SATB2 drives glioblastoma growth by recruiting CBP to promote FOXM1 expression in glioma stem cells
2020; Springer Nature; Volume: 12; Issue: 12 Linguagem: Inglês
10.15252/emmm.202012291
ISSN1757-4684
AutoresWeiwei Tao, Aili Zhang, Kui Zhai, Zhi Huang, Haidong Huang, Wenchao Zhou, Qian Huang, Xiaoguang Fang, Briana C. Prager, Xiuxing Wang, Qiulian Wu, Andrew E. Sloan, Manmeet S. Ahluwalia, Justin D. Lathia, Jennifer S. Yu, Jeremy N. Rich, Shideng Bao,
Tópico(s)MicroRNA in disease regulation
ResumoArticle30 October 2020Open Access Transparent process SATB2 drives glioblastoma growth by recruiting CBP to promote FOXM1 expression in glioma stem cells Weiwei Tao Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Aili Zhang Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Kui Zhai Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Zhi Huang Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Haidong Huang Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Wenchao Zhou Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Qian Huang Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Xiaoguang Fang orcid.org/0000-0002-3368-4133 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Briana C Prager Division of Regenerative Medicine, Department of Medicine, University of California, San Diego, San Diego, CA, USA Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Xiuxing Wang Division of Regenerative Medicine, Department of Medicine, University of California, San Diego, San Diego, CA, USA Search for more papers by this author Qiulian Wu Division of Regenerative Medicine, Department of Medicine, University of California, San Diego, San Diego, CA, USA Search for more papers by this author Andrew E Sloan Brain Tumor and Neuro-Oncology Center & Center of Excellence for Translational Neuro-Oncology, University Hospitals Seidman Cancer Center, Case Western Reserve University, Cleveland, OH, USA Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Manmeet S Ahluwalia Brain Tumor and Neuro-Oncology Center, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Justin D Lathia Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Brain Tumor and Neuro-Oncology Center, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Jennifer S Yu Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Center for Cancer Stem Cell Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Department of Radiation Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Jeremy N Rich Division of Regenerative Medicine, Department of Medicine, University of California, San Diego, San Diego, CA, USA Search for more papers by this author Shideng Bao Corresponding Author [email protected] orcid.org/0000-0002-4236-2662 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Center for Cancer Stem Cell Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Weiwei Tao Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Aili Zhang Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Kui Zhai Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Zhi Huang Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Haidong Huang Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Wenchao Zhou Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Qian Huang Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Xiaoguang Fang orcid.org/0000-0002-3368-4133 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Briana C Prager Division of Regenerative Medicine, Department of Medicine, University of California, San Diego, San Diego, CA, USA Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Xiuxing Wang Division of Regenerative Medicine, Department of Medicine, University of California, San Diego, San Diego, CA, USA Search for more papers by this author Qiulian Wu Division of Regenerative Medicine, Department of Medicine, University of California, San Diego, San Diego, CA, USA Search for more papers by this author Andrew E Sloan Brain Tumor and Neuro-Oncology Center & Center of Excellence for Translational Neuro-Oncology, University Hospitals Seidman Cancer Center, Case Western Reserve University, Cleveland, OH, USA Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Manmeet S Ahluwalia Brain Tumor and Neuro-Oncology Center, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Justin D Lathia Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Brain Tumor and Neuro-Oncology Center, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Jennifer S Yu Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Center