Disruption of protein neddylation with MLN4924 attenuates paclitaxel-induced apoptosis and microtubule polymerization in ovarian cancer cells

Surgery and chemotherapy are the gold-standard treatments for ovarian cancer. The major cause of treatment failure in patients with ovarian cancer is tumoral heterogeneity and drug resistance. Paclitaxel (PTX) is one of the most commonly used first-line drugs for ovarian cancer chemotherapy. Unfortunately, the mechanisms of PTX chemoresistance remain unclear. Here, we examined the effects of post-translational neddylation on the sensitivity of ovarian cancer cells (OCCs) to PTX-induced apoptosis. Disruption of protein neddylation with the first-in-class inhibitor MLN4924 dramatically neutralized PTX-mediated antiproliferative, antimigration, and apoptotic effects in human OCCs. Moreover, MLN4924 treatment interrupted PTX-induced microtubule polymerization. Importantly, two neddylation conjugating E2 enzymes, UBE2M and UBE2F, were found to play essential roles in PTX-induced cytotoxicity and tubulin polymerization in OCCs. In summary, our findings demonstrated that disruption of protein neddylation by MLN4924 conferred resistance to PTX and provided insights into the potential mechanisms of PTX chemoresistance in ovarian cancer.     

Inactivation of the Cullin (CUL)-RING E3 ligase by the NEDD8-activating enzyme inhibitor MLN4924 triggers protective autophagy in cancer cells

The multiunit Cullin (CUL)-RING E3 ligase (CRL) controls diverse biological processes by targeting a mass of substrates for ubiquitination and degradation, whereas its dysfunction causes carcinogenesis. Post-translational neddylation of CUL, a process triggered by the NEDD8-activating enzyme E1 subunit 1 (NAE1), is required for CRL activation. Recently, MLN4924 was discovered via a high-throughput screen as a specific NAE1 inhibitor and first-in-class anticancer drug. By blocking CUL neddylation, MLN4924 inactivates CRL and causes the accumulation of CRL substrates that trigger cell cycle arrest, senescence and/or apoptosis to suppress the growth of cancer cells in vitro and in vivo. Recently, we found that MLN4924 also triggers protective autophagy in response to CRL inactivation. MLN4924-induced autophagy is attributed partially to the inhibition of mechanistic target of rapamycin (also known as mammalian target of rapamycin, MTOR) activity by the accumulation of the MTOR inhibitory protein DEPTOR, as well as reactive oxygen species (ROS)-induced stress. Moreover, the blockage of autophagy response enhances apoptosis in MLN4924-treated cells. Together, our findings not only reveal autophagy as a novel cellular response to CRL inactivation by MLN4924, but also provide a piece of proof-of-concept evidence for the combination of MLN4924 with autophagy inhibitors to enhance therapeutic efficacy.     

Inactivation of the Cullin (CUL)-RING E3 ligase by the NEDD8-activating enzyme inhibitor MLN4924 triggers protective autophagy in cancer cells

The multiunit Cullin (CUL)-RING E3 ligase (CRL) controls diverse biological processes by targeting a mass of substrates for ubiquitination and degradation, whereas its dysfunction causes carcinogenesis. Post-translational neddylation of CUL, a process triggered by the NEDD8-activating enzyme E1 subunit 1 (NAE1), is required for CRL activation. Recently, MLN4924 was discovered via a high-throughput screen as a specific NAE1 inhibitor and first-in-class anticancer drug. By blocking CUL neddylation, MLN4924 inactivates CRL and causes the accumulation of CRL substrates that trigger cell cycle arrest, senescence and/or apoptosis to suppress the growth of cancer cells in vitro and in vivo. Recently, we found that MLN4924 also triggers protective autophagy in response to CRL inactivation. MLN4924-induced autophagy is attributed partially to the inhibition of mechanistic target of rapamycin (also known as mammalian target of rapamycin, MTOR) activity by the accumulation of the MTOR inhibitory protein DEPTOR, as well as reactive oxygen species (ROS)-induced stress. Moreover, the blockage of autophagy response enhances apoptosis in MLN4924-treated cells. Together, our findings not only reveal autophagy as a novel cellular response to CRL inactivation by MLN4924, but also provide a piece of proof-of-concept evidence for the combination of MLN4924 with autophagy inhibitors to enhance therapeutic efficacy.     

