EZH2 is a component of the epigenetic regulator PRC2 that suppresses gene expression. Elevated expression of EZH2 is common in human cancers and is associated with tumor progression and poor prognosis. In this study, we show that EZH2 elevation is associated with epigenetic modifications of Kaposi sarcoma–associated herpesvirus (KSHV), an oncogenic virus that promotes the development of Kaposi sarcoma and other malignancies that occur in patients with chronic HIV infections. KSHV induction of EZH2 expression was essential for KSHV-induced angiogenesis. High expression of EZH2 was observed in Kaposi sarcoma tumors. In cell culture, latent KSHV infection upregulated the expression of EZH2 in human endothelial cells through the expression of vFLIP and LANA, two KSHV-latent genes that activate the NF-κB pathway. KSHV-mediated upregulation of EZH2 was required for the induction of Ephrin-B2, an essential proangiogenic factor that drives endothelial cell tubule formation. Taken together, our findings indicate that KSHV regulates the host epigenetic modifier EZH2 to promote angiogenesis. Cancer Res; 72(14); 3582–92. ©2012 AACR.
Kaposi sarcoma–associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma, the most common cancer in HIV-infected subjects. Kaposi sarcoma is a multifocal neovascular tumor of proliferating spindle endothelial cells latently infected by KSHV (1). In cell culture, KSHV infection converts human primary endothelial cells into spindle shapes with altered expression of cell-surface markers and a distinct cellular gene expression program (2–6). Extensive studies have identified a number of cellular factors that mediate KSHV-induced malignant cellular proliferation (1).
Kaposi sarcoma is a highly vascularized and angiogenic tumor. Indeed, KSHV infection promotes the angiogenic phenotypes of endothelial cells, which include induction of tubule formation on Matrigel, capillary morphogenesis in low growth factor conditions, and enhanced migration, invasion, and permeability (7–11). Several proangiogenic factors are induced following KSHV infection of human endothelial cells. These include VEGF-A and VEGF-C, angiopoietin-2, interleukin (IL)-6, IL-8, Ephrin-B, matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 (7, 8, 12–14). Upregulation of these proangiogenic cytokines is, in part, mediated by multiple mitogen-activated protein kinase pathways, Notch pathway, and NF-κB pathway (7, 8, 12, 13, 15).
Enhancer of Zeste Homolog 2 (EZH2) is a main component of polycomb repressive complex 2 (PRC2), which regulates chromatin structure and gene expression through posttranslational modifications of histone (16). EZH2 has attracted intense interest because of its role in maintaining cell stemness and development of cancer (16). When the PRC2 complex is recruited to chromatin, EZH2 catalyses the trimethylation of the lysine 27 of histone H3 (H3K27me3), which leads to subsequent recruitment of the PRC1 complex that monoubiquitylates the lysine 119 of histone H2A (H2AK119ub1) to prevent RNA polymerase II–dependent transcriptional elongation leading to transcriptional repression (16, 17). Several reports suggest that EZH2 directly interacts with DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and that EZH2 is necessary for the maintenance of DNA methylation and repression of specific genes, including tumor-suppressor genes (16, 17). EZH2 is highly expressed in numerous human cancers, including breast and prostate, with high EZH2 levels often correlating with advanced stages of tumor progression and poor prognosis (18, 19). EZH2 connects the estrogen and Wnt signaling pathways by directly interacting with estrogen receptor α and β-catenin, resulting in transcriptional upregulation of c-myc and cyclin D1 in breast cancer cells (20). Moreover, mutation of EZH2 Y641, which results in gain-of-function, has been described in lymphomas (21). Recent studies have shown that both VEGF and FGF2 induce angiogenesis through upregulation of EZH2 expression, and inhibition of EZH2 attenuated the cell proliferation, migration, and tubule formation of glioblastoma cells (18, 22–24).
Because of important biologic roles of EZH2 in tumorigenesis and angiogenesis and the results of recent studies that have shown the connection of EZH2 with the epigenetic modifications of the KSHV genome (25, 26), we sought to investigate how KSHV infection might regulate EZH2 and whether such regulation might contribute to KSHV-induced angiogenesis. In this work, we have found that EZH2 is highly expressed in Kaposi sarcoma tumors, and KSHV infection of endothelial cells upregulates the expression of EZH2. Furthermore, KSHV upregulation of EZH2 is partially mediated by the NF-κB pathway and the viral latent genes vFLIP (ORF71) and LANA (ORF73). Finally, we have shown that EZH2 mediates KSHV-induced endothelial cell tubule formation on Matrigel by regulating the expression of proangiogenic gene Ephrin-B2.
