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. 2014 Feb 13;5(2):e1059.
doi: 10.1038/cddis.2014.21.

Suppression of tumor angiogenesis by targeting the protein neddylation pathway

W-T Yao  1 , J-F Wu  2 , G-Y Yu  2 , R Wang  3 , K Wang  4 , L-H Li  5 , P Chen  5 , Y-N Jiang  2 , H Cheng  6 , H W Lee  7 , J Yu  7 , H Qi  8 , X-J Yu  6 , P Wang  3 , Y-W Chu  9 , M Yang  8 , Z-C Hua  4 , H-Q Ying  10 , R M Hoffman  11 , L S Jeong  7 , L-J Jia  2
Affiliations

V体育平台登录 - Suppression of tumor angiogenesis by targeting the protein neddylation pathway

W-T Yao (VSports手机版) et al. Cell Death Dis. .

Abstract

Inhibition of protein neddylation, particularly cullin neddylation, has emerged as a promising anticancer strategy, as evidenced by the antitumor activity in preclinical studies of the Nedd8-activating enzyme (NAE) inhibitor MLN4924. This small molecule can block the protein neddylation pathway and is now in clinical trials VSports手机版. We and others have previously shown that the antitumor activity of MLN4924 is mediated by its ability to induce apoptosis, autophagy and senescence in a cell context-dependent manner. However, whether MLN4924 has any effect on tumor angiogenesis remains unexplored. Here we report that MLN4924 inhibits angiogenesis in various in vitro and in vivo models, leading to the suppression of tumor growth and metastasis in highly malignant pancreatic cancer, indicating that blockage of angiogenesis is yet another mechanism contributing to its antitumor activity. At the molecular level, MLN4924 inhibits Cullin-RING E3 ligases (CRLs) by cullin deneddylation, causing accumulation of RhoA at an early stage to impair angiogenic activity of vascular endothelial cells and subsequently DNA damage response, cell cycle arrest and apoptosis due to accumulation of other tumor-suppressive substrates of CRLs. Furthermore, we showed that inactivation of CRLs, via small interfering RNA (siRNA) silencing of its essential subunit ROC1/RBX1, recapitulates the antiangiogenic effect of MLN4924. Taken together, our study demonstrates a previously unrecognized role of neddylation in the regulation of tumor angiogenesis using both pharmaceutical and genetic approaches, and provides proof of concept evidence for future development of neddylation inhibitors (such as MLN4924) as a novel class of antiangiogenic agents. .

