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Yao W.-T.,Fudan University | Wu J.-F.,Fudan University | Yu G.-Y.,Fudan University | Wang R.,East China Normal University | And 18 more authors.
Cell Death and Disease | Year: 2014

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. 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 CullinRING 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. © 2014 Macmillan Publishers Limited.


Chen P.,Fudan University | Chen P.,Zhengzhou University | Hu T.,Zhengzhou University | Liang Y.,Fudan University | And 16 more authors.
Oncotarget | Year: 2015

Liver cancer is the second-most frequent cause of cancer death in the world and is highly treatment resistant. We reported previously that inhibition of neddylation pathway with specific NAE inhibitor MLN4924, suppressed the malignant phenotypes of liver cancer. However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance. Here, we report that blockage of autophagy with clinically-available autophagy inhibitors (e.g. chloroquine) significantly enhanced the efficacy of MLN4924 on liver cancer cells by triggering apoptosis. Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins. Importantly, the down-regulation of NOXA expression via siRNA silencing substantially attenuated apoptosis of liver cancer cells. Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation. The elimination of DNA damage or blockage of ROS production significantly reduced the expression of NOXA, and thereby attenuated apoptosis and reduced growth inhibition of liver cancer cells. Moreover, blockage of autophagy enhanced the efficacy of MLN4924 in an orthotopic model of human liver cancer, with induction of NOXA and apoptosis in tumor tissues. These findings provide important preclinical evidence for clinical investigation of synergistic inhibition of neddylation and autophagy in liver cancer.


Li L.,Fudan University | Li L.,Shanghai Medical College | Wang M.,Shanghai JiaoTong University | Yu G.,Fudan University | And 33 more authors.
Journal of the National Cancer Institute | Year: 2014

Background A number of oncoproteins and tumor suppressors are known to be neddylated, but whether the neddylation pathway is entirely activated in human cancer remains unexplored. Methods NEDD8-activating enzyme (NAE) (E1) and NEDD8-conjugating enzyme (E2) expression and global-protein neddylation were examined by immunohistochemistry, immunoblotting, and real-time polymerase chain reaction analysis. Cell proliferation, clonogenic survival, migration, and motility in vitro, as well as tumor formation and metastasis in vivo, were determined upon neddylation inhibition by MLN4924, an investigational NEDD8-activating enzyme inhibitor. Survival was analyzed with Kaplan-Meier methods and compared by the log-rank test. All statistical tests were two-sided. Results The entire neddylation pathway, including NEDD8-activating enzyme E1, NEDD8-conjugating enzyme E2, and global-protein neddylation, is overactivated in both lung adenocarcinoma and squamous-cell carcinoma. Compared with lung adenocarcinoma patients with low expression, those with high expression had worse overall survival (NEDD8-activating enzyme E1 subunit 1 [NAE1]: hazard ratio [HR] = 2.07, 95% confidence interval [CI] = 0.95 to 4.52, P =. 07; ubiquitin-conjugating enzyme E2M (UBC12): HR = 13.26, 95% CI = 1.77 to 99.35, P =. 01; global protein neddylation: HR = 3.74, 95% CI = 1.65 to 8.47, P =. 002). Moreover, inhibition of neddylation by the NAE inhibitor MLN4924 statistically significantly suppressed proliferation, survival, migration, and motility of lung cancer cells in vitro and tumor formation and metastasis in vivo. At the molecular level, MLN4924 inactivated Cullin-RING E3 ligases, led to accumulation of tumor-suppressive Cullin-RING E3 ligase substrates and induced phorbol-12-myristate-13-acetate-induced protein 1 (NOXA)-dependent apoptosis or cellular senescence. Conclusions Our study highlights the overactivated neddylation pathway in lung cancer development and as a promising therapeutic target. © 2014 The Author.


