doi:10.1093/jmcb/mjy007 Journal of Molecular Cell Biology (2018), 10(2), 147–160 j 147 Published online March 12, 2018 Article PHF20 collaborates with PARP1 to promote stemness and aggressiveness of neuroblastoma cells through activation of SOX2 and OCT4 1,2,† 2,3,† 2,4 2 3 2 Wenyong Long , Wei Zhao , Bo Ning , Jing Huang , Junjun Chu , Linfeng Li , 1,2 2 2 1 2,5,6, Qianquan Ma , Changsheng Xing , Helen Y. Wang , Qing Liu , and Rong-Fu Wang Department of Neurosurgery in Xiangya Hospital, Central South University, Changsha 410008, China Center for Inﬂammation and Epigenetics, Houston Methodist Research Institute, Houston, TX 77030, USA Key Laboratory of Stem Cells and Tissue Engineering, Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China Institute Center for Molecular Design and Biomimetics, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY 10065, USA Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX 77030, USA These authors contributed equally to this work. * Correspondence to: Rong-Fu Wang, E-mail: email@example.com Edited by Hua Lu The differentiation status of neuroblastoma (NB) strongly correlates with its clinical outcomes; however, the molecular mechan- isms driving maintenance of stemness and differentiation remain poorly understood. Here, we show that plant homeodomain ﬁnger-containing protein 20 (PHF20) functions as a critical epigenetic regulator in sustaining stem cell-like phenotype of NB by using CRISPR/Cas9-based targeted knockout (KO) for high-throughput screening of gene function in NB cell differentiation. The expression of PHF20 in NB was signiﬁcantly associated with high aggressiveness of the tumor and poor outcomes for NB patients. Deletion of PHF20 inhibited NB cell proliferation, invasive migration, and stem cell-like traits. Mechanistically, PHF20 interacts with poly(ADP-ribose) polymerase 1 (PARP1) and directly binds to promoter regions of octamer-binding transcription factor 4 (OCT4) and sex determining region Y-box 2 (SOX2) to modulate a histone mark associated with active transcription, trimethyla- tion of lysine 4 on histone H3 protein subunit (H3K4me3). Overexpression of OCT4 and SOX2 restored growth and progression of PHF20 KO tumor cells. Consistently, OCT4 and SOX2 protein levels in clinical NB specimens were positively correlated with PHF20 expression. Our results establish PHF20 as a key driver of NB stem cell-like properties and aggressive behaviors, with implications for prognosis and therapy. Keywords: PHF20, neuroblastoma, PARP1, cancer stem cell-like traits, epigenetic regulation Introduction (Molenaar et al., 2012). Therefore, the identiﬁcation of key Neuroblastoma (NB) is the most common extracranial solid regulators that control NB risk stratiﬁcation is critically tumor of childhood, accounting for the largest number of cancer- important for developing more effective therapeutics. related deaths in children (Louis and Shohet, 2015). This tumor Several markers that predict a good or poor treatment out- arises from the developing neural crest cells, which possess self- come have been reported (Janoueix-Lerosey et al., 2009). renewal and multipotency characteristics (Huang and Weiss, 2013), Currently, the most validated prognostic marker of high-risk dis- and aberrations in normal developmental processes are most likely ease and poor prognosis is ampliﬁcation of the NB-derived its primary cause (Takahashi et al., 2013). Notably, patients with v-myc avian (MYCN) oncogene (Powers et al., 2016), which is undifferentiated or poorly differentiated NB have signiﬁcantly present in ∼25% of cases (Huang and Weiss, 2013). MYCN is worse outcomes than those with well-differentiated NB involved in the regulation of self-renewal and can substitute for MYC in reprogramming ﬁbroblasts into induced pluripotent stem Received October 6, 2017. Revised January 22, 2018. Accepted February 9, 2018. cells (iPSC) (Smith et al., 2010). Emerging evidence from several © The Author (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. tumor types, including NB, points to the potential active role of Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/147/4857401 by Ed 'DeepDyve' Gillespie user on 20 June 2018 148 j Long et al. the cancer initiating cells in disease progression, relapse, and poor PHF20, ﬁbroblast growth factor receptor 2 (FGFR2), myeloid dif- outcomes (Suva et al., 2013). Thus, MYCN drives NB into a stem ferentiation primary response gene 88 (MYD88), and notch cell-like state by blockade of differentiation pathways and expres- homolog 1 (NOTCH1) involved in the maintenance of the sion of self-renewal and pluripotency factors (Kaneko et al., 2015). de-differentiated state of SH-SY5Y cells. There are signiﬁcantly Some NB cells retain multipotency and overexpress stem cell- increased neurite density and more differentiated cells in related genes, such as OCT4 and SOX2 (Singovski et al., 2016). SH-SY5Y cells infected with one of these sgRNAs compared with OCT4, SOX2, and NANOG have been demonstrated to play crit- the control cells (Figure 1D and Supplementary Figure S1). ical roles in stem cell self-renewal and have been proposed to Importantly, FGFR2 and NOTCH1 identiﬁed in our screening have promote the self-renewal of cancer cells with stem cell-like prop- been reported to play a role in the maintenance of NB stem cell erties (Mu et al., 2017). Despite these correlative studies and malignant phenotypes (Zweidler-McKay, 2008; Katoh and between OCT4 and SOX2 expression and the stem cell-like state Nakagama, 2014). We further validated the screening results of NB, how OCT4 and SOX2 are reactivated for conferring NB with two sgRNAs against PHF20 using western blotting analysis, stem cell-like traits remains unclear. and showed marked KO efﬁciency of PHF20 sgRNA1 and Systematic search and analysis for genomic alterations using sgRNA2, compared with control