Nuclear receptor modulators inhibit osteosarcoma cell proliferation and tumour growth by regulating the mTOR signaling pathway

Abstract

Osteosarcoma is the most common primary malignant bone tumour in children and adolescents. Chemoresistance leads to poor responses to conventional therapy in patients with osteosarcoma. The discovery of novel effective therapeutic targets and drugs is still the main focus of osteosarcoma research. Nuclear receptors (NRs) have shown substantial promise as novel therapeutic targets for various cancers. In the present study, we performed a drug screen using 29 chemicals that specifically target 17 NRs in several different human osteosarcoma and osteoblast cell lines. The retinoic acid receptor beta (RARb) antagonist LE135, peroxisome proliferator activated receptor gamma (PPARg) antagonist T0070907, liver X receptor (LXR) agonist T0901317 and Rev-Erba agonist SR9011 significantly inhibited the proliferation of malignant osteosarcoma cells (U2OS, HOS-MNNG and Saos-2 cells) but did not inhibit the growth of normal osteoblasts. The effects of these NR modulators on osteosarcoma cells occurred in a dose-dependent manner and were not observed in NR-knockout osteosarcoma cells. These NR modulators also significantly inhibited osteosarcoma growth in vivo and enhanced the antitumour effect of doxorubicin (DOX). Transcriptomic and immunoblotting results showed that these NR modulators may inhibit the growth of osteosarcoma cells by regulating the PI3K/AKT/mTOR and ERK/mTOR pathways. DDIT4, which blocks mTOR activation, was identified as one of the common downstream target genes of these NRs. DDIT4 knockout significantly attenuated the inhibitory effects of these NR modulators on osteosarcoma cell growth. Together, our results revealed that modulators of RARb, PPARg, LXRs and Rev-Erba inhibit osteosarcoma growth both in vitro and in vivo through the mTOR signaling pathway, suggesting that treatment with these NR modulators is a novel potential therapeutic strategy.

Fig. 1. Identification of candidate NR modulators for inhibiting osteosarcoma cell growth.

U2OS and HOS-MNNG cells were treated with the 29 indicated NR modulators that target 17 NRs at a concentration of 10 μM. A–C Eleven NR modulators significantly inhibited U2OS cell growth. D–F Ten NR modulators significantly inhibited HOS-MNNG cell growth. Saos-2 (G) and hFOB 1.19 (H) cells were treated with the 5 indicated modulators that targeted a single nuclear receptor at a concentration of 10 μM. The cell confluence was evaluated in real time by the IncuCyte Zoom living cell imaging system. Mean ± SD, n = 6; *P < 0.05, the NR modulator group versus the Ctrl group.

Fig. 2. NR modulators inhibited osteosarcoma cell growth by specifically targeting NRs in a dose-dependent manner.

A–D U2OS, E–H HOS-MNNG, and Saos-2 (I–L) cells were treated with the RARb antagonist, PPARg antagonist, LXR agonist and Rev-Erba agonist at gradient concentrations (1, 5, or 10 μM). The cell confluence was evaluated in real time by the IncuCyte zoom living cell imaging system. n = 6; *P < 0.05, the NR modulator group versus the Ctrl group. M–P Wild-type (Wt) and NR-knockout (KO) U2OS cells were treated with the corresponding NR modulators at a concentration of 10 μM. The cell confluence was evaluated in real time by the IncuCyte zoom living cell imaging system. Mean ± SD, n = 6; *P < 0.05, the NR-KO group versus the Wt group. ^#P < 0.05, the NR modulator group versus the saline group. △Confluence represents the difference in confluence between the saline group and the NR modulator group. The violin plots show the distribution of the △Confluence of the indicated genotypes. Paired t-tests were used to evaluate the significance. *P < 0.05, the NR KO group versus the Wt group.

Fig. 3. Osteosarcoma cell proliferation was repressed by NR modulator treatments.