Materials and Methods
Cell lines and reagents
Uninfected and KSHV-infected SLK cells, named SLKn and SLKp, respectively, were provided by Dr. Adam Grundhoff at Heinrich Dr. Adam Grundhoff's location is Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg Germany Pette Institute, Hamburg, Germany, in September, 2010, and cultured as described (27). The status of KSHV infection was verified in this study (Fig. 2C). Several batches of primary human blood outgrowth endothelial cells (BOEC) were obtained from Dr. Robert Hebbel at the University of Minnesota, Minneapolis, MN, between 2009 and 2010 and maintained in EGM-2 plus Bullet Kit (Lonza) on collagen-coated plates or dishes as previously described (28). BOECs can be passaged in vitro for more than 30 passages while maintaining their phenotypes. We used cells at passage 7 to 8 for the experiments.
NF-κB super suppressor IκBα mutant (IκBαM) lentiviral construct (p156RRLsinPPTCMV-IκBαM) was provided by Dr. Inder Verma at Salk Institute, San Diego, CA. vFLIP lentiviral construct was obtained from Dr. Chris Boshoff at UCL Cancer Institute, London, UK. EZH2 promoter firefly luciferase reporter construct, which contains a DNA fragment from −1,093 bp to +48 bp of the transcriptional start site, was provided by Dr. Felix Hoppe-Seyler at German Cancer Research Center, Heidelberg, Germany. The thymidine kinase promoter Renilla luciferase reporter plasmid (pRL-TK) was used as a control for transfection efficiency (Promega). Lentivirus clone TRCN000040076 for one of the EZH2 short hairpin RNA (shRNA) constructs was obtained from Open Biosystems. A second lentiviral EZH2 shRNA construct was previously described (26). Lentivirus production and infection were carried out as previously described (29). Ephrin-B2 shRNA lentiviral particles containing 3 different target-specific constructs were from Santa Cruz Biotechnology (sc-39438-V).
KSHV virus production and cell infection
Recombinant KSHV-GFP was isolated as previously described (27). Isolated virus was purified by centrifugation at 24,000 rpm for 2 hours with a 20/35% Nycodenz gradient (Thermo Fisher Scientific). The gradient junction band containing KSHV was collected. The purified virus preparation was aliquoted and stored at −80°C until use. The purified KSHV was diluted in Opti-MEM I Reduced Serum Medium before use. For KSHV infection, BOECs were plated on collagen-coated 12-well plate at 3 × 104 cells per well overnight and mock infected or infected with KSHV at more than 80% infection rates based on the percentages GFP-positive cells at 2 days postinfection. The concentration of infectious viral particles was determined before infection as published (3). To enhance infection efficiency, plates were centrifuged at 3,000 rpm for 1 hour at 25°C immediately after addition of KSHV to cells. At 3 hours postinfection, cells were washed 3 times with PBS and cultured in full EGM-2 medium.
Immunofluorescence and immunohistochemistry
Immunofluorescence was done as previously described (29). An anti-LANA rat monoclonal antibody (Advanced Biotechnologies Inc) and a Rhodamine Red-X–conjugated Affinipure Donkey Anti-Rat secondary antibody (Jackson ImmunoResearch Laboratories, Inc.,) were used for LANA staining. Immunohistochemistry for EZH2 was carried out on a formalin-fixed paraffin-embedded tissue microarray obtained from the AIDS and Cancer Specimen Resource (ACSR) of the National Cancer Institute. The tissue microarray slide was subjected to citrate antigen retrieval for 30 minutes and blocked for nonspecific protein binding with Universal Protein Block (Dako). Slides were incubated with an anti-EZH2 antibody (#36-6300; Invitrogen) at 1:200 followed by an anti-rabbit IgG secondary antibody and a DAB chromogen for color development (Envision; Dako). LANA staining and hematoxylin and eosin (H&E) staining of tissue microarray sections from the same block were carried out by ACSR. Images were captured using a Nikon E800M microscope equipped with a Nikon DXM1200 digital camera and the Nikon ACT-1 imaging software system (Nikon Instruments Inc.).