for Cancer Stem Cell Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Department of Radiation Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Jeremy N Rich Division of Regenerative Medicine, Department of Medicine, University of California, San Diego, San Diego, CA, USA Search for more papers by this author Shideng Bao Corresponding Author [email protected] orcid.org/0000-0002-4236-2662 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Center for Cancer Stem Cell Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Author Information Weiwei Tao1, Aili Zhang1, Kui Zhai1, Zhi Huang1, Haidong Huang1, Wenchao Zhou1, Qian Huang1, Xiaoguang Fang1, Briana C Prager2,3, Xiuxing Wang2, Qiulian Wu2, Andrew E Sloan4,5, Manmeet S Ahluwalia6, Justin D Lathia5,6,7, Jennifer S Yu1,5,8,9, Jeremy N Rich2 and Shideng Bao *,1,5,8 1Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA 2Division of Regenerative Medicine, Department of Medicine, University of California, San Diego, San Diego, CA, USA 3Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA 4Brain Tumor and Neuro-Oncology Center & Center of Excellence for Translational Neuro-Oncology, University Hospitals Seidman Cancer Center, Case Western Reserve University, Cleveland, OH, USA 5Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA 6Brain Tumor and Neuro-Oncology Center, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA 7Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, OH, USA 8Center for Cancer Stem Cell Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA 9Department of Radiation Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA *Corresponding author. Tel: +1 216 636 1009; Fax: +1 216 636 5454; E-mail: [email protected] EMBO Mol Med (2020)12:e12291https://doi.org/10.15252/emmm.202012291 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Nuclear matrix-associated proteins (NMPs) play critical roles in regulating chromatin organization and gene transcription by binding to the matrix attachment regions (MARs) of DNA. However, the functional significance of NMPs in glioblastoma (GBM) progression remains unclear. Here, we show that the Special AT-rich Binding Protein-2 (SATB2), one of crucial NMPs, recruits histone acetyltransferase CBP to promote the FOXM1-mediated cell proliferation and tumor growth of GBM. SATB2 is preferentially expressed by glioma stem cells (GSCs) in GBM. Disrupting SATB2 markedly inhibited GSC proliferation and GBM malignant growth by down-regulating expression of key genes involved in cell proliferation program. SATB2 activates FOXM1 expression to promote GSC proliferation through binding to the MAR sequence of FOXM1 gene locus and recruiting CBP to the MAR. Importantly, pharmacological inhibition of SATB2/CBP transcriptional activity by the CBP inhibitor C646 suppressed GSC proliferation in vitro and GBM growth in vivo. Our study uncovers a crucial role of the SATB2/CBP-mediated transcriptional regulation in GBM growth, indicating that targeting SATB2/CBP may effectively improve GBM treatment. SYNOPSIS Aberrant expression of nuclear matrix-associated proteins (NMPs) has been shown to associate with tumor growth in various human cancers. This study shows that SATB2, a key NMP, and its coactivator CBP critically contribute to glioblastoma (GBM) growth, suggesting SATB2/CBP is a therapeutic target. SATB2 and CBP are preferentially expressed by glioma stem cells (GSCs) in GBMs. SATB2 recruits CBP to activate FOXM1 transcription, promoting GSC proliferation and GBM tumor growth. Inhibition of SATB2/CBP transcriptional activity by the CBP inhibitor C646 suppressed GBM growth. The paper explained Problem Glioblastoma (GBM) is the most frequent and malignant type of human primary brain tumor. The prognosis of GBM remains extremely poor despite significant advances in the treatment of other solid cancers. Accumulating evidence supports that glioma stem cells (GSCs) are responsible for GBM initiation, progression, and therapeutic resistance. Therefore, better understanding of the molecular mechanisms driving GSC proliferation and self-renewal may offer new therapeutic strategies targeting GSCs to inhibit GBM malignant growth and overcome the therapeutic resistance. Results Our study identified SATB2/CBP as a critical regulatory complex that activates FOXM1 expression in GSCs to promote the GSC maintenance and GBM malignant growth. Both SATB2 and CBP are preferentially