The Protein Neddylation Inhibitor MLN4924 Suppresses Patient-Derived Glioblastoma Cells via Inhibition of ERK and AKT Signaling

Glioblastoma is a highly aggressive and lethal brain tumor, with limited treatment options. Abnormal activation of the neddylation pathway is observed in glioblastoma, and the NEDD8-activating enzyme (NAE) inhibitor, MLN4924, was previously shown to be effective in glioblastoma cell line models. However, its effect has not been tested in patient-derived glioblastoma stem cells. We first analyzed public data to determine whether NEDD8 pathway proteins are important in glioblastoma development and patient survival. NAE1 and UBA3 levels increased in glioblastoma patients; high NEDD8 levels were associated with poor clinical outcomes. Immunohistochemistry results also supported this result. The effects of MLN4924 were evaluated in 4 glioblastoma cell lines and 15 patient-derived glioblastoma stem cells using high content analysis. Glioblastoma cell lines and patient-derived stem cells were highly susceptible to MLN4924, while normal human astrocytes were resistant. In addition, there were various responses in 15 patient-derived glioblastoma stem cells upon MLN4924 treatment. Genomic analyses indicated that MLN4924 sensitive cells exhibited enrichment of Extracellular Signal Regulated Kinase (ERK) and Protein kinase B (AKT, also known as PKB) signaling. We verified that MLN4924 inhibits ERK and AKT phosphorylation in MLN4924 sensitive cells. Our findings suggest that patient-derived glioblastoma stem cells in the context of ERK and AKT activation are sensitive and highly regulated by neddylation inhibition.

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Anti-vimentin, anti-TUFM, anti-NAP1L1 and anti-DPYSL2 nanobodies display cytotoxic effect and reduce glioblastoma cell migration

Therapeutic Advances in Medical Oncology ◽

10.1177/1758835920915302 ◽

2020 ◽

Vol 12 ◽

pp. 175883592091530 ◽

Cited By ~ 1

Author(s):

Alja Zottel ◽

Ivana Jovčevska ◽

Neja Šamec ◽

Jernej Mlakar ◽

Jernej Šribar ◽

...

Keyword(s):

Stem Cells ◽

Cell Migration ◽

Cell Lines ◽

Water Soluble ◽

Glioblastoma Cell ◽

Glioblastoma Cells ◽

Mitochondrial Translation ◽

Cytotoxic Effects ◽

Glioblastoma Stem Cells ◽

Glioblastoma Cell Lines

Background: Glioblastoma is a particularly common and very aggressive primary brain tumour. One of the main causes of therapy failure is the presence of glioblastoma stem cells that are resistant to chemotherapy and radiotherapy, and that have the potential to form new tumours. This study focuses on validation of eight novel antigens, TRIM28, nucleolin, vimentin, nucleosome assembly protein 1-like 1 (NAP1L1), mitochondrial translation elongation factor (EF-TU) (TUFM), dihydropyrimidinase-related protein 2 (DPYSL2), collapsin response mediator protein 1 (CRMP1) and Aly/REF export factor (ALYREF), as putative glioblastoma targets, using nanobodies. Methods: Expression of these eight antigens was analysed at the cellular level by qPCR, ELISA and immunocytochemistry, and in tissues by immunohistochemistry. The cytotoxic effects of the nanobodies were determined using AlamarBlue and water-soluble tetrazolium tests. Annexin V/propidium iodide tests were used to determine apoptotsis/necrosis of the cells in the presence of the nanobodies. Cell migration assays were performed to determine the effects of the nanobodies on cell migration. Results: NAP1L1 and CRMP1 were significantly overexpressed in glioblastoma stem cells in comparison with astrocytes and glioblastoma cell lines at the mRNA and protein levels. Vimentin, DPYSL2 and ALYREF were overexpressed in glioblastoma cell lines only at the protein level. The functional part of the study examined the cytotoxic effects of the nanobodies on glioblastoma cell lines. Four of the nanobodies were selected in terms of their specificity towards glioblastoma cells and protein overexpression: anti-vimentin (Nb79), anti-NAP1L1 (Nb179), anti-TUFM (Nb225) and anti-DPYSL2 (Nb314). In further experiments to optimise the nanobody treatment schemes, to increase their effects, and to determine their impact on migration of glioblastoma cells, the anti-TUFM nanobody showed large cytotoxic effects on glioblastoma stem cells, while the anti-vimentin, anti-NAP1L1 and anti-DPYSL2 nanobodies were indicated as agents to target mature glioblastoma cells. The anti-vimentin nanobody also had significant effects on migration of mature glioblastoma cells. Conclusion: Nb79 (anti-vimentin), Nb179 (anti-NAP1L1), Nb225 (anti-TUFM) and Nb314 (anti-DPYSL2) nanobodies are indicated for further examination for cell targeting. The anti-TUFM nanobody, Nb225, is particularly potent for inhibition of cell growth after long-term exposure of glioblastoma stem cells, with minor effects seen for astrocytes. The anti-vimentin nanobody represents an agent for inhibition of cell migration.

