Targeting the CK1α/CBX4 axis for metastasis in osteosarcoma

Abstract

Osteosarcoma, an aggressive malignant cancer, has a high lung metastasis rate and lacks therapeutic target. Here, we reported that chromobox homolog 4 (CBX4) was overexpressed in osteosarcoma cell lines and tissues. CBX4 promoted metastasis by transcriptionally up-regulating Runx2 via the recruitment of GCN5 to the Runx2 promoter. The phosphorylation of CBX4 at T437 by casein kinase 1α (CK1α) facilitated its ubiquitination at both K178 and K280 and subsequent degradation by CHIP, and this phosphorylation of CBX4 could be reduced by TNFα. Consistently, CK1α suppressed cell migration and invasion through inhibition of CBX4. There was a reverse correlation between CK1α and CBX4 in osteosarcoma tissues, and CK1α was a valuable marker to predict clinical outcomes in osteosarcoma patients with metastasis. Pyrvinium pamoate (PP) as a selective activator of CK1α could inhibit osteosarcoma metastasis via the CK1α/CBX4 axis. Our findings indicate that targeting the CK1α/CBX4 axis may benefit osteosarcoma patients with metastasis.

Fig. 1. CBX4 promotes cell migration and invasion by increasing Runx2 in osteosarcoma cells.

a–f, o The indicated proteins were analyzed by Western blotting in the indicated stable cells of three independent experiments. g–l, q Migration and invasion abilities were determined using the indicated stable cells as described in Methods section. m, n, p The relative mRNA levels of Runx2 were normalized to the GAPDH level in the indicated stable cells as determined by qRT-PCR. The bars indicate the SD. The results are expressed as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 using the two-sided Student’s t-test. n.s, no significance. Source data are provided as a Source Data file.

Fig. 2. CBX4 increases Runx2 via recruiting GCN5 to the Runx2 promoter in osteosarcoma cells.

a, b The indicated stable cells transfected with the Runx2-Luc reporter for 48 h were subjected to the luciferase activity assay as described in Methods section. c The ChIP assay was performed in U2OS cells using anti-CBX4 antibody or IgG antibody, as indicated, of three independent experiments. The p16 promoter was used as the positive control. d, e ChIP-qPCR analysis of the occupancies of CBX4, GCN5, H3K27Ac, and Pol II on the Runx2 promoter in the indicated stable cells. f U2OS/MTX300 cells were subjected to immunoprecipitation (IP) using anti-CBX4 antibody or anti-IgG antibody followed by Western blotting as indicated of three independent experiments. g, h U2OS cells expressing pSIN-Vector or pSIN-CBX4 were transiently transfected with siGCN5 as indicated and then analyzed by qRT-PCR (g) and Western blotting (h). i, j The indicated stable cells co-transfected with the Runx2-Luc reporter and HA-GCN5 for 48 h were subjected to the luciferase activity assay as described in Methods section. k–m The indicated U2OS/MTX300 cells were subjected to Western blotting, cell migration and invasion assays. The bars indicate the SD. The results are expressed as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 using the two-sided Student’s t-test. n.s no significance. Source data are provided as a Source Data file.

Fig. 3. CHIP is the E3 ligase for the ubiquitination and degradation of CBX4.

a Lysates from HEK293T cells were IP with anti-Flag agarose and subjected to SDS-PAGE and Coomassie staining. The indicated bands were sequenced by mass spectrometry (MS) analysis. b–d HEK293T cells transfected with the indicated plasmids or siRNAs for 48 h were analyzed Western blotting. These resluts are repeated of three independent experiments. e, h, k HEK293T cells co-transfected with the indicated plasmids for 36 h were incubated with 20 μg/ml cycloheximide (CHX) for the indicated periods and then analyzed by Western blotting. Quantitation of Flag-CBX4 protein levels was based on the Western blotting results. n = 1. f, g, j HEK293T cells were co-transfected with the indicated plasmids for 48 h and then subjected to IP using anti-FLAG antibody followed by Western blotting. i U2OS/MTX300 cells were subjected to IP using anti-CBX4 antibody or anti-IgG antibody followed by Western blotting as indicated. These resluts are repeated of three independent experiments. Source data are provided as a Source Data file.