sgRNA (Figure 1E). PHF20 KO in whole-genome or whole-exome sequencing show that NB has SH-SY5Y cells substantially changed cell morphology and remarkably low genetic complexity along with few genes that have downregulated the core pluripotent genes (i.e. SOX2, OCT4, and signiﬁcant mutations (Chmielecki et al., 2017). These ﬁndings indi- NANOG), compared with those in control cells (Figure 1F and G). cate that aberrant epigenetic modiﬁcations, including DNA methyla- These data suggest that PHF20 is an important regulator to tion and histone modiﬁcation (Olsson et al., 2016), are important maintain the stemness of NB cells. features of both development and progression of NB. However, their functional relevance is largely unknown. In this study, we PHF20 is highly expressed, and the expression is associated developed a targeted knockout (KO) strategy and conducted a with the aggressiveness in NB screening of 573 transcriptional and epigenetic factors required for To determine the relationship between PHF20 expression and NB differentiation. Among the genes identiﬁed, we found that plant NB aggressiveness, we investigated the PHF20 expression in the homeodomain ﬁnger-containing protein 20 (PHF20) was a key epi- Asgharzadeh NB dataset and found that 92.3% of the 117 NBs genetic factor controlling the stem cell-like phenotype of NB. had higher PHF20 expression compared with normal tissues Ablation of PHF20 led to inhibition of proliferation and malignancy, (Supplementary Figure S2A). We then determined the protein while ectopic expression of PHF20 enhanced the expression of level of PHF20 in different NB cell lines, including CHLA-255, JF, OCT4 and SOX2, suggesting that PHF20 is a pivotal regulator of NB LA-N-6, NB-19, NGP, SK-N-AS, SH-EP, SH-SY5Y, and IMR-32, and initiation and progression. Thus, our ﬁndings have identiﬁed PHF20 found higher levels of PHF20 expression in all nine NB cell lines, as a therapeutic target for NB treatment. but not in control cells (Figure 2A). Normal peripheral blood mononuclear cells (PBMCs) were used as a control since normal Results neural cell lines or tissues were not available for western blot Identiﬁcation of PHF20 as a driver of stem cell-like phenotype analysis. To determine whether PHF20 overexpression is corre- in NB lated with high histologic grade in NB tissues, we examined Identiﬁcation of key factors that regulate cancer initiation and PHF20 expression in parafﬁn-embedded human NB samples by progression may help develop novel and effective strategies to immunohistochemistry, and found a marked increase in the overcome the chemoresistance associated with NB therapy. expression of PHF20 in all NB tumor samples when compared Thus, we designed a high-throughput screening based on a with the normal peripheral nerve tissue (Figure 2B) and normal CRISPR/Cas9 library of 573 sgRNAs to identify potential targets human tissue from different organs (Figure 2C and Supplementary (Supplementary Table S1). This screening targeted 288 genes, Figure S2B). The association between PHF20 expression in NB which included frequently mutated genes and epigenetic regula- and tumor-free survival time of selected patients was analyzed tors. As shown in Figure 1A, retinoic acid (RA)-treated SH-SY5Y through Kaplan–Meier survival analysis with two different cells showed intense neurite networking by Day 3, while datasets (Figure 2D and Supplementary Figure S2C). In both The untreated SH-SY5Y cells formed aggregates over time. Stem cell Cancer Genome Atlas (TCGA) and Texas Children’s Hospital data- pluripotent genes, such as SOX2, OCT4, NANOG, and NESTIN, sets, high PHF20 expression was inversely correlated with poor were dramatically downregulated in a time-dependent manner median overall survival (OS) of NB patients (P < 0.016) after RA induced neuritogenesis (Figure 1B), indicating that SH- (Figure 2D and Supplementary Figure S2C). Collectively, these SY5Y cells can serve a useful cellular model to identify the key results demonstrate the pivotal role of PHF20 in the aggressive regulators of NB differentiation. We transduced SH-SY5Y cells behavior of NB and patient overall survival. with 573 sgRNA lentiviral supernatants (one sgRNA per well) with puromycin selection on Day 3 to eliminate the uninfected PHF20 increases cellular viability and proliferative capacity cells (Figure 1C and Supplementary Table S1). Infected cells of NB cells both in vitro and in vivo were cultured for differentiation and imaged at Day 10 with crys- Human NB cell lines can be divided into three categories: N-type tal violet staining. After three rounds of screening, we identiﬁed (neuroblastic), S-type (substrate-adherent and the non-neuronal), Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/147/4857401 by Ed 'DeepDyve' Gillespie user on 20 June 2018 PHF20 confers neuroblastoma stem cell-like traits j 149 Figure 1 High-throughput screening of key regulators for NB differentiation using a CRISPR/Cas9 sgRNA library. (A) Bright-ﬁeld microscopy of crystal violet staining of SH-SY5Y cells with and without RA treatment. Neurite outgrowth (arrows) began at Day 3 post-RA treatment. Scale bar, 50 μm. (B) The mRNA expression of SOX2, OCT4, NANOG, and NESTIN of SH-SY5Y cells at 0, 36, and 72 h post-RA treatment. (C) A schematic diagram of the sgRNA library screening system. (D) Heat maps generated from sgRNA library screening of SH-SY5Y cell differen- tiation analysis. (E) Western blot analysis of PHF20 expression in control cells by non-speciﬁc sgRNA and PHF20 KO SH-SY5Y cells by two dif- ferent PHF20-speciﬁc sgRNAs. (F) Crystal violet staining in control cells and PHF20 KO SH-SY5Y cells. Dense neurite networks (arrows) were found in PHF20 KO SH-SY5Y cells. (G) The mRNA expression of SOX2, OCT4, and NANOG in control cells and PHF20 KO SH-SY5Y cells from two different sgRNAs. Data are plotted as mean ± SD of three