expressed by GSCs in GBMs. SATB2/CBP binds to the matrix attachment region of FOXM1 gene locus to stimulate FOXM1 transcription in GSCs. Importantly, pharmacological inhibition of SATB2/CBP transcriptional activity by the CBP inhibitor C646 potently inhibited GBM tumor growth. Impact Our data demonstrate that targeting the SATB2/CBP-FOXM1 axis markedly inhibited GSC proliferation and GBM tumor growth, offering an effective therapeutic strategy through the inhibition of SATB2/CBP to improve GBM treatment. Introduction Glioblastoma (GBM; WHO grade IV glioma) is the most frequent and malignant type of human primary brain tumor. The prognosis of GBM is extremely poor despite significant advances in the treatment of other solid cancers. The median survival of GBM patients remains less than 16 months (Furnari et al, 2007; Stupp et al, 2009). The standard therapies including surgical resection, radiation therapy, and chemotherapy are largely ineffective for GBMs due to universal therapeutic resistance and rapid tumor recurrence (Wen & Kesari, 2008). Therefore, it is crucial to identify new treatments to improve the anti-cancer efficacy. GBM displays striking cellular heterogeneity and hierarchy within a tumor containing a fraction of stem cell-like cancer cells called glioma stem cells (GSCs) at the apex of differentiation hierarchy. GSCs exhibit remarkable capacities of proliferation and self-renewal and play critical roles in modulating the tumor microenvironment, neovascularization, cancer invasion, and immune evasion (Magee et al, 2012; Lathia et al, 2015; Finocchiaro, 2017; Tao et al, 2020). Accumulating evidence supports that GSCs are responsible for tumor initiation, progression, and therapeutic resistance (Bao et al, 2006; Jin et al, 2017). Thus, better understanding of the molecular mechanisms driving GSC proliferation and self-renewal may offer new insights into GBM tumorigenesis, which may lead to effective therapeutic approaches to improve GBM treatment. Altered chromatin organization is one of hallmarks in cancer cells. Abnormalities in chromatin architecture and transcriptional regulation occur in most cancer cells (He et al, 2008), but our knowledge regarding the potential mechanisms driving the changes in chromatin organization and transcription activity in cancer cells is limited. The alteration of chromatin organization in cancer cells leads to dysregulation of gene expression, which contributes to the malignant transformation of cells (Schuster-Bockler & Lehner, 2012). Nuclear matrix-associated proteins (NMPs) are a family of proteins that specifically bind to the matrix attachment regions (MAR) of genomic DNA to regulate chromatin organization and gene expression (Dunn et al, 2003; Wang et al, 2010; Yamaguchi & Takanashi, 2016). Aberrant expression of NMPs has been shown in various human cancers, including breast cancer, lymphoma, colon cancer, non-small cell lung cancer, gastric cancer, and liver cancer (Lever & Sheer, 2010). However, whether NMPs are aberrantly expressed in glioma cells particularly GSCs is not clear, and whether the altered NMP expression contributes to GBM malignant growth has not been defined. To interrogate the potential relationship between the expression of NMPs and GBM tumor development, we queried the expression pattern of NMPs in clinical database and found that SATB2 (the Special AT-rich Binding Protein-2) is enriched in GBMs. SATB2 is a transcription factor that was originally identified as a protein interacting with the nuclear matrix attachment regions (MAR) of DNA. STAB2 regulates gene expression by modulating chromatin architecture and functioning as a transcriptional cofactor (Dobreva et al, 2003; Britanova et al, 2008; Diaz-Alonso et al, 2012). When SATB2 is localized to the matrix attachment region, it promotes chromatin rearrangement by recruiting chromatin-remodeling proteins to these DNA sequences to either activate or repress gene transcription (Britanova et al, 2008; Gyorgy et al, 2008; Zhou et al, 2012). SATB2 is an evolutionarily conserved protein in vertebrates from zebrafish to mammals (Sheehan-Rooney et al, 2010). SATB2 as one of critical NMPs has multiple roles in osteoblast differentiation, craniofacial patterning, cleft palate formation, and neuronal development (Dobreva et al, 2006; Leoyklang et al, 2007; Britanova et al, 2008; Zarate & Fish, 2017). SATB2 affects craniofacial morphogenesis via repression of HOXA2 and regulates