Modulation of Stemness and Differentiation Regulators by Valproic Acid in Medulloblastoma Neurospheres

Abstract

Changes in epigenetic processes such as histone acetylation are proposed as key events influencing cancer cell function and the initiation and progression of pediatric brain tumors. Valproic acid (VPA) is an antiepileptic drug that acts partially by inhibiting histone deacetylases (HDACs) and could be repurposed as an epigenetic anticancer therapy. Here, we show that VPA reduced medulloblastoma (MB) cell viability and led to cell cycle arrest. These effects were accompanied by enhanced H3K9 histone acetylation (H3K9ac) and decreased expression of the MYC oncogene. VPA impaired the expansion of MB neurospheres enriched in stemness markers and reduced MYC while increasing TP53 expression in these neurospheres. In addition, VPA induced morphological changes consistent with neuronal differentiation and the increased expression of differentiation marker genes TUBB3 and ENO2. The expression of stemness genes SOX2, NES, and PRTG was differentially affected by VPA in MB cells with different TP53 status. VPA increased H3K9 occupancy of the promoter region of TP53. Among the genes regulated by VPA, the stemness regulators MYC and NES showed an association with patient survival in specific MB subgroups. Our results indicate that VPA may exert antitumor effects in MB by influencing histone acetylation, which may result in the modulation of stemness, neuronal differentiation, and the expression of genes associated with patient prognosis in specific molecular subgroups. Importantly, the actions of VPA in MB cells and neurospheres include a reduction in the expression of MYC and an increase in TP53.

Keywords: MYC; TP53; differentiation; medulloblastoma; stemness; valproic acid.

Figure 1.

VPA reduces MB cell viability. (A) D283 and Daoy human MB cells were treated with a range of VPA concentrations (0.5, 1.0, 2.5, 5.0, 10.0, and 20.0 mM) for 48 h, and cell viability was measured by a trypan exclusion assay. IC₅₀ concentrations of VPA for MB cells with a 95% confidence interval (CI) were 2.3 mM for D283 and 2.2 mM for Daoy cells. (B) Immunofluorescence assay using antibodies against H3K9ac, histone H3, and nuclei marker (DAPI) in the MB control and VPA-treated cells. The IC₅₀ concentration of VPA (2.3 mM for D283 and 2.2 mM for Daoy) was used. Semi-quantification of the H3K9ac signal intensity relative to DAPI is shown. Results are the mean ± SD of three independent experiments; * p < 0.05, *** p < 0.001 and **** p < 0.0001 compared to the controls.

Figure 2.

VPA leads to cell cycle arrest in MB cells. (A) Cell cycle analysis of the D283 and Daoy cells treated with VPA and controls. (B) Relative mRNA levels of CDKN1A and MYC in MB cells were assessed using RT-qPCR. (C) Western blot analysis of the p21 protein in MB cells after VPA exposure. The relative densitometric unit (RDU) analysis normalized by β-actin and corrected by the control is shown. All experiments were conducted using the IC₅₀ concentration of VPA (2.3 mM for D283 and 2.2 mM for Daoy) for 48 h. Results are the mean ± SD of three independent experiments; * p < 0.05, ** p < 0.01 and **** p < 0.0001 compared to the controls.

Figure 3.

VPA impairs the expansion of MB neurospheres enriched with stemness marker genes. (A) Representative images of D283 and Daoy MB cells and derived neurospheres. (B) Relative mRNA levels of SOX2, NES, and PRTG in the monolayer cells and neurospheres were verified using RT-qPCR. (C) VPA effects on MB neurosphere formation after 5 days of drug exposure. Semi-quantitative analysis of neurosphere size in the VPA-treated and control neurospheres is shown. (D) After 5 days of MB neurosphere growth, VPA was added and the neurosphere size and number were evaluated after 48 h. Semi-quantitative analyses of the neurosphere size and number in the VPA-treated and control neurospheres are shown. Images were taken in an inverted microscope with 5 X magnification and the scale bar corresponded to 500 μm. (E) An immunofluorescence assay against H3K9ac, histone H3, and nuclei marker (DAPI) was performed in the VPA-treated and control neurospheres. Fluorescent images were taken with an inverted microscope with 20 X magnification and the scale bar corresponded to 100 μm. (F) Relative mRNA levels of SOX2, NES, and PRTG in the control and VPA-treated neurospheres were verified using RT-qPCR. Experiments were conducted using the IC₅₀ concentration (2.3 mM for D283 and 2.2 mM for Daoy) of VPA. Results represent the mean ± SD of three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001 compared to the controls.

Figure 4.

VPA increases CDKN1A and TP53 while reducing MYC in MB-derived neurospheres. (A) Relative mRNA levels of CDKN1A and MYC in the D283- and Daoy-derived MB neurospheres were verified using RT-qPCR. (B) Relative mRNA levels of TP53 in the D283-derived MB neurospheres were verified using RT-qPCR. (C) ChIP-qPCR for H3K9ac occupancy of the promoter region of TP53 in the D283-derived neurospheres. Experiments were conducted using the IC₅₀ concentration (2.3 mM for D283 and 2.2 mM for Daoy) of VPA for 48 h. Results are the mean ± SD of three independent experiments; * p < 0.05, ** p < 0.01, and **** p < 0.0001 compared to the controls. For the ChIP results * p < 0.05 and ** p < 0.01 comparing the VPA-treated cells versus the controls immunoprecipitated with anti-IgG or anti-H3K9ac; # p < 0.05 comparing the samples immunoprecipitated with the same antibody (IgG versus H3K9ac) between the VPA-treated and control groups.

Figure 5.

VPA effects on features of neuronal differentiation in MB cells. (A) Representative images of morphological changes in the VPA-treated and control D283 and Daoy MB cells. (B) Relative mRNA levels of TUBB3 and ENO2 in the MB cells were verified using RT-qPCR. (C) Immunofluorescence assay against neuronal differentiation marker protein beta III tubulin and nuclei marker (DAPI). (D) Relative mRNA levels of TUBB3 and ENO2 in the VPA-treated and control neurospheres derived from either D283 or Daoy cells were measured using RT-qPCR. (E) Immunofluorescence assay against neuronal differentiation marker beta III tubulin and nuclei marker (DAPI) in the VPA-treated and control neurospheres. The IC₅₀ concentration of VPA (2.3 mM for D283 and 2.2 mM for Daoy) was used for treatment. Cells were exposed to VPA for 48 h. Fluorescent images were taken with an inverted microscope with a magnification of 10 X (monolayer) or 20 X (neurospheres). Results are the mean ± SD of three independent experiments; ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared to the controls.

Figure 6.

Schematic model highlighting selected aspects of VPA-induced molecular and functional effects in MB. Exposure to VPA leads to a reduction in MB cell cycle arrest and differentiation in the MB monolayer and neurospheres. VPA can increase TP53 while reducing MYC expression, potentially inhibiting stemness and promoting features of differentiation.