Western blotting was carried out as previously described (29) using antibodies to EZH2 (Cell Signaling Technology), Ephrin-B2 (Abcam), and IκBα (Santa Cruz Biotechnology). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), detected by an antibody (Santa Cruz Biotechnology), was used as a loading control. Immunoreactive bands were visualized by autoradiography following development with an enhanced chemiluminescence system (Amersham).
RNA extraction, reverse transcription, and real-time quantitative PCR
Total RNA was isolated using the TRIzol kit (Invitrogen) and treated with DNase I (Ambion) following the manufacturer's instructions. RNA was reverse transcribed into cDNA using Superscript II reverse transcriptase as described in the protocol (Invitrogen). Amplification reactions were carried out in a 25 μL reaction volume containing 50 ng total RNA, specific primers of EZH2, GAPDH, Ephrin-B2 or vFLIP, and SYBR Advantage qPCR Premix (Clontech). The specificity of the amplified products was controlled by postamplification dissociation curve analyses and agarose gel electrophoresis of the amplified products. The primer sequences are as follows: EZH2-F: 5′-TTGTTGGCGGAAGCGTGTAAAATC-3′, EZH2-R: 5′-TCCCTAGTCCCGCGCAATGAGC-3′; Ephrin-B2-F: 5′- GGAAGAAGTTCGACAACAAGTCC-3′, Ephrin-B2-R: 5′- TTCAGCAAGAGGACCACCAGCGT-3′; GAPDH-F: 5′-CGGAGTCAACGGATTTGGTCGTAT-3′, GAPDH-R: 5′-AGCCTTCTCCATGGTGGTGAAGAC; vFLIP-F: 5′-CGTCTACGTGGAGAACAGTGAGCT-3′, vFLIP-R: 5′-CTGGGCACGGATGACAGGGAAGTG-3′. Results were presented as mean ± SD from 3 independent experiments.
Tubule formation assay
Chilled growth factor-reduced Matrigel (BD Biosciences) was added to the wells of prechilled 96-well plate at 70 μL per well. The plate was placed in a 37°C incubator for 30 minutes to allow the Matrigel to solidify. BOECs were serum starved overnight, washed with PBS twice, detached with Accutase (Invitrogen), resuspended in full culture medium, and plated at 3,000 cells per well in the 96-well plates containing the solidified Matrigel. Cells were cultured in CO2 incubator at 37°C for 7 hours, and images were taken with the Zeiss Axiovert microscope with a ×10 objective (Carl Zeiss Microimaging Inc.). Tubule length was counted using the Axiovision software (Carl Zeiss Microimaging Inc.). Similar procedures were carried out with the SLK cells, except that the step of serum starvation was omitted, and cells were plated at 10,000 cells per well in the Matrigel-coated plate.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assay was carried out to detect EZH2 binding to the Ephrin-B2 promoter locus, as previously described, using antibody to EZH2 and IgG as a control (26). The enrichment of EZH2 on Ephrin-B2 promoter locus was calculated as the fold increase of the immunoprecipitated DNA compared with IgG-precipitated DNA. Loci of LANA and RTA promoters were used as low and high binding controls, as previously described (26). Each data points in ChIP figures were averages of at least 3 independent ChIPs using 3 independent chromatin preparations. The primers for LANA promoter locus are 5′-GTTTATAAGTCAGCCGGACCAA-3′ (LANA-F) and 5′-GATATAACTCCGCCCTCCACTA-3′ (LANA-R), which amplify a DNA fragment at 0.5-kb upstream of transcriptional start site. The primers for RTA promoter locus are 5′-CCCCAACACAAGGACCTTTA-3′ (RTA-F) and 5′-GCTTTTGGATACCCTGGTGA-3′ (RTA-R), which amplify a DNA fragment at 1.0-kb upstream of transcriptional start site. The primers for Ephrin-B2 promoter locus are 5′-TCGCCATGGCTGTGAGAAGGGA-3′ (Ephrin-B2-F) and 5′-GGATCGCGGACGCCACTTACT-3′ (Ephrin-B2-R), which amplify a DNA fragment from −5 bp to +142 bp of the transcriptional start site.
