Background
Angiogenesis is a tightly regulated process that is essential for normal organ development, tissue repair and regeneration, and tumor growth. Growth, maturation and migration of endothelial cells and vessel formation are controlled by environmental cues such as nitric oxide (NO) and hypoxia, and a network of growth factors, receptors and transcription factors [
1-
3]. Among the transcription factors that control angiogenesis, the most extensively studied include hypoxia-inducible factors (HIF), nuclear factor κB (NF-κB) and members of the Signal Transducer and Activator of Transcription (STAT) and E-twenty-six (ETS) families of transcription factors [
3-
6].
The homeobox gene super-family comprises more than 200 vertebrate genes and encodes transcription factors, termed homeoproteins, that are characterized by their conserved helix-turn-helix DNA-binding domain [
7]. Homeoproteins are expressed in a temporal- and tissue- specific manner and play essential roles in controlling cell lineage-specification and tissue morphogenesis [
7-
9]. Increasing evidence indicates that angiogenesis is tightly regulated by specific sets of homeoproteins. HOXA9 promotes endothelial cell migration and tube formation by activating transcription of the gene encoding ephrin B4 [
10]. HOXB5 promotes endothelial sprouting by inducing angiopoietin-2 expression [
11]. In contrast, HOXA5, HOXD10 and GAX exert inhibitory effects on angiogenesis by various mechanisms such as inducing expression of thrombospondin-2 and p21WAF1/CIP1 [
12-
14].
Many homeobox genes have been found to be up- or down- regulated in a variety of tumors [
8,
9]. The mechanisms of most of these genes in tumor growth and progression are poorly understood as only few transcriptional targets have been identified. Notably, several homeoproteins that are aberrantly expressed in tumor cells have been found to control expression of pro-angiogenic growth factors. HOXB7 is overexpressed in more than 50% of breast cancers and activates the gene that encodes fibroblast growth factor-2 [
15]. Conversely, loss of NKX3.1, which occurs in 80% of prostate cancers, increases expression of vascular endothelial growth factor (VEGF)-C [
16]. High tumor microvessel density is strongly predictive of poor outcomes in patients with ovarian cancer [
17,
18]. We previously identified that DLX4, a homeoprotein that is absent from most normal adult tissues, is expressed in approximately 50% of ovarian cancers and is strongly associated with reduced survival [
19]. Studies using xenograft models revealed that DLX4 expression in ovarian cancer cells increases tumor microvessel density, implicating a pro-angiogenic function for DLX4 [
19]. Inducible nitric oxide synthase (iNOS) is an enzyme that promotes angiogenesis by generating NO [
2,
20] and its expression in ovarian cancers is strongly associated with poor outcomes [
21,
22]. In this study, we identified that DLX4 induces iNOS expression in part by stimulating STAT1 activity and promotes ovarian tumor angiogenesis by inducing iNOS expression. These findings raise the possibility that specific sets of homeoproteins promote tumor angiogenesis not only by directly regulating transcription of angiogenic growth factors, but also by modulating intracellular signaling and environmental cues.
Discussion
Several homeoproteins that are expressed in endothelial cells play important roles in transcriptional programs that control normal endothelial cell growth, migration and tube formation [
10-
14]. Increasing evidence indicates that aberrant expression of other sets of homeoproteins in tumor cells increases tumor angiogenesis (reviewed in Ref. [
9]), but their mechanisms are poorly understood. In this study, we identified that DLX4, a homeoprotein that is overexpressed in ovarian cancer, stimulates ovarian tumor angiogenesis by inducing iNOS expression. To our knowledge, our study is the first to identify a role for a homeoprotein in controlling NO generation. NO is generated from L-arginine, NADPH and oxygen by NO synthases of which there are three isoforms [
2,
20]. nNOS and eNOS are constitutively expressed, predominantly in neuronal cells and endothelial cells, respectively, and are also expressed in several types of tumors including a subset of ovarian cancers [
2,
20,
34]. iNOS produces substantially more NO than the other NO synthase isoforms [
2]. We found that high DLX4 expression is associated with increased expression of iNOS but not of nNOS or eNOS in clinical specimens of ovarian cancer, and that DLX4 induces expression of iNOS but not of nNOS or eNOS in ovarian tumor cells. A principal mechanism by which NO promotes angiogenesis is by increasing VEGF-A production [
32,
33]. Our findings that the stimulatory effects of DLX4 on VEGF-A production and tumor angiogenesis are abrogated when iNOS is inhibited support the conclusion that these stimulatory effects of DLX4 are primarily mediated via its induction of iNOS. In addition to its potent angiogenic property, VEGF-A causes ascites formation in ovarian cancer [
35]. DLX4 expression in ovarian tumor cells induced ascites formation in mice and this induction was abrogated when iNOS was inhibited. Agents that neutralize VEGF-A or inhibit VEGF receptor tyrosine kinase activity have been extensively evaluated in clinical trials of ovarian cancer patients and a limitation of these agents is acquired resistance [
36]. Inhibition of iNOS might be an effective approach to inhibit tumor angiogenesis and also ascites formation in ovarian cancer.
