Glioblastomas are highly lethal brain tumors containing tumor-propagating glioma stem cells (GSCs). The molecular mechanisms underlying the maintenance of the GSC phenotype are not fully defined. Here we demonstrate that the zinc finger and X-linked transcription factor (ZFX) maintains GSC self-renewal and tumorigenic potential by upregulating c-Myc expression. ZFX is differentially expressed in GSCs relative to non-stem glioma cells and neural progenitor cells. Disrupting ZFX by shRNA reduced c-Myc expression and potently inhibited GSC self-renewal and tumor growth. Ectopic expression of c-Myc to its endogenous level rescued the effects caused by ZFX disruption, supporting that ZFX controls GSC properties through c-Myc. Furthermore, ZFX binds to a specific sequence (GGGCCCCG) on the human c-Myc promoter to upregulate c-Myc expression. These data demonstrate that ZFX functions as a critical upstream regulator of c-Myc and plays essential roles in the maintenance of the GSC phenotype. This study also supports that c-Myc is a dominant driver linking self-renewal to malignancy. Stem Cells2014;32:2033–2047
Glioblastoma (GBM) is the most prevalent and lethal type of primary brain tumor with a median survival less than 15 months [1, 2]. Despite recent therapeutic advances in the treatment of other cancers, current therapies for GBM remain largely ineffective due to therapeutic resistance and rapid tumor recurrence . GBM displays remarkable cellular heterogeneity and hierarchy with self-renewing glioma stem cells (GSCs) at the apex of the hierarchical organization [4-7]. GSCs are functionally defined by their self-renewal potential, multilineage differentiation potency, and in vivo tumorigenic capacity to propagate tumors that recapitulate the cellular hierarchy and tissue architecture of the parental tumor [8-14]. We and others have shown that GSCs actively interact with niches to promote tumor angiogenesis, cancer invasion, immune evasion, and resistance to current therapies [8, 15-19]. Recently, we found that GSCs are able to generate the majority of vascular pericytes to support vessel function and tumor growth . Thus, therapeutic targeting of GSCs may suppress malignant behaviors and significantly improve GBM treatment. To target GSCs specifically and effectively, it is critical to understand better the molecular mechanisms underlying the maintenance of GSC self-renewal and tumorigenic potential.
The maintenance of stem cell properties in embryonic stem cells (ESCs), adult stem cells, and cancer stem cells (CSCs) is controlled by similar but distinct sets of key transcription factors. An ESC-like gene expression signature has been found in poorly differentiated aggressive human tumors including GBMs that show preferential overexpression of a subset of the stem cell transcription regulators . Molecular targets of c-Myc, SOX2, Nanog, and Oct4 are more frequently overexpressed in poorly differentiated tumors than in well-differentiated tumors . The presence of an ESC-specific signature in human malignant tumors suggests that the ESC expression signature contributes to the stem cell-like phenotype in cancers [21, 22]. Although the molecular links between stemness and neoplasia are not fully defined, it has been demonstrated that a c-Myc network actually accounts for similarities between ESC and cancer cell transcription programs , suggesting that the apparent similarity of cancer and ESC signatures largely reflects the pervasive nature of c-Myc regulatory networks. A genetic study also validated c-Myc as a critical regulator linking the cooperative actions of p53 and Pten in the control of normal and malignant stem cell differentiation, self-renewal, and tumorigenic potential . Thus, c-Myc appears to be the dominant driver connecting the stem cell phenotype to malignancy. While c-Myc is required for maintaining the proliferation and self-renewal of ESCs [25, 26], c-Myc also plays crucial roles in maintaining proliferation of cancer cells . Upregulation or increased activation of c-Myc has been found in 70% of human cancers . A recent study demonstrated that stabilization of c-Myc caused by mutations in the ubiquitin ligase FBXW7 drives leukemia-initiating activity of T cell acute lymphoblastic leukemia (T-ALL) . Moreover, c-Myc is critically important for the maintenance of GSC self-renewal and proliferation in GBMs [24, 30]. c-Myc is preferentially expressed in GSCs relative to non-stem tumor cells in GBMs . However, the transcriptional control of c-Myc expression in GSCs is poorly understood. A better understanding of the transcriptional regulation of c-Myc in GSCs may provide new insights into the molecular link between stemness and malignancy in GBM. In this study, we identified the zinc finger and X-linked transcription factor (ZFX) as a critical upstream regulator of c-Myc expression in GSCs.
