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Prohibitin Promotes De-Differentiation and Is a Potential Therapeutic Target in Neuroblastoma

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Prohibitin Promotes De-Differentiation and Is a Potential Therapeutic Target in Neuroblastoma

Ian C MacArthur et al. JCI Insight.

Abstract

Gain of the long arm of chromosome 17 (17q) is a cytogenetic hallmark of high-risk neuroblastoma, yet its contribution to neuroblastoma pathogenesis remains incompletely understood. Combining whole-genome and RNA sequencing of neuroblastomas, we identified the prohibitin (PHB) gene as highly expressed in tumors with 17q gain. High PHB expression correlated with poor prognosis and was associated with loss of gene expression programs promoting neuronal development and differentiation. PHB depletion induced differentiation and apoptosis and slowed cell cycle progression of neuroblastoma cells, at least in part through impaired ERK1/2 activation. Conversely, ectopic expression of PHB was sufficient to increase proliferation of neuroblastoma cells and was associated with suppression of markers associated with neuronal differentiation and favorable neuroblastoma outcome. Thus, PHB is a 17q oncogene in neuroblastoma that promotes tumor cell proliferation, and de-differentiation.

Keywords: Cancer; Genetics; Oncogenes; Oncology.

Conflict of interest statement

Conflict of interest: AK is a consultant for Novartis.

