ONC201 Suppresses Neuroblastoma Growth by Interrupting Mitochondrial Function and Reactivating Nuclear ATRX Expression While Decreasing MYCN

Abstract

Neuroblastoma (NB) is characterized by several malignant phenotypes that are difficult to treat effectively without combination therapy. The therapeutic implication of mitochondrial ClpXP protease ClpP and ClpX has been verified in several malignancies, but is unknown in NB. Firstly, we observed a significant increase in ClpP and ClpX expression in immature and mature ganglion cells as compared to more malignant neuroblasts and less malignant Schwannian-stroma-dominant cell types in human neuroblastoma tissues. We used ONC201 targeting ClpXP to treat NB cells, and found a significant suppression of mitochondrial protease, i.e., ClpP and ClpX, expression and downregulation of mitochondrial respiratory chain subunits SDHB and NDUFS1. The latter was associated with a state of energy depletion, increased reactive oxygen species, and decreased mitochondrial membrane potential, consequently promoting apoptosis and suppressing cell growth of NB. Treatment of NB cells with ONC201 as well as the genetic attenuation of ClpP and ClpX through specific short interfering RNA (siRNA) resulted in the significant upregulation of the tumor suppressor alpha thalassemia/mental retardation X-linked (ATRX) and promotion of neurite outgrowth, implicating mitochondrial ClpXP proteases in MYCN-amplified NB cell differentiation. Furthermore, ONC201 treatment significantly decreased MYCN protein expression and suppressed tumor formation with the reactivation of ATRX expression in MYCN-amplified NB-cell-derived xenograft tumors. Taken together, ONC201 could be the potential agent to provide diversified therapeutic application in NB, particularly in NB with MYCN amplification.

Keywords: ATRX; ClpPX protease; MYCN; ONC201; apoptosis; mitochondria; neuroblastoma.

Figure 1

Comparison of ClpP and ClpX expressions in subtypes of human NBs: (A) Representative profiles of ClpP and ClpX immunostaining in differentiated NBs. (B,C) Histological scoring for ClpP and ClpX intensity in NBs tissues. Data in each bar chart are represented as mean ± SD. Primitive neuroblast (n = 4), differentiated neuroblast (n = 12), immature ganglion cell (n = 10), mature ganglion cell (n = 4), and Schwannian stroma (n = 12). Upper panel, ×200 magnification, scale bar 100 μm; lower panel, ×400 magnification, scale bar 50 μm. * p < 0.05.

Figure 2

Effects of ONC201 on cell survival in NB cells: (A) SK-N-AS and BE(2)M17 cells were treated with ONC201 (5 μM) for 48 h and 96 h. The cell survival was detected by Resazurin assay. (B) Representative phase contrast images demonstrating cell density and morphological changes in NBs exposed to ONC201 for 48 h. Scale bar 50 μm. (C) After incubation with ONC201 (5 μM) for 48 h, cells were collected and stained by trypan blue solution. The percentage of trypan-blue-positive cells was quantified using flow cytometry. (D,E) Representative profiles of TUNEL staining (red) in control and ONC201-treated NB cells. DAPI (blue) was used as a nuclear counterstain. Scale bar 20 μm. (D) The apoptotic cells are expressed as the percentage of the number of TUNEL-positive cells over the number of DAPI-positive cells. All data are expressed as mean ± SD from triplicate experiments. * p < 0.05.

Figure 3

Effects of ONC201 on mitochondrial functions in NB cells: (A) The relative protein levels of ClpP, ClpX, SDHB, and NDUFS1 were determined by Western blot and quantified by Image Pro-plus analysis software. (B,C) OCR in ONC201-treated cells were detected by the Seahorse XF24 analyzer in the absence or presence of oligomycin (1 μM), FCCP (0.5 μM), and rotenone (1 μM) by counting 1 × 10⁵ cells. (D,E) Profiling of ECAR in control and ONC201-treated cells was measured by the Seahorse XF24 analyzer. (F) Cells were labeled with TMRM (100 nM) to examine mitochondrial membrane potential and analyzed by flow cytometry. (G) Total cellular ATP levels were measured using the luciferase-based luminescence assay kit. All data were obtained from three independent experiments, and are expressed as mean ± SD. * p < 0.05.