ZFX is a critical transcription factor that controls the self-renewal potential of both human and murine ESCs [31-33]. ZFX and c-Myc belong to the same regulatory network of ESC self-renewal [25, 34, 35]. Mammalian ZFX protein contains several functional domains including an acidic transcriptional activation domain, a nuclear localization signal sequence, and a DNA binding domain consisting of 13 C2H2 zinc fingers . Recent studies demonstrated that ZFX also plays important roles in the nucleosome [37, 38]. Deletion of ZFX in mice impaired the self-renewal capacity of ESCs and hematopoietic stem cells . ZFX is also required for the maintenance of the self-renewal potential of human ESCs . ZFX overexpression promotes ESC self-renewal by inhibiting cell differentiation [31, 32]. In addition, loss of ZFX resulted in a significant increase of apoptotic cell death and the upregulation of stress inducible factors in ESCs . These studies suggest that ZFX plays a crucial role in the maintenance of ESCs. Interestingly, ZFX has been implicated in the Myc-mediated tumorigenesis of liver cancer , suggesting that ZFX may also play a critical role in the pathogenesis of human cancers. However, the functional significance of ZFX in the maintenance of GSCs in GBM has not been defined, although ZFX has been shown to be associated with proliferation and survival of glioma cell lines [40, 41]. Thus, defining the potential role of ZFX in the maintenance of self-renewal and tumorigenic potential of GSCs may offer new insights into the molecular mechanisms underlying the regulation of the stem cell-like phenotype. In this study, we interrogated the potential role of ZFX in the maintenance of the GSC phenotype. We demonstrated that ZFX controls c-Myc expression to maintain the self-renewal and tumorigenic potential of GSCs. Moreover, we found that ZFX binds to a specific sequence (GGGCCCCG) on the human c-Myc promoter to mediate the upregulation of c-Myc in GSCs.
Materials and Methods
Isolation of GSCs and Non-Stem Tumor Cells from GBM
De-identified GBM surgical specimens were collected from the Brain Tumor and Neuro-Oncology Center at Cleveland Clinic or University Hospitals of Case Western Reserve University in accordance with an Institutional Review Board-approved protocol. GSCs and matched non-stem tumor cells were isolated from GBM patient specimens or xenografts through cell sorting and functionally characterized as previously described [8, 20, 42, 43] with minor modification. Briefly, GBM tumors were disaggregated using the Papain Dissociation System (Worthington Biochemical) according to the manufacturer's instructions. Isolated cells were recovered in stem cell medium (Neurobasal-A medium with B27 supplement, 10 ng/ml epidermal growth factor (EGF), and 10 ng/ml basic fibroblast growth factor (bFGF)) for at least 6 hours to allow re-expression of surface markers and then sorted by fluorescence-activated cell sorting (FACS) or magnetic cell sorting for GSCs using at least two surface markers (CD15/CD133). The enriched GSCs were maintained in the stem cell medium, and the identity was confirmed by SOX2 and OLIG2 expression and the activation of SOX2 promoter-driven green fluorescent protein (GFP) expression. The CSC phenotype of GSCs was validated by functional assays of self-renewal (serial neurosphere formation at the clonal density), in vitro induction of differentiation, and tumor propagation (in vivo limiting dilution assay) as previously described [42, 43]. After validation of the CSC phenotype by these functional assays, the sorted GSCs (D456, T387, T4302, T4121, T3359, T3691, T3742, CW702, CW1336, and CCF2170) were used for the in vitro and in vivo experiments.
Culture of Human Neural Progenitor Cells
Human neural progenitor cell (NPC) lines derived from fetal brains (Lonza, Allendale, NJ, http://www.lonza.com) were cultured and maintained as suspension cultures or propagated as monolayers attached on the BD stem cell Matrigel-coated dishes or coverslips in Neurobasal stem cell media supplemented with B27 (lifetechnologies, Grand Island, NY, http://www.lifetechnologies.com) and EGF/bFGF (20 ng/ml each, R&D, Pittsburgh, PA, http://www.rndsystems.com). These NPC lines have been validated for their potential to differentiate into neurons, astrocytes, and oligodendrocytes upon induction as demonstrated in our previous studies [43, 44].
