/^ astrocytes to have tumorigenic potential (Supplementary Fig. S7A), indicating an oncogenic effect of SOX2. A1207-SOX2, A172-SOX2, and Ink4a/Arj~/~ astrocyte-SOX2 accelerated tumor sphere formation—a hallmark of GSCs (Fig. 5A and B; Supplementary Fig. S7B; refs. 6, 7)—when grown in serum-free medium supplemented with EGF and bFGF, which is a reliable suspension culture condition for NSCs and GSCs to form neuro-spheres and maintain their sternness (33). Furthermore, these results imply that SOX2 might be causally connected to ID4-mediated switching of glioma cells to iGSCs. To address this possibility, we examined the tumor sphere-forming ability of ID4-overexpressing glioma cells with or without SOX2 knockdown. Consistent with our previous study (23), tumor sphere numbers were significantly increased in A1207-ID4 and A172-ID4 compared with their controls. However, SOX2 depletion in these cells dramatically repressed their tumor sphere-forming abilities, partially attributed to a decreased proliferation as determined by BrdU incorporation assay (Fig. 5C; Supplementary Fig. S8A and B). To further investigate the functional role of SOX2 in the ID4-driven acquisition of sternness of iGSCs, we carried out qRT-PCR to examine stem cell markers and their differentiated progeny markers in ID4-overexpressing glioma cells with or without SOX2 knockdown. Consequently, we observed that stem cell markers (Nestin, CD133, and CD15) were markedly increased in A1207-ID4 and A172-ID4 compared with their controls (Fig. 5C). However, SOX2 knockdown in these cells dramatically repressed stem cell marker expression and, instead, induced differentiated lineage marker expression, including GFAP and S100|3 (astrocyte), Tujl (neuron), as well as NG2 and 01ig2 (oligodendrocyte; Fig. 5C). Notably. GFAP expression was predominantly elevated by SOX2 depletion in A1207-ID4 whereas Tujl, 01ig2, and NG2 levels were dramatically increased in A172-ID4-shSOX2 (Fig. 5C), suggesting that aberrant differentiation of GSCs into a particular cell type is not likely to be determined by loss of SOX2. A serial passage analysis revealed that the tumor sphere-forming ability of A1207-SOX2 is gradually increased after 3 consecutive passages, although it is less effective than that in A1207-ID4. Furthermore, FACS analysis showed that, compared with controls, stem cell marker-positive cell populations increased in A1207-ID4 and A1207-SOX2 grown in NSC culture conditions (NBE medium supplemented with EGF and bFGF) whereas differentiation marker-positive populations increased in these cells grown in differentiation culture conditions (NBE medium supplemented with 5% FBS) for 7 days (Fig. 5D). In addition, we found that differentiation marker-positive cell populations decreased in A1207-ID4 and A1207-SOX2 in NSC cell culture conditions (Supplementary Fig. S9). Tumors derived from A1207-ID4 and A1207-SOX2 contained increased stem cell marker-positive cells compared with tumors derived from controls (Supplementary Fig. S10A-C). Together, these data indicate that SOX2 regulates, at least in part, ID4-driven iGSC genesis and maintenance and that SOX2 can reprogram glioma cells into iGSCs. The ID4-miR-9*-SOX2 regulatory axis primes ABCC3/6-mediated chemoresistance in hGSCs derived from GBM patients Consistent with previous observations that serum-free medium supplemented with EGF and bFGF facilitates maintenance of GSC features whereas media supplemented with FBS robustly differentiates GSCs into several differentiated lineages (33), we found that the 5% FBS-containing medium allowed our hGSCs (hGSCl, hGSC2, and hGSC3; derived from GBM patients; ref. 24) to be cells expressing various differentiated lineage markers (Fig. 6A; Supplementary Fig. Sll). Furthermore, ID4 and SOX2 protein were markedly reduced in these differentiated hGSCs (Fig. 6A), indicating a plausible connection between loss of ID4 and SOX2 and hGSC differentiation. To investigate whether the ID4-miR-9*-SOX2 3416 Cancer Res; 71 (9) May 1, 2011 Cancer Research Downloaded from cancerres.aacrjournals.org on April 12, 2014. © 2011 American Association for Cancer Research. ID4-miR-9*-SOX2 Pathway in Glioma Stem Cell Traits Figure 5. SOX2 induces self-renewal and tumorigenicity of glioma cells. A and B, representative tumor images and tumor weights obtained from nude mice (n = 6) injected subcutaneously with A1207-control and A1207-SOX2 (A) and A172-control and A172-SOX2 (B). Representative tumor sphere images and their numbers (>10 jim) attained from these cells grown for 14 days in NBE supplemented with EGF and bFGF. C, tumor spheres (>10 ^m) were markedly reduced in 2 A1207-ID4-shSOX2 cell lines and 2 A172-ID4-shSOX2 cell lines compared with A1207-ID4 and A172-ID4, respectively. The mRNA levels of GSC and their differentiated lineage markers were examined by qRT-PCR analysis. D, number of tumor spheres (>10 ^m) derived from 3 consecutive serial passages of A1207-control, A1207-ID4, and A1207-SOX2 grown for 14 days in NBE medium supplemented with EGF and bFGF. FACS analysis revealed GSC and their differentiated progeny populations of A1207-control, A1207-ID4, and A1207-SOX2 grown in the stem cell and differentiation-culture conditions, respectively; *, P < 0.05; " P < 0.01. A A1207 Control SOX2 0.1 rŤi m Control SOX2 60 X I 40 Q. o 20 E ß o Control SOX2 B A172 Control SOX2 Control SOX2 Control SOX2 60 w m I 40 - CL o 20 E ß o Control SOX2 shSOX2 1 7 ° S CO J U] £ 4 Cl X o d) J z 2 í 1 0 A1207 ■ Control □ ID4-shScr ■ID4-shSOX2-1 ■ ID4-shSOX2-2 ■ Control □ ID4-shScr ■ID4-shSOX2-1 ■ ID4-shSOX2-2 shSOX2 CD133 CD15 GFAP 25 ■ 20 ■ 1.5 ■Control □ ID4—-■ SC X2 Mil passage 2' passage 3' passage Stem cell culture condition 3 ~2.5 2 1.5 - 1 0.5 0 ■ Control DID4 «SOX2 Nestin SOX2 CD133 Stem cell culture condition 2.5 £ 2 ■ 1.5 = 0.5 I Control DID4 BSOX2 Üb Tuji NG2 GFAP S100ß Differentiation culture condition regulatory axis is also required for ABCC3 and ABCC6 expression in these hGSCs, we depleted ID4 in hGSCl and hGSC2 and found that ID4 knockdown resulted in a significant increase in miR-9* levels but a marked reduction in ABCC3 and ABCC6 expression (Fig. 6B). Similar to verapamil treatment, ID4 depletion led to decrease in side population of hGSCs (Fig. 6C). Both hGSCl-shID4 and hGSC2-shID4 showed substantial increases in BCNU-induced apoptosis as compared with their controls (Fig. 6D). Together, these findings suggest that the ID4-miR-9*-SOX2 regulatory axis plays a crucial role in controlling ABC transporter-mediated chemoresistance in both patient-derived hGSCs and human iGSCs. Tumor growth and chemoresistance in ID4- and SOX2-overexpressing glioma cells in vivo, and correlation of ID4 and ABCC3/ABCC6 expressions in human GBM patients Compared with the A1207-control, both A1207-ID4 and A1207-SOX2 showed an increased tumor growth and chemoresistance in vivo when tumors were intratumorally injected with BCNU (Fig. 7A). Furthermore, immunofluorescence analysis revealed that proliferating cell nuclear antigen (PCNA)-positive cells (proliferating cells) were increased in A1207-ID4 and A1207-SOX2-driven tumors whereas cleaved caspase-3-positive cells (apoptotic cells) were evidently www.aacrjournals.org Cancer Res; 71(9) May 1, 2011 3417 Downloaded from cancerres.aacrjournals.org on April 12, 2014. © 2011 American Association for Cancer Research. Jeon et al. hGSC-1 hGSC-3 hGSC-2 hGSC-1 hGSC-3 hGSC-2 ID4 SOX2 |3-Actin ■shScrambled □shlD4 ■shScr. + Vera B 3 ? 2.5 CD ■Control □ shlD4 hGSC-1 rh 41 i j ID4 ABCC3 ABCC6 miR-9* ■ Control hGSC-2 □ shlD4 ID4 ABCC3 ABCC6 miR-9* ■ Control □ shlD4 hGSC-1 hGSC-2 hGSC-1 hGSC-2 Figure 6. Significance of the ID4-miR-9*-SOX2-ABCC3/ABCC6 regulatory pathway in chemoresistance of hGSCs derived from patients with glioma. A, representative images showing morphology of 3 hGSCs grown under NBE medium supplemented with EGF and bFGF (NBE; N) and NBE medium supplemented with 5% FBS (FBS; F). ID4 and SOX2 protein levels decreased in 3 hGSCs grown in NBE medium supplemented with 5% FBS. B, qRT-PCR analysis revealed that ID4 knockdown in hGSC1 and hGSC2 reduced ABCC3 and ABCC6 mRNA levels but increased miR-9* expression. C, percentages of side population in hGSC-1 and hGSC-2. Vera, verapamil. D, percentage of dead cells in the hGSCs described in (B) treated with BCNU was determined by AnnexinV/PI-mediated FACS analysis; \ P < 0.05; **, P < 0.01. elevated in A1207-control-driven tumors that were treated with BCNU every 3 days for a total of 9 days (Fig. 7B; Supplementary Fig. S12). In addition, we found that ABCC3 and ABCC6 mRNA levels significantly correlated with ID4 levels in human GBM specimens (Fig. 7C). Discussion In the