SLKp cells were cotransfected with both EZH2 promoter firefly luciferase reporter construct and pRL-TK Renilla luciferase reporter construct using Lipofectamine 2000 (Invitrogen). Cells were split at 24 hours posttransfection at 4 × 104 cells per well on 12-well plate. Cells were treated with Bay-11 at 38 hours posttransfection for 10 hours. Dual luciferase assay was then carried out following the instructions of the manufacturer (Promega).
EZH2 is overexpressed in KSHV tumors
EZH2 deregulation is observed in various malignancies and associated with tumor cell proliferation and angiogenesis (18, 19, 22). We examined the expression of EZH2 in Kaposi sarcoma tumors in a tissue microarray consisting of 38 normal biopsies and 173 Kaposi sarcoma biopsies from different organs. EZH2 was highly expressed in Kaposi sarcoma specimens compared with normal controls (Table 1). Of 38 normal specimens, only 4 (11%) had weak EZH2 staining. In contrast, among the 173 Kaposi sarcoma specimens, 157 (91%) had strong EZH2 staining. Figure 1 shows the representative staining EZH2 patterns in skin and lymph node Kaposi sarcoma specimens. Of 25 lymph node Kaposi sarcoma specimens, 24 (96%) had strong EZH2 staining (Table 1). In contrast, only 1 of the 28 (4%) normal control lymph node specimens had weak EZH2 staining. Of 77 skin Kaposi sarcoma specimens, 75 (97%) were positive for EZH2, whereas only 1 of 5 (20%) normal skin controls showed very light EZH2 staining (Table 1). The weak staining of this normal skin sample was due to nonspecific uptake by the macrophages. Insufficient clinical data on the Kaposi sarcoma specimens was available for further analysis of the correlation of the EZH2 staining pattern and intensity with Kaposi sarcoma disease status.
KSHV infection upregulates EZH2 in endothelial cells
To determine whether KSHV infection induced EZH2 expression in endothelial cells in culture, we infected BOECs with KSHV. BOECs are derived from blood circulating precursor cells and have typical endothelial cobblestone morphology and features (28). BOECs harbor KSHV in Kaposi sarcoma patients and support KSHV infection (30). Almost all cells in the KSHV-infected BOEC cultures were LANA positive 5 days after infection (Fig. 2A) without detectable expression of viral lytic protein ORF65 (data not shown), indicating that these cells were latently infected by KSHV. As expected, KSHV infection converted the BOECs from cobblestone shape to spindle shape (Supplementary Fig. S1A), with an increase in the expression of lymphatic markers FLT1, PROX1, LYVE1, and podoplanin (Supplementary Fig. S1B). Thus, similar to vascular endothelial cells (4–6), KSHV infection reprograms BOECs to acquire markers of lymphatic endothelial cells, which are features of Kaposi sarcoma tumor cells. As shown in Fig. 2B, KSHV infection of BOECs increased the expression of EZH2 mRNA and protein. Histone H3 is a known substrate of the PRC2. One of the common marks targeted by PRC2 is H3K27me3. Consistent with the upregulation of EZH2, KSHV infection also increased the level of H3K27me3 in BOECs (Fig. 2B). To further confirm these observations, we examined the EZH2 expression in SLK cells with and without KSHV infection. SLK cells are KSHV-negative endothelial cells isolated from a Kaposi sarcoma tumor. Similar to BOECs, KSHV established latent infection in SLK cells based on the detection of LANA protein (Fig. 2C) but not lytic protein ORF65 (data not shown). Compared with uninfected cells (SLKn), KSHV infection of SLK cells (SLKp) led to the upregulation of EZH2 protein and an increase in H3K27me3 (Fig. 2D). Together these data indicated that KSHV infection upregulated EZH2 and caused an increase of its substrate H3K27me3 in endothelial cells.