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Abstract 4235: Anti-proliferative effects of ZR2002, a novel combi-molecule with EGFR/DNA binary targeting properties compared to Gefitinib in glioblastoma cell lines and brain tumor stem cells

10.1158/1538-7445.am2015-4235 ◽

2015 ◽

Author(s):

Zeinab Sharifi ◽

Jean-Claude Bertrand ◽

Kevin Petrecca ◽

Elliot Goodfellow ◽

Bassam Abdulkarim ◽

...

Keyword(s):

Stem Cells ◽

Brain Tumor ◽

Cell Lines ◽

Glioblastoma Cell ◽

Tumor Stem Cells ◽

Brain Tumor Stem Cells ◽

Glioblastoma Cell Lines

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Embryonal Carcinoma and Glioblastoma Cell Lines Derived from Monkey Induced Pluripotent Stem Cells

10.1007/7651_2020_303 ◽

2020 ◽

Author(s):

Hirohito Ishigaki ◽

Yasushi Itoh

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Embryonal Carcinoma ◽

Glioblastoma Cell ◽

Induced Pluripotent ◽

Glioblastoma Cell Lines

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Isolation of Cancer Stem Cells from Three Human Glioblastoma Cell Lines: Characterization of Two Selected Clones

PLoS ONE ◽

10.1371/journal.pone.0105166 ◽

2014 ◽

Vol 9 (8) ◽

pp. e105166 ◽

Cited By ~ 35

Author(s):

Fortunata Iacopino ◽

Cristiana Angelucci ◽

Roberto Piacentini ◽

Filippo Biamonte ◽

Annunziato Mangiola ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Cell Lines ◽

Glioblastoma Cell ◽

Human Glioblastoma ◽

Human Glioblastoma Cell ◽

Glioblastoma Cell Lines

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Effects of Single or Combined Treatments with Radiation and Chemotherapy on Survival and Danger Signals Expression in Glioblastoma Cell Lines

BioMed Research International ◽

10.1155/2014/453497 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 14

Author(s):

Francesca Pasi ◽

Alessandro Paolini ◽

Rosanna Nano ◽

Riccardo Di Liberto ◽

Enrica Capelli

Keyword(s):

Cell Lines ◽

Treatment Options ◽

Glioblastoma Cell ◽

X Rays ◽

Combined Treatments ◽

Adjuvant Effect ◽

Danger Signals ◽

Multiple Treatment ◽

Limited Effectiveness ◽

Glioblastoma Cell Lines

The success of chemo- and radiotherapy in glioblastoma multiforme, the most common and lethal primary brain tumour, could rely on the induction of immunogenic tumour cell death and on the induction of anticancer immune response. In this study we investigated cell survival to single treatments or combination of X-rays and temozolomide in glioblastoma cell lines (T98G and U251MG) and we attempted to identify danger signals (HMGB1 and HSP70) released by dying cells in the microenvironment that could activate antitumour immunity contributing to the therapeutic efficacy of conventional treatments. Our data suggest that HSP70 translocates from cytoplasm to extracellular environment after an increase in radiation dose and HMGB1 translocates from the nucleus to the cytoplasm and subsequently is released into the extracellular space, confirming a role of these proteins as signals released after radiation-induced damage in glioblastoma cells. We also could state that TMZ had limited effectiveness in activating HMGB1 and HSP70 signalling and, instead, an adjuvant effect was observed in some combined treatments, depending on schedule, cell line, and timing. A big challenge in tumour therapy is, therefore, to identify the most beneficial combination and chronology of multiple treatment options to contribute to the improvement of the therapeutic outcome.

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