The p21-dependent radiosensitization of human breast cancer cells by MLN4924, an investigational inhibitor of NEDD8 activating enzyme

Radiotherapy is a treatment choice for local control of breast cancer. However, intrinsic radioresistance of cancer cells limits therapeutic efficacy. We have recently validated that SCF (SKP1, Cullins, and F-box protein) E3 ubiquitin ligase is an attractive radiosensitizing target. Here we tested our hypothesis that MLN4924, a newly discovered investigational small molecule inhibitor of NAE (NEDD8 Activating Enzyme) that inactivates SCF E3 ligase, could act as a novel radiosensitizing agent in breast cancer cells. Indeed, we found that MLN4924 effectively inhibited cullin neddylation, and sensitized breast cancer cells to radiation with a sensitivity enhancement ratio (SER) of 1.75 for SK-BR-3 cells and 1.32 for MCF7 cells, respectively. Mechanistically, MLN4924 significantly enhanced radiation-induced G2/M arrest in SK-BR-3 cells, but not in MCF7 cells at early time point, and enhanced radiation-induced apoptosis in both lines at later time point. However, blockage of apoptosis by Z-VAD failed to abrogate MLN4924 radiosensitization, suggesting that apoptosis was not causally related. We further showed that MLN4924 failed to enhance radiation-induced DNA damage response, but did cause minor delay in DNA damage repair. Among a number of tested SCF E3 substrates known to regulate growth arrest, apoptosis and DNA damage response, p21 was the only one showing an enhanced accumulation in MLN4924-radiation combination group, as compared to the single treatment groups. Importantly, p21 knockdown via siRNA partialy inhibited MLN4924-induced G2/M arrest and radiosensitization, indicating a causal role played by p21. Our study suggested that MLN4924 could be further developed as a novel class of radiosensitizer for the treatment of breast cancer.     

Chk1 inhibitor SCH 900776 enhances the antitumor activity of MLN4924 on pancreatic cancer

MLN4924 inhibits the cullin-RING ligases mediated ubiquitin-proteasome system, and has showed antitumor activities in preclinical studies, but its effects and mechanisms on pancreatic cancer (PC) remains elusive. We found that MLN4924 inhibited the proliferation and clonogenicity of PC cells, caused DNA damage, particularly double-strand breaks, and leaded to Chk1 activation and cell-cycle arrest. Chk1 inhibitor SCH 900776 alone exhibited minimal cytotoxicity, and caused no DNA damage on PC cells. But in the combination therapy, SCH 900776 enhanced the cytotoxicity and DNA damage caused by MLN4924, likely by abrogating G2/M arrest and promoting DNA re-replication. In vivo study on a xenograft PC mouse model also showed that SCH 900776 increased the efficacy of MLN4924. We also evaluated the level of NEDD8-activating enzyme (NAE), the direct target of MLN4924, and found that NAE level was elevated in PC tissues compared with normal pancreas, but was irrelevant with prognosis. Our findings provide the preclinical evidence and the rationale of the combination therapy of MLN4924 with SCH 900776 or other Chk1 inhibitors to treat PC.     