Figure 4

Effects of ONC201 on mitochondria-mediated apoptosis signaling in NB cells: (A) The intracellular ROS production of ONC201-treated cells was detected by DHE staining. The relative intensity of DHE fluorescence was analyzed by flow cytometry. (B) Mitochondrial ROS was detected by MitoSOX Red and immediately analyzed by flow cytometry. Data are expressed as fold change compared with control group. (C,D) To detect cytochrome C release, cytosolic and mitochondrial fractions of NB cells were subjected into 15% SDS-PAGE and probed with anti-cytochrome c antibody by Western blot analysis. Alpha-tubulin and VDAC1 were used as the cytosolic and mitochondrial internal control, respectively. (E) The protein levels of cleaved caspase 3 were detected by Western blot. The relative protein ratio was normalized with β-actin using Image Pro-plus analysis software. All data were obtained from three independent experiments, and are expressed as mean ± SD. * p < 0.05.

Figure 5

Effects of ONC201 on neurite outgrowth and the express profiles of ATRX in NB cells: (A) Representative of Giemsa staining in ONC201-treated SK-N-AS and BE(2)M17 cells. Scale bar 20 μm (upper panel) and 10 μm (lower panel), respectively. Red arrow indicates the neurite extension. (B) Western blot analysis of MYCN and ATRX protein in ONC201-treated BE(2)M17 cells. The relative protein ratio was normalized with β-actin using Image Pro-plus analysis software. (C) Representative images demonstrating ATRX enhancement (red) in the nucleus of SK-N-AS and BE(2)M17 cells when exposed to ONC201 (5 μM) for 48 h. The mitochondria were labeled with anti-TOM20 antibody (green). The nuclei were labeled with DAPI (blue). Scale bar 10 µm. (D) Western blot analysis of ATRX protein in nuclear and cytosolic fractions in BE(2)M17 cells. The relative ATRX ratio was normalized with lamin b and alpha-tubulin, respectively, and expressed as mean ± SD. (E) Analysis of ATRX expression in siClpP- and siClpX-treated SK-N-AS cells by Western blot. The relative protein ratio was normalized with β-actin, and is expressed as mean ± SD. All data were obtained from four to six independent experiments. * p < 0.05.

Figure 6

Effect of ONC201 administration on tumor growth in NB xenograft animal model and ClpXP and ATRX expression in tumor tissues: (A) Images of the resected tumors in the three groups. Tumor weight (g) was measured at sacrifice by microbalance and expressed as mean ± SD. Control = 3, treatment = 5. Scale bar 1 cm. (B) Representative pictures of ATRX expression in control and ONC201-treated neuroblastoma tissues. Scale bar 100 μm (upper panel) and 30 μm (lower panel), respectively. The percentage of nuclear ATRX-positive cells was calculated from five random images at 400x magnification per tissue, and are expressed as mean ± SD (n = 3). (C,D) Immunohistochemistry analysis of ClpP and ClpX expression in controls and ONC201-treated neuroblastoma tissues. Scale bar 30 μm. The intensities of ClpP and ClpX staining were calculated from five random images at 400× magnification per tissue, and are expressed as mean ± SD (n = 3). (E) Analysis of MYCN expression in ONC201-treated BE(2)M17 cells by Western blot. The relative protein ratio was normalized with β-actin using Image Pro-plus analysis software. * p < 0.05.

Figure 7

The proposed working model for interruption of mitochondrial functions, induced apoptosis, and differentiation in NB cells: ONC201 treatment targets mitochondria to perturb mitochondrial functions, e.g., the treatment disrupts the mitochondrial ClpXP complex, impairs respiratory chain activity, and leads to mitochondrial function loss and ROS accumulation. The enhancement of ROS production finally induces apoptosis in NB cells. In addition to cell death, ONC201 treatment also promotes the nuclear expression of ATRX, which supports neuronal differentiation and the suppression of NB growth.