The findings of the present study indicate that DLX4 interacts with STAT1 and induces iNOS expression at least in part by stimulating STAT1 activity. Although dominant-negative STAT1 abrogated the ability of DLX4 to induce iNOS expression, we cannot eliminate the possibility that DLX4 might also stimulate activity of other transcription factors that activate
NOS2 transcription such as NF-κB and HIF-1α [
27,
37]. To our knowledge, our study is the first to identify an interaction between a homeoprotein and a member of the STAT family. Although the precise mechanism by which DLX4 stimulates STAT1 activity remains to be determined, it is possible that DLX4 stabilizes STAT1-containing transcriptional complexes. Whereas inflammatory cytokines induce
NOS2 transcription, transforming growth factor-β (TGF-β) decreases
NOS2 mRNA stability and translation and increases degradation of iNOS protein [
38]. We previously identified that DLX4 blocks TGF-β/Smad signaling [
25]. It is therefore possible that DLX4 might also increase levels of iNOS by blocking TGF-β-mediated
NOS2 mRNA destabilization and iNOS protein degradation. Whereas iNOS induces VEGF-A production, VEGF-A stimulation reciprocally increases expression of iNOS [
39]. DLX4 might additionally, through its induction of iNOS, enhance the iNOS-VEGF-A regulatory loop in tumors.
Whereas NO has a pro-tumorigenic effect by stimulating angiogenesis, continuous exposure to NO at high concentrations exerts genotoxic effects [
20]. Metabolites of NO can cause DNA damage and lead to cell death [
20]. NO has been reported to induce expression of the catalytic subunit of DNA-dependent protein kinase (DNA-PK), an essential component of the non-homologous end-joining (NHEJ) DNA repair machinery [
40]. This NO-mediated induction of DNA-PK has been found to protect cells from the deleterious effects of NO and other DNA damaging agents [
40]. We recently identified that DLX4 stimulates DNA-PK activity and NHEJ-mediated DNA repair by interacting with Ku70 and Ku80 that bind to DNA double-strand break ends [
29]. This activity of DLX4 protected tumor cells against DNA damaging chemotherapeutic agents such as doxorubicin and etoposide [
29]. It is possible that DLX4 might similarly protect tumor cells against the genotoxic effects of high NO levels by stimulating repair of DNA damage caused by NO metabolites. In addition, several studies have identified that STAT1 confers resistance to DNA damage (reviewed in Ref. [
41]). DLX4 might also confer resistance to NO-induced DNA damage by stimulating STAT1 activity.
Because relatively few transcriptional targets of homeoproteins have been identified, it is unclear how the tumor-promoting properties of these factors are related to their normal developmental functions. The
DLX4 gene was originally identified in a screen of a placental cDNA library [
42] but its normal function is poorly understood. Preeclampsia is characterized by reduced placental blood flow and elevated maternal blood pressure, and is a major cause of fetal and maternal morbidity and mortality [
43]. Substantial evidence indicates that disruption of NO bioavailability contributes to the pathophysiology of preeclampsia and that expression of iNOS is reduced in preeclamptic placentas [
43-
45]. Interestingly, it has also been reported that DLX4 expression is down-regulated in preeclamptic placentas [
46]. Our findings that DLX4 induces iNOS expression raise the intriguing possibility that down-regulation of DLX4 in the placenta might promote endothelial dysfunction in preeclampsia by causing a reduction in iNOS levels.
