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Combined inhibition of RAC1 and Bcl-2/Bcl-xL synergistically induces glioblastoma cell death through down-regulation of the Usp9X/Mcl-1 axis

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Abstract

Purpose

Anti-apoptotic and pro-migratory phenotypes are hallmarks of neoplastic diseases, including primary brain malignancies. In this work, we examined whether reprogramming of the apoptotic and migratory machineries through a multi-targeting approach would induce enhanced cell death and enhanced inhibition of the migratory capacity of glioblastoma cells.

Methods

Preclinical testing and molecular analyses of combined inhibition of Bcl-2/Bcl-xL and RAC1 were performed in established, primary cultured and stem-like glioblastoma cell systems.

Results

We found that the combined inhibition of Bcl-2/Bcl-xL and RAC1 resulted in synergistic pro-apoptotic and anti-migratory effects in a broad range of different glioblastoma cells. At the molecular level, we found that RAC1 inhibition led to a decreased expression of the deubiquitinase Usp9X, followed by a decreased stability of Mcl-1. We also found that the combined inhibition led to a significantly decreased migratory activity and that tumor formation of glioblastoma cells on chorion allantoic membranes of chicken embryos was markedly impaired following the combined inhibition.

Conclusions

Our data indicate that concomitant inhibition of RAC1 and Bcl-2/Bcl-xL induces pro-apoptotic and anti-migratory glioblastoma phenotypes as well as synergistic anti-neoplastic activities. The clinical efficacy of this inhibitory therapeutic strategy warrants further evaluation.

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Acknowledgements

We thank Andrea Schuster for her excellent technical support with the CAM assay and Angelika Vollmer for her assistance with the time-lapse analyses. MDS is supported by grants NIH NINDS K08 NS083732, NIH NINDS R01 NS095848 and NIH NINDS R01 NS102366.

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Authors and Affiliations

  1. Department of Neurological Surgery, Ulm University Medical Center, Albert-Einstein-Allee 23, D-89081, Ulm, Germany

    Michal Hlavac, Annika Dwucet, Jens Engelke, Christian Rainer Wirtz, Marc-Eric Halatsch & Georg Karpel-Massler

  2. IIAIG, Study Center, Burlington, VT, USA

    Richard Eric Kast

  3. Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany

    Mike-Andrew Westhoff & Klaus-Michael Debatin

  4. Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA

    Markus D. Siegelin

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  1. Michal Hlavac
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Correspondence to Georg Karpel-Massler.

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Supplementary figure 1

A, In silico analysis based on the TCGA glioblastoma (GBM) dataset showing Kaplan-Meier curves for patients with high or low RAC1 mRNA expression (www.oncolnc.org, last accessed 12/11/2018). B, Chemical structures of ABT263 and NSC23766 (ChemDraw Professional 16.0, Perkin Elmer). C, ULM-GBM-PC35 cells were treated for 48 h with NSC23766 (NSC), ABT263 or the combination at indicated concentrations. Staining with annexin V/propidium iodide was performed prior to flow cytometric analysis. Representative flow plots are shown. Data are representative for three independent experiments. D, Quantitative representation of U251, ULM-GBM-PC38, T98G and ULM-GBM-PC35 glioblastoma cells treated as described for C and Fig. 2A-C. Columns, mean; bars, SEM. N = 3. Statistical significance was assessed by Student’s t test. (PPTX 647 kb)

Supplementary figure 2

A, U251 cells were treated for 48 h with NSC23766 and/ or ABT263 as indicated prior to staining with TMRE and flow cytometric analysis. The fold increase of cells with a reduced mitochondrial membrane potential (MMP) was calculated in comparison to control. Columns, mean; bars, SD. N = 3. Statistical significance was assessed by Student’s t test. B, U251 cells were subjected for 48 h to the combination treatment in the presence or absence of zVAD.fmk prior to staining with propidium iodide and flow cytometric analysis. Columns, mean; bars, SD. N = 3. Statistical significance was assessed by Student’s t test. C, U251 cells were treated for 48 h with the combination of 20 μM NSC23766 (NSC) and 2 μM ABT263 (ABT) in the presence or absence of 20 μM Necrostatin. Staining with propidium iodide was performed prior to flow cytometric analysis. Representative histograms are shown. D, Quantitative representation of U251 cells treated as described for C. Columns, mean; bars, SD. N = 3. Statistical significance was assessed by Student’s t test. E, U87MG cells were treated for 6 h or 24 h with increasing concentrations of NSC23766 under serum starvation. Whole-cell extracts were examined by Western blot for Mcl-1, Bcl-2 and Bcl-xL. Actin served as a loading control. Densitometric analysis was perfomed using ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Normalized data are presented on top of the respective Western blots. Data are representative for two independent experiments. (PPTX 2139 kb)

Supplementary figure 3

A, U251 cells were treated for 48 h with non-targeting (n.t.)-siRNA, or Mcl-1-siRNA followed by treatment with ABT263 or solvent for 24 h. Staining with propidium iodide was performed prior to flow cytometric analysis. Columns, mean; bars, SD. N = 3. Statistical significance was assessed by Student’s t test. B, U251 cells were treated for 48 h with NSC23766, the selective Bcl-2 inhibitor ABT199 or the combination. Staining with propidium iodide was performed prior to flow cytometric analysis. Representative histograms are shown. C, U251 cells were treated for 6 h or 24 h with NSC23766 (20 μM), ABT263 (2 μM) or both prior to performing rtPCR for Mcl-1. 18S served as housekeeping gene. Columns, mean. Bars, SD. N = 3. Statistical significance was assessed by Student’s t test. D, ULM-GBM-PC38 were treated with NSC23766 or solvent for 24 h before adding 10 μg/mL cycloheximide and Western blot analysis for Mcl-1 and Actin. Data are representative for two independent experiments. (PPTX 1908 kb)

Supplementary figure 4

A, A172 cells were seeded on 24-well plates followed by sequential microscopic imaging (magnification, ×10) over a total time period of 24 h. Single-cell tracking was performed using the MtrackJ software (see Materials and Methods). Wind-rose plots displaying the paths of 15 single cells per treatment condition during the 24 h observation period. The tracks were aligned to start from the same initial position to facilitate comparisons. B, Total distance of 45 cells covered within 24 h per treatment condition. Columns, mean; bars, SEM. Data are representative for 3 independent experiments. (PPTX 112 kb)

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Hlavac, M., Dwucet, A., Kast, R.E. et al. Combined inhibition of RAC1 and Bcl-2/Bcl-xL synergistically induces glioblastoma cell death through down-regulation of the Usp9X/Mcl-1 axis. Cell Oncol. 42, 287–301 (2019). https://doi.org/10.1007/s13402-019-00425-3

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