independent experiments. *P < 0.05;**P < 0.01; ***P < 0.001 compared with controls using Student’s t-test. and I-type (the intermediate) (Spengler et al., 1997). N-type PHF20, we decided to continue our work using S-type NB cells. (SH-SY5Y) and I-type [BE(2)-C] usually express MYCN at high- To determine the role of PHF20 in S-type NB tumorigenesis, we er levels. Surprisingly, S-type NB cells include both malignant established PHF20 KO cell clones of SH-EP and SK-N-AS. PHF20 cellssuchasSK-N-AS andinnocent cells such as SH-EP that KO in these cells was demonstrated by western blot analysis express low levels of MYCN (Ross et al., 2003), suggesting (Figure 3A) and used for subsequent experiments. Both PHF20 that other survival mechanisms are important. As N-type and KO SH-EP and SK-N-AS cells showed signiﬁcantly reduced cell I-type cells express high levels of MYCN, which has been asso- viability compared with cells expressing CRISPR/Cas9 non- ciated to NB aggressiveness. Since our focus was on the role of speciﬁc control sgRNA (Figure 3B). To extend these in vitro Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/147/4857401 by Ed 'DeepDyve' Gillespie user on 20 June 2018 150 j Long et al. Figure 2 PHF20 is highly expressed in NB and correlates with the poor outcome of NB patients. (A) Western blot analysis of PHF20 expres- sion in nine NB cell lines and normal PBMCs. (B) IHC staining of PHF20 in NB of Grades 1–3 from patients and comparison with normal per- ipheral nervous tissue. (C) The statistical results showed the proportion of PHF20-positive cells in each group. (D) The association between PHF20 expression in NB and tumor-free survival time of selected patients was analyzed by Kaplan–Meier analysis in TCGA dataset. Scale bar, 50 μm. Data are plotted as mean ± SD of three independent experiments. *P < 0.05;**P < 0.01; ***P < 0.001 compared with controls using Student’s t-test. observations, we investigated whether PHF20 KO could inhibit assay also showed that the number of apoptotic cells increased the tumorigenic capacity of NB cells in vivo. We injected PHF20 in the PHF20 KO groups (Figure 3F and G). Together, these results KO SH-EP, PHF20 KO SK-N-AS, and their corresponding control suggest that PHF20 promotes tumor growth and proliferation, cells subcutaneously into NOD-SCID IL2Rγ-null (NSG) mice. The and reduces cell apoptosis. tumor volumes were measured every other day up to 28 days. PHF20 KO in both SH-EP and SK-N-AS cells remarkably decreased PHF20 promotes migratory and invasive capacities of NB cells the tumor volume and weight (Figure 3C–E). Furthermore, the To investigate the effect of PHF20 on cell migration, we ﬁrst xenograft tumors were excised and processed for hematoxylin performed the wound healing assay using PHF20 KO SH-EP and eosin (H&E) staining, immunohistochemical staining (IHC), and PHF20 KO SK-N-AS cells, and found that their migration and a terminal deoxynucleotidyl transferase dUTP nick-end label- abilities were signiﬁcantly reduced compared with control cells ing (TUNEL) assay (Figure 3F). Unlike the high PHF20 expression (Supplementary Figure S3A). Consistently, we showed that seen in the control group, IHC staining indicated that PHF20 was PHF20 KO SH-EP and PHF20 KO SK-N-AS cells had reduced deleted in the KO group. The signiﬁcant reduction in the cell pro- invasive capacity compared with control cells (Figure 4Aand liferation rate in PHF20 KO cells-derived tumors was further con- B), suggesting that PHF20 is required for the invasive ability ﬁrmed by measuring Ki-67 levels (Figure 3F and G). The TUNEL of NB cells. Since Wnt/β-catenin (Blanc et al., 2005), MYCN Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/147/4857401 by Ed 'DeepDyve' Gillespie user on 20 June 2018 PHF20 confers neuroblastoma stem cell-like traits j 151 Figure 3 PHF20 promotes proliferation of NB cells in vitro and in vivo.(A) Demonstration of ablation of PHF20 in NB PHF20 KO cells by west- ern blotting analysis. PHF20 KO clones were generated with PHF20 sgRNA #1 and #2.(B) A total of 5000 wild-type (WT) and PHF20 KO SH- EP cells and 50000 WT and PHF20 KO SK-N-AS cells were plated in a 96-well plate using 200 μl medium. Cell viability was assayed using CellTiter-Glo®.(C) Representative xenografts excised from NSG mice. The number of mouse xenografts and tumor incidence in each group is noted on the right. (D) Growth of tumors following subcutaneous injection of PHF20 KO or control cells. (E) The tumor weight of subcutane- ous xenografts formed by NB WT and PHF20 KO cells is illustrated. (F) Hematoxylin and eosin (H&E) staining and IHC staining of PHF20 and Ki-67, as well as the TUNEL assay of xenografts. (G) The statistical results showing the proportion of Ki-67-positive cells in each ﬁeld and the proportion of apoptotic cells in the TUNEL assay. Scale bar, 50 μm. Data are plotted as mean ± SD of three independent experiments. *P < 0.05;**P < 0.01; ***P < 0.001 compared with controls using Student’s t-test. (Kaneko et al., 2015), and epithelial–mesenchymal transition the expression of Wnt3a, Mycn, N-cadherin,and Vimentin genes (EMT) signaling (Nieto et al., 2016) drive invasive and metastatic at the mRNA level, while dramatically increased E-cadherin behavior in NB cells, we next examined the expression proﬁles of expression (Figure 4C and D). These ﬁndings indicate that these genes on PHF20 KO SH-EP and PHF20 KO SK-N-AS cells. PHF20 promotes invasiveness of NB cells by regulating Wnt/ PHF20 KO SH-EP and PHF20 KO SK-N-AS cells remarkably reduced β-catenin, MYCN, and EMT signaling expression. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/147/4857401 by Ed 'DeepDyve' Gillespie user on 20 June 2018 152 j Long et al. Figure 4 PHF20 promotes migration and invasion of NB cells. (A) PHF20 KO and its control cells were subjected to transwell matrigel inva- sion assays. (B) Quantiﬁcation of migrated cells through Matrigel for each cell line is shown. (C) Expression levels of Egfr, Wnt3a, Mycn, and Bmi1 were analyzed by quantitative real-time PCR (qPCR) in PHF20-deﬁcient and control NB cells. (D) Expression levels of N-cadherin (N- cad), E-cadherin (E-cad), Vimentin, and Slug were analyzed by qPCR in PHF20-deﬁcient and control NB cells. Scale bar, 50 μm. Data are plot- ted as mean ± SD of three independent experiments. *P < 0.05;**P < 0.01; ***P < 0.001 compared with controls using Student’s t-test. PHF20 enhances properties of stem cell-like behavior in NB cells we measured gene expression in PHF20 KO SH-EP and PHF20 It has been proposed that tumor-initiating cells (TICs) exhibit KO SK-N-AS cells using real-time PCR and western blotting, and stem cell-like properties (Schwitalla et al., 2013). Therefore, we found that PHF20 KO dramatically decreased the expression of next sought to determine the contribution of PHF20 in the acqui- SOX2, OCT4, and NANOG at the mRNA and protein levels (Figure sition of cancer stem-like properties. Because neurospheres are 5C and D). We further examined the expression of SOX2, OCT4, the clonal cell clusters of neural stem cells, neurosphere forma- and NANOG in PHF20 KO SH-EP cell- and control cell-derived tion assays are well established in neurobiological research tumors. IHC staining results revealed a signiﬁcant reduction in (Galli, 2013). Using the neurosphere formation assay, we SOX2, OCT4, and NANOG expression in PHF20 KO SH-EP and observed that PHF20 KO markedly decreased clonogenic cap- PHF20 KO SH-N-AS cell- and control cell-derived tumors (Figure acity and sphere size in SH-EP and SK-N-AS cells (Figure 5A). To 5E and F), suggesting that PHF20 regulates SOX2, OCT4, and determine in vivo tumor-initiating efﬁciency of NB cells, we sub- NANOG expression in NB cells. Further, we performed gain-of- cutaneously transplanted PHF20 KO SH-EP cells and control function experiments using lentiviral constructs expressing cells into NSG mice at serial dilutions and estimated TIC fre- PHF20 in PHF20 KO SH-EP cells, as shown in Supplementary quencies using L-Calc software. We found that PHF20 KO cells Figure S4A. Importantly, ectopic expression of PHF20 rescued possessed signiﬁcantly lower TICs and initiated tumor activity SOX2, OCT4, and NANOG expression in PHF20 KO SH-EP cells less efﬁciently compared with the control cells (Figure 5B), sug- (Supplementary Figure S4A), suggesting that PHF20 is necessary gesting that PHF20 is required for maintaining the TIC-like to upregulate stem cell core factor expression. properties. Pluripotency genes SOX2, OCT4, and NANOG are the most fre- PHF20 regulates the expression of SOX2 and OCT4 through quently enumerated stem cell factors and are responsible for interaction with PARP1 and SSRP1 self-renewal in a variety of tumor types (Cao et al., 2015; To further understand how PHF20 regulates SOX2, OCT4, and Gawlik-Rzemieniewska and Bednarek, 2016; Villodre et al., NANOG expression, we performed chromatin immunoprecipitation– 2016). The expression of stemness genes, such as SOX2, OCT4, quantitative PCR (ChIP–qPCR) assays using KO SH-EP and con- and NANOG, decreased when SH-SY5Y cells were differentiated trol cells. Antibodies against PHF20 were used to pull down into neural-like cells induced by PHF20 KO. We reasoned that the chromatin complex, and two pairs of primers against SOX2, PHF20 promotes NB stem cell-like properties by regulating the OCT4, and NANOG promoter regions were used. The ChIP–qPCR expression of SOX2, OCT4, and NANOG. To test this prediction, experiments revealed that PHF20 was strongly bound to SOX2 Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/147/4857401 by Ed 'DeepDyve' Gillespie user on 20 June 2018 PHF20 confers neuroblastoma stem cell-like traits j 153 Figure 5 PHF20 confers stem cell-like behavior to NB cells. (A) A tumor sphere formation assay was performed to assess the self-renewal capacity of WT and PHF20 KO cells. Five random wells were photographed. The sphere number was counted after 7 days. (B) A summary of the tumor incidence data for animals after subcutaneous injection of PHF20 KO or control cells is shown. (C) Expression levels of SOX2, OCT4, and NANOG were analyzed by qPCR in PHF20 KO and control NB cells. (D) Western blot analysis of SOX2, OCT4, and NANOG expres- sion in PHF20 KO and control NB cells. β-actin served as a loading control. (E) IHC staining of SOX2, OCT4, and NANOG from xenografts. (F) The statistical results showing proportion of SOX2-, OCT4-, and NANOG-positive cells in each ﬁeld. Scale bar, 50 μm. Data are plotted as mean ± SD of three independent experiments. *P < 0.05;**P < 0.01; ***P < 0.001 compared with controls using Student’s t-test. and OCT4 promoters (Figure 6A). However, PHF20 was not (Barski et al., 2007). We next tested whether the modulation of detected at NANOG promoter sites. The trimethylation of H3K4 SOX2, OCT4, and NANOG expression by PHF20 is correlated with in promoter regions is associated with active gene expression, H3K4me3 and H3K27me3 modiﬁcation at their gene promoters. while the trimethylation of H3K27 represses gene expression The ChIP–qPCR assay using H3K4me3 antibody showed that Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/147/4857401 by Ed 'DeepDyve' Gillespie user on 20 June 2018 154 j Long et al. Figure 6 PHF20 regulates SOX2 and OCT4 expression and H3K4 trimethylation by interacting with PARP1.