Synergistic effect of JQ1 and rapamycin for treatment of human osteosarcoma

Abstract

Bromodomain and extra terminal domain (BET) proteins are important epigenetic regulators facilitating the transcription of genes in chromatin areas linked to acetylated histones. JQ1, a BET protein inhibitor, has antiproliferative activity against many cancers, mainly through inhibition of c-MYC and upregulation of p21. In this research, we investigated the use of JQ1 for human osteosarcoma (OS) treatment. JQ1 significantly inhibited the proliferation and survival of OS cells inducing G1 cell cycle arrest, premature senescence, but little effect on apoptosis. Interestingly, c-MYC protein levels in JQ1-treated cells remained unchanged, whereas the upregulation of p21 protein was still observable. Although effective in vitro, JQ1 alone failed to reduce the size of the MNNG/HOS xenografts in immunocompromised mice. To overcome the resistance of OS cells to JQ1 treatment, we combined JQ1 with rapamycin, an mammalian target of rapamycin (mTOR) inhibitor. JQ1 and rapamycin synergistically inhibited the growth and survival of OS cells in vitro and in vivo. We also identified that RUNX2 is a direct target of bromodomain-containing protein 4 (BRD4) inhibition by JQ1 in OS cells. Chromatin immunoprecipitation (ChIP) showed that enrichment of BRD4 protein around RUNX2 transcription start sites diminished with JQ1 treatment in MNNG/HOS cells. Overexpression of RUNX2 protected JQ1-sensitive OS cells from the effect of JQ1, and siRNA-mediated inhibition of RUNX2 sensitized the same cells to JQ1. In conclusion, our findings suggest that JQ1, in combination with rapamycin, is an effective chemotherapeutic option for OS treatment. We also show that inhibition of RUNX2 expression by JQ1 partly explains the antiproliferative activity of JQ1 in OS cells.

Keywords: JQ1; RUNX2; osteosarcoma; rapamycin; synergism.

© 2014 UICC.

Figures

Figure 1

Effect of JQ1 on the proliferation and survival of human osteosarcoma (OS) cells in vitro. (a) Dose-dependent antiproliferative activity of JQ1 against seven human OS cells in vitro as measured by LDH cytotoxicity assay after 72 hr exposure to JQ1. Data represent mean % LDH activity ± standard deviation (SD; error bars). (b) Time-dependent antiproliferative activity of JQ1. Cells were exposed to 7.5 µM JQ1 for 2, 12 and 24 hr, washed extensively, and cultured in JQ1-free media. Cell growth at 24–96 hr was measured by MTT assay. Data represent mean % cell growth ± SD. (c) Soft agar colony formation assay results. Cells were mixed in soft agar and cultured for 2 weeks in the presence or absence of 7.5 µM JQ1 followed by counting. Data represent mean number of colonies ± SD of three experiments done in triplicates. Asterisks (*) indicate p-value less than 0.05 (*) or 0.001 (**) by t-test. NS: not significant.

Figure 2

Effect of JQ1 on cell cycle, apoptosis and senescence of human OS cells in vitro. (a) G1 cell cycle arrest after 24 hr exposure to 7.5 µM JQ1. Cells were stained with propidium iodide (PI) and analyzed by FACS. (b) Apoptosis of OS cells after 72 hr exposure to 7.5 µM JQ1. Cells were stained with PI and Annexin V-FITC and analyzed by FACS. Numbers indicate % gated cells in early (top) and late (bottom) apoptosis. (c) Senescence of OS cells after 72 hr exposure to 7.5 µM JQ1. Senescence-associated β-galatosidase (SA-β-gal) activity was measured by X-gal staining. Senescent cells stained dark green. Magnification ×200.

Figure 3

Effect of JQ1 on expression of c-MYC, p21 and SIRT1 in human OS cells. Cells were treated with either DMSO (control) or 7.5 µM JQ1 subjected to qRT-PCR (6 hr) and Western blot analyses (12 hr). (a) mRNA expression level of c-MYC and p21 genes in JQ1-treated cells. Dotted lines indicate the normalized expression level of each gene in DMSO-treated control cells. GAPDH was used as a loading control. Measurements were repeated in triplicates. Data represent the mean relative expression ± standard deviation (SD; error bars). Asterisks (*) indicate p-value less than 0.05 (*) or 0.001 (**) by t-test. (b) Representative images showing changes in protein level of c-MYC, SIRT1 and p21 in JQ1-treated cells. Numbers indicate relative band intensity of each gene normalized to the intensity of the genes in control (1.0). GAPDH was used as a loading control.

