It has been reported that the progression of osteosarcoma was closely associated with the aberrant activation of canonical Wnt signaling. Wnt inhibitory factor-1 (WIF-1) is a secreted Wnt inhibitor whose role in human osteosarcoma remains unknown. In this study, WIF-1 expression in NHOst and osteosarcoma cell lines was determined by real-time reverse transcription-PCR, methylation-specific PCR, and Western blotting analysis. In addition, tissue array from patient samples was examined for WIF-1 expression by immunohistochemistry. Compared with normal human osteoblasts, WIF-1 mRNA and protein levels were significantly downregulated in several osteosarcoma cell lines. The downregulation of WIF-1 mRNA expression is associated with its promoter hypermethylation in these tested cell lines. Importantly, WIF-1 expression was also downregulated in 76% of examined osteosarcoma cases. These results suggest that the downregulation of WIF-1 expression plays a role in osteosarcoma progression. To further study the potential tumor suppressor function of WIF-1 in osteosarcoma, we established stable 143B cell lines overexpressing WIF-1. WIF-1 overexpression significantly decreased tumor growth rate in nude mice as examined by the s.c. injection of 143B cells stably transfected with WIF-1 and vector control. WIF-1 overexpression also markedly reduced the number of lung metastasis in vivo in an orthotopic mouse model of osteosarcoma. Together, these data suggest that WIF-1 exerts potent antiosteosarcoma effect in vivo in mouse models. Therefore, the reexpression of WIF-1 in WIF-1-deficient osteosarcoma represents a potential novel treatment and preventive strategy. Mol Cancer Ther; 9(3); 731-41. ©2010 AACR.
Purpose of Study: To investigate the role of the Neuropilin-1 (NRP-1) in angiogenesis and regulation by Wnt-signaling pathway in osteosarcoma (OS) cell lines. Methods: In vitro tube formation assay using HUVEC cells to examine the effect of Frzb, WIF1, and DKK3 on angiogenesis. Microarray and real-time PCR to assess the mRNA level of NRP-1 in SaOS-2 cells transfected with Dominant Negative LRP5 (DNLRP5) and PcDNA vector control. Western blots were performed to evaluate the expression of NRP-1 in 8 OS cell lines and normal osteoblast (NHOst), as well as the effect of Wnt inhibitors