pevonedistat and Breast-Neoplasms

Topics: Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Cyclopentanes; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; NEDD8 Protein; Prognosis; Pyrimidines; Survival Rate; Tumor Cells, Cultured; Ubiquitin-Activating Enzymes

Activation of sterol regulatory element-binding protein 1 (SREBP-1), a master lipogenic transcription factor, is associated with cancer metabolism and metabolic disorders. Neddylation, the process of adding NEDD8 to its substrate, contributes to diverse biological processes. Here, we identified SREBP-1 as a substrate for neddylation by UBC12 and explored its impact on tumor aggressiveness. In cell-based assays, SREBP-1 neddylation prolonged SREBP-1 stability with a decrease in ubiquitination. Consequently, NEDD8 overexpression facilitated proliferation, migration, and invasion of SK-Hep1 liver tumor cells. MLN4924 (an inhibitor of the NEDD8-activating enzyme-E1) treatment or UBC12 knockdown prevented SREBP-1 neddylation and tumor cell phenotype change. This effect was corroborated in an in vivo xenograft model. In human specimens, SREBP-1, UBC12, and NEDD8 were all upregulated in hepatocellular carcinoma (HCC) compared to nontumorous regions. Moreover, SREBP-1 levels positively correlated with UBC12. In GEO database analyses, SREBP-1 levels were greater in metastatic HCC samples accompanying UBC12 upregulation. In HCC analysis, tumoral SREBP-1 and UBC12 levels discriminated overall patient survival rates. Additionally, MLN4924 treatment destabilized SREBP-1 in MDA-MB-231 breast cancer cells and in the tumor cell xenograft. SREBP-1 and UBC12 were also highly expressed in human breast cancer tissues. Moreover, most breast cancers with lymph node metastasis displayed predominant SREBP-1 and UBC12 expressions, which compromised overall patient survival rates. In summary, SREBP-1 is neddylated by UBC12, which may contribute to HCC and breast cancer aggressiveness through SREBP-1 stabilization, and these events can be intervented by MLN4924 therapy. Our findings may also provide potential reliable prognostic markers for tumor metastasis.

Topics: Animals; Breast; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclopentanes; Female; Humans; Liver; Liver Neoplasms; Lymphatic Metastasis; Mice; NEDD8 Protein; Prognosis; Protein Stability; Pyrimidines; Sterol Regulatory Element Binding Protein 1; Survival Rate; Ubiquitin-Activating Enzymes; Ubiquitin-Conjugating Enzymes; Ubiquitination; Up-Regulation; Xenograft Model Antitumor Assays

Estrogen-related receptor beta (ERRβ) is downregulated in breast cancer cells and its overexpression in breast cancer patients is positively correlated with an improved prognosis and prolonged relapse-free survival. Here, we unravelled a molecular mechanism for ERRβ downregulation in breast cancer. We found that ERRβ is a key substrate of the SCF complex and that NEDDylation can activate the Cullin subunits of the SCF complex to target ERRβ for degradation in breast cancer. Consistently, using in vitro and in vivo models, we demonstrated that MLN4924, a specific small molecule inhibitor of NEDDylation, can restore ERRβ expression and culminate in a reduction in cell proliferation and migration of breast cancer cells. We also showed that increased ERRβ expression promotes the upregulation of its target genes, including the tumour suppressors p21

Topics: Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cullin Proteins; Cyclopentanes; Disease Progression; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression Regulation, Neoplastic; Humans; NEDD8 Protein; Neoplasm Recurrence, Local; Pyrimidines; Receptors, Estrogen; Ubiquitins

How the neddylation pathway functions in breast tumor and regulation of estrogen receptor (ER) expression is rarely reported. The purpose of this study was to identify the role of neddylation in breast cancer and ER expression, and further explore the underlying mechanisms.. Expression patterns of nedd8-activating enzyme (NAE) and nedd8, two key proteins in the neddylation pathway, were examined in human breast specimens. ER-α expression was investigated using animal 18F-FES-PET/CT and immunoblotting upon NAE inhibitor MLN4924 treatment. Chromatin immunoprecipitation assay, luciferase reporter promoter assay, and the CRISPR-Cas9 system were used to elucidate the mechanism of ER-α regulation by MLN4924. The ER-positive breast cancer mouse model was used to determine the synergetic effect of MLN4924 and fulvestrant on tumor growth. All statistical tests were two-sided.. Both NAE1 and nedd8 expressions were higher in the ER-positive subgroup. Higher expressions of NAE1 and nedd8 indicated poorer prognosis. Importantly, ER-α expression was significantly downregulated upon MLN4924 treatment. Our proof-of-principle study determines the activation of neddylation in breast tumor tissues for the first time and reveals a new ER-α regulatory mechanism, as well as further explores an effective approach to improve fulvestrant sensitivity through a neddylation inactivation combination.