Methods
Plasmids
The pIRES-EGFP2 plasmid encoding FLAG-tagged DLX4 has been previously described [
25]. The
DLX4 cDNA was subcloned into the pRetroQ-AcGFP retroviral vector (Clontech). Other plasmids were as follows: pGFP-V-RS plasmids containing non-targeting and
DLX4 shRNAs (OriGene Technologies), pGIPZ plasmids containing
NOS2 shRNAs (GE Healthcare), eGFP-STAT1 (provided by Alan Perantoni, National Cancer Institute, Frederick, MD; Addgene plasmid 12301) [
48], pRc/CMV-Flag STAT1α Y701F [
28] (provided by James Darnell, Rockefeller University, New York, NY; Addgene plasmid 8702). A firefly luciferase reporter construct driven by tandem GAS elements was purchased from SABiosciences.
Antibodies (Abs) and other reagents
Sources of Abs were as follows: DLX4, iNOS, CD34 (Abcam), STAT1, phosphorylated STAT1 (Y701) (Cell Signaling Technology), FLAG, actin (Sigma-Aldrich), secondary Abs (BD Biosciences, Invitrogen). Recombinant IFN-γ was purchased from R&D Systems.
Cell culture and transfection
Culture media were purchased from Invitrogen and were supplemented with penicillin-streptomycin and 10% fetal bovine serum. Vector-control and DLX4-overexpressing ES2 cell lines have been previously described [
19] and were cultured in McCoys’ 5A medium. The parental 2008 cell line was provided by Zahid Siddik (MD Anderson Cancer Center, Houston, TX) and cultured in RPMI 1640 medium. Parental A2780, OVCAR8 and OVCA429 cell lines were provided by Gordon Mills (MD Anderson Cancer Center) and cultured in RPMI 1640 medium (A2780, OVCAR8) and Dulbecco’s Modified Eagle’s Medium (DMEM) (OVCA429). The parental TOV112D cell line was provided by Ju-Seog Lee (MD Anderson Cancer Center) and cultured in a 1:1 mixture of MCDB 105 medium and Medium 199. The U3A cell line [
31] was provided by George Stark (Cleveland Clinic Lerner Research Institute, Cleveland, OH) and cultured in DMEM. Immortalized mouse ovarian endothelial cells [
49] were provided by Isaiah Fidler (MD Anderson Cancer Center) and cultured in DMEM medium. Ampho-293 cells were provided by Douglas Boyd (MD Anderson Cancer Center) and cultured in DMEM. Ampho-293 cells were transfected with pRetroQ-AcGFP retroviral constructs by using Lipofectamine®2000 reagent (Invitrogen). At 48 h thereafter, viral supernatants were harvested and used to infect U3A and A2780 cells. In other experiments, cultured cells were directly transfected with plasmids by using Lipofectamine®2000 reagent or FuGENE®6 reagent (Promega). Cells were selected by addition of G418 (400 μg/mL) or puromycin (0.5 μg/mL).
NO assay
Cells were cultured overnight in medium that contained no FBS or Phenol Red. Culture supernatants were thereafter depleted of proteins that were >10 kD in size by centrifugation at 14,000 × g for 10 mins through Amicon Ultra 0.5 mL centrifugal filters (Millipore). NO levels in supernatants were assayed by using Griess reagent (Total NO and Nitrate/Nitrite Parameter Assay Kit, R&D Systems) following manufacturer’s instructions. Three independent experiments were performed for each assay.