KSHV upregulation of EZH2 is mediated by the NF-κB pathway and viral latent genes vFLIP and LANA
Activation of NF-κB pathway has been shown to upregulate the expression of EZH2 (31). Because the NF-κB pathway is constitutively activated in KSHV-infected cells (32), we determined whether it might mediate KSHV induction of EZH2 expression. Indeed, inhibition of the NF-κB pathway with specific inhibitor Bay-11 reduced the expression of EZH2 protein in a dose-dependent manner in SLKp cells (Fig. 3A). Similar results were also observed in BOECs. To confirm these results, we inhibited the NF-κB pathway with a super suppressor IκBαM. Expression of IκBαM reduced the expression of EZH2 protein (Fig. 3A). Bay-11 also inhibited the expression of EZH2 mRNA by 80% (Fig. 3B); however, it only inhibited EZH2 promoter activity by 40% in a reporter assay, suggesting that the NF-κB pathway might only partially regulate the expression of EZH2 at the transcriptional level. Alternative mechanism such as mRNA stability might also be involved in the regulation of EZH2 mRNA level by this pathway.
Because KSHV established latent infection in both BOECs and SLK cells, we searched for latent genes that might activate the NF-κB pathway and cause the upregulation of EZH2. We focused on vFLIP and LANA because both of them have been shown to activate the NF-κB pathway (33, 34). Indeed, expression of vFLIP in BOECs upregulated the expression of EZH2 at both protein and mRNA levels, which was inhibited by Bay-11 (Fig. 3C). Because we were not able to obtain a functional antibody to vFLIP, we examined the expression of vFLIP mRNA by quantitative reverse transcription PCR (qRT-PCR). We detected robust expression of vFLIP mRNA in the vFLIP-transduced cells, which was not affected by Bay-11 (Fig. 3C). Similar to vFLIP, expression of LANA also increased the expression of EZH2, however, to a less extent, by less than 2-fold, in BOECs (Fig. 3C). In contrast to vFLIP and LANA, we did not observe any increase in EZH2 expression following the expression of KSHV-latent gene vCyclin (ORF72, data not shown). KSHV infection induces inflammatory cytokines, some of which can activate the NF-κB pathway. Conditioned medium from KSHV-infected BOECs only marginally increased the expression of EZH2 protein (Fig. 3D). Together, these results indicated that KSHV upregulation of EZH2 is mainly mediated by vFLIP and LANA through the NF-κB pathway in latent KSHV-infected endothelial cells.
Induction of EZH2 is essential for KSHV-induced angiogenesis
Overexpression of EZH2 is often associated with advanced stage of tumors (19, 35). EZH2 has been shown to regulate cancer angiogenesis (22, 24). We determined whether KSHV-induced upregulation of EZH2 contributed to KSHV-induced angiogenesis. We used an in vitro Matrigel tubulogenesis assay to measure the angiogenic activity of endothelial cells. KSHV infection of BOECs increased the expression of EZH2 as well as tubule formation, whereas knockdown of EZH2 reduced tubule formation (Fig. 4A). Similar results were also observed in SLK cells (Fig. 4B). These results indicated that EZH2 mediated KSHV-induced angiogenesis.
EZH2 regulation of KSHV-induced angiogenesis is mediated by Ephrin-B2
To delineate the mechanism involved in EZH2-mediated KSHV-induced tubule formation, we screened a panel of proangiogenic genes that could be regulated by EZH2 in KSHV-infected endothelial cells. We found that Ephrin ligand Ephrin-B2 protein was upregulated in KSHV-infected BOECs and constitutively expressed in SLKp cells (Fig. 5A). Knockdown of EZH2 reduced the expression of Ephrin-B2 protein (Fig. 5A). Upregulation of Ephrin-B2 by KSHV was also observed at the mRNA level (Fig. 5B). Knockdown of EZH2 reduced the expression of Ephrin-B2 mRNA in KSHV-infected BOECs (Fig. 5B). To further determine whether EZH2 directly binds to the Ephrin-B2 promoter to regulate its expression, we carried out ChIP assay in KSHV-infected SLKp cells. A previous study has shown that EZH2 binds strongly to the RTA promoter chromatin but weakly to the LANA promoter chromatin in KSHV-infected cells. We used these KSHV loci as controls for our experiments. Indeed, compared with IgG control, an EZH2-specific antibody enriched 18-fold of the RTA promoter chromatin but only 3.1-fold of the LANA promoter chromatin (Fig. 5C). Knockdown of EZH2 reduced its binding to the RTA promoter chromatin by 82% but had marginal effect on the LANA promoter chromatin (Fig. 5C). Similar to the RTA promoter, EZH2 enriched Ephrin-B2 promoter chromatin by 13-fold (Fig. 5C). Knockdown of EZH2 reduced its binding to the Ephrin-B2 promoter chromatin by 70% (Fig. 5C). These results indicated that EZH2 regulates the expression of Ephrin-B2 by directly binding to its promoter.