MLN4924 is an efficient inhibitor of NEDD8 conjugation in plants

The conjugation of the ubiquitin-like modifier NEURAL PRECURSOR CELL-EXPRESSED DEVELOPMENTALLY DOWN-REGULATED PROTEIN8/RELATED TO UBIQUITIN1 (NEDD8/RUB1; neddylation) is best known as an important posttranslational modification of the cullin subunits of cullin-RING-type E3 ubiquitin ligases (CRLs). MLN4924 has recently been described as an inhibitor of NEDD8-ACTIVATING ENZYME1 (NAE1) in human. Here, we show that MLN4924 is also an effective and specific inhibitor of NAE1 enzymes from Arabidopsis (Arabidopsis thaliana) and other plant species. We found that MLN4924-treated wild-type seedlings have phenotypes that are highly similar to phenotypes of mutants with a partial defect in neddylation and that such neddylation-defective mutants are hypersensitive to MLN4924 treatment. We further found that MLN4924 efficiently blocks the neddylation of cullins in Arabidopsis and that MLN4924 thereby interferes with the degradation of CRL substrates and their downstream responses. MLN4924 treatments also induce characteristic phenotypes in tomato (Solanum lycopersicum), Cardamine hirsuta, and Brachypodium distachyon. Interestingly, MLN4924 also blocks the neddylation of a number of other NEDD8-modified proteins. In summary, we show that MLN4924 is a versatile and specific neddylation inhibitor that will be a useful tool to examine the role of NEDD8- and CRL-dependent processes in a wide range of plant species.     

The novel role and function of LINC01235 in metastasis of gastric cancer cells by inducing epithelial-mesenchymal transition

LncRNAs play a vital role in the tumorigenesis of gastric cancer (GC). This study determined that LINC01235 expression has greater fold changes by analyzing TCGA RNA-Seq data. The qRT-PCR assay confirmed that LINC01235 is significantly over-expressed in GC cells and tissues. Additionally, the overall survival analysis showed that patients with a higher LINC01235 expression had a poorer prognosis than those with a lower LINC01235 expression. Univariate Cox regression analysis indicated that high LINC01235 expression is positively correlated with poor prognosis. Moreover, LINC01235 was an independent poor prognostic marker for GC in multivariate Cox analysis. In vitro assays suggested that LINC01235 knockdown suppresses GC cell migration and invasion. GSEA revealed that high LINC01235 expression is strongly enriched in the EMT pathway. Western blotting results revealed that LINC01235 silencing decreases the expression of EMT-induced proteins. In conclusion, LINC01235 can promote GC cell metastasis via EMT and function as a prognostic biomarker.     

Effect of UV-irradiation, spleen cells and MLN cells on protective immunity against Angiostrongylus cantonensis infection in mice

The UV-irradiated larvae of Angiostrongylus cantonensis were used for inducing immunity in mice. A single oral dose of 100 infective UV-irradiated larvae exposed for 5 min or 15 min induced 91% and 97% protection against subsequent infection. Adoptive protection against A. cantonensis could also be transferred by spleen and mesenteric lymph node cells obtained from vaccinated mice.     

miR-148a promoted cell proliferation by targeting p27 in gastric cancer cells

Accumulating evidence has shown that miRNAs are aberrantly expressed in human gastric cancer and crucial to tumorigenesis. Herein, we identified the role of miR-148a in gastric cell proliferation. miR-148a knockdown inhibited cell proliferation in gastric cancer cell lines. Conversely, miR-148a overexpression promoted cell proliferation and cell cycle progression. p27, a key inhibitor of cell cycle, was verified as the target of miR-148a, indicating miR-148a might downregulate p27 expression to promote gastric cell proliferation. Moreover, we confirmed that miR-148a expression was frequently and dramatically downregulated in human advanced gastric cancer tissues, and observed a good inverse correlation between miR-148a and p27 expression in tumor samples. Thus, our results demonstrated that miR-148a downregulation might exert some sort of antagonistic function in cell proliferation, rather than promote cell proliferation in gastric cancer.     

A protective role of 27-kDa heat shock protein in glucocorticoid-evoked apoptotic cell death of hippocampal progenitor cells

Hippocampus is one of the most vulnerable tissues to glucocorticoid (GC). In the present study, we demonstrate that dexamethasone (DEX), a synthetic GC, induces apoptotic cell death in hippocampal progenitor HiB5 cells without any additional insult. Interestingly, expression of 27-kDa heat shock protein (HSP27) was markedly induced by DEX in time- and dose-dependent manners. This induction was dependent on the production of reactive oxygen species (ROS), suggesting that DEX-evoked oxidative damage to HiB5 cells is responsible for the HSP27 induction. To evaluate a possible role of HSP27, we generated two mutant HiB5 cell lines, in which expression of HSP27 was inhibited or enhanced by the over-expression of HSP27 cDNA with antisense or sense orientation (AS-HSP27 and S-HSP27, respectively). DEX-induced apoptotic cell population was significantly increased in AS-HSP27 HiB5 cells and evidently decreased in S-HSP27 cells. These results indicate that HSP27 protects hippocampal progenitor cells from GC-induced apoptotic cell death.     

