Angiogenesis, which is the sprouting of new blood vessels from pre-existing vasculature, is a crucial process in tumour pathogenesis and invasive tumour growth as well as development, wound repair and reproduction (Risau, 1997). Thus, anti-angiogenesis-based therapies are useful modalities for the treatment of cancer. Angiogenesis is essential for the growth and progression of tumours because it enables the delivery of oxygen and nutrients to the growing tumour (Li
VEGF is activated under hypoxic conditions via the oxygen-sensing factor HIF-1α. HIF-1 is a heterodimer consisting of HIF-1α and HIF-1β subunits and is an important transcription factor that mediates new blood vessel formation by regulating the expression of the angiogenic factor VEGF. Additionally, HIF-1 plays a major role in cellular responses to hypoxia in healthy and carcinoma cells (Masoud and Li, 2015). Hypoxia plays a pivotal role in tumour development, progression of vascular-related diseases and subsequent pathological angiogenesis involving upregulation of VEGF and VEGF-related target molecules. HIF-1α is a well-validated therapeutic target protein found in many solid tumours. It is the main regulator of several factors pivotal for tumour angiogenesis, involving those that modulate tumour progression, embryonic development, cell metabolism, apoptotic cell death, and metastasis (Wigerup
NOEY2, a small GTP-binding protein belonging to the Ras superfamily, shares 60% homology with Ras and Rap (Gasper
In this study, we confirmed the anti-tumour and anti-angiogenic effects of NOEY2 in a mouse model of ovarian cancer. We also validated the anti-tumour and anti-angiogenic effects of NOEY2-N
Human ovarian carcinoma cell lines (SKOV-3 and OVCAR-3) and human embryonic kidney 293T (HEK293T) cells were maintained in accordance with American Type Culture Collection (ATCC, Manassas, VA, USA) instructions. HUVECs were obtained from Clonetics (Walkersville, MD, USA), and were grown in EGM-2 BulletKit medium (Clonetics) on 0.3% gelatin (Sigma, St. Louis, MO, USA) coated dishes. Cells were seeded in a humidity chamber at 37°C with 5% carbon dioxide. Specific pathogen-free BALB/c-nu/nu mice (approximately 5-6 weeks old) were supplied by Orientbio (Seongnam, Korea). All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the Research Institute of the National Cancer Center (Goyang, Korea). The primary antibodies used were anti-caspase-3, anti-Bcl-2 anti-Bcl-xL, anti-Bax, anti-p53, anti-VEGFR-1, anti-VEGFR-2, anti-phospho-VEGFR-2 (Tyr-951), anti-phospho-VEGFR-2 (Tyr-1175), anti-HIF-1α, anti-survivin, anti-COX-2, anti-PI3K, anti-phospho-PI3K, anti-PDK-1, anti-phospho-PDK-1 (Ser241), anti-Akt, anti-phospho-Akt (Ser473 and Thr308), anti-mTOR, anti-phospho-mTOR (Ser2448), anti-TSC2, anti-phospho-TSC2 (Ser1462), anti-p70S6K, anti-phospho-p70S6K (Thr421) (Cell Signaling, Beverly, MA, USA), anti-p27, anti-p21, anti-VEGF, anti-Ki67, anti-CD31 (PECAM-1) (Ab-1; Oncogene, Cambridge, MA, USA), anti-PARP, anti-XIAP (BD Biosciences, San Jose, CA, USA), anti-cyclin D1, anti-CDK4, anti-NF-κB, anti-GSK-3β, anti-phospho-GSK-3β (Ser-9) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PCNA (Dako Denmark A/S, Glostrup, Denmark), anti-NOEY2 (BD Pharmingen, San Diego, CA, USA), and anti-NOEY2 and anti-β-actin (Sigma).
The full length of NOEY2 was amplified by PCR and cloned into the
In brief, human SKOV-3 ovarian carcinoma cells (2.3×106), stably transfected with NOEY2 or empty vector, were injected subcutaneously into 5- to 6-week-old BALB/c-nu/nu mice and allowed to form tumours 70-100 mm3 in size. Tumour volume was calculated in three dimensions using callipers and evaluated by the following formula: tumour volume (mm3)=(
For immunohistochemistry, tumour samples from xenografted mice were collected, fixed and serially sectioned as previously reported (Yu
For western blotting analysis, protein lysates were prepared from homogenised frozen tumour tissues, and a Bradford protein assay was used to determine protein concentration. Equal amounts of protein (20 μg) were loaded onto 8-12% SDS-PAGE.