Conditioned medium and cell proliferation assays
Equivalent numbers of tumor cells (1.5×106) were seeded in 10 cm dishes and cultured in medium containing 2% FBS for 2 d. Tumor-conditioned medium was assayed for VEGF-A levels by ELISA (R&D Systems) and added to endothelial cells. Endothelial cell growth was measured by using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay (Roche). Three independent experiments were performed for each assay.
qRT-PCR
NOS1, NOS2 and NOS3 mRNA levels were analyzed by using primers and SYBR®Green qPCR Master Mix that were purchased from SABiosciences. RPL32 transcript levels were used as controls for normalization and were detected by using the following primers: forward: 5′-ACAAAGCACATGCTGCCCAGTG-3′, reverse: 5′- TTCCACGATGGCTTTGCGGTTC-3′.
Immunofluorescence staining of cultured cells
For analysis of staining by flow cytometry, cells were fixed in 1% paraformaldehyde (20 min at 4°C) and permeabilized in 0.1% saponin (15 min at room temperature). Thereafter, cells were incubated with Abs to DLX4 (1:20) or to iNOS (1:100) for 30 min at 4°C, washed and incubated with peridinin-chlorophyll-protein complex-conjugated secondary Ab. Staining was detected by flow cytometry (FACS Calibur, BD Biosciences). For analysis of staining by fluorescence microscopy, cells were plated in chamber slides, fixed in 4% paraformaldehyde (20 min at 4°C) and permeabilized in 0.1% Triton-X100 (15 min at room temperature). Thereafter, cells were incubated with STAT1 Ab (1:100) for 30 min at 4°C, washed and incubated with Alexa Fluor 594-conjugated secondary Ab. Cells were stained with diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) and then viewed using an Eclipse 80i fluorescence microscope (Nikon).
IP and immunoblotting
Cell extracts were prepared by lysing cells in M-PER buffer (Pierce Biotechnology). For IP, 2 mg of cell extract was pre-cleared with protein G agarose and incubated with FLAG Ab or with control Ig using lysis buffer (20 mM Tris–HCl pH 8.0, 100 mM NaCl, 1% Nonidet P-40, 10% glycerol, 2 mM EDTA). Cell extracts and immunoprecipitates were subjected to SDS-PAGE and immunoblotting using polyvinylidene fluoride membrane (GE Healthcare).
Reporter assays
Cells were co-transfected with plasmids containing
DLX4 cDNA or shRNAs, firefly luciferase reporter plasmid and Renilla luciferase reporter plasmid to normalize transfection efficiency as previously described [
25]. Where indicated, recombinant IFN-γ (10 ng/mL) was added to cells at 24 h after transfection. At 16 h thereafter, luciferase activities were assayed by using the Dual-luciferase reporter assay kit (Promega). Three independent experiments were performed for each assay.
Mouse i.p. xenografts
All animal studies were approved by the University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee. Four-week-old female nude mice were purchased from Charles River and inoculated i.p. with 1 × 106 cells of ES2 lines (n = 5 mice per group). At 3 weeks thereafter, mice were euthanized by CO2 asphyxiation. Volume of ascites was measured. Sections of formalin-fixed, paraffin-embedded tissues were stained with Abs to iNOS and CD34 and staining detected by streptavidin-biotin-peroxidase and 3,3′-diaminobenzidine (Dako). Microvessels were counted in 5 independent and random 100x microscopic fields in stained tumor tissue sections of each mouse.
Published gene expression data from Australian Ovarian Cancer Group Study [
23] (GSE9891, n = 285) and from the Japanese Serous Ovarian Cancer Group Study [
24] (GSE32062, n = 260) were downloaded from the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database (
http://www.ncbi.nlm.nih.gov/geo). Patients were stratified according to expression of
DLX4 in tumors, where
DLX4 transcript levels were defined as High (≥ upper quartile) and Low (≤ lower quartile).
Statistical analysis
STATISTICA6 software (StatSoft Inc.) was used for statistical analysis. Statistical significance of data of in vitro and in vivo assays was assessed by unpaired two-tailed Student’s t-test. Significance of differences in gene expression between groups of patients was assessed by Mann–Whitney U-test. P values of < 0.05 were considered significant.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BQT and HN designed and performed experiments and wrote the manuscript. SYK designed and performed experiments. DH and NB performed experiments. All authors read and approved the final manuscript.