present study, we show a novel ID4-miR-9*-SOX2 regulatory pathway, where SOX2 plays crucial roles in controlling (i) the chemoresistance of GSCs by inducing ABCC3 and ABCC6, (ii) acquisition of self-renewal potential in glioma cells, and (iii) maintenance of sternness in ID4-driven iGSCs and GSCs derived from GBM patients (Fig. 7D). In contrast to most ABC transporters (ABCC1, ABCC2, ABCC4, and ABCC5) that are abundantly expressed in various anatomic regions of the normal brain, ABCC3 and ABCC6 expressions are rarely detectable in normal brain tissues (34). A few studies have reported that ABCC3 and ABCC6 dimin- ished the sensitivity of cancer cells to anticancer drugs, such as etoposide, doxorubicin, and daunorubicin, in a cell-type-dependent manner. However, no previous report has described the substrate specificity and role of ABCC3 and ABCC6 in the anticancer drug resistance of GSCs. miRNAs determine fundamental biological processes and human cancer initiation and progression through negative regulation of gene expression (15, 35). In particular, several miRNAs are involved in the development of anticancer drug resistance of many human malignancies by repressing ABC transporters. For example, miR-328, hsa-miR-519c, and hsa-miR-520h can repress ABCG2 expression by directly targeting its 3'-UTR in breast and pancreatic cancer cells (36-38). However, our findings revealed that ABCC3 and ABCC6 are not direct targets for miR-9*; instead, their expression is transcriptionally upregulated by SOX2, which is elevated by ID4-mediated suppression of miR-9*. Moreover, we found that ABCG2 is upregulated in several ID4-driven iGSCs and GSCs derived from patients. Thus, ABCG2 might probably be involved in 3418 Cancer Res; 71 (9) May 1, 2011 Cancer Research Downloaded from cancerres.aacrjournals.org on April 12, 2014. © 2011 American Association for Cancer Research. ID4-miR-9*-SOX2 Pathway in Glioma Stem Cell Traits Figure 7. Acceleration of tumorigenicity and chemoresistance in vivo by ID4 and SOX2, and expression of ID4 and ABCC3/ABCC6 in human GBM patients. A, representative tumor images (left) and tumor weights (right). As subcutaneously transplanted in nude mice (n = 18), A1207-ID4 and A1207-SOX2 showed marked increases in tumorigenicity and chemoresistance compared with A1207-control. B, cell proliferation (PCNA-positive ones) was significantly elevated in A1207-ID4 and A1207-SOX2-driven tumors. Compared with control tumors, A1207-ID4 and A1207-SOX2-driven tumors possessed reduced apoptotic cells (cleaved caspase-3-positive ones) as treated with BCNU. C, ABCC3 and ABCC6 mRNA levels correlated with ID4 mRNA levels in human GBM specimens (n = 32). D, a schematic diagram showing ID4-miR-9*-SOX2-ABCC3/ABCC6 signaling axis responsible for GSC genesis and chemoresistance; \ P < 0.05; " P < 0.01. BCNU Vehicle B 100 80 60 40 20 I BCNU □ Vehicle lull Control S0X2 ID4 A1207 p = 0.633259 P< 0.001 120 100 — 80 H '53 60 5 I 40 H 20 0 2 4 ID4 expression (log) ] I BCNU □ Vehicle m Control S0X2 ID4 A1207 BCNU □ Vehicle Control S0X2 A1207 Ol O 7 p = 0.78875 sin ( 6 P< 0.001 , ♦ ♦ 5 Q. 4 C6 es ♦ 5 ♦ O ♦ ♦ ♦ CD - < -"^ ID4 miR9* SOX2 ABCC3.6 0 2 4 6 ID4 expression (log) ► Chemoresistance Sternness ID4-mediated chemoresistance of iGSCs and GSCs to BCNU. However, the expression of ABCG2 in primary GSCs and ID4-driven iGSCs tested in this study differs from that in a previous report showing that ABCG2 activity in GSCs is determined by its cell-membrane localization and not by its expression levels (14). Our findings suggest that ABCC3 and ABCC6 may be bona fide GSC-relevant ABC transporters that are associated with chemoresistance because their expression was found to be elevated in most ID4- and SOX2-driven iGSCs and patient-driven GSCs tested but not in glioma cell lines. SOX2 and other reprogramming factors, such as Nanog 0CT4, and c-MYC, are frequently upregulated in poorly differentiated tumors (39) and have a high correlation with tumor regrowth and metastatic recurrence (40). SOX2 depletion in GSCs promotes differentiation and losses of sternness and tumorigenicity (22). TGF-|3 signaling enhances GSC sternness and tumorigenicity by inducing SOX4-mediated SOX2 expres- sion (32). Our findings revealed that SOX2 can reprogram Ink4a/Arf~/~ astrocytes and glioma cells to cells displaying GSC features and