Targeting the Wnt/β-catenin pathway in human osteosarcoma cells

Abstract

Aberrant activation of Wnt signaling has been implicated in human osteosarcoma, which may provide a genetic vulnerability that can be targeted in osteosarcoma treatment. To test whether Wnt activation is necessary for osteosarcoma growth, colony formation, invasion, and metastasis, we treated human osteosarcoma cells with a small molecule inhibitor of Wnt/β-catenin, PRI-724, which suppresses Wnt/β-catenin-mediated transcription. We found increased protein levels of endogenous active-β-catenin in five human osteosarcoma cell lines. Treatment with PRI-724 was sufficient to inhibit human osteosarcoma 143B and SJSA-1 cell proliferation. Suppressed Wnt signaling was confirmed by decreased protein levels of the Wnt target Cyclin D1. Furthermore, we revealed significant inhibitory effects on cell migration, invasion, and colony formation in the human osteosarcoma cells. Using deposited data from next generation sequencing studies, we analyzed somatic mutations and gene expression of components in the Wnt/β-catenin pathway. We found somatic mutations and upregulated gene expression of many components in the Wnt/ β-catenin pathway, indicating activated Wnt signaling. Taken together, our results illustrate the critical role of Wnt/β-catenin signaling in human osteosarcoma pathogenesis and growth, as well as the therapeutic potential of Wnt inhibitors in the treatment of human osteosarcoma.

Keywords: Cyclin D1; PRI-724; Wnt/β-catenin signaling; osteosarcoma.

Figure 1. Expression analysis of Wnt pathway genes in human OS cells

Western blot analysis of active β-catenin, total β-catenin and β-actin proteins in human OS cell lines (143B, Saos-2, SJSA-1 , U-2 OS, MG-63) with human breast cancer cell high-level-expression controls (MCF7, MDA-MB-231) and additional human mesenchymal stem cell low-level-expression controls (hMSC). Representative blots are shown in the top panels, and densitometric quantification of the blots is shown in the bottom graph. N.D., not detectable.

Figure 2. Effect of PRI-724 on 143B cell proliferation

(A) Chemical structure of PRI-724 (modified from https://www.selleckchem.com/). (B) 143B cell proliferation assay upon PRI-724 treatment. (C) Western blotting shows CyclinD1 protein level following PRI-724 treatment in 143B cells. (D) Quantification of (C).

Figure 3. Effect of PRI-724 on 143B cell migration and invasion

(A) 143B cell wound closure assay upon PRI-724 treatment. Red dashed lines indicate cell migration fronts. (C) 143B cell migration in Transwell system with or without PRI-724 treatment. (E) 143B cell invasion through Transwells coated with Matrigel, with or without PRI-724 treatment. (B, D, F) Quantification of (A, C, E).

Figure 4. Effect of PRI-724 on SJSA-1 cell proliferation, migration, and invasion

(A) SJSA-1 cell proliferation assay upon PRI-724 treatment. (B) Western blotting shows CyclinD1 protein level following PRI-724 treatment in SJSA-1 cells. (C) SJSA-1 cell wound closure assay upon PRI-724 treatment. Red dashed lines indicate cell migration fronts. (D) SJSA-1 cell migration in Transwell system with or without PRI-724 treatment. (E) Quantification of (D). (F) SJSA-1 cell invasion through Transwells coated with Matrigel, with or without PRI-724 treatment. (G) Quantification of (F).

Figure 5. Effects of PRI-724 on 143B and SJSA-1 cell colony formation

(A) Images of crystal violet stained 143B and SJSA-1 colonies in wells. (B) Images of colonies under 2X magnification.

Figure 6. Gene expression of Wnt pathway components in human OS tumor samples

(A) Volcano-plot analysis of gene expression in human OS and normal tissue control. (n=18 paired samples). Differential transcripts with statistical significance (FDR < 0.05, -log10 of p-value, y-axis) are shown in red (< 1.5-fold change) or green (> 1.5-fold change) dots. Non-significant genes (FDR > 0.05) are shown in black or orange dots (1.5-fold cutoff). Arrows label select genes with significant differential expression. (B) Median RPKM values of differentially expressed genes between OS and normal tissue control (n=18 paired samples, median, Wilcoxon signed-rank test, ^*, P< 0.05; ^**, P<0.01; ^***, P<0.001).

Figure 7. A hypothetical model of Wnt signaling in OS genesis and development

Constitutive Wnt/β-catenin signal activation is common in human osteosarcoma while activating genetic mutations of Wnt pathway components in osteosarcoma are rare. Wnt/β-catenin signaling may play critical roles in OS proliferation, metastasis and OS cancer stem cell (CSC) maintenance.