Immunoblot, Immunofluorescence, and Immunohistochemistry
GSCs or NPCs were induced to differentiate in vitro by the addition of serum or withdrawal of growth factors as previously described [8, 43, 44]. The cells were cultured on the Matrigel-coated coverslips or dishes and then induced to differentiate in the serum-containing medium (10% Fetal Bovine Serum in α-Minimum Essential Media) or in Neurobasal-A medium with B27 but without EGF and bFGF. At indicated time points, cells were harvested for IB analysis or fixed for IF staining.
DNA Constructs and Lentiviral Transfection
Lentiviral clones expressing ZFX shRNA (shZFX) or nontargeting (NT) shRNA (SHC002) were acquired from Sigma-Aldrich. Two of five shZFX clones (sh09 and sh10) displayed high efficiency of knockdown (75%–85% reduction) and were used for all related experiments. A lentiviral construct expressing Flag-tagged ZFX (Flag-ZFX) or Flag-tagged c-Myc (Flag-Myc) was generated by cloning the human ZFX or c-Myc2 open reading frame with the N-terminal Flag sequence into the pCDH-MCS-T2A-Puro-MSCV vector (System Biosciences, Mountain View, CA, http://www.systembio.com). Viral particles were produced in 293T cells with the pACK set of helper plasmids (System Biosciences) in stem cell media. Viral stocks were concentrated by precipitation with PEG-8,000 and titered according to the manufacturer's instructions.
Intracranial Tumor Formation and In Vivo Bioluminescence Imaging
Intracranial transplantation of GSCs to establish GBM xenografts was performed as previously described [8, 42, 43]. To monitor tumor growth in living animals, all GSCs used for the animal studies were transduced with firefly luciferase through lentiviral infection. GSCs expressing firefly luciferase were then transduced with NT shRNA or shZFX and/or Flag-Myc (expression similar to endogenous level) or control vector through lentiviral infection twice at 24-hour intervals. 24 hours after the second transduction, viable cells (5 × 103 cells per animal or as indicated) were intracranially transplanted into athymic immunocompromised mice. For the survival experiments, animals were maintained until manifestation of neurological signs or for 180 days, whichever occurred sooner. To examine tumor growth, mouse brains implanted with T387 GSCs expressing shZFX or NT shRNA and/or Flag-c-Myc or control vector were monitored by bioluminescence imaging or harvested simultaneously after GSC transplantation. For the imaging analysis, animals were administrated with d-luciferin intraperitoneally and anesthetized with isoflurane. The tumor luciferase images were captured by using the IVIS imaging system (Xenogen-100). All animal procedures conformed to the Cleveland Clinic IACUC approved protocol.
c-Myc Promoter Reporter Assay
To identify the activating element of c-Myc promoter in response to ZFX, the DNA fragments containing different regions of the human c-Myc promoter were cloned into a luciferase report vector pGL4.16-Luc2CP-Hygro (Promega, Madison, WI, http://www.promega.com). Mutagenesis of the potential ZFX binding site was performed using site-directed polymerase chain reaction (PCR) mutagenesis and confirmed by sequencing. The luciferase reporter construct or the ZFX-expression construct was introduced into GSCs by using the specific transfection reagent X-tremeGENE (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com). Two days after transfection, the luciferase activity under the control of different promoter regions was measured using a bioluminescence reader.
ZFX Promoter and the Promoter-Driven GFP Expression
Human ZFX promoter (−774/+228) sequence (Switchgear Genomics) was cloned from human genomic DNA by PCR using the following primers and then confirmed by sequencing. ZFXpro-Forward: 5′-A AAA TCA GTT TGG CTC TGA CTG C; ZFXpro-Reverse: 5′-CCG GGC CTA ACT GCG GCC TCC. The cloned ZFX promoter was inserted into pCDH-EF1-MCS-T2A-Puro lentiviral vector to replace the internal EF1 promoter, and GFP open reading frame was cloned into MCS sites to construct the ZFX promoter-driven GFP expression.
Chromatin Immunoprecipitation Assay
The chromatin immunoprecipitation (ChIP) assay was performed using the EZ-CHIP kit (Millipore). Briefly, cell lysates were sonicated on ice to obtain DNA fragments of 200–1,000 bps. The potential ZFX-bound DNA complex was immunoprecipitated with the anti-ZFX antibody (Cell Signaling) and protein G agarose beads. After the agarose beads were washed for four times, the ZFX-bound DNA fragments were eluted and then amplified by real-time quantitative PCR.
Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL) assay detecting apoptotic cell death on tumor sections was performed with the ApopTag Plus Peroxidase In Situ Apoptosis Kit (Millipore) according to the manufacturer's instructions. The intensity of apoptotic cells was quantified with Image J software.