U2OS, HOS-MNNG and Saos-2 cells were treated with the RARb antagonist, PPARg antagonist, LXR agonist and Rev-Erba agonist at 10 μM for 2 days. A The KI-67 protein levels were evaluated by immunofluorescence. Scale bar: 100 μm. B The relative KI-67-positive area was normalized to the costained DAPI-positive area. Mean ± SD, n = 5; *P < 0.05, the NR modulator group versus the Ctrl group. C Cell lysates were collected. The protein levels of PCNA were evaluated by immunoblotting. D An MTT viability assay was performed to analyse the cell viability of each group. Mean ± SD, n = 5; *P < 0.05, the NR modulator group versus the Ctrl group. E Cell cycle analysis was performed after cell collection, fixation and PI staining. The proportions of cells in the G0/G1, S, and M/G2 phases in each group are shown. Mean ± SD, n = 3; *P < 0.05, the NR modulator group versus the Ctrl group.

Fig. 4. In vivo antitumour effects of the NR modulators.

HOS-MNNG cells (5 × 10⁶) were subcutaneously injected into BALB/c nude mice. Beginning on the 3^rd day after tumour inoculation, the mice were subcutaneously injected with NR modulators (1 μg dissolved in 100 μl saline) or 100 μl saline in situ every 3 days. A Representative images show tumour formation after NR modulator treatment. B The tumour volumes were calculated every day after inoculation. C, D After the mice were sacrificed, the tumours were harvested and weighed. Mean ± SD, n = 8; *P < 0.05, the NR modulator group versus the Ctrl group. To evaluate the synergistic effect of NR with DOX, mice were intraperitoneally injected with DOX (5 mg/kg) or saline and subcutaneously injected in situ with the NR modulators (1 μg dissolved in 100 μl saline) or saline every 3 days beginning on the 6^th day after tumour inoculation. E Representative images show tumour formation in each group. F The tumour volumes were calculated every day after inoculation. G, H After the mice were sacrificed, the tumours were harvested and weighed. Mean ± SD, n = 8; *P < 0.05, the DOX or DOX + NR modulator group versus the Ctrl group. ^#P < 0.05, the DOX + NR modulator group versus the DOX group.

Fig. 5. mTOR acted as a core downstream pathway by which the NR modulators inhibited the growth of osteosarcoma.

U2OS cells were treated with the RARb antagonist, PPARg antagonist, LXR agonist and Rev-Erba agonist at 10 μM for 24 h. The total RNA of cells was extracted for RNA-seq analysis, and the DEGs of each group were identified (n = 3). A Volcano plots show all the genes that were statistically upregulated or downregulated and the number of DEGs in each group. B Functional enrichment analysis was performed to identify significantly enriched KEGG pathways in each group. C Immunoblots show the levels of phosphorylated ERK1/2, p38, JNK, AKT, AMPK and mTOR in cells.

Fig. 6. Identification of DDIT4 as a key common target by which the NR modulators inhibited osteosarcoma growth.

A Plot shows the four genes that were commonly regulated by the NR modulators (upregulated in black, downregulated in white). B FPKM of the common targeted genes in each group. Mean ± SD, n = 3; *P < 0.05, the NR modulator group versus the Ctrl group. Wt and DDIT4-KO U2OS cells were treated with the RARb antagonist, PPARg antagonist, LXR agonist and Rev-Erba agonist at 10 μM. C The levels of phosphorylated mTOR and S6 were evaluated. D–G Cell growth was evaluated by the IncuCyte zoom living cell imaging system. Mean ± SD, n = 6; *P < 0.05, the DDIT4 KO group versus the Wt group. ^#P < 0.05, the NR modulator group versus the saline group. △Confluence represents the difference in confluence between the saline group and the NR modulator group. The violin plots show the distribution of the △Confluence of the indicated genotypes. Paired t-tests were used to evaluate the significance. *P < 0.05, the DDIT4 KO group versus the Wt group.