(A)Analysis ofPHF20 binding to the promoter regions of SOX2, OCT4,and NANOG in NB cells by ChIP–qPCR assay with PHF20-speciﬁc antibody. The data are presented as fold enrichment relative to input DNA. (B)ChIP–qPCR analysis of H3K4me3 and H3K27me3 of the SOX2, OCT4,and NANOG promoters in PHF20 KO and control cells. (C) Celllysateofthe PHF20 KO cells with ectopic ﬂag-PHF20 expression cell lysate was subjected to immunoprecipitation with PHF20-speciﬁc antibody. The resolved proteins were subjected to Coomassie blue staining and excised for mass spectrometry. The top scored proteins that may interact with PHF20 were listed. (D) 293T cells were transfected with Flag-PHF20 or HA-tagged SSRP1 or PARP1.Cell extracts were immunoprecipitated with anti-Flag beads, followed by immunoblotting with anti-HA and anti-Flag antibodies. (E) Western blot analysis of PHF20,SOX2,OCT4, and NANOG expression in SSRP1 and PARP1 knockdown (KD) SH-EP cells using shRNA. Data are plotted as mean ± SD of three independent experiments. *P < 0.05;**P < 0.01;***P < 0.001 compared with controls using Student’s t-test. decreased SOX2 and OCT4 expression in PHF20 KO SH-EP cells not. Moreover, we did not observe appreciable difference in was associated with decreased H3K4me3 levels at the gene pro- H3K27me3 occupancy at the gene promoter regions of SOX2 moter regions, compared with the control cells (Figure 6B, left). and OCT4 in PHF20 KO cells and control cells (Figure 6B, right), OCT4 expression was reduced in #2 but not #1 primer set. We suggesting that the transcriptional activation of SOX2 and OCT4 postulated that primer #2 was associated with a critical region induced by PHF20 is mainly regulated through modulating for PHF20 regulation of OCT4 transcription, while primer #1 did H3K4me3 at the gene promoter region in NB cells. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/147/4857401 by Ed 'DeepDyve' Gillespie user on 20 June 2018 PHF20 confers neuroblastoma stem cell-like traits j 155 Because PHF20 is a histone methylation binding protein, we Further, PHF20 KO cells overexpressing SOX2 and OCT4 gener- reasoned that PHF20 regulates SOX2 and OCT4 expression by ated signiﬁcantly larger tumors compared to PHF20 KO cells interacting with other epigenetic factors. To identify the candi- alone (Figure 7C). Using IHC staining, we showed that SOX2 and dates of the PHF20 complex in NB cells, we performed PHF20 OCT4 were indeed highly expressed in a variety of groups immunoprecipitation (IP) on cell lysates of PHF20 KO SH-EP cells (Figure 7D). Moreover, SOX2 and OCT4 overexpression increased with PHF20 overexpression. Cell lysates of PHF20 KO SH-EP cells the percentage of Ki-67-positive cells compared to the PHF20 KO were used as a negative control. Discrete bands stained with group (Figure 7D). These results clearly demonstrate that SOX2 Coomassie blue were excised and subjected to mass spectro- and OCT4 are critical mediators of PHF20-promoted stem cell-like metric analysis (Figure 6C). More than 200 candidate proteins phenotypes in NB cells. were identiﬁed, with PARP1 yielding the highest score in mass To further substantiate these ﬁndings, we next analyzed spectrometry data (Figure 6C and Supplementary Table S2). SOX2 and OCT4 expression in human NB tissue arrays, and Many of the candidate proteins were nucleic-acid-binding found both SOX2 and OCT4 overexpression in patients with NB proteins (21%), which is in agreement with the function of tumor grade ranging from 1 to 3 (Figure 7E). Therefore, our PHF20 binding to SOX2 and OCT4 promoters (Supplementary study identiﬁes PHF20 as a key regulator of NB growth and Figure S5A).Wesubjected thetop 30 candidate proteins to metastasis through upregulation of SOX2 and OCT4 expression, the ingenuity pathway analysis (IPA), and found PARP1 to be thus serving as an important biomarker for NB diagnosis and closely related to the other proteins (Supplementary Figure S5B). therapeutics. To validate whether PARP1 interacts with PHF20, we immuno- precipitated Flag-tagged PHF20 and detected HA-tagged PARP1 Discussion and its partner SSRP1 (Chiou et al., 2013), respectively. Western The current therapy of high-risk NB patients includes surgery, blot results showed that PHF20 interacts with PARP1 and SSRP1 intensive myeloablative chemotherapy with autologous periph- (Figure 6D). Moreover, we found that PHF20 interacted with eral blood stem cell reinfusion, retinoid treatment, and antibody WDR5 but not H2AFY (Supplementary Figure S5C). Next, we therapy (Cheung and Dyer, 2013). Even with intensive therapies, addressed the question of whether PARP1 indeed had any func- due to the aggressive nature of this disease, most NB patients tion in regulating PHF20-driven gene expression. We knocked relapse with therapy-resistant tumors (Meacham and Morrison, down PARP1 and SSRP1 using two different shRNAs and ana- 2013). Incomplete understanding of NB differentiation has ham- lyzed the expression of SOX2, OCT4, and NANOG in SH-EP cells pered the development of new therapeutic approaches for by western blotting, and found that the expression of pluripotency aggressive NB. One potential therapeutic option is to develop genes signiﬁcantly decreased in PARP1 and SSRP1 KD SH-EP cells immunotherapy against NB by targeting overexpressed proteins compared with control cells (Figure 6E). Moreover, silencing PARP1 like PHF20 (Fischer et al., 2001). Our present study has identi- or SSRP1 also inhibited the neurosphere formation capacities of ﬁed a novel function of PHF20 in promoting an essential charac- SH-EP cells (Supplementary Figure S5D). ChIP–qPCR experiments teristic of malignant NB, namely the cancer stemness. To revealed that PARP1 directly bound to SOX2 and OCT4 promoters, elucidate the mechanisms of how PHF20 promotes stem cell-like while SSRP1 only bound to OCT4 promoter (Supplementary traits in NB cells, we show that SOX2 and OCT4 are regulated by Figure S5E). Furthermore, PHF20 binding to SOX2 and OCT4 PHF20 and function as effective mediators of PHF20-induced NB promoters decreased in PARP1 KD SH-EP cells but not in initiation and progression. SSRP1 KD cells, compared with the control cells (Supplementary Our recent study shows that PHF20 functions as a key regula- Figure S5F). These data demonstrate that the binding