Quantitative Reverse Transcriptase PCR
Total cellular RNA was isolated with the RNeasy Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) and reverse transcribed into cDNA using the Superscript III Kit (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Real time PCR was performed on an Applied Biosystems 7900HT cycler using SYBR-Green Mastermix (SA Biosciences) with the following primers: c-Myc-Forward: 5′-TCA AGA GGC GAA CAC ACA AC; c-Myc-Reverse: 5′-GGC CTT TTC ATT GTT TTC CA. ZFX-Forward: 5′-GGC AGT CCA CAG CAA GAA C; ZFX-Reverse: 5′-TTG GTA TCC GAG AAA GTC AGA AG. GAPDH-Forward: 5′-GGT CTC CTC TGA CTT CAA CA; GAPDH-Reverse: 5′-GTG AGG GTC TCT CTC TTC CT. SOX2-Forward: 5′-CAA GAT GCA CAA CTC GGA GA; SOX2-Reverse: 5′-CGG GGC CGG TAT TTA TAA TC. OLIG2-Forward: 5′-GGT AAG TGC GCA ATG CTA AGC TGT; OLIG2-Reverse: 5′-TAC AAA GCC CAG TTT GCA ACG CAG. c-Myc promoter (−156/−1)-Forward: 5′-GAG CTG TGC TGC TCG CGG CCG C; c-Myc promoter (−156/−1)-Reverse: 5′-TAG ATA AAG CCC CGA AAA CCG GC. c-Myc promoter (−83/+124)-Forward: 5′-agg gct tct cag agg cttg; c-Myc promoter (−83/+124)-Reverse: 5′-cct att cgc tcc gga tct c. c-Myc promoter (−1,024/−874)-Forward: 5′-cct ccc tct cgc cct agc cca g; c-Myc promoter (−1,024/−874)-Reverse: 5′-gac tga gtc ccc caa ttt gct.
All grouped data are presented as mean ± SD. The difference between groups was assessed by one-way ANOVA or one-way ANOVA on ranks tests. For the in vivo experiments, log rank survival analysis was performed. Sigma Stat Software (Version 3.5) was used for all statistical analyses.
ZFX Is Preferentially Expressed by GSCs in Human GBMs
To determine expression pattern of ZFX in GSC populations and non-stem glioma cells in human GBMs, we performed coimmunofluorescence staining of ZFX with several GSC markers on GBM tumor sections. ZFX was preferentially expressed in the cancer cells coexpressing the GSC markers (SOX2, OLIG2, and CD133) in several human primary GBMs (Fig. 1A–1C; Supporting Information Fig. S1A) and GBM xenografts (Supporting Information Fig. S1B, S1C). In addition, ZFX-expressing cells were often found proximal to blood vessels marked by CD31 staining in primary GBMs (Fig. 1D), consistent with the fact that GSCs are mainly localized in perivascular niches [16, 45]. To determine whether ZFX is commonly expressed in human GBM tumors, we examined ZFX expression in GBM tissue microarrays containing 74 human primary GBMs. ZFX was expressed in a subpopulation of cancer cells in the majority (69 of 74 cases, 93.2%) of GBM samples (Fig. 1E; Supporting Information Table S1). In contrast, ZFX was not detected in human normal brain tissues adjacent to GBM tumors (Fig. 1E) or normal mouse brains (Supporting Information Fig. S1D). These data demonstrate that ZFX is preferentially expressed in GSCs in the majority of human GBMs.