osteoblast differentiation by interacting with transcription factors RUNX2 and ATF4 to enhance their activity (Dobreva et al, 2006). Moreover, SATB2 regulates neuronal specification during development by recruiting the chromatin-remodeling complexes to CTIP2 locus to regulate its expression (Britanova et al, 2008). Recent studies demonstrated that SATB2 is associated with tumor growth or suppression. SATB2 has been shown to suppress tumor progression in colorectal cancer, non-small cell lung cancer and gastric cancer, and high SATB2 expression is associated with a favorable prognosis (Mansour et al, 2015; Wu et al, 2016; Ma et al, 2018). However, SATB2 promotes tumor growth in hepatocellular carcinoma, osteosarcoma, and triple-negative breast cancer (Jiang et al, 2015; Luo et al, 2016; Xu et al, 2017). Our analysis of clinical database indicated that SATB2 is enriched in GBMs, suggesting that SATB2 may play a tumor-promoting role in GBMs. Therefore, we investigated the potential role of SATB2 in regulating GSC properties and GBM tumor growth, and found that SATB2 augmented GSC proliferation by recruiting histone acetyltransferase CBP to promote FOXM1 expression in GSCs. FOXM1 is a member of the forkhead box transcription factor family, which is evolutionarily conserved and contains a common DNA-binding domain called forkhead box domain (Laoukili et al, 2007; Liao et al, 2018). FOXM1 is a typical transcription factor related to cell proliferation and involved in cancer growth (Laoukili et al, 2007; Nandi et al, 2018). It regulates cell cycle process by modulating the expression of various cell cycle-related genes required for G1/S, G2/M progression (Li et al, 2012). FOXM1 is highly expressed in GBM and informs poor prognosis of GBM patients (Liu et al, 2006; Lee et al, 2015; Zhong et al, 2016). However, the functional significance and the molecular mechanisms underlying FOXM1 regulation in GSCs are poorly understood. In this study, we found that FOXM1 expression is activated by SATB2 in GSCs. SATB2 binds to the MAR sequence of the FOXM1 gene locus and recruiting CBP to the MAR site to promote FOXM1 expression. Our study uncovers a critical role of the SATB2/CBP complex in regulating FOXM1 expression to promote GSC proliferation and GBM malignant growth. Importantly, inhibition of SATB2/CBP transcriptional activity by the CBP inhibitor C646 significantly suppressed GSC proliferation and GBM tumor growth, indicating that targeting SATB2/CBP may be an effective therapeutic strategy to improve GBM treatment. Results SATB2 is preferentially expressed by GSCs To determine the potential relationship between the nuclear matrix-associated proteins (NMPs) and GBM malignant growth, we mapped the expression of NMPs in TCGA GBM and low-grade glioma (LGG) databases, with consideration of tumor transcriptional subtype, IDH1/P53/PTEN mutation status, tumor grade, patient age, and performance status. Our analyses focused on several key NMPs including SATB1/2, SAFB1/2, EZH2, SUZ12, BMI1, PCL3, RAE28, and CTCF, as these NMPs have been shown to be aberrantly expressed in cancers (Lever & Sheer, 2010). The analyses revealed that SATB2, EZH2, SUZ12, and PCL3 are enriched in older patients with glioblastoma (GBM) with worse performance status (Appendix Fig S1). Among these four genes, SATB2's role in GBM progression has not been defined. To interrogate the functional significance of SATB2 expression in GBM malignant growth, we initially examined SATB2 expression pattern in several human GBM specimens and found that SATB2 is preferentially expressed in nuclei of glioma cells expressing the GSC markers SOX2 and OLIG2 (Fig 1A and B; Appendix Fig S2A). Further experiments demonstrated that SATB2 is rarely expressed in glioma cells expressing the differentiation markers (GFAP, TUBB3, and GALC) in human GBMs (Appendix Fig S2B–G). To confirm the preferential expression of SATB2 in GSCs, we assessed SATB2 expression in isolated GSCs and matched non-stem tumor cells (NSTCs) that were functionally validated as described in Materials and Methods. The results showed that SATB2 and the GSC markers SOX2 and OLIG2 were preferentially expressed in all isolated GSC populations relative to matched NSTCs (Fig 1C–E; Appendix Fig S2H). In addition, SATB2 was expressed at much higher levels in GSCs than in neural progenitor cells (NPCs) (Fig 1F and G). As GSC population decreases during differentiation, we examined the expression