It has been shown that Ephrin-B2 is highly expressed in Kaposi sarcoma tissues, and KSHV infection upregulated the expression of Ephrin-B2 in human endothelial cells (13). Furthermore, inhibition of the Eph pathway is sufficient to decrease cell growth, migration, and angiogenesis of Kaposi sarcoma cells (36). Indeed, knockdown of either Ephrin-B2 or EZH2 inhibited the proliferation of KSHV-infected BOECs and SLK cells, as indicated by the significant lower numbers of cells per field at 3 days posttransduction, with the specific shRNAs (Fig. 6A and B). Knockdown of Ephrin-B2 also inhibited tubule formation of KSHV-infected BOECs and SLK cells (Fig. 6A and B).
EZH2 was initially described as a controller of cell differentiation and organism development. It is highly expressed in stem cells (16). Expression of EZH2 is required for the inhibition of differentiated genes and maintenance of the identity of stem cells (37). The expression of EZH2 decreases once stem cells differentiated into specific type of cells (38). Subsequent studies revealed roles of EZH2 in normal cell proliferation and cell-cycle control as well as progression of cancer. In cell culture, overexpression of EZH2 induces cell invasion, growth in soft agar, and motility, whereas knockdown of EZH2 inhibits cell proliferation and cell invasion (18, 35). Dysregulation of EZH2 has been associated with tumor aggressiveness, therapeutic refractory, and tumor angiogenesis in prostate, breast, and ovarian cancer cells (18, 19, 22). EZH2 is regulated by several pathways including retinoblastoma-E2F (39), c-Myc (40), NF-κB (31), and p53 (41) pathways, as well as several miRNAs (23, 40). In this study, we have found that EZH2 is highly expressed in Kaposi sarcoma tumors and highly upregulated in KSHV-infected endothelial cells. Furthermore, we have shown that KSHV-induced upregulation of EZH2 is mediated by the NF-κB pathway and KSHV-latent genes vFLIP and LANA.
Like all herpesvirus, KSHV has both latent and lytic phases in its life cycle. Most tumor cells in Kaposi sarcoma lesions display KSHV-latent replication program, expressing only a handful of latent gene products that are involved in host cell survival, proliferation, and manipulation of the tumor microenvironment. Among KSHV-latent genes, vFLIP and LANA have been shown to activate the NF-κB pathway (33, 34). Our results have shown that KSHV upregulation of EZH2 is mainly mediated by vFLIP, and to a less extent, by LANA. Our previous study has shown that vFLIP inhibits KSHV lytic replication program to promote viral latency through activation of the NF-κB pathway and inhibition of the AP-1 pathway (42). A recent study has shown that EZH2 mediates KSHV latency in primary effusion lymphomas and in KSHV-infected endothelial cells by regulating latency-specific histone-repressive modifications such as H3K27me3 (26). Thus, in addition to the AP-1 pathway, this study has identified EZH2 as a new cellular target that mediates vFLIP regulation of KSHV latency through the NF-κB pathway.
Because Kaposi sarcoma is a highly angiogenic tumor and KSHV infection of endothelial cells enhances the angiogenic phenotype of these cells, we examined the effect of upregulation of EZH2 on KSHV-induced tubule formation on Matrigel. EZH2 is an epigenetic repressor and inhibited the expression of several tumor suppressor genes, including p16, E-cadherin, DAB2IP, and RUNX3 among others (35). Upregulation of EZH2 by VEGF promotes angiogenesis by inhibiting the expression of angiogenesis inhibitor VASH1 through epigenetic silencing (22). Maspin, an angiogenesis inhibitor, is also downregulated by EZH2 in a microarray analysis (38). Examination of KSHV-infected BOECs showed that the expression of both genes were at the lower detection limit of qRT-PCR, albeit VASH1 seemed to be downregulated. Interestingly, we did not observe any significant increases in the expression of VASH1 following EZH2 knockdown (data not shown). In addition to angiogenesis inhibitors, we have also examined proangiogenenic factors including Eph ligand Ephrin-B2, Notch ligands Jag1 and DLL4, VEGFR3, and MMP-2 and MMP-9. Among these factors, Ephrin-B2 is the only one consistently upregulated following KSHV infection. Knockdown of EZH2 downregulated the expression of Ephrin-B2 in KSHV-infected BOECs and SLK cells.