To further confirm the specific expression of ZFX in GSCs, we examined ZFX protein levels in matched GSCs and non-stem tumor cells isolated from six primary GBMs or xenografts. GSCs were isolated from GBM tumors through dual labeled FACS using at least two putative GSC surface markers (CD15+/CD133+) and validated by expression of GSC transcription factors (SOX2 and OLIG2) and the ability to activate the SOX2 promoter-driven GFP expression (data not shown). The enrichment for GSCs was further confirmed through three functional assays: (a) self-renewal potential measured by tumorsphere formation at clonal (single cell) density; (b) multipotency tested by cell differentiation induction in vitro; and (c) tumor formation capacity evaluated by limiting dilution assay [8, 15, 42, 43, 46]. IB analysis showed that ZFX protein levels were commonly higher in GSCs than matched non-stem tumor cells (Fig. 2A). This result was validated by IF costaining of ZFX and several GSC markers (SOX2, OLIG2, and c-Myc) in matched T387 GSCs and non-stem glioma cells (Fig. 2B, 2C; Supporting Information Fig. S2A). ZFX was also coexpressed with GSC markers such as OLIG2 in T4121 GSC tumorspheres (Fig. 2D). As GSCs share similar properties with NPCs, we examined ZFX expression in human NPC lines. IB analysis indicated that ZFX and c-Myc levels in NPCs were much lower than that in GSCs, although both GSCs and NPCs expressed similar levels of SOX2 (Supporting Information Fig. S2B). This result was validated by IF costaining of ZFX, c-Myc, and SOX2 in NPCs (Supporting Information Fig. S2C, S2D). Consistently, the subventricular zone or dentate gyrus region that is enriched with NPCs in mouse brain displayed abundant SOX2 expression but low ZFX expression (Supporting Information Fig. S2E). Collectively, these data demonstrate that ZFX is preferentially expressed in GSCs relative to non-stem tumor cells and NPCs.
ZFX Is Required for Self-Renewal and Proliferation of GSCs
To define the functional significance of the preferential expression of ZFX in GSCs, we examined the effect of ZFX downregulation by shRNA on the maintenance of GSCs. Targeting ZFX in T387 GSCs by lentiviral-mediated shRNA (sh09 or sh10) reduced ZFX protein by 75%–85% (Fig. 3A). Disrupting ZFX by the shRNA markedly reduced tumorsphere formation of the GSCs as the sphere size and number dramatically decreased after ZFX knockdown (Fig. 3B–3D). In addition, ZFX knockdown significantly reduced proliferation of the GSCs (Fig. 3E) but showed little effect on non-stem glioma cells and NPCs (Supporting Information Fig. S3A, S3B). These results were validated in four GSC populations and matched non-stem tumor cells derived from different GBM tumors (data not shown). To further confirm the functional link between ZFX expression and GSC maintenance, we examined the change in ZFX expression as GSCs differentiate. ZFX and other GSC markers including c-Myc and SOX2 gradually decreased during GSC differentiation, while the expression of the astrocyte marker GFAP and the neuronal marker MAP2 increased (Fig. 3F). IF confirmed that ZFX expression was abolished on day 6 after induction of GSC differentiation (Fig. 3G). To further trace ZFX promoter activity in GSCs and the differentiated cells, we cloned the human ZFX promoter and constructed a ZFX promoter-driven GFP expression (ZFXpro-GFP) in a lentiviral vector (Supporting Information Fig. S3C). Most cells in the tumorspheres derived from the GSCs transduced with the ZFXpro-GFP expressed high levels of GFP (Supporting Information Fig. S3D), while the differentiated cells derived from the ZFXpro-GFP-GSCs largely lost GFP expression but increased the fraction of cells expressing GFAP or TUJ1 (a neuronal marker) after induction of differentiation (Supporting Information Fig. S3E, S3F), suggesting that the ZFX promoter is activated in GSCs but silenced in the differentiated cells. These data demonstrate that ZFX is required for maintaining the self-renewal and proliferation potential of GSCs.
Targeting ZFX Disrupted GBM Tumor Growth and Increased the Survival of Mice Bearing GSC-Derived Xenografts
One of defining functional phenotypes of CSCs is their potent tumor propagation ability. To address the functional requirement for ZFX in the maintenance of the tumorigenic potential of GSCs, we examined the effect of ZFX disruption by shRNA on tumor formation of GSCs. The sorted T387 GSCs were transduced with firefly luciferase and ZFX shRNA (shZFX-09 or shZFX-10) or NT shRNA and then transplanted into the brains of immunocompromised mice. Bioluminescence imaging analysis monitoring tumor development indicated that animals bearing the GSCs expressing shZFX markedly delayed the tumor progression relative to the control group bearing the GSCs expressing NT shRNA (Fig. 4A). Necropsy of animals sacrificed simultaneously 21 days after implantation revealed that GSCs transduced with shZFX (sh-09 or sh-10) frequently failed to generate tumors or had reduced tumor size relative to the GSCs expressing NT shRNA (Fig. 4B). Moreover, mice intracranially implanted with the GSCs transduced with shZFX survived significantly longer than the control mice implanted with the GSCs transduced with NT shRNA (Fig. 4C). TUNEL assay demonstrated a significant increase in apoptotic cell death occurred in the infrequent GBM tumors derived from the GSCs expressing shZFX (Fig. 4D, 4E). Taken together, these data demonstrate that ZFX is required for maintaining the tumorigenic capacity of GSCs in vivo, suggesting that ZFX might be a potential target that may be disrupted to inhibit the tumorigenic potential of GSCs.