Yang M.,Anticancer, Inc. | Yang M.,AntiCancer Biotech Beijing Co. | Jiang P.,AntiCancer Biotech Beijing Co. | Hoffman R.M.,Anticancer, Inc. | Hoffman R.M.,University of California at San Diego
Anticancer Research | Year: 2015

Background/Aim: We previously developed PC-3 human prostate cancer cells expressing red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) linked to histone H2B expressed in the nucleus. We demonstrate in the present report the use of these dual-color cells for early detection of apoptosis in the presence of cancer chemotherapy agents. Materials and Methods: Induction of apoptosis was observed by real-time imaging of cytoplasmic and nuclear size and shape changes and nuclear fragmentation using fluorescence microscopy. Apoptosis was also detected by measuring DNA fragmentation. The cancer chemotherapy agents paclitaxel and vinblastine were used for induction of apoptosis. Results: When the PC-3 dual-color cells were treated with paclitaxel or vinblastine, cytoplasmic and nuclear size and shape changes and nuclear fragmentation were observed by 24 hours. The paclitaxel-treated PC-3 dual-color cells exhibited ring-like structures formed by the fragmented nuclei, which could be brightly visualized by H2B-GFP fluorescence. Apoptosis was also detected by the dual-color PC-3 cells by 24 hours when treated with vinblastine. However, no nuclear ring-like structures were formed in the PC-3 cells by vinblastine treatment. In contrast, DNA fragmentation could not be observed in PC-3 cells until 48 hours after exposure to paclitaxel. Conclusion: Dual-color PC-3 cells can serve as a simple real-time early reporter of apoptosis and as a screen for novel cancer therapeutics or genotoxic agents. The dual-color cell real-time imaging assay is a more sensitive and earlier reporter for apoptosis than the DNA fragmentation assay.


PubMed | Zhengzhou University, Anticancer, Inc., P.A. College, P-Com and 2 more.
Type: Comparative Study | Journal: Anticancer research | Year: 2015

Esophageal squamous cell carcinoma (ESCC) is a recalcitrant cancer. Mouse models of this disease could be used for discovery of more effective therapy for ESCC.The green fluorescent protein (GFP)-expressing human esophageal cancer EC1 cell line was established with a lentiviral expression system. Subsequently, nude mice were injected subcutaneously, intracardiac or intravenously, or orthotopically implanted with EC1-GFP cells. Tumor growth and metastasis were examined by fluorescence in vivo imaging or by open fluorescence imaging after autopsy.Four different mouse xenograft models of ESCC expressing GFP were established. In the subcutaneous model, primary tumor growth was monitored in real-time by whole-body fluorescence imaging. No metastasis was observed in the subcutaneous or surgical orthotopic implantation model. By 55 days after implantation, all mice had developed orthotopic esophageal cancer, but without detectable metastasis. In contrast, experimental metastasis occurred in the intracardiac and intravenous models. In the intravenous injection model, the lung was the sole organ of experimental metastasis. In the intracardiac model, extensive experimental metastases occurred in the bone, brain and lung.The mouse xenograft models of ESCC developed in the present study can provide a means of discovering more effective therapy of this recalcitrant type of cancer.


Hua W.,Fudan University | Li C.,Fudan University | Yang Z.,Fudan University | Li L.,Fudan University | And 10 more authors.
Neuro-Oncology | Year: 2015