of PHF20 tor of stem cell self-renewal and cellular reprogramming (Zhao to SOX2 and OCT4 promoters is mainly dependent on PARP1 in et al., 2013). The role and molecular mechanisms by which NB cells. PHF20 contributes to tumor stem cell-like properties remain largely unknown. Originally, discovered as an autoantibody in SOX2 and OCT4 play dominant roles in PHF20-induced stemness glioblastoma patients (Pallasch et al., 2005), PHF20 has been in NB cells found to be abundantly expressed in several other cancer types To test whether the stem cell-like traits conferred by PHF20 (Pallasch et al., 2005; Bankovic et al., 2010; Zaatar et al., 2012). were mediated through SOX2 and OCT4, we ectopically expressed Furthermore, PHF20 plays a vital role in carcinogenesis by sig- SOX2 and OCT4 alone or together in PHF20 KO SH-EP cells. niﬁcantly enhancing the self-renewal and tumor-initiating cap- We conﬁrmed the expression of SOX2 and OCT4 in PHF20 KO ability of lung cancer cells (Klein et al., 2016). In this study, we SH-EP cells using western blot analysis (Figure 7A).Next,we demonstrated that the elevated PHF20 expression promotes implanted PHF20 KO NB cells, with and without enforced expres- tumor cell growth and proliferation and is inversely correlated sion of SOX2, OCT4, or both, into NSG mice. SH-EP cells with with poor outcome in NB patients. empty vector and PHF20 KO SH-EP cells with MYCN overexpres- It has been shown that PHF20 exhibits high selectivity sion were used as negative and positive controls, respectively. for H3K4me2 as an epigenetic reader for further modiﬁcation We observed that overexpressing SOX2 and OCT4,alone or (Klein et al., 2016). Moreover, PHF20 could recruit the H3K4 together, signiﬁcantly rescued the tumor incidence in the xeno- methyltransferase complex to modulate H3K4me3 of the OCT4 graft model induced by PHF20 KO SH-EP cells (Figure 7B). promoter during cellular reprogramming through interaction of Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/147/4857401 by Ed 'DeepDyve' Gillespie user on 20 June 2018 156 j Long et al. Figure 7 OCT4 and SOX2 play dominant roles in PHF20-induced stemness in NB cells. (A) Western blot analysis of SOX2 and OCT4 overex- pressed either individually or in combination in PHF20 KO cells. (B) Representative xenografts excised from different groups of NSG mice are shown. Incidence was calculated by the number of tumors formed divided by total number of mice for each group. (C) The average tumor weight of varied groups. (D) H&E and IHC staining of PHF20,Ki-67,SOX2,OCT4, and NANOG expression in different groups. (E) IHC staining of SOX2 and OCT4 in NB tissue samples of Grades 1–3 from patients and in normal peripheral nervous tissue. Scale bar, 50 μm. Data are plotted as mean ± SD of three independent experiments. *P < 0.05;**P < 0.01;***P < 0.001 compared with controls using Student’s t-test. WDR5 (Zhao et al., 2013). Our current study showed that dele- process consequently reduced expression of NANOG, as OCT4, tion of PHF20 expression signiﬁcantly decreased the trimethyla- SOX2, and NANOG (OSN) form a positive feedback regulation tion status of H3K4 at the SOX2 and OCT4 gene promoters. This loop at the transcriptional level. Our data identiﬁed a previously Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/147/4857401 by Ed 'DeepDyve' Gillespie user on 20 June 2018 PHF20 confers neuroblastoma stem cell-like traits j 157 unknown epigenetic connection between PHF20 and stem cell CRISPR/Cas9 sgRNA library screening core factors (i.e. OSN) in NB. Furthermore, PHF20 interacts with SH-SY5Y cells were cultured in 96-well plates at 1 × 10 cells PARP1 to promote the accumulation of H3K4me3 for epigenetic per well. After 24 h, the cells were infected with lentivirus har- control of SOX2 and OCT4 activity in NB cells. PARP1 has been boring different sgRNAs at appropriate concentrations. For each reported to control H3K4me3 through PARylation to exclude his- differentiation screening, three wells were grouped together as tone demethylase KDM5B(Krishnakumar and Kraus, 2010). one triplicate. Cells were grown in RPMI 1640 medium contain- Previous studies support a role of PARP1 in the regulation of pluri- ing 2 μg/ml puromycin for 3 days, then switched to fresh RPMI potency networks in stem cells (Roper et al., 2014). In the 1640 medium. After growing for an additional 7 days, the puro- absence of PARP1, embryonic stem (ES) cells exhibit a decrease mycin resistant cells were stained with crystal violet to ascertain in ground state pluripotency (Doege et al., 2012). PARP1 can be cell morphology and degree of differentiation. Differentiated recruited to the NANOG and Esrrb loci to establish early epigen- cells, those with a neurite length of greater than 10 μm, were etic marks during somatic cell reprogramming (Doege et al., counted using the Metamorph™ software. 2012). In addition, the epigenetic modulation function of PARP1 is involved in regulating expression for a wide variety of genes Generation of PHF20 KO cell lines (Jiang et al., 2015). As such, PARP1 may be a prerequisite for the SH-EP or SH-N-AS cells were stably transduced with a PHF20 NB stem-cell fate. Further studies are needed to investigate the sgRNA and cloned by limiting dilution cloning. Brieﬂy, cells were role of PARP1 in NB. Given the low frequency of mutations in NB, plated at a density of 2 × 10 cells per 6-well plate. Cells were PHF20-induced epigenetic regulation through its interaction with individually transduced with non-speciﬁc sgRNA or PHF20 PARP1 may play an essential role in the control of NB stemness. sgRNA expression lentivirus. Two days after transduction, cells We show that expression of SOX2 and OCT4 is correlated to were cultured with the 2 μg/ml puromycin for 3 days. Then, the the clinical stages of NB. SOX2 is an important regulator of cel- cells were re-seeded