Ectopic Expression of ZFX Augmented GSC Proliferation and Tumorigenic Potential
Because ZFX is critical for maintaining GSC proliferation and tumorigenic potential, we examined whether ectopic expression of ZFX impacts GSC proliferation, tumorsphere formation, and tumor formation in vivo. Forced expression of ZFX (Flag-ZFX) in T387 GSCs increased c-Myc expression as demonstrated by IB analysis and IF (Fig. 5A, 5B). As expected, ectopic expression of ZFX enhanced GSC tumorsphere formation (Fig. 5C–5E) and cell growth in vitro (Fig. 5F). To examine whether forced expression of ZFX enhances the tumorigenic potential of GSCs, we monitored the tumor growth of GBM xenografts derived from the luciferase-labeled GSCs that were transduced with Flag-ZFX or vector control. Bioluminescence imaging of the intracranial tumors indicated that ectopic expression of ZFX significantly augmented tumor growth of the GSC-derived xenografts (Fig. 5G, 5H). As a consequence, ectopic expression of ZFX in the GSCs significantly reduced the survival of mice bearing the GSC-derived xenografts (Fig. 5I). Collectively, these data demonstrate that forced expression of ZFX enhanced GSC proliferation and tumorigenic potential, further supporting that ZFX plays a critical role in maintaining GSC malignant properties.
ZFX Controls c-Myc Expression in GSCs by Binding to a Specific Sequence on the c-Myc Promoter
To determine the molecular mechanisms underlying ZFX-mediated GSC maintenance, we screened for key regulators of GSCs temporally related to ZFX downregulation or the downstream targets immediately affected by ZFX knockdown. Surprisingly, Tbx3 and Tcl1, two well-known ZFX downstream targets in ESCs , were not affected by ZFX knockdown in T387 GSCs (Supporting Information Fig. S4). Instead, we identified c-Myc as an early downstream effector of ZFX in GSCs (Fig. 3A; Supporting Information Fig. S4). Real time PCR analysis confirmed that ZFX disruption caused a rapid and significant downregulation of c-Myc expression in the GSCs before the expression change of other GSC markers (SOX2 and OLIG2) (Fig. 6A). IF validated that ZFX disruption in the GSCs resulted in a rapid decrease of c-Myc protein levels (Fig. 6B). Consistently, forced expression of ZFX (Flag-ZFX) increased expression of c-Myc as demonstrated by both IB analysis and IF (Fig. 6C, 6D). To determine whether ZFX directly controls c-Myc expression in GSCs, we cloned different regions of the human c-Myc promoter  into a luciferase reporter construct and tested promoter activity in response to ectopic expression of ZFX (Fig. 6E). Forced expression of ZFX significantly increased the reporter activity driven by all promoter fragments containing the P2 region (−147 to +51) but not the promoter fragment lacking the P2 region (Fig. 6E), suggesting that the P2 region of the c-Myc promoter contains a ZFX response element. Sequence analysis indicated the P2 region of the c-Myc promoter contains a putative ZFX-binding sequence (GGGCCCCG) [31, 48]. A mutation of the putative ZFX-binding site (GGGCCCCG → GGATCCTA) on the P2 region abolished the promoter activity in response to ZFX ectopic expression (Fig. 6F, 6G). To determine whether ZFX binds to the P2 region of c-Myc promoter, we performed ChIP assay with the anti-ZFX specific antibody. PCR analysis confirmed that the anti-ZFX antibody specifically pulled down the c-Myc promoter fragment containing the P2 region but not the upstream fragment containing the P1 region and lacking the P2 region (Fig. 6H). These data demonstrate that ZFX upregulates c-Myc expression by binding to a specific sequence (GGGCCCCG) on the P2 region of the c-Myc promoter. As c-Myc is an important factor required for the maintenance of self-renewal and proliferation potential of ESCs and GSCs, our data suggest that ZFX may control the maintenance of the GSC phenotype through regulation of c-Myc expression.
