β-Catenin transcriptional activity is minimal in canine osteosarcoma and its targeted inhibition results in minimal changes to cell line behaviour

Abstract

Canine osteosarcoma (OS) is an aggressive malignancy associated with poor outcomes. Therapeutic improvements are likely to develop from an improved understanding of signalling pathways contributing to OS development and progression. The Wnt signalling pathway is of interest for its role in osteoblast differentiation, its dysregulation in numerous cancer types, and the relative frequency of cytoplasmic accumulation of β-catenin in canine OS. This study aimed to determine the biological impact of inhibiting canonical Wnt signalling in canine OS, by utilizing either β-catenin siRNA or a dominant-negative T-cell factor (TCF) construct. There were no consistent, significant changes in cell line behaviour with either method compared to parental cell lines. Interestingly, β-catenin transcriptional activity was three-fold higher in normal canine primary osteoblasts compared to canine OS cell lines. These results suggest canonical Wnt signalling is minimally active in canine OS and its targeted inhibition is not a relevant therapeutic strategy.

Keywords: cell signalling; comparative oncology; in vitro models; oncology; tumour biology.

Figure 1

Figure 1A–C. Evaluation of siRNA Efficacy in targeting β-catenin. Significant reductions in (A) β-catenin mRNA expression, (B) β-catenin protein expression, and (C) β-catenin protein as measured by densitometry were achieved following treatment with siRNA1 or siRNA4 compared to a negative control siRNA. HDAC was used as a loading control for the western blot and densitometry measurements. All calculations are a 2-way ANOVA with Sidak’s multiple comparison post-test.

Figure 2

Figure 2A–G. Evaluation of siRNA targeting β-catenin behavioral effects. There were no consistent differences between siRNA-treated cells and negative control siRNA-treated cells for (A) proliferation at 24 hours, (B) proliferation over a 72-hour time course, (C) apoptosis, (D) doxorubicin sensitivity, (E) invasion, or (F) migration. There was also no change in β-catenin transcriptional activity as measured by TCF-responsive luciferase reporter assay (G). All calculations are a 2-way ANOVA with Sidak’s post-test.

Figure 3

Figure 3A–G. Evaluation of D17 cells stably expressing dnTCF4. (A) Following incorporation of the dnTCF4 construct, both clones exhibited reduced β-catenin transcriptional activity by TCF-responsive luciferase reporter assay. This was matched with reductions in proliferation (B) and doxorubicin sensitivity (E), but not alterations in apoptosis (C), carboplatin sensitivity (D), invasion (F), or migration (G). All calculations are a 1-way ANOVA with Dunnett’s post-test, except D and E, which are 2-way ANOVA with Sidak’s post-test.

Figure 4

Evaluation of β-catenin transcriptional activity in canine normal primary osteoblasts compared to canine OS cells. Canine normal primary osteoblasts (k9Ob) exhibit a 2.7-fold higher β-catenin transcription activation than D17 cells (p=0.0055), and 2.9-fold higher than Abrams cells (p=0.0042). Calculation is a 1-way ANOVA with Dunnett’s post-test.