at a density of 0.3 cells per well in 96-well lular processes for cancer development, including Wnt/β- plates and grown in medium containing 2 μg/ml puromycin. Catenin and EMT signaling (Li et al., 2013). PHF20 ablation After 3 weeks, 10−30 monoclones per sgRNA were picked and reduced SOX2 expression and blocked the Wnt and EMT- expanded. The PHF20 KO efﬁciency of monoclones was evalu- induced expression of N-cadherin and Vimentin. We propose ated by western blot analysis. that the PHF20−SOX2 axis is involved in activation of the Wnt signaling and the metastatic EMT factors for the maintenance of PARP1 and SSRP1 shRNA gene silencing NB invasive phenotype. These ﬁndings provide a mechanistic PARP1, SSRP1, and non-speciﬁc control lentiviral shRNAs framework explaining clinical observations that NB tumors were purchased from GIPZ shRNA library (GE Dharmacon). expressing high levels of PHF20 also highly express SOX2 and PARP1 shRNA 1 clone ID: V2LHS_196007; PARP1 shRNA 2 clone OCT4 and are associated with poor patient survival. Previous ID: V2LHS_201984; SSRP1 shRNA1 clone ID: V2LHS_153561; studies have shown that OCT4 induced MYCN expression in SSRP1 shRNA2 clone ID: V3LHS_363710. SH-EP cells were trans- human NB cells (Kaneko et al., 2015). In this study, we found duced with lentivirus harboring different shRNAs. Prior to use, that PHF20 ablation decreased transcription of both OCT4 and shRNA-positive cells were selected for by culturing in medium MYCN. Thus, OCT4 may serve as a critical link between PHF20 containing 2 μg/ml puromycin for 1 week. and MYCN in NB. It has been established that MYCN-positive NB cells are potently resistant to differentiation (Loven et al., 2010). Cell viability assay Therefore, functional interplay between PHF20 and MYCN may CellTiter-Glo Luminescent Cell Viability Assay (Promega) was contribute to maintaining the stem cell-like status of NB cells. used to determine NB cell viability, as per manufacturer’s direc- Based our ﬁndings, we propose a working model showing that tions. Cells were seeded in 96-well plates at a density of 5 × 10 PHF20, together with PARP1, modulates the trimethylation of SH-EP cells/well or 5 × 10 SK-N-AS cells/well, and then were H3K4 of SOX2 and OCT4 promoters, leading to their activation incubated at 37°C in a humidiﬁed 5%CO atmosphere. The cul- and subsequent induction of NANOG. This further alters the ture medium was discarded at 0, 12, 24, 36,and 48 h; cell lysis expression of Wnt, MYCN, and EMT signaling (Supplementary was induced by adding 40 μl of CTG solution to each well and Figure S6). Collectively, these events direct NB cell malignancy as incubating for 2 min at 37°C on an orbital shaker. Staining inten- evidenced by outgrowth, increased cell motility and invasion, and sity in the medium was determined by measuring absorbance upregulated capacity to self-renew. Our ﬁndings provide novel (optical density, OD) at 450 nm. therapeutic targets by pharmacological inhibition of PHF20 or for developing immunotherapy through targeting PHF20 against NB. Wound healing assay Materials and methods Cells were seeded in 6-well plates and grown to 90% con- Cell culture and reagents ﬂuency. The cell monolayers were scraped using a sterile plastic Human NB cell lines were grown in Gibco RPMI 1640 tip, followed by culturing in RPMI 1640 medium with 1% FBS at medium containing 10% fetal bovine serum (FBS) at 37°Cina 37°C in a humidiﬁed 5%CO atmosphere. Micrographs were humidiﬁed 5%CO atmosphere. taken to assess cell migration at 0, 12, 24, 36, and 48 h. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/147/4857401 by Ed 'DeepDyve' Gillespie user on 20 June 2018 158 j Long et al. Transwell invasion and migration assay Meier analysis, we used the Texas Children’s Hospital dataset The invasiveness of NB cells was assessed by their ability to (Houston, TX; n = 88) and the TCGA dataset (RNA-seq data pass through Corning Matrigel Matrix (CORNING)-coated Transwell offered by Therapeutically Applicable Research to Generate inserts (Millipore). The upper surface of the polycarbonic mem- Effective Treatments [TARGETS], n = 149). The TCGA dataset can branes (8.0 μm pore size) of the transwell chambers was coated be accessed via http://cancergenome.nih.gov/. Brieﬂy, the sam- with Matrigel (1:4 diluted with RPMI 1640). Cells (1 × 10 )in ples within each dataset were sorted according to PHF20 100 μl of serum-free RPMI 1640 medium were seeded into the expression. Based on the X-tile cutoff expression value, we upper compartments of the chambers. The lower compart- divided the samples into two groups, PHF20 low and PHF20 ments of the chambers were ﬁlled with 500 μlofRPMI 1640 high. All cutoff expression levels and their resulting groups were containing 10% FBS. After 48 h, invasive cells that migrated analyzed for survival. We selected the strongest signiﬁcance from the Matrigel to the lower surface of the ﬁlters were ﬁxed based on the P-value and the corresponding cutoff value to gen- in 70% ethanol, stained with 0.2% crystal violet, and counted erate the Kaplan–Meir graphs. under an inverted microscope at 100× magniﬁcation. Cell inva- sion was determined by averaging the number of cells counted Subcutaneous tumor model in four randomly selected visual ﬁelds per ﬁlter. Six-week-old male and female NSG mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed in individu- Neurosphere assay ally ventilated microisolator cages. All animal experiments were Cells were seeded at a concentration of 2000 cells/100 μlin approved by the Institutional Animal Care and Use Committee complete neural stem cell (NSC) basal medium [9:1 mixture of (Houston Methodist Research Institute). See Supplementary NSC basal medium and NSC proliferation supplement containing material for details. 