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Figures

Figure 1
Figure 1
MLN4924 inhibits angiogenesis in multiple angiogenic assays. (a) Rat aortic ring angiogenesis assay. The aortic rings from 6-week-old male Sprague–Dawley rats were randomly seeded into Matrigel-coated wells and sealed with an overlay of Matrigel. VEGF in serum-free ECGM, with or without MLN4924, was added into the wells. After 6 days, microvessel sprouting was fixed and photographed using an inverted microscope (Olympus; magnification × 100) and vascular areas of each treatment were calculated (n=6). (b) Chick embryo chorioallantoic membrane (CAM) assay. The top air sac portions of fertilized chicken eggs were opened and sterilized filter disks with various concentrations of MLN4924 were placed on regions of the CAM relatively deficient in preexisting blood vessels. At the end of analysis, CAMs were photographed with a digital camera and angiogenesis inhibition was assessed by the number of visible blood vessel branch points in each group and expressed as a percentage of negative control (n=6). (c) In vivo Matrigel plug assay. Matrigel containing indicated amounts of MLN4924, VEGF and heparin were subcutaneously injected into the ventral area of 6-week-old C57BL/6 mice. After 6 days, Matrigel plugs were collected and photographed (c, upper panel). Plug sections were immunostained with a specific anti-CD31 antibody for microvessel density assay (c, bottom panel) (n=6). Data were presented as mean±S.E.M. *P<0.05, **P<0.01 and ***P<0.001
Figure 2
Figure 2
MLN4924 suppresses tumor angiogenesis and progression in a mouse footpad model of human pancreatic cancer. Human MiaPaCa-2-RFP pancreatic cancer cells were inoculated into the footpads of GFP transgenic nude mice, treated with 60 mg/kg MLN4924 or 10% HPBCD as negative control subcutaneously twice a day, and subjected to angiogenesis and tumor growth assays over time. At 10 days post treatment, the status of tumor angiogenesis of treated mice was determined by noninvasive real-time optical imaging (white arrows show blood vessels) (a) and the tumor volume was measured (b). At the end of treatment, the tumors were collected, photographed and weighed (left, bright field; right, fluorescent imaging) (c) and tumor tissue sections were immunostained with a specific anti-CD31 antibody for microvessel density assay (d). Data are presented as mean±S.E.M. **P<0.01 and ***P<0.001
Figure 3
Figure 3
MLN4924 suppresses tumor angiogenesis, progression and metastasis in an orthotopic pancreatic cancer model. To establish the orthotopic pancreatic cancer model, small pieces of human MiaPaCa-2-RFP pancreatic cancer tissue that originated from subcutaneous tumors of nude mice were inserted into the pancreases of nude mice, as described in the Materials and Methods. The tumor-bearing mice were then treated with MLN4924 or 10% HPBCD as a negative control. Tumor growth, metastasis and angiogenesis were determined (n=10 for each group). (a) Real-time optical imaging of the progression of MiaPaCa-2-RFP orthotopic pancreatic cancer (left panel) and tumor growth curves derived from the intensity of tumor fluorescence signals by ModFit LT software (right panel). (b) At the end of treatment, the primary pancreatic tumors were harvested, photographed under fluorescence (left panel) and weighed (right panel). (c) Lymph node metastases were harvested, photographed (left panel) and weighed (right panel). (d) CD31 staining of primary tumor sections for microvessel density analysis. Data are presented as mean±S.E.M. *P<0.05 and ***P<0.001
Figure 4
Figure 4
MLN4924 inhibits protein neddylation and multiple angiogenic phenotypes of HUVECs. HUVECs were treated with MLN4924 for 12 h and subjected to immunoblotting analysis of indicated proteins using GAPDH as a loading control. (a) The expression of neddylation enzymes in HUVECs. (b) The inhibition of global protein neddylation and cullin neddylation by MLN4924 in HUVECs. (c) Capillary tube formation of HUVECs was inhibited by MLN4924. (d) MLN4924 inhibited the transwell migration of HUVECs. (e) MLN4924 inhibited HUVEC motility, recorded using time-lapse microscopy. Data are presented as mean±S.E.M. *P<0.05, **P<0.01 and ***P<0.001
Figure 5
Figure 5
Short-term treatment with MLN4924 induces RhoA accumulation which inhibits angiogenic activity of HUVECs. (a) Effect of short-term MLN4924 treatment on cell viability. HUVECs seeded in 96-well plates were cultured overnight and treated with MLN4924 at indicated concentrations for 12 h, followed by the ATPlite cell viability assay. (b) MLN4924 treatment induced accumulation of RhoA. HUVECs were treated with MLN4924 for 12 h and subjected to immunoblotting analysis of indicated proteins. (c) Downregulation of RhoA expression via siRNA silencing (siRhoA). (d and e) Knockdown of RhoA rescued the inhibitory effects of MLN4924 on capillary tube formation (d) and transwell migration (e) of HUVECs. HUVECs transfected with siRhoA or siControl were treated with MLN4924 and subjected to the capillary tube formation assay and transwell migration assay
Figure 6
Figure 6
Long-term inactivation of neddylation with MLN4924 induces cell cycle arrest, reduces cell viability and increases apoptosis in HUVECs. (a) The accumulation of cell cycle regulators and DNA replication licensing proteins upon MLN4924 treatment for 48 h. (b) HUVECs were treated with MLN4924 for 48 h and subjected to PI staining and FACS analysis. The percentage of cells in G2 phase and sub-G1 phase was indicated. (c) HUVECs were treated with MLN4924 for 48 h and subjected to IB analysis using antibodies against cleaved PARP and cleaved Caspase 3 with GAPDH as a loading control. (d) HUVECs seeded in 96-well plates were cultured overnight and treated with MLN4924 at different concentrations for 48 h, followed by the ATPlite cell viability assay. (e) HUVECs were treated with MLN4924 for 48 h and subjected to IB analysis using antibodies against apoptosis-regulatory proteins with GAPDH as a loading control. (f and g) Knockdown of NOXA attenuated apoptotic activation (f) and proliferation inhibition (g) upon MLN4924 treatment. HUVECs transfected with siNOXA or siControl were treated with MLN4924 for 48 h and subjected to IB analysis for indicated proteins and the ATPlite cell viability assay. Data are presented as mean±S.E.M. **P<0.01 and ***P<0.001
Figure 7
Figure 7
CRL inactivation via ROC1/RBX1 siRNA silencing inhibits angiogenic activity of HUVECs. HUVECs were transfected with siROC1 or siControl and subjected to the capillary tube formation assay (a), transwell migration assay (b), PI staining and FACS analysis (c), PARP activation detection (d), ATPlite cell viability assay (e) and IB analysis of indicated proteins (f). Data was presented as mean±S.E.M. **P<0.01 and ***P<0.001
Figure 8
Figure 8
Working model. Inhibition of neddylation pathway with MLN4924 suppresses angiogenesis by inducing cullin deneddylation, CRL inactivation and accumulation of CRL substrates that impairs migration, proliferation and survival of vascular endothelial cells. Genetic inactivation of CRL via ROC1/RBX1 siRNA silencing recapitulates the antiangiogenic effect of MLN4924. N8, Nedd8; SR, substrate receptor; DDR, DNA damage response
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References

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