plays a crucial role in the maintenance of cancer sternness in ID4-driven iGSCs and GSCs derived from GBM patients. Furthermore, similar to a previous study revealing that the EWS-FLI-1 oncogene can reprogram primary cells to display the cancer stem cell phenotype by derepressing miR-145-mediated inhibition of SOX2 (41). our findings elucidate the induction of SOX2 by ID4-mediated suppression of miR-9* in GSCs and the sufficiency of SOX2 in reprogramming glioma cells to generate GSCs. Although several studies have reported that miR-145 is ubiquitously expressed in many tissues and functions as a potent SOX2 repressor in ESCs and iPSCs (17,42,43), we believe that miR-9* is a bona fide SOX2 repressor in NSCs and GSCs, because miR-9* expression is predominantly restricted in differentiated brain cells in vivo and in vitro, and is inversely associated www.aacrjournals.org Cancer Res; 71(9) May 1, 2011 3419 Downloaded from cancerres.aacrjournals.org on April 12, 2014. © 2011 American Association for Cancer Research. Jeon et al. with SOX2 expression in NSCs and differentiated neuronal cells (27, 28, 44). Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Acknowledgments We thank Kenneth S. Kosik (University of California, Santa Barbara, CA) for providing the pMir-Report-SOX2-3'-UTR plasmid. Grant Support This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST; no. 2008-0058785). H-M. Jeon is a recipient of the Best Graduate Student Scholarship from Korea University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must, therefore, be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received September 10, 2010; revised February 17, 2011; accepted March 3, 2011; published online April 29, 2011. References 1. Wen PY, Kesari S. Malignant gliomas in adults. N Engl J Med 2008;359:492-507. 2. Khasraw M, Lassman AB. Advances in the treatment of malignant gliomas. 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Correction Cancer Research Correction: Online Publication Dates for Cancer Research May 1, 2011 Articles The 4 articles noted below, which were published in the May 1, 2011 issue of Cancer Research, were published with incorrect online publication dates. Corrected versions of these articles have been published online. Ertl HCJ, ZaiaJ, Rosenberg SA, June CH, Dotti G, Kahn J, et al. Considerations for the clinical application of chimeric antigen receptor T cells: observations from a Recombinant DNA Advisory Committee symposium held June 15, 2010. Cancer Res 2011;71:3175-81. Ren N, Wu J-C, Dong Q-Z, Sun H-J, Jia H-L, Li G-C, et al. Association of specific genotypes in metastatic suppressor HTPAP with tumor metastasis and clinical prognosis in hepatocellular carcinoma. Cancer Res 2011;71:3278-86. Jeon H-M, Sohn Y-W, Oh S-Y, Kim S-H, Beck S, Kim S, et al. ID4 imparts chemore-sistance and cancer sternness to glioma cells by derepressing miR-9*-mediated suppression of SOX2. Cancer Res 2011;71:3410-21. Calvo V, Beato M. BRCA1 counteracts progesterone action by ubiquitination leading to progesterone receptor degradation and epigenetic silencing of target promoters. Cancer Res 2011;71:3422-31. Also, the OnlineFirst date is missing from the following article; it was published OnlineFirst on March 17, 2011. Lou Y, McDonald PC, Oloumi A, Chia S, Ostlund C, Ahmadi A, et al. Targeting tumor hypoxia: suppression of breast tumor growth and metastasis by novel carbonic anhydrase LX inhibitors. Cancer Res 2011;71:3364-76. Published OnlineFirst May 31, 2011. ©2011 American Association for Cancer Research. doi: 10.1158/0008-5472.CAN-11 -1396 www.aacrjournals.org 4325 Correction Cancer Research Correction: ID4 Imparts Chemoresistance and Cancer Sternness to Glioma Cells by Derepressing miR-9*-Mediated Suppression of SOX2 In this article (Cancer Res 2011;71:3410-21), which was published in the May 1, 2011, issue of Cancer Research (1), the name of the third author is incorrect. The correct name is Se-Yeong Oh. Reference 1. Jeon H-M, Sohn Y-W, Oh S-Y, Kim S-H, Beck S, Kim S, et al. ID4 imparts chemoresistance and cancer sternness to glioma cells by derepressing miR-9*-mediated suppression of SOX2. Cancer Res 2011;71:3410-21. Published OnlineFirst August 30, 2011. ©2011 American Association for Cancer Research. doi: 10.1158/0008-5472.CAN-11 -2545 www.aacrjournals.org for Cancer Research 6085