Ectopic Expression of c-Myc to its Endogenous Level Rescued the Phenotype Caused by ZFX Disruption
To confirm c-Myc upregulation as a critical molecular mechanism mediating the effects of ZFX on the maintenance of GSC properties and tumorigenic potential, we examined whether ectopic expression of c-Myc to its endogenous level could rescue the effects caused by ZFX disruption in vitro and in vivo. GSCs derived from GBM tumors (CCF2170 or T387) were transduced with Flag-tagged human c-Myc (Flag-c-Myc) or vector control and shZFX (sh09 or sh10) or NT shRNA. IB analysis confirmed that the level of ectopic expression of Flag-c-Myc was similar to its endogenous level in GSCs (Supporting Information Fig. S5A). The ectopic expression of c-Myc was not affected by ZFX knockdown, while the endogenous c-Myc was markedly reduced by ZFX disruption. Ectopic expression of c-Myc to its endogenous level in the GSCs was able to rescue the impaired tumorsphere formation caused by ZFX knockdown (Supporting Information Fig. S5B–S5D) and restored the cell growth affected by ZFX disruption (Supporting Information Fig. S5E). Because ZFX disruption in the GSCs significantly inhibited GBM tumor growth and increased animal survival, we examined whether ectopic expression of c-Myc could restore the GSC tumor growth. Expression of c-Myc to its endogenous level indeed rescued GSC tumor growth impaired by ZFX downregulation as demonstrated by in vivo bioluminescence imaging (Fig. 7A, 7B). Consistently, ectopic expression of c-Myc also attenuated the increased survival of mice bearing the GSC-derived tumors expressing shZFX (Fig. 7C). Furthermore, TUNEL assay indicated that expression of c-Myc attenuated the increased apoptosis caused by ZFX knockdown in GSC-derived GBM tumors (Fig. 7D, 7E). These data demonstrate that ectopic expression of c-Myc to its endogenous level in GSCs largely attenuated the effects of ZFX disruption on GSC growth in vitro and tumor progression in vivo, indicating that ZFX controls c-Myc expression to maintain the GSC phenotype and tumorigenic potential.
The zinc finger transcription factor ZFX plays a critical role in modulating the self-renewal of both murine and human ESCs (hESCs) [31, 32]. ZFX inactivation in mice resulted in small animal size and reduced number of germ cells in both male and female animals . Reduced ZFX expression led to a loss of self-renewal of hESCs while ZFX overexpression enhanced the clonogenicity and decreased spontaneous differentiation of hESCs . These studies suggest that ZFX is an essential self-renewal regulator that has a conserved role in the maintenance of hESCs and murine ESCs. Although CSCs are distinct from ESCs, it is possible that CSCs and ESCs share a similar molecular program for maintaining the self-renewal potential [21-23]. Thus, it is important to determine the functional significance of ZFX in the maintenance of the stem cell-like properties in GBMs. As disrupting ZFX in GSCs markedly inhibited GSC proliferation and survival and potently induced apoptotic cell death in the GSC-derived GBM tumor, ZFX also has a role in the maintenance of GSC survival. However, in ESCs, ZFX maintains stem cells by suppressing differentiation and thus targeting ZFX in ESCs induced cell differentiation [31, 32]. It seems that disrupting ZFX in ESCs and GSCs resulted in similar but distinct phenotypes, suggesting that ZFX may mediate through different mechanisms to control the stem cell phenotype in ESCs and GSCs. It is likely that ZFX differentially regulates downstream targets in ESCs and GSCs. ZFX has been shown to regulate expression of a network of downstream targets involved in the self-renewal and proliferation of ESCs [33, 34]. Two of downstream direct targets of ZFX in ESCs are Tbx3 and Tcl1 . However, ZFX knockdown or overexpression in GSCs did not shown an effect on expression of both Tbx3 and Tcl1 (Supporting Information Fig. S4). Instead of Tbx3 and Tcl1, c-Myc is the main downstream target of ZFX in GSCs. Our rescue experiments demonstrated that ZFX controls c-Myc upregulation to maintain the GSC phenotype and tumorigenic potential, as ectopic expression of c-Myc to its endogenous level in GSCs was able to rescue the effects caused by ZFX disruption in vitro and in vivo. Thus, ZFX may signal through distinct downstream targets to exert its differential roles in the maintenance of ESCs and GSCs.