For analysis of the DNA content using flow cytometry, SKOV-3 and OVCAR-3 cancer cells were grown at a density of approximately 3.5-4.0×105 in 60-mm plates. Cells were detached using trypsin and rinsed twice with phosphate-buffered saline (PBS). The pellets were re-suspended with binding buffer and incubated with fluorescein isothiocyanate (FITC)-labelled annexin V and propidium iodide (PI) for 15 min according to the supplier’s instructions (BD Pharmingen, Mississauga, ON, Canada). The labelled cells were analysed using a fluorescence activated cell sorting (FACS) Vantage BD FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). Cell viability was measured using the CellTiter-Glo luminescent assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. In brief, cells were maintained at a density of 4.0×103 per well in 96-well plates. After 24 h, cells were transfected/treated with control (empty-inserted vector only), NOEY2(wt), or three truncated mutants, respectively. Cell viability was calculated using CellTiter-Glo reagent (Promega, Madison, WI, USA).
For caspase-3 activity, cells (2.4×106) were maintained in either the absence or presence of NOEY2 for 24 h at 37°C. Caspase-3 activity was calculated using actyl-DEVD-7-amino-4-trifluoromethyl coumarin as the substrate, according to the supplier’s instructions (BD Pharmingen). In brief, the cells were placed with VP-16 (100 μg/mL) for 24 h, lysed in lysis buffer, and centrifuged at 4°C for 25 min at 12,000
For bait construction with human NOEY2, cDNA encoding full-length human NOEY2 was cloned into the
Co-IP was carried out as described previously (Rho
The VEGFR-2 promoter fragment was cloned from human placental complementary DNA (cDNA) to evaluate VEGFR-2 promoter activity by PCR using primers: 5′-TAGCGAGCTCTGCCACAAGAAGTCCACACA-3′ (sense); 5′-CACCCGACCTGTCTGCCTTCC-3′ (antisense). The domain including the VEGFR-2 promoter (from −887 to +295) region was introduced between the
To measure endothelial cell proliferation, cells were seeded at a density of 3.0×104 per well of gelatinised plates in standard medium on day 0. Next, [3H] thymidine incorporation analysis was carried out as reported previously (Kim
For the main body, except for N-terminal 34 amino acids, three x-ray crystal structures from the Ras superfamily were used as a template structure: human Rap1A (Protein Data Bank code: 1GUA), Rap2A (3RAP) and H-Ras (1K8R) structures. However, for the N-terminal domain, only one NMR structure was available: human interleukin-6 (1IL6). For homology-modelling procedures, the MODELER module in the INSIGHT II program (Accelrys, Inc., San Diego, CA, USA) was used to manipulate sequence and structure and to perform the calculations.
All EM and MD calculations were performed with the DISCOVER module in the INSIGHT II program using the consistent-valence force-field (Dauber-Osguthorpe
Data values presented as mean ± standard deviation (SD) and statistical comparisons were determined statistically using Student’s
To explore the direct anti-tumour and anti-angiogenic activities of NOEY2, we investigated the effects of NOEY2 on tumour growth in ovarian tumour xenografts
We also confirmed the anti-angiogenic effects of NOEY2 in the tumours by counting the number of blood vessels stained with an antibody against CD31, a typical marker of endothelial cells. A fourfold reduction in the number of CD31-positive blood vessels was found in the tumour sections from the NOEY2-overexpressing mice (Fig. 1F). Western blotting results also demonstrated that the expression of VEGF and HIF-1α, and phosphorylation of VEGFR-2 (Tyr-1175) were markedly decreased in the NOEY2-overexpressing tumours versus the controls (Fig. 1G). Interestingly, the level of VEGFR-1 phosphorylation (Tyr-951) was not changed (Fig. 1G).