Fig. 4. CK1α promotes the turnover of CBX4 by CHIP.

a, c HEK293T cells were cotransfected with the indicated plasmids for 48 h and then subjected to IP using anti-FLAG antibody followed by Western blotting analysis of three independent experiments. b HEK293T cells cotransfected with the indicated plasmids for 36 h were incubated with 20 μg/ml CHX for the indicated periods and then analyzed by Western blotting. Quantitation of Flag-CBX4 protein levels was based on the Western blotting results. n = 1. d U2OS/MTX300 cells were subjected to IP using anti-CBX4 antibody or anti-IgG antibody followed by Western blotting as indicated. e HEK293T cells cotransfected with the indicated plasmids for 48 h were analyzed by Western blotting. f HEK293T cells cotransfected with the indicated plasmids for 40 h were incubated with 10 μM MG132 for 8 h and then subjected to IP using anti-FLAG antibody followed by Western blotting. g Flag-CBX4 WT or T437A mutant was purified from HEK293T cells, incubated with or without the purified V5-CK1α in vitro as described in Methods, and then analyzed by Western blotting. h, i The co-IP assay was performed using the indicated U2OS/MTX300 stable cells with anti-CBX4 antibody or anti-IgG antibody as indicated. j–m The indicated cells were starved by excluding fetal bovine serum (FBS) from the medium for 24 h, and then the cells were treated with TNFα, RANKL, and M-CSF for 24 h as indicated and subjected to Western blotting (j–l) or the co-IP assay using anti-CBX4 antibody or anti-IgG antibody as indicated (m). These resluts are repeated of three independent experiments. Source data are provided as a Source Data file.

Fig. 5. Inhibition of cell migration and invasion by CK1α primarily depends on the decrease of CBX4 in osteosarcoma cells.

a–c, g–i The indicated proteins were analyzed by Western blotting in the indicated stable cells. d–f, j–l The migration and invasion abilities were determined in the indicated stable cells as described in Methods section. m The indicated U2OS/MTX300 cells were subjected to Western blotting, cell migration and invasion assays. The bars indicate the SD. The results are expressed as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 using the two-sided Student’s t-test. n.s no significance. Source data are provided as a Source Data file.

Fig. 6. Reverse correlation between CK1α and CBX4 in osteosarcoma tissues.

a Representative immunohistochemical staining images of both CBX4 and CK1α for 55 paraffin-embedded osteosarcoma tissues. Scale bar: 100 μm. b A positive correlation was observed between CBX4 and CK1α protein levels in the osteosarcoma tissues used in a (p = 0.022, χ² tests. R: Spearman correlation coefficient). *p < 0.05 using the two-sided Pearson chi-squared tests. c–f Overall survival curves were generated based on the protein levels of CK1α (c, e) or CBX4 (d, f) in the osteosarcoma tissues used in a. *p < 0.05 using Kaplan–Meier plots and compared with the log-rank test.

Fig. 7. Pyrvinium suppresses lung metastasis of osteosarcoma by inhibiting CBX4 via CK1α activation.

a, b The indicated cells were incubated with or without PP for 48 h and then subjected to Western blotting (a) and cell migration and invasion assays (b). c The indicated cells were incubated with or without PP for 48 h and then subjected to IP using anti-CBX4 antibody or anti-IgG antibody followed by Western blotting. d HEK293T cells transfected with the indicated plasmids 24 h were incubated with PP for 24 h and then subjected to IP using Flag-agarose followed by Western blotting. These resluts are repeated of three independent experiments. e, f The indicated U2OS/MTX300 stable cells were subjected to Western blotting (e) and cell migration and invasion assays (f). g, h The indicated U2OS/MTX300 stable cells were incubated with or without PP for 48 h and then subjected to Western blotting (g) and cell migration and invasion assays (h). n = 3. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 using the two-sided Student’s t-test. n.s no significance. i, j The indicated U2OS/MTX300-luc stable cells were used in the orthotopic osteosarcoma metastasis model with or without pp (0.5 mg/kg, three times a week) as described in Methods section; n = 6. Data are presented as mean values ± SD. *p < 0.05 using the two-sided Student’s t-test. n.s: no significance. Source data are provided as a Source Data file.

Fig. 8. A proposed model for both function and regulation of CBX4 in osteosarcoma.

CBX4 is overexpressed in osteosarcoma and recruits GCN5 to sustain H3K27Ac in the Runx2 promoter to transcriptionally up-regulateRunx2 and then to promote lung metastasis; the phosphorylation of CBX4 at T437 by CK1α, which is a tumor suppressor in osteosarcoma, facilitates its ubiquitination and degradation by CHIP; PP as a selective activator of CK1α promotes the degradation of CBX4, which may benefit osteosarcoma patients with high expression levels of CBX4.