Ephrin-B2 is a transmembrane ligand and regulates angiogenic growth of the blood vessel and lymphatic endothelium (43). Eph-ephrin binding leads to bidirectional signaling in cells expressing the receptor (forward signaling) or ligand (reverse signaling). Ephrin-B2 is upregulated during physiologic and pathologic angiogenesis in the adult, and loss of Ephrin-B2 leads to vascular defects (44). Intriguingly, Ephrin-B2–mediated reverse signaling controls VEGFR internalization. Endothelial cells lacking Ephrin-B2 reverse signaling are unable to internalize VEGFR2 and VEGFR3 and cannot properly transmit VEGF signals, resulting in impairment of angiogenesis (45, 46). Previous studies have shown that Ephrin-B2 is highly expressed in Kaposi sarcoma tissues, KSHV infection upregulates Ephrin-B2 in endothelial cells (13), and the inhibition of Ephrin-B2 blocked tumor growth and reduced tumor vessel density and perfusion in a Kaposi sarcoma tumor xenograft model (36). Our results have confirmed that Ephrin-B2 expression is increased following KSHV infection. Furthermore, we have shown that knockdown of EZH2 inhibits Ephrin-B2 expression at both protein and mRNA levels, and knockdown of either Ephrin-B2 or EZH2 inhibits cell growth and tubule formation in KSHV-infected endothelial cells.
EZH2 and its associated PRC2 complex typically repress or maintain the repression status of their target genes (37, 47, 48). EZH2 is associated with the promoters of a number of tumor suppressor genes and therefore inhibits their expression (35, 47). However, EZH2 also upregulates some positive regulators, including CCND1, CCNE1, CCNA2, and c-Myb among others (20, 39). A recent study has shown that EZH2, through interacting with RelA/RelB, is required for the establishment of constitutive activation of a subset of NF-κB–targeted genes (49). Our results have shown that EZH2 binds to the promoter of angiogenesis enhancer Ephrin-B2 and upregulated its expression, indicating EZH2 might also be required for either the activation or the maintenance of the activated state of this positive angiogenesis regulator. Further studies are required to delineate the mechanism mediating this positive regulation.
In summary, we have shown that KSHV promotes angiogenesis by upregulating a proangiogenic Ephrin-B2 through EZH2. These findings suggest that both EZH2 and Ephrin-B2 are promising novel targets for KSHV-induced angiogenesis.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: M. He, M. Cannon, S.-J. Gao
Development of methodology: M. He, M. Schutten, P. Gill, M. Cannon
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Bakken
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. He, M. Schutten, Z. Toth, M. Cannon, S.-J. Gao
Writing, review, and/or revision of the manuscript: M. He, M. Schutten, Z. Toth, P. Gill, M. Cannon, S.-J. Gao
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Schutten, Z. Toth, J.U. Jung, S.-J. Gao
Study supervision: M. Cannon, S.-J. Gao
Performed RT-qPCR for the Bay-11 study: W. Zhang
This work was funded by an American Cancer Society Research Scholar grant (RSG-10-060-01-MPC) to M. Cannon and NIH grants to S.-J. Gao (R01CA132637, R01CA124332, and R01CA096512).
The authors thank Drs. Adam Grundhoff, Robert Hebbel, Inder Verma, Chris Boshoff, and Felix Hoppe-Seyler for providing us critical reagents, Paula Overn in the Comparative Pathology Shared Resource, Masonic Cancer Center at the University of Minnesota for her technical assistance for the IHC experiments, and members of Dr. Gao's laboratory for technical assistance and helpful discussions.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received August 29, 2011.
- Revision received May 3, 2012.
- Accepted May 5, 2012.
- ©2012 American Association for Cancer Research.