Finally, we assessed the effects of NOEY2 on the activation of PI3K since the pro-angiogenic effects of VEGF/VEGFR-2 are mediated by the PI3K/Akt signalling (Byrne
NOEY2 can be divided into an N-terminal region unique to NOEY2, the main body having five conserved GTP binding sites, and the CAAX motif of the C-terminal region (Luo
To identify the specific region responsible for the activity of anti-tumour effects, we generated three truncated NOEY2 expressing constructs (deletion of the GTP-binding, C-terminal, and N-terminal domains) (Fig. 2A,
To determine which regions of NOEY2 are important to induce cytotoxicity in SKOV-3 and OVCAR-3 ovarian cancer cells, the cells were transfected with the control (empty-vector only), NOEY2(wt), NOEY2 (ΔGTP BD), NOEY2 (ΔC-terminal), and NOEY2(ΔN-terminal), respectively. We first investigated the colony formation ability. The numbers of colonies formed were noticeably enhanced in NOEY2(ΔN-terminal)-only-transfected SKOV-3 and OVCAR-3 cancer cells, whereas two constructs, including NOEY2(wt), inhibited the colony formation (Fig. 2A,
Flow cytometric detection was then performed using labelled annexin V (Fig. 2B). The cells transfected with NOEY2(ΔN-terminal) showed no change in the early-stage or late-stage apoptotic fraction, as in the control. These observations suggest that cell growth suppression by NOEY2 results from increased apoptotic cell death and the N-terminus of NOEY2 (NOEY2-N) is essential to induce apoptosis. We next assessed the effects of NOEY2-N on cell viability by using the CellTiter-Glo assay kit as described in the methodology. As seen in Fig. 2C, the viability of the ovarian cancer cells was gradually diminished by treatment with NOEY2-N in a concentration-dependent manner. Subsequently, we examined whether this effect of NOEY2-N is related to the activation of caspase-3. NOEY2-N induced caspase-3 activity as much as NOEY2(wt) and significantly more than the control (Fig. 2D).
Since cleaved caspase-3 can induce the degradation of pro-PARP, we investigated whether the activation of caspase-3 induced by NOEY2(wt) or NOEY2-N ultimately induces the cleavage of pro-PARP. As shown in Fig. 2E, NOEY2-N induced the degradation of Pro-PARP as much as NOEY2(wt) (Fig. 2E).
Collectively, these results indicate that the N-terminal domain consisting of 34 amino acids might contribute to the apoptotic and anti-proliferative effects of NOEY2 in ovarian cancer cells.
NOEY2 inhibits tumour angiogenesis. We investigated whether NOEY2-N, which mimics the apoptotic effects of NOEY2, can also mimic the anti-angiogenic effect of NOEY2 on tumours. We first assessed the intracellular binding of NOEY2-N to VEGFR-1 and VEGFR-2. As presented in Fig. 3A, β-galactosidase was fully activated (92.19 ± 0.91) upon the interaction of NOEY2-N with VEGFR-2 but not with the empty vector (vector only: 1.92 ± 0.69) or VEGFR-1 (2.01 ± 0.72) (Fig. 3A). Therefore, in subsequent experiments, VEGFR-1 was used as a negative control. We next employed co-immunoprecipitation to confirm the direct interaction between NOEY2-N and VEGFR-2. DNA constructs expressing
Collectively, we found that administration of NOEY2-N reduced the levels of VEGF, HIF-1α, and phosphorylation of VEGFR-2 (Tyr-1175) in ovarian cancer.
Homology-modelled structures for the main protein body and N-terminal domain of the human NOEY2 were firstly constructed using MODELER with four available template structures--three for the main protein body and one for the N-terminal domain (Fig. 4A-4C). Initially, the two separately constructed structures were manually connected for the next step (Fig. 4D). To generate many different starting conformers, the homology-modelled and manually connected structure was equilibrated for 50 ps at an abnormally high temperature (350K). The five selected conformers are shown with a starting structure (green coloured in Fig. 4C), and from them, the final five conformations were obtained. We compared the five structures with each other and found three structures with a relatively similar shape. One of these three structures was finally chosen as the native conformer. We selected the structure whose entire N-terminus interacted with the main protein-body regions (Fig. 4D). This final structure is the very first three-dimensional structure of the human NOEY2 protein.
To find or predict the key residues in the N-terminal domain, the selected final structure (Fig. 4D) was carefully investigated. Two different views of the final native structure (Fig. 4E, 4F) provide the relative position between the N-terminal domain and the main body. By checking the position of each residue in the N-terminal domain, residues 15 and 16 can easily be seen in the hinge region of the N-terminal domain. Therefore, Lys15 and Arg16 can be regarded as potential key residues in the N-terminal domain. Lys15 interacts with the side chains of the two main body residues through the backbone oxygen atom. The distances from the oxygen atom of Lys15, to NE2 of Gln189 and to CZ2 of Trp57 are 3.14 Å and 3.27 Å, respectively (Fig. 4G). Arg16 interacts with the main body in a very different way than Lys15 does. Arg16 uses the side-chain atom, whereas Lys15 uses the backbone atom for the interaction. Additionally, Arg16 interacts with the backbone atoms from the main-body residues, whereas Lys15 interacts with the side-chain atoms from the main-body residues (Fig. 4H). For Arg16, the distance between the NH2 and oxygen of Gly79 is 2.80 Å, and the distance between NE and oxygen of His82 is also 2.80 Å (Fig. 4H).
