CHIR99021, trough GSK-3β Targeting, Reduces Epithelioid Sarcoma Cell Proliferation by Activating Mitotic Catastrophe and Autophagy

Abstract

Epithelioid sarcoma (ES) is a rare disease representing <1% of soft tissue sarcomas. Current therapies are based on anthracycline alone or in combination with ifosfamide or other cytotoxic drugs. ES is still characterized by a poor prognosis with high rates of recurrence. Indeed, for years, ES survival rates have remained stagnant, suggesting that conventional treatments should be revised and improved. New therapeutic approaches are focused to target the key regulators of signaling pathways, the causative markers of tumor pathophysiology. To this end, we selected, among the drugs to which an ES cell line is highly sensitive, those that target signaling pathways known to be dysregulated in ES. In particular, we found a key role for GSK-3β, which results in up-regulation in tumor versus normal tissue samples and associated to poor prognosis in sarcoma patients. Following this evidence, we evaluated CHIR99021, a GSK-3 inhibitor, as a potential drug for use in ES therapy. Our data highlight that, in ES cells, CHIR99021 induces cell cycle arrest, mitotic catastrophe (MC) and autophagic response, resulting in reduced cell proliferation. Our results support the potential efficacy of CHIR99021 in ES treatment and encourage further preclinical and clinical studies.

Keywords: CHIR99021; GSK-3β inhibition; autophagy; epithelioid sarcoma; mitotic catastrophe.

Figure 1

Overall survival (OS) curves, based on mRNA expression levels of the nine drug targets’ genes in the TCGA Sarcoma dataset. High (red) and low (black) expression cohorts were divided through maximally selected rank statistics. The significance of survival differences was estimated by log-rank tests. Number of patients: TGFBR1: low 98, high 161; ACVR1B: low 189, high 70; ACVR1C: low 231, high 28; GSK3A: low 25, high 234; GSK3B: low 117, high 142; NAE1: low 112, high 147; CDK4: low 185, high 74; CDK6: low 179, high 80; HDAC1: low 223, high 36.

Figure 2

Progression-free interval (PFI) curves, based on mRNA expression levels of the nine drugs’ target genes in the TCGA Sarcoma dataset. High (red) and low (black) expression cohorts were divided through maximally selected rank statistics. The significance of survival differences was estimated by log-rank tests. Number of patients: TGFBR1: low 104, high 155; ACVR1B: low 182, high 77; ACVR1C: low 191, high 68; GSK3A: low 232, high 27; GSK3B: low 121, high 138; NAE1: low 149, high 110; CDK4: low 195, high 64; CDK6: low 25, high 234; HDAC1: low 36, high 223.

Figure 3

Expression of the nine target genes in tumor and normal samples (TCGA Sarcoma dataset). Expression values of genes are log₂–transformed. Statistical significance was assessed by ANOVA. Number of patients: tumor = 262, normal = 2.

Figure 4

CHIR99021 affects the proliferation of as ES cell line. (A) CHIR99021’s effect on the viability of the VA-ES-BJ cell line evaluated using a trypan blue exclusion assay. Cell line was exposed to 100 µM for 24 and 48 h. Cell viability is presented as percentages distinguishing dead cells (light blue) from live cells (blue). The graph represents means ± SE of three independent experiments; differences between treatments groups were estimated by paired t tests. (B) Dot plot diagrams following 24 and 48 h treatments of VA-ES-BJ cells with CHIR99021 analysed by FACS, after Annexin V-FITC and PI labelling. Representative dot plots present intact cells in the lower-left quadrant, early apoptotic cells in the lower-right quadrant, late apoptotic or necrotic cells in the upper-right quadrant and necrotic cells in the upper-left quadrant. Histogram reported the mean ± SE of apoptotic cell percentage from three separate experiments. (C) Representative plots of DNA content distribution of cell cycle phases of VA-ES-BJ cell treated with CHIR99021 100 µM for 24 and 48 h. Histogram represents the percentage of cell number in the G₀/G₁, S, and G₂/M phases after treatment. The values are representative of means ± SE of three separate experiments using paired t tests (*: p < 0.05; **: p < 0.001). (D) Western blot analysis showing the expression of GSK-3α/β and p-GSK-3α/β proteins in VA-ES-BJ cell after treatment with CHIR99021 or vehicle for 24 and 48 h. The same blot was reprobed with anti-β-Actin to confirm the equal loading of each lane (40 μg of whole lysate). (E) Western blot analysis reported the expression levels of protein related to cell cycle signals at 24 and 48 h after CHIR99021 100 µM treatment. The blot was first incubated with anti-Cdk1/Cdk2, anti-CyclinB1, anti-phospho-HistoneH3, anti-HistoneH3, anti-phospho-β-Catenin and then reprobed with anti-β-Actin to confirm the equal loading of each lane (40 μg of whole lysate).