of SATB2 during GSC differentiation induced by the serum. A gradual reduction of SATB2 and the GSC marker SOX2 was observed during GSC differentiation, which was accompanied by the increased expression of the differentiation marker GFAP (Fig 1H), indicating a potential link between SATB2 expression and GSC status. Collectively, these data demonstrate that SATB2 is preferentially expressed by GSCs in GBMs, suggesting a potential role of SATB2 in the GSC maintenance. Figure 1. SATB2 is preferentially expressed by GSCs Immunofluorescence of SATB2 (green) and the GSC marker SOX2 or OLIG2 (red) on frozen tissue sections of human GBM surgical specimens. SATB2 is preferentially expressed by GSCs in human GBMs. Scale bar, 25 μm. Quantification of the fraction of SATB2+ cells in SOX2+ or OLIG2+ cells in human GBMs. More than 90% SOX2+ or OLIG2+ cells showed SATB2 staining. n = 3 GBMs. qPCR analysis of SATB2 mRNA expression in GSCs and matched non-stem tumor cells (NSTCs) (n = 5). Immunoblot analysis of SATB2, SOX2, and OLIG2 expression in cell lysates of GSCs and matched NSTCs. Immunofluorescence of SATB2 (green) and SOX2 (red) in T3359 GSCs and matched NSTCs. Scale bar, 50 μm. qPCR analysis of SATB2 mRNA expression in GSCs and neural progenitor cells (NPCs) (n = 3). Immunoblot analysis of SATB2 and SOX2 expression in cell lysates of GSCs and NPCs. Immunoblot analysis of SATB2, GSC marker SOX2, and differentiation marker GFAP expression during serum-induced GSC differentiation. Data information: Data are represented as mean ± SD. **P < 0.01, ***P < 0.001, Mann–Whitney test (C) or one-way ANOVA analysis followed by Tukey's test (F). Exact P values are reported in Appendix Table S3. Download figure Download PowerPoint SATB2 is required for GSC proliferation and self-renewal As SATB2 is preferentially expressed in GSCs, we next investigated the functional significance of SATB2 in the GSC maintenance by using two distinct shRNAs targeting SATB2. Lentivirus-mediated expression of shSATB2-1 or shSATB2-2 markedly reduced SATB2 protein levels in GSCs (Fig 2A). We found that disruption of SATB2 significantly inhibited GSC growth as measured by cell titer assay (Fig 2B) and reduced DNA replication as assayed by EDU incorporation assay (Fig 2C and D). In contrast, disruption of SATB2 had little effect on the growth and survival of NSTCs (Appendix Fig S3A) and NPCs (Appendix Fig S3B). In addition, silencing SATB2 impaired GSC self-renewal as assessed by tumorsphere formation assays (Fig 2E–G) and in vitro limiting dilution assays (Fig 2H). Consistently, disrupting SATB2 also reduced expression of the GSC markers including SOX2 and OLIG2 (Appendix Fig S3C). Taken together, these results indicate that SATB2 is essential for GSC proliferation and self-renewal. Figure 2. Disrupting SATB2 impaired GSC proliferation and self-renewal A. Immunoblot analysis of SATB2 expression in GSCs transduced with lentiviral-mediated non-targeting shRNA (shNT) or SATB2 shRNA (shSATB2). B. Cell viability of GSCs transduced with shNT or shSATB2 (n = 5). C. EdU incorporation assay of GSCs transduced with shNT or shSATB2. Scale bar: 50 μm. D. Quantification of (C) showing the percentage of EdU+ cells (n = 5). E. Tumorsphere images of GSCs transduced with shNT or shSATB2. Scale bar: 100 μm. F, G. Quantification of the diameter (F) or number (G) of tumorspheres formed by GSCs expressing shNT or shSATB2 (F: n = 9; G: n = 5). H. In vitro limiting dilution analysis of the tumorsphere formations of GSCs expressing shNT or shSATB2. Silencing SATB2 attenuated the self-renewal capacity of GSCs. Data information: Data are represented as mean ± SD. ****P < 0.0001, two-way ANOVA analysis followed by Tukey's test (B), one-way ANOVA analysis followed by Tukey's test (D, F, and G) or ELDA analysis for differences in stem cell frequencies (H). Exact P values are reported in Appendix Table S3. Download figure Download PowerPoint Silencing SATB2 suppresses GSC-driven tumor growth As the most important property of GSCs is their potent capacity to propagate tumors in vivo, we then examined the impact of SATB2 inhibition on the GSC-driven intracranial tumor growth. GSCs (T3359 or H2S) expressing firefly luciferase and shSATB2 (shSATB2-1 or shSATB2-2) or shNT were transplanted into the brains of immunocompromised mice by intracranial injection. In vivo bioluminescent imaging of orthotopic tumors showed that silencing SATB2 dramatically inhibited