The mutants 15KR16 of NOEY2-N by Ala, Asn, Asp, and Ser scanning mutagenesis were first determined by testing their effects on the proliferation and death of endothelial cells. In general, VEGF enhanced the DNA synthesis of the un-transfected cells, and the cells transfected with the empty vector were comparable to non-induced cells (Plate
As seen in Fig. 6A, the un-transfected or control cells incubated with VEGF formed a capillary-like tubule structure on Matrigel. In contrast, the K15A, K15A, K15D, K15N, K15S, R16D, R16N, and R16S mutants, including NOEY2-N (positive control), completely disrupted the VEGF-induced capillary-like tubular structure, but the R16A mutant did not (Fig. 6B). Finally, we examined the effects of the NOEY2 mutants on the expression of VEGF and HIF-1α by immunoblotting. As expected, the N-termini of the NOEY2 mutants, including K15A, K15D, K15N, K15S, R16D, R16N, and R16S, dramatically suppressed the expression of both VEGF and HIF-1α, whereas R16A did not (Fig. 6C).
Taken together, these results strongly suggest that the R16 residue within the N-terminal region is a key residue involved in the apoptotic and anti-angiogenic activities of NOEY2.
Currently, ovarian cancer remains the most fatal of the gynaecological malignant tumours in women worldwide, and its incidence increases every year (Sung
NOEY2 enhances autophagy by suppressing the Akt/mTOR-signalling pathway or by directly participating in the autophagy initiation complex through regulation of the nuclear localisation of the autophagy-associated transcription factor FOXO3 (Lu
In xenograft mouse models, tumour growth and angiogenesis are associated with HIF-1α expression. In addition, HIF-1α protein synthesis is controlled by promoting the PI3K/Akt and extracellular signal-regulated kinase (ERK)/MAPK-signalling pathways and is involved in tumour growth (Majmundar
The over-expression of the tumour suppressor p53 under hypoxia may be linked to the HIF-1α-dependent pathway that initiates apoptosis. Furthermore, p53-independent pathways may provoke apoptotic cell death via the Bcl-2 pathway (Meadows
Finally, we used western blotting analysis to assess the expression of PI3K, a signalling factor downstream of VEGFR-2 in NOEY2-treated tumours. Treatment with NOEY2 noticeably decreased the phosphorylation, but not total levels, of PDK1, Akt, mTOR, TSC-2, p70S6K, and GSK-3β (Fig. 1H). These results strongly indicate that NOEY2 induces changes in the VEGFR-2 related PI3K/Akt signalling pathway.
Expression of VEGF has been proposed to be associated with the prognosis in ovarian cancer (Horikawa
Through homology modelling of NOEY2, we predicted that K15 and R16, which reside in the hinge region of the N-terminal domain, are important for the intramolecular interaction with the main domain (Fig. 4). The importance of these two residues for the apoptosis and anti-angiogenic effects of NOEY2-N was evaluated through site-directed mutagenesis (Fig. 5, 6). We found that the R16 residue of NOEY2-N is important for the apoptotic and anti-angiogenic effects.
It has been reported that NOEY2-N can inhibit the RAS/MAPK signalling by directly binding to K-ras and H-ras (Sutton
In summary, we propose that NOEY2 may overcome the deficiencies of the existing therapies that target VEGF, HIF-1α, p53, or the associated signalling pathways. With over-expression of NOEY2-N and its binding with VEGFR-2, it was observed that VEGF-induced endothelial cell was significantly diminished via inhibition of the VEGFR-2/mTOR/HIF-1α signalling pathway. NOEY2-N can have a greater inhibitory effect on tumour cells and tumour vessels with over-activated VEGF and HIF-1α signalling pathways. In particular, the R16 residue within the N-terminal region of NOEY2 is involved in the activation of apoptosis. The interaction of NOEY2-N with VEGFR-2 offers a useful mechanism for inhibiting oncogenesis. Further research on NOEY2-N and its tumour-suppressing characteristics may lead to effective anticancer therapies in the future.
This study was partially supported by a grant from the National Cancer Center (NCC-0810410-3), BK21 FOUR program, the Basic Science Research Program, through the National Research Foundation (NRF) of Korea (NRF-2020R1A2C3004973, NRF-2018R1A5A2023127, NRF-2020M3E5E2038356), and Global PhD. Fellowship through the NRF of Korea (NRF-2018H1A2A1061990).
The authors have declared that no competing interest exists.