Figure 5

CHIR99021 induces mitotic catastrophe in the ES cell line. (A) Cells were treated with CHIR99021 100 µM for 24 or 48 h, stained with Hoechst-33342, and examined under fluorescence microscopy at 63X magnification. As shown, many cells have characteristics peculiar of mitotic catastrophe. The aberrant morphology of cells was based on the evaluation of at least 30 nuclei in each sample. Each bar is the mean ± S.E. of three separate experiments; * p < 0.05, versus the relative control. (B) Western blot analysis reported the expression levels of protein related to mitotic or spindle checkpoint arrest at 24 and 48 h after CHIR99021 100 µM treatment on VA–ES–BJ. The blot was first incubated with anti-MAD2, anti-p21 Waf1/Cip1 and anti-γ-H2A.X and then reprobed with anti-β-Actin to confirm the equal loading of each lane (40 μg of whole lysate).

Figure 6

CHIR99021 induces autophagy in the VA-ES-BJ cell line. Cells were treated with CHIR99021 100 µM for 24 or 48 h and detecting LC3A/B levels by immunofluorescences. (A) Increase of LC3-positive puncta formation supported the involvement of autophagy in VA-ES-BJ cells. (B) Western blot analysis reporting the expression levels of protein related to autophagic process at 24 and 48 h after CHIR99021 100 µM treatment. The blot was first incubated with anti-LC3A/B and then reprobed with anti-β-Actin to confirm the equal loading of each lane (60 μg of whole lysate).

Figure 7

CHIR99021 promotes autophagy through AMPK/mTORC1/ULK1 axis. Western blots showing levels of specific autophagy markers in VA-ES-BJ cells measured at 24 and 48 h after exposure to CHIR99021 100 µM. The blot was first incubated with anti-mTOR, anti-phospho-mTOR, anti-phospho-RAPTOR, anti-AMPK, anti-phospho-AMPK, anti-phopho-ULK1 and then reprobed with anti-β-Actin to confirm the equal loading of each lane (60 μg of whole lysate).

Figure 8

CHIR99021 affects the proliferation of the NEPS cell line. (A) Western blot analysis showing the expression of GSK-3α/β and phospho-GSK-3α/β proteins in NEPS cells after treatment with CHIR99021 or vehicle for 24 and 48 h. The same blot was reprobed with anti-β-Actin to confirm the equal loading of each lane (40 μg of whole lysate). (B) CHIR99021 effect on the viability of the NEPS cell line, evaluated using a trypan blue exclusion assay. Cells were exposed to 50 µM for 24 and 48 h. Cell viability is presented as percentages distinguishing dead cells (light blue) from live cells (blue). The graph represents means ± SE of three independent experiments; differences between treatments groups were estimated by paired t tests. (C) Distribution of cell cycle phases of NEPS cell treated with CHIR99021 50 µM for 24 and 48 h. Histogram represents the percentage of cell number in the G₀/G₁, S, and G₂/M phases after treatment. The values are representative of means ± SE of three separate experiments using paired t test (*: p < 0.05; **: p < 0.001). (D) CHIR99021 induces mitotic catastrophe in the NEPS cell line. Cells were treated with CHIR99021 50 µM for 24 or 48 h, stained with Hoechst-33342, and examined with fluorescence microscopy at 63X magnification. The aberrant morphology of cells was based on the evaluation of at least 30 nuclei in each sample. Each bar is the mean ± S.E. value from three separate experiments; (* p < 0.05, ** p < 0.001 versus the relative controls. (E) Cells were treated with CHIR99021 50µM for 24 or 48 h and LC3A/B-fluorescent puncta were detected.