GSC-driven tumor growth in mouse brains (Fig 3A and B; Appendix Fig S3D and E). Consequently, animals bearing xenografts derived from GSCs expressing shSATB2 survived significantly longer than the control animals (Fig 3C; Appendix Fig S3F). Further experiment demonstrated that silencing SATB2 reduced the tumorogenic potential of GSCs in an in vivo limiting dilution assay (Appendix Table S1). Given that SATB2 promotes GSC proliferation in vitro, we next examined the impact of SATB2 disruption on cell proliferation in GSC-derived xenografts. Immunofluorescent staining confirmed that the expression of SATB2 was significantly decreased in xenografts expressing shSATB2 (Fig 3D and E; Appendix Fig S3G and H). We found that cell proliferation was markedly reduced in the GBM xenografts derived from GSCs expressing shSATB2 than control tumors as demonstrated by Ki67 immunofluorescence (Fig 3F and G; Appendix Fig S3I and J). In addition, silencing SATB2 significantly reduced GSC population as revealed by SOX2 immunofluorescence (Fig 3H and I; Appendix Fig S3K and L). Collectively, these data demonstrate that SATB2 disruption potently suppresses the GSC-driven tumor growth, indicating that SATB2 is critical for maintaining the tumorigenic potential of GSCs in vivo. Figure 3. Disrupting SATB2 inhibited tumor growth and prolonged mice survival Bioluminescent images of the GBM xenografts derived from the luciferase-labeled T3359 GSCs expressing NT or SATB2 shRNA. Representative images on day 21 posttransplantation are shown (n = 5 mice per group). Silencing SATB2 significantly delayed GBM growth. Quantification of the bioluminescence of xenografts derived from the luciferase-labeled T3359 GSCs expressing shNT or shSATB2 on day 21 posttransplantation (n = 5 mice per group). Kaplan–Meier survival curves of mice intracranially implanted with T3359 GSCs expressing shNT or shSATB2 (shNT: n = 7 mice; shSATB2-1 or shSATB2-2: n = 5 mice). Median survival: shNT, 28 days; shSATB2-1, 48 days; shSATB2-2, 45 days. Animals bearing GSC-derived xenografts expressing SATB2 shRNA survived longer than the control animals. Immunofluorescence of SATB2 (Green) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n = 5 tumors per group). Scale bar: 40 μm. Quantification of SATB2 intensity in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n = 5 tumors per group). Immunofluorescence of Ki67 (Green) in tumor xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n = 5 tumors per group). Scale bar: 40 μm. Quantification of Ki67 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n = 5 tumors per group). Immunofluorescence of SOX2 (Red) in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n = 4 tumors per group). Scale bar: 40 μm. Quantification of SOX2 positive cells in xenografts derived from T3359 GSCs expressing shNT or shSATB2 (n = 4 tumors per group). Data information: Data are shown as mean ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001 compared with shNT group, Kruskal–Wallis test followed by Dunn's test (B), log-rank test (C), or one-way ANOVA analysis followed by Tukey's test (E, G, and I). Exact P values are reported in Appendix Table S3. Download figure Download PowerPoint SATB2 is required for the expression of genes involved in cell cycle progression To understand the molecular mechanisms by which SATB2 promotes GSC proliferation, we performed transcriptional profiling on GSCs expressing shSATB2 or shNT and found that GSCs expressing shSATB2 exhibited distinct gene expression profiles relative to control GSCs with shNT (Fig 4A). Gene ontology analysis suggested that the most significantly down-regulated expression of genes in GSCs after SATB2 disruption is associated with cell cycle progression and chromosome organization (Fig 4B), while there was no significant functional relevance for the up-regulated genes in GSCs expressing shSATB2. Among the genes involved in cell cycle process, FOXM1 appeared to be the most important downstream target of SATB2 due to several reasons. First, FOXM1 is a master transcription factor in cell cycle regulation and proliferation (Wierstra & Alves, 2007; Li et al, 2012). Second, FOXM1 is overexpressed in GBMs and informs poor survival of GBM patients (Lee et al, 2015; Zhong et al, 2016). Lastly, FOXM1 is co-localized with GSC markers SOX2 and Nestin in primary GBM specimens (Joshi et al, 2013). Therefore, we validated the effect of SATB2 disruption on FOXM
Referência(s)