Topics: Active Transport, Cell Nucleus; Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclopentanes; Disease-Free Survival; Drug Resistance, Neoplasm; Enzyme Inhibitors; Estrogen Receptor alpha; Estrogen Receptor Antagonists; Female; Forkhead Box Protein O3; Fulvestrant; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; NEDD8 Protein; Pyrimidines; Ubiquitin-Activating Enzymes; Ubiquitins; Xenograft Model Antitumor Assays

Breast cancer is a common type of cancer among female cancer patients and the main cause of cancer-related deaths. During the last decades, targeted therapies for breast cancer have been rapidly developing. Among them, MLN4924, a first-in-class NEDD8-activating enzyme (NAE) inhibitor, has performed antitumor activity by inactivating the cullin-RING ligases and causing the accumulation of their substrates to induce apoptosis in a number of studies. In this study, we found that MLN4924 activates the AKT pathway in both HER2-positive and triple-negative breast cancer (TNBC) cell lines. Given that AKT signaling is responsible for tumor progression and drug resistance in some types of cancers, we hypothesized that the AKT inhibitor may synergistically enhance the tumor suppression capability in breast cancer by MLN4924. To demonstrate the sensitizing effect, MK-2206 was chosen as the adjuvant treatment, and cell growth, migration and apoptosis were detected. The results showed that MLN4924 treatment inhibited cell growth and migration and induced apoptosis in both SK-BR3 and MDA-MB231 breast cancer cell lines. More importantly, the combined treatment of MLN4924 and MK-2206 indeed caused stronger cytotoxicity and inhibition of migration and a much higher induction of apoptosis compared with MLN4924 treatment alone. Our study provides the proof-of-concept evidence for strategic drug combination of MLN4924 with an AKT inhibitor for maximal killing of breast cancer cells via the enhancement of apoptosis.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclopentanes; Female; Heterocyclic Compounds, 3-Ring; Humans; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrimidines; Signal Transduction; Ubiquitin-Activating Enzymes

Two-deoxy-D-glucose (2DG) causes cytotoxicity in the cancer cells by disrupting the thiol metabolism, and MLN4924 inactivates the SCF E3 ligase and so causes the accumulation of its substrates which trigger apoptosis and hence might enhance the efficiency of radiotherapy and overcame on the radioresistance of the cancer cells.. SKBR3 and MCF-7 breast cancer cells were treated with 500 μM 2DG and/or MLN4924 (30, 100, 200 and 300 nM), and in combination in the presence and absence of 1, 1.5 and 2 Gy gamma irradiation. The effects of the treatments - 2DG, MLN4924, irradiation alone and combined - on MCF-7 and SKBR3 cell lines were evaluated by MTT assay, TUNEL assay, cell death detection, Q-PCR for caspase-3 and Bcl-2 expression analysis, and finally clonogenic survival assay.. The treatments enhanced the further radio cytotoxicity via inducing the apoptosis cell signaling gene, caspase-3. The 2DG and MLN4924 treatments could act as a radiosensitizer, especially on the SKBR3 cells, and further sensitized the cells with a sensitivity enhancement ratio (SER) of 1.41 and 1.27 in SKBR3 and MCF-7 cells, respectively.. The combined chemo-radiotherapy might improve the breast cancer treatment outcome.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Chemoradiotherapy; Cyclopentanes; Deoxyglucose; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Humans; MCF-7 Cells; Pyrimidines; Radiotherapy Dosage; Treatment Outcome

2-deoxy-D-Glucose (2DG) causes cytotoxicity in cancer cells by disrupting thiol metabolism. It is an effective component in therapeutic strategies. It targets the metabolism of cancer cells with glycolysis inhibitory activity. On the other hand, MLN4924, a newly discovered investigational small molecule inhibitor of NAE (NEDD8 activating enzyme), inactivates SCF E3 ligase and causes accumulation of its substrates which triggers apoptosis. Combination of these components might provide a more efficient approach to treatment. In this research, 2DG and MLN4924 were co-applied to breast cancer cells (MCF-7 and SKBR-3) and cytotoxic and apoptotic activity were evaluated the by Micro culture tetrazolium test (MTT), TUNEL and ELISA methods. Caspase3 and Bcl2 genes expression were evaluated by real time Q-PCR methods. The results showed that MLN4924 and MLN4924/2DG dose-dependently suppressed the proliferation of MCF7 and SKBR-3 cells. Cell survival of breast cancer cells exposed to the combination of 2DG/MLN4924 was decreased significantly compared to controls (p<0.05), while 2DG and MLN4924 alone had less pronounced effects on the cells. The obtained results suggest that 2DG/MLN4924 is much more efficient in breast cancer cell lines with enhanced cytotoxicity via inducing a apoptosis cell signaling gene, caspase-3.

Topics: Antimetabolites; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Proliferation; Cyclopentanes; Deoxyglucose; Drug Therapy, Combination; Female; Humans; In Situ Nick-End Labeling; Pyrimidines; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Ubiquitin-Activating Enzymes

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.

Topics: Apoptosis; Base Sequence; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cyclopentanes; Female; Flow Cytometry; Humans; NEDD8 Protein; Oncogene Protein p21(ras); Pyrimidines; Radiation-Sensitizing Agents; RNA, Small Interfering; Ubiquitins