Background The neddylation pathway has been recently identified as an attractive anticancer target, and MLN4924, a specific NEDD8-activating enzyme inhibitor, has been developed as a first-in-class anticancer agent. However, neither the status of the neddylation pathway in glioblastoma (GBM) nor the effect of MLN4924 against GBM has been systematically investigated yet. Methods To measure the activation state of the neddylation pathway in GBM, expression of the NEDD8-activating enzyme (E1), NEDD8-conjugating enzyme (E2), and global protein neddylation in GBM tumor tissues versus adjacent tissues were examined by immunoblotting analysis and immunohistochemistry staining. To assess the therapeutic efficacy of neddylation inhibition in GBM, cell proliferation in vitro and tumor growth in vivo were determined upon neddylation inhibition by MLN4924, an investigational NEDD8-activating enzyme inhibitor. Results The neddylation pathway was overactivated in a majority of GBM tumor tissues when compared with adjacent normal tissues. The upregulation of this pathway in GBM tissues was positively correlated with high-grade disease and postoperative recurrence but was negatively associated with patient overall survival. MLN4924 treatment inhibited cullin neddylation, inactivated cullin-RING E3 ligase, and led to the accumulation of tumor-suppressive cullin-RING E3 ligase substrates to trigger cell-cycle arrest and senescence or apoptosis in a cell-line dependent manner. Moreover, inhibition of neddylation by MLN4924 significantly suppressed tumor growth in an orthotopic xenograft model of human GBM. Conclusion Our study indicates that an overactivated neddylation pathway may be involved in GBM progression and that inhibition of this oncogenic pathway is a potentially new therapeutic approach for GBM. © 2015 The Author(s).


PubMed | Fudan University, University of California at San Diego, Zhengzhou University, AntiCancer Biotech Beijing Co. and Seoul National University
Type: Journal Article | Journal: Oncotarget | Year: 2015

Liver cancer is the second-most frequent cause of cancer death in the world and is highly treatment resistant. We reported previously that inhibition of neddylation pathway with specific NAE inhibitor MLN4924, suppressed the malignant phenotypes of liver cancer. However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance. Here, we report that blockage of autophagy with clinically-available autophagy inhibitors (e.g. chloroquine) significantly enhanced the efficacy of MLN4924 on liver cancer cells by triggering apoptosis. Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins. Importantly, the down-regulation of NOXA expression via siRNA silencing substantially attenuated apoptosis of liver cancer cells. Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation. The elimination of DNA damage or blockage of ROS production significantly reduced the expression of NOXA, and thereby attenuated apoptosis and reduced growth inhibition of liver cancer cells. Moreover, blockage of autophagy enhanced the efficacy of MLN4924 in an orthotopic model of human liver cancer, with induction of NOXA and apoptosis in tumor tissues. These findings provide important preclinical evidence for clinical investigation of synergistic inhibition of neddylation and autophagy in liver cancer.


PubMed | Seoul National University, Zhengzhou University, Linzhou Cancer Hospital, AntiCancer Biotech Beijing Co. and 2 more.
Type: Journal Article | Journal: Clinical cancer research : an official journal of the American Association for Cancer Research | Year: 2016

Targeting the protein neddylation pathway has become an attractive anticancer strategy; however, the role of death receptor-mediated extrinsic apoptosis during treatment remained to be determined.The activation of extrinsic apoptosis and its role in MLN4924 treatment of human esophageal squamous cell carcinoma (ESCC) were evaluated both in vitro and in vivo The expression of the components of extrinsic apoptotic pathway was determined by immunoblotting analysis and downregulated by siRNA silencing for mechanistic studies.Pharmaceutical or genetic inactivation of neddylation pathway induced death receptor 5 (DR5)-mediated apoptosis and led to the suppression of ESCC in murine models. Mechanistically, neddylation inhibition stabilized activating transcription factor 4 (ATF4), a Cullin-Ring E3 ubiquitin ligases (CRL) substrate. Transcription factor CHOP was subsequently transactivated by ATF4 and further induced the expression of DR5 to activate caspase-8 and induce extrinsic apoptosis. Moreover, the entire neddylation pathway was hyperactivated in ESCC and was negatively associated with patient overall survival.Our findings highlight a critical role of ATF4-CHOP-DR5 axis-mediated extrinsic apoptosis in neddylation-targeted cancer therapy and support the clinical investigation of neddylation inhibitors (e.g., MLN4924) for the treatment of ESCC, a currently treatment-resistant disease with neddylation hyperactivation. Clin Cancer Res; 22(16); 4145-57. 2016 AACR.


PubMed | University of Houston, Seoul National University, East China Normal University, AntiCancer Biotech Beijing Co. and 3 more.
Type: | Journal: Cell death & disease | Year: 2014

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. 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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