20 ng/ml EGF, 10 ng/ml basic ﬁbroblast growth factor (bFGF), and 1 μl/ml of 0.2% heparin] in each well of a 24-well ultralow- Immunohistochemistry attachment plate (Corning Life Sciences). One milliliter of the Parafﬁn-embedded human NB and peripheral nerve tissue NSC basal medium was used in each of the 24 wells. After 7 array (MC602) was purchased from US Biomax. The study cohort days of incubation, sphere number and sphere size were consisted of 30 cases (60 cores), comprised of 5 cases with nor- counted and analyzed. mal peripheral nerve tissue and 25 cases with tumor lymph node metastasis (TNM) grading that ranged from 1 to 3. The Real-time PCR and ChIP−qPCR analysis xenograft tissues were formalin-ﬁxed, processed, and parafﬁn- Real-time PCR and ChIP assay were performed with speciﬁc embedded. The H&E staining was performed in the Cancer kit. See Supplementary material for details. Pathology Laboratory at Houston Methodist Hospital. Antigens were retrieved by autoclaving in 0.01 mol/L sodium citrate buf- Western blot, IP, and mass spectrometry fer (pH 6.0)at 121°C, 20 psi for 3−5 min. Endogenous peroxid- The cells were lysed in RIPA buffer, and analyzed. See ase activity and non-speciﬁc binding sites were blocked using Supplementary material for details. 3% hydrogen peroxide and 10% goat serum, respectively. The blocked sections were incubated overnight at 4°C with primary Ingenuity pathway analysis antibody in 1% BSA in phosphate buffer saline with Tween 20 The top 30 candidate genes identiﬁed by mass spectrometry (PBST), followed by 1 h incubation with secondary antibody. were uploaded into the IPA software (Qiagen). The core analysis The slides were stained with diaminobenzidine (DAB) for 2 min, function included in the software was used to interpret the dif- counterstained with hematoxylin, and mounted with Immuno- ferentially expressed data, including gene networks. Each gene mount (Thermo Fisher Scientiﬁc). The scoring criterion was the identiﬁer was mapped to its corresponding gene object in the average percentage of positively stained cells counted in 10 ran- Ingenuity Pathway Knowledge Base (IPKB). Nodes (proteins) domly selected visual ﬁelds. and edges (the biological relations between the nodes) were generated on the basis of their functional and biological con- TUNEL assay nectivity. The length of an edge reﬂects the evidence in the lit- TUNEL assay was performed using TumorTACS™ In Situ erature supporting that node-to-node relation. Apoptosis Detection Kit 4815-30-K (TREVIGEN) as per manu- facturer’s instructions. Brieﬂy, 4-μM thick formalin-ﬁxed, Human datasets and survival analysis parafﬁn-embedded tissue sections were de-parafﬁnized and Three publically accessible online data repositories for cancer re-hydrated. Endogenous peroxidase activity was quenched by were used in this study: Gene Expression Omnibus (GEO), The hydrogen peroxide, and tissue protein was hydrolyzed with Cancer Genome Atlas (TCGA), and Texas Children’s Hospital. For proteinase K. All sections were incubated with 50 μl/sample the determination of PHF20 expression in human NB tissue and labeling reaction mix at 37°Cfor 1 h in a humidity chamber. After normal adrenal tissue, we analyzed series GSE3446, titled ‘Gene labeling reaction stopped, samples were covered with Strep-HRP expression proﬁles of primary tumors from patients with meta- solution and incubated for 10 min at 37°C, treated with DAB solu- static NB lacking MYCN ampliﬁcation’ (n = 117). For the Kaplan– tion, and counterstained with methyl green. 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CHD1L regulated PARP1-driven pluripotency and chromatin remodeling during the early-stage cell repro- Funding gramming. Stem Cells 33, 2961–2972. This work was supported by grants from the National Natural Kaneko, Y., Suenaga, Y., Islam, S.M., et al. (2015). Functional interplay between MYCN, NCYM, and OCT4 promotes aggressiveness of human neu- Science Foundation of China (81572766 and 31771630), the roblastomas. Cancer Sci. 106, 840–847. National Key Research and Development Program of China Katoh, M., and Nakagama, H. (2014). FGF receptors: cancer biology and ther- (2017YFA0103800), Guangdong Innovative and Entrepreneurial apeutics. Med. Res. Rev. 34, 280–300. Research Team Program (2016ZT06S029), Guangdong Natural Klein, B.J., Wang, X., Cui, G., et al. (2016). PHF20 readers link methylation of Science Foundation (2016A030313215 and 2016A030313238), histone H3K4 and p53 with H4K16 acetylation. Cell Rep. 17, 1158–1170. Krishnakumar, R., and Kraus, W.L. (2010). PARP-1 regulates chromatin struc- SYSU Young Teachers Training Program (16YKZD14) and grants ture and transcription through a KDM5B-dependent pathway. Mol. Cell 39, (CA101795 and 1U54CA210181) from U.S. National Cancer 736–749. Institute, National Institutes of Health (NIH), DOD (W81XWH-16- Li, X., Xu, Y., Chen, Y., et al. (2013). SOX2 promotes tumor metastasis by 1-0417), and CPRIT (DP150099,RP170537, and RP150611). stimulating epithelial-to-mesenchymal transition via regulation of WNT/β- catenin signal network. Cancer Lett. 336, 379–389. Louis, C.U., and Shohet, J.M. (2015). Neuroblastoma: molecular pathogenesis Conﬂict of interest: none declared. and therapy. Annu. Rev. Med. 66, 49–63. Loven, J., Zinin, N., Wahlstrom, T., et al. (2010). MYCN-regulated microRNAs repress estrogen receptor-alpha (ESR1) expression and neuronal differenti- Author contributions: W.L. and W.Z. performed most experi- ation in human neuroblastoma. Proc. Natl Acad. Sci. 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Notch signaling in pediatric malignancies. Curr. ferentiation state are primary determinants of MYCN gene expression and Oncol. Rep. 10, 459–468. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/147/4857401 by Ed 'DeepDyve' Gillespie user on 20 June 2018
Journal of Molecular Cell Biology – Oxford University Press
Published: Mar 12, 2018
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