Figures

Figure 1
Figure 1. Chromosome 17q gain in neuroblastoma is associated with high PHB copy number and expression and correlates with adverse patient prognosis.
(A) Plot of chromosome 17 with regions of copy number gain (shown in blue) as detected in 56 patient samples of tumor-normal paired whole-genome sequencing. PHB locus indicated (black arrowhead). (B) Kaplan-Meier curve displaying overall survival for patients with low versus high PHB mRNA expression measured using RNA-Seq. P value calculated by log-rank test. (C) PHB expression in patients with 17q versus whole 17 or no gain. **P < 0.05, 2-tailed t test. (D) Heatmap of the significantly differentially expressed genes in patients with high versus low PHB expression. Statistical significance assessed with the Mann-Whitney U test. (E) Volcano plot showing differentially expressed genes in low- versus high-PHB groups. Neuronal differentiation genes NEGR1, HES1, and NGF marked (red circles). (F) GO analysis showing most GO categories significantly enriched in differentially expressed genes in low versus high PHB–expressing tumors. P values calculated with Fisher’s exact test. (G) GSEA indicating enrichment and upregulation of genes involved in neuronal differentiation, neuron development, and neurogenesis in patient tumors expressing PHB at low levels. P values calculated with Fisher’s exact test.
Figure 2
Figure 2. Prohibitin is expressed in neuroblastoma cell lines, has diverse subcellular localization, and is posttranslationally modified to influence RAS/MAPK signaling.
(A) Immunofluorescence showing subcellular localization of PHB (shown in green). Mitochondrial staining indicated by COX4 (shown in red). Nuclear staining indicated by DAPI (shown in blue). Scale bar: 50 μm. (B) Fraction of PHB signal quantified in each compartment. Data represent 4 technical replicates. (C) Western blot analysis demonstrating detection of PHB phosphorylated at T258, as well as ERK1/2 phosphorylated at T202/Y204.
Figure 3
Figure 3. PHB knockdown impairs ERK activation, reduces cell viability, slows cell cycle progression, and induces apoptosis in neuroblastoma cells.
(A) Western blot analysis displaying quantities of phosphorylated ERK1/2 at T202/Y204 in IMR-5/75 cells. ERK1/2 results are representative of 3 independent experiments. (B) IMR-5/75 cell proliferation following PHB knockdown as measured with the RTCA iCelligence system. Each condition was tested in duplicate. (C) FACS plots showing IMR-5/75 cell cycle distribution following PHB knockdown. (D) Quantification of cells in S phase after PHB knockdown. Data represent mean ± SD. *P < 0.001, 2-tailed t test. (E) Cell viability of IMR-5/75 cells expressing ectopic PHB-V5 following transduction of an shRNA against the 3′ UTR of PHB. Viability measured with the CellTiter-Glo luminescent viability assay. Data represent mean ± SEM. *P < 0.001, 2-tailed t test. (F) Representative FACS plots of TUNEL-stained IMR-5/75 neuroblastoma cells after PHB knockdown. (G) Quantification of TUNEL-positive cells after PHB knockdown. Data represent mean ± SD. *P < 0.001, 2-tailed t test. Three technical replicates shown. Bonferroni’s correction was applied to account for multiple comparisons.
Figure 4
Figure 4. PHB knockdown promotes differentiation of neuroblastoma cells.
(A) Photomicrographs of Kelly cells after transduction with shRNAs against either PHB or GFP. Photomicrographs representative of 3 independent samples. (B) NTRK1 and NGFR mRNA expression measured by quantitative reverse transcription PCR (qRT-PCR) after PHB knockdown. Data represent mean ± SEM. n = 3, *P < 0.001, 2-tailed t test. (C) Heatmap showing significantly differentially expressed genes in IMR-5/75 after PHB knockdown. PHB, NTRK1, and NGFR are labeled in bold and denoted by arrowheads. n = 3, and P < 0.001. (D) GO analysis of differentially expressed genes after PHB knockdown. n = 3, Fisher’s exact test. (E) GSEA plot of neuronal differentiation after PHB knockdown. n = 3, Fisher’s exact test. Stated n values indicate number of biological replicates.
Figure 5
Figure 5. Ectopic expression of PHB promotes proliferation, migration, and dedifferentiation of neuroblastoma cells.
(A) Western blot analysis of cells after stable expression of PHB-V5 compared with empty vector control cells. Contrast of blots was enhanced equally for clarity. (B) Proliferation of IMR-5/75 cells stably expressing PHB-V5 compared with empty vector control cells as measured with the RTCA iCelligence system. Each condition was tested in triplicate. (C) Migration of IMR-5/75 and SH-SY5Y cells stably expressing PHB-V5 compared with empty vector control cells as measured by scratch assay. Data represent mean ± SD. n = 3, and **P < 0.05 for IMR-5/75; n = 1, and P > 0.05 for SH-SY5Y; 2-tailed t test. (D) Number of mounds formed by SH-SY5Y cells stably expressing PHB-V5 compared with empty vector control cells. Data represent mean ± SD. n = 3, and **P < 0.001; 2-tailed t test. (E) Expression of NTRK1 and NGFR measured by qRT-PCR. Data represent mean ± SD. ***P < 0.0003, and **P < 0.005; 2-tailed t test. Stated n values represent number of biological replicates.
Figure 6
Figure 6. RocA treatment impairs ERK activation and promotes differentiation of neuroblastoma cells in vitro.
(A) Dose-response curve of cells treated with RocA as measured with CellTiter-Glo luminescent viability assay 72 hours after treatment. Data represent 3 technical replicates. (B) Western blot analysis of IMR-5/75 cells 6, 24, and 48 hours after treatment with 50 nM RocA compared with DMSO-treated cells. Data represent a single experiment. (C) Heatmap showing differentially expressed genes in IMR-5/75 cells 24 hours following treatment with RocA (50 nM) compared with DMSO-treated cells. n = 3. (D) GO analysis of differentially expressed genes in IMR-5/75 cells 24 hours following treatment with RocA (50 nM) compared with DMSO-treated cells. n = 3, Fisher’s exact test. (E) GSEA analysis in IMR-5/75 cells 24 hours following treatment with RocA (50 nM) compared with DMSO-treated cells. n = 3, Fisher’s exact test. Stated n values indicate number of biological replicates.
Figure 7
Figure 7. RocA treatment impairs ERK activation in an ALK-mutant, patient-derived xenograft in vivo.
(A) Heatmaps showing excess over Bliss synergy scores for combination treatment with RocA and trametinib in neuroblastoma cells. Values greater than 1 (shown in red) denote synergistic combinations while values less than 1 (shown in green) denote antagonistic combinations. Data represent 3 technical triplicates. (B) Dosing schedule of RocA, trametinib, and vehicle controls for patient-derived xenograft treatment. (C) Western blot analysis of patient-derived xenografts of mice treated with RocA, trametinib, or RocA and trametinib combination treatment compared with vehicle control–treated mice.

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