Figure 4

Effect of combination of JQ1 and rapamycin on OS cells in vitro. MNNG/HOS and SJSA cells were grown in various concentrations of JQ1 and rapamycin, and the dose–response relationship was determined after 48 hr by LDH assay (Supporting Information Fig. S2a and Supporting Information Table S3). (a) Fraction affected (Fa)-Combination index (CI) plot for MNNG/HOS and SJSA cells exposed to JQ1 and rapamycin. Fa-CI plots were generated using selected data points with constant concentration ratio of JQ1 and rapamycin (1:1). (b) Changes in CI values at different drug concentrations (ED50, ED75 and ED90) of JQ1 and rapamycin in MNNG/HOS and SJSA cells. Data represent mean CI ± standard deviation (SD). (c) The effect of JQ1 (7.5 µM) and/or rapamycin (12.5 nM) on the mRNA level of c-MYC and p21 genes of MNNG/HOS and SJSA cells at 12 hr exposure. Expression levels of c-MYC and p21 were normalized to the expression level of GAPDH. Data represent mean relative expression ± SD. Asterisks (*) indicate p-value less than 0.05 (*) or 0.001 (**) by t-test. (d) Representative Western blot images showing changes in protein levels of the selected genes in the same condition as described in panel (c). Numbers indicate relative band intensity of each gene normalized to the intensity of the genes in control (1.0). GAPDH was used as a loading control. NS: not significant.

Figure 5

Effect of combination of JQ1 and rapamycin on human OS xenografts in vivo. MNNG/HOS xenografts were grown in athymic nude mice and treated with JQ1 and/or rapamycin 72 hr after cell injection (Day 0). Ctrl, diluent control; JQ1, JQ1 (50 mg/kg body weight, i.p. daily); Rapa, rapamycin (1.5 mg/kg, i.p. every other day). (a) Volumetric growth of MNNG/HOS xenografts. Data show mean tumor volume ± standard deviation (SD, error bars) of five mice per group. (b) Comparison of tumor weight of each group. At Day 24, mice were sacrificed and tumors were dissected and weighed. Data represent mean tumor weight ± SD of ten tumors per group. Asterisks (*) indicate p-value less than 0.05 (*) or 0.001 (**) by t-test. (c) H&E staining show representative tumor sections of each group. Arrows indicate the boundaries of necrotic tumor cells in the combination group. Magnification ×200. NS: not significant.

Figure 6

RUNX2 as a mediator of JQ1 activity in human OS cells. (a) Gene set enrichment analysis (GESA) plot showing downregulation of genes with RUNX2 binding motifs by JQ1 treatment in MNNG/HOS cells. Green lines represent the running enrichment score for the gene set as it progresses down the ranked list (x-axis) according to their differential expression score. Black vertical lines show the location of the members of the gene set. GESAs were done using either the differentially expressed genes (DEGs, left) or the whole transcriptome (right) in the gene set. (b) Endogenous mRNA expression level of RUNX2 of seven human OS cell lines measured by qRT-PCR. Data indicate relative mean expression ± standard deviation (SD, error bars). RUNX2 expression level of each cell was normalized to the RUNX2 expression level of hFOB1.19 human osteoblast cells (set to 1.0). GAPDH was used as a loading control. (c) Changes in RUNX2 mRNA expression level of seven human OS cell lines upon JQ1 treatment (7.5 µM, 6 hr). Dotted line indicates the expression level of RUNX2 mRNA of each cell without JQ1 treatment (set to 1.0). GAPDH was used as a loading control. (d) Schematic diagram showing RUNX2 gene and the location of ChIP primers used in the study. Each short, black vertical bar with a number represents one of the seven exons of RUNX2. Two arrows with Roman numerals (I and II) display the location of two transcription start sites. Bar graph below shows enrichment of PCR products from MNNG/HOS cells treated with or without JQ1 (7.5 µM, 6 hr). Data represent relative fold enrichment ± SD. Asterisk (*) indicates p-value less than 0.05 by t-test. (e) Changes in p21 protein level in RUNX2-overexpressing MNNG/HOS cells. Cells were transfected with either empty vector (EV) or pEF-BOS-RUNX2 expression vector (RUNX2). After 48 hr, cells were treated with 7.5 µM JQ1 for 12 hr and harvested for Western blot analysis. Numbers indicate relative band intensity of each gene normalized to the intensity of the genes in control (1.0). GAPDH was used as a loading control. (f) Changes in p21 protein level in MNNG/HOS cells transfected with siRNA oligos against RUNX2. Cells were transfected with either scrambled control oligos (SCR) or two kinds of siRUNX2 oligos (oligo1 and oligo2). After 48 hr, cells were treated with 7.5 µM JQ1 for 12 hr and harvested for Western blot analysis. Numbers indicate relative band intensity of each gene normalized to the intensity of the genes in control (1.0). GAPDH was used as a loading control. NES: normalized enrichment score.