Abstract
Although osteosarcoma represents the most common bone malignancy, the molecular and cellular mechanisms influencing its pathogenesis have remained elusive. Recent evidence has suggested that the Wnt signaling pathway may play a crucial role in osteosarcoma. This study employed a microarray approach to discover novel genes and pathways involved in Wnt signaling in osteosarcoma. We developed a Wnt10b-expressing cell line using the human U2OS osteosarcoma model (U2OS-Wnt10b) and performed microarray and pathway analyses using parental U2OS cells as control. Differential expression of 1,003 genes encompassing 28 pathways was noted. The Wnt, NFκB, and Notch pathways were chosen for further study based on their known importance in bone biology. Known Wnt-responsive genes Axin-2 (4.9-fold), CD44 (2.1-fold), endothelin-1 (4.2-fold) and sclerostin domain containing-1 (43-fold) were regulated by Wnt10b. The proinflammatory cytokines interleukin-1α and tumor necrosis factor-α, known inducers of NFκB, were upregulated both at the transcript and protein level, and NFκB reporter activity was stimulated 3.8-fold, confirming NFκB activation. Interestingly, genes involved in Notch signaling [Notch-1 (2.4-fold) and Jagged-1 (3.1-fold)] were upregulated, whereas the Notch inhibitor, lunatic fringe, was downregulated (8.2-fold). This resulted in the activation of the classic Notch-responsive genes, hairy and enhancer of split-1 (Hes-1; 2.2-fold) and hairy/enhancer-of-split related with YRPW motif-1 (Hey-1; 2.5-fold). A Hey-1 reporter construct was regulated 9.1-fold in U2OS-Wnt10b cells, confirming Notch activation. Interestingly, Wnt3a failed to induce the Notch and NFκB pathways, demonstrating Wnt-specificity. In conclusion, our data demonstrate that Wnt10b, but not Wnt3a, stimulates the NFκB and Notch pathways in U2OS osteosarcoma cells.