c-Myc has been studied extensively for its instrumental role in proliferation and growth of normal and neoplastic cells. Abnormal expression or regulation of c-Myc has been found in diverse human tumors and often correlates with poor prognosis and advanced stage of malignancies [28, 50]. c-Myc is critically required for the maintenance of pluripotent stem cells [25, 26]. Introduction of c-Myc with other key transcription factors (SOX2/Oct4/Klf4) generates induced pluripotent stem cells [51, 52]. Moreover, several studies have demonstrated that c-Myc also functions as an oncogenic protein and plays crucial roles in maintaining the self-renewal, proliferation, and tumorigenic potential of CSCs including GSCs [24, 30, 53]. A recent study demonstrated that abnormal upregulation of c-Myc caused by the inactivation of its negative regulator FBXW7 (a c-Myc ubiquitin ligase) led to proliferation of leukemia stem cells in T-ALL . Thus, c-Myc serves as a functional link connecting stemness and malignancy. It has been shown that the proliferation, growth, and survival of GSCs critically depend on c-Myc expression [24, 30]. Targeting c-Myc impaired proliferation and survival of GSCs and significantly suppressed GSC tumor growth . Consistently, c-Myc is significantly upregulated in GSCs relative to matched non-stem tumor cells. However, the molecular mechanisms associated with the upregulation of c-Myc in GSCs were not fully understood. Our study revealed that c-Myc expression in GSCs is controlled by ZFX that is preferentially expressed in GSCs. In addition, we found that ZFX binds to a specific sequence on the c-Myc promoter to regulate c-Myc expression. ZFX-regulated c-Myc expression is required for maintaining the GSC phenotype and tumorigenic potential. A recent study demonstrated that ZFX controls leukemia stem cell self-renewal and propagation and prevents differentiation of acute myeloid leukemia and T-lymphoblastic leukemia (T-ALL) . This study also showed that ZFX contributes to gene induction and transformation by Myc overexpression in myeloid progenitors , suggesting a molecular link between ZFX and Myc in different cancer stem or progenitor cells. Interestingly, under hypoxic conditions HIF-2α has been shown to bind to c-Myc promoter and enhance c-Myc expression to promote tumor progression [55, 56]. Our previous study showed that HIF-2α is differentially expressed in GSCs and is preferentially stabilized in GSCs in response to hypoxia . Thus, c-Myc is a critical regulatory node that may mediate different signals for the maintenance of self-renewal, proliferation, survival, and tumorigenic potential in varied conditions. As c-Myc is universally important to survival of both normal and neoplastic cells, therapeutic targeting of c-Myc is not practical. However, manipulation of its upstream regulator ZFX may effectively eliminate GSCs with fewer side effects. As disrupting ZFX in vivo potently induced apoptosis, suppressed tumor growth, and increased the survival of animals bearing the GBM xenografts, therapeutic targeting of ZFX may significantly improve patient survival.
Our data demonstrate that the zinc finger transcription factor ZFX controls c-Myc expression to maintain the self-renewal and tumorigenic potential of GSCs. ZFX is preferentially expressed in GSCs. Silencing ZFX by shRNA reduced c-Myc expression and potently inhibited GSC self-renewal and tumor growth, indicating that ZFX is required for the maintenance of the GSC phenotype. Ectopic expression of c-Myc to its endogenous level was able to rescue the phenotypes caused by ZFX disruption, suggesting that ZFX maintains GSC properties through c-Myc upregulation. We also found that ZFX binds to a specific sequence (GGGCCCCG) on the human c-Myc promoter to regulate c-Myc expression in GSCs. Our studies reveal that ZFX-mediated c-Myc regulation plays a critical role in connecting stemness to neoplasia. Thus, ZFX is a potential molecular target of GSCs for future development of anti-GBM therapeutics.
We thank the Brain Tumor and Neuro-Oncology Centers at Cleveland Clinic and University Hospitals of Case Western Reserve University for providing GBM surgical specimens for this study; Cathy Shemo and Sage O'Bryant of the Flow Cytometry Core, Judith Drazba of the Imaging Core at Cleveland Clinic Lerner Research Institute for their assistance. We are grateful to members in Dr. Rich's laboratory for the scientific discussion. This work was supported by the Cleveland Clinic Foundation and a NIH R01 grant (NS070315) to S.B.
X.F. and S.B.: designed the experiments, analyzed the data, and wrote the manuscript; X.F., Z.H., W.Z., and Q.W.: performed the experiments; A.E.S. and R.E.M.: provided GBM surgical specimens; R.E.M.: performed pathological analyses; J.S.Y., G.O., and J.N.R.: provided scientific inputs and helped to edit the manuscript.
Disclosure of Potential Conflicts of Interests
The authors indicate no potential conflicts of interests.