Inhibition of histone/lysine acetyltransferase activity kills CoCl2-treated and hypoxia-exposed gastric cancer cells and reduces their invasiveness

Hypoxia enhances immortality and metastatic properties of solid tumors. Deregulation of histone acetylation has been associated with several metastatic cancers but its effect on hypoxic responses of cancer cells is not known. This study aimed at understanding the effectiveness of the hydrazinocurcumin, CTK7A, an inhibitor of p300 lysine/histone acetyltransferase (KAT/HAT) activity, in inducing apoptosis of gastric cancer cells (GCCs) exposed to cobalt chloride (CoCl₂), a hypoxia-mimetic chemical, or 1% O₂. Here, we show that CTK7A-induced hydrogen peroxide (H₂O₂) generation in CoCl₂-exposed and invasive gastric cancer cells (GCCs) leads to p38 MAPK-mediated Noxa expression and thereafter, mitochondrial apoptotic events. Noxa induction in normal immortalized gastric epithelial cells after CTK7A and hypoxia-exposure is remarkably less in comparison to similarly-treated GCCs. Moreover, hypoxia-exposed GCCs, which have acquired invasive properties, become apoptotic after CTK7A treatment to a significantly higher extent than normoxic cells. Thus, we show the potential of CTK7A in sensitizing hypoxic and metastatic GCCs to apoptosis induction.     

In silico identification of novel lncRNAs with a potential role in diagnosis of gastric cancer

Abstract

Gastric cancer (GC) is the second leading cause of cancer-related deaths in the world. Due to the shortage of adequate symptoms in the early stages, it is diagnosed when the tumor has spread to distant organs. Early recognition of GC enhances the chance of successful treatment. Molecular mechanisms of GC are still poorly understood. LncRNAs are emerging as new players in cancer in both oncogene and tumor suppressor roles. High-throughput technologies such as RNA-Seq, have revealed thousands of lncRNAs which are dysregulated in GC. In this study, we retrieved lncRNAs obtained by High-throughput technologies from OncoLnc database. Consequently, retrieved lncRNAs were compared in literature-based databases including PubMed. As a result, two lists, including experimentally validated lncRNAs and predicted lncRNAs were provided. We found 43 predicted lncRNAs that had not been experimentally validated in GC, so far. Further Bioinformatics analyses were performed to obtain the expression profile of predicted lncRNAs in tumor and normal tissues. Also, the roles and targets of predicted lncRNAs in GC were identified by related databases. Finally, using the GEPIA database was reviewed the significant relationship of predicted lncRNAs with the survival of GC patients. By recognizing the lncRNAs involved in initiation and progression of GC, they may be considered as potential biomarkers in the GC early diagnosis or targeted treatment and lead to novel therapeutic strategies.

Communicated by Ramaswamy H. Sarma     

The protective role of myeloid-derived suppressor cells in concanavalin A-induced hepatic injury

The mechanism underlying T cell-mediated fulminant hepatitis is not fully understood. In this study, we investigated whether myeloid derived suppressor cells (MDSCs) could prevent the concanavalin A (ConA)- induced hepatitis through suppressing T cell proliferation. We observed an increase in the frequencies of MDSCs in mouse spleen and liver at early stage of ConA treatment, implicating that the MDSCs might be involved in the initial resistance of mice against ConAmediated inflammation. Subpopulation analysis showed that the MDSCs in liver of ConA-induced mice were mainly granulocytic MDSCs. Adoptive transfer of the bone marrow-derived MDSCs into ConA-treated mice showed that the MDSCs migrated into the liver and spleen where they suppressed T cell proliferation through ROS pathway. In addition, the frequencies of MDSCs in mice were also significantly increased by the treatment with immune suppressor glucocorticoids. Transfer of MDSCs into the regulatory T cell (Treg)- depleted mice showed that the protective effect of MDSCs on ConA-induced hepatitis is Treg-independent. In conclusion, our results demonstrate that MDSCs possess a direct protective role in T cell-mediated hepatitis, and increasing the frequency of MDSCs by either adoptive transfer or glucocorticoid treatment represents a potential cell-based therapeutic strategy for the acute inflammatory disease.     

Upregulation of periostin prevents P53-mediated apoptosis in SGC-7901 gastric cancer cells

Periostin is frequently upregulated in human cancers including gastric cancer and implicated in cancer cell proliferation, invasion, and epithelial–mesenchymal transition. This study was undertaken to investigate the effects of periostin overexpression on the chemosensitivity of gastric cancer cells. We constructed a stable cell line overexpressing periostin in SGC-7901 human gastric cancer cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that periostin had no influence on the proliferation of SGC-7901 cells. Compared to empty vector-transfected cells, overexpression of periostin rendered SGC-7901 cells more resistant to cisplatin or 5-fluorouracil (5-FU)-induced apoptosis, accompanying with less release of cytochrome c from mitochondria and diminished cleavage of caspase-3 and poly (ADP-ribose) polymerase. Periostin-overexpressing cells treated with cisplatin or 5-FU showed significantly (p < 0.05) decreased expression of Bax and p53 proteins and increased expression of Bcl-2 protein, when compared to drug-treated mock counterparts. Restoration of p53 expression by delivering wild-type p53 gene resulted in a marked increase in drug-induced apoptosis in periostin-overexpressing SGC-7901 cells. Periostin overexpression elevated the phosphorylation of Akt. Pretreatment of periostin-overexpressing cells with an Akt inhibitor, MK-2206, partially rescued periostin-mediated inhibition of p53 expression and drug resistance. Taken together, our data indicate that periostin confers protection against cisplatin or 5-FU-induced apoptosis in SGC-7901 cells, likely through modulating the Akt/p53 pathway, and thus represents a potential therapeutic target in gastric cancer.     

The protective role of selenium on genetic damage and on cancer

Collectively, results from epidemiologic studies, laboratory bioassays, and human clinical intervention trials clearly support a protective role of selenium against cancer development. Several hypotheses have been proposed to explain these observations. Increased genomic instability, either inherent or induced by exogenous agents (mutagens or carcinogens), has been considered as a primary event leading to neoplastic transformation. This report deals specifically with the evidence for a role of selenium in the inhibition of carcinogen-induced covalent DNA adduct formation and retardation of oxidative damage to DNA, lipids and proteins, and for modulating cellular and molecular events that are critical in cell growth inhibition and in the multi-step carcinogenesis process. At present, the bulk of our knowledge on the role of selenium on genetic stability is based primarily on animal data and from studies conducted in in vitro systems. Studies performed in vitro showed that the dose and form of selenium compounds are critical factors with regard to cellular responses. Inorganic (at doses up to 10microM) and organic selenium compounds (at doses equal to or greater than 10microM) elicit distinctly different cellular responses. The recommended daily allowance (RDA) is 50-70 microgramSe per day for healthy adults; with 40 microgramSe as minimum requirement. Less than 11 microgramSe will definitely put people at risk of deficiency that would be expected to cause genetic damage. Daily doses of 100-200 microgramSe inhibited genetic damage and cancer development in humans. About 400 microgramSe per day is considered an upper limit. Clearly, doses above the RDA are needed to inhibit genetic damage and cancer. However, it has been hypothesized that the intake of excessive doses of selenium may cause oxidative damage, leading to genomic instability. The use of a cocktail consisting of selenium, and other vitamins and minerals appears to be a promising approach to inhibit genetic damage and the development of cancer. It is the author's recommendation that development of mechanism-based hypotheses that can be tested in pilot studies in different populations prior to a large-scale clinical trial in humans, is of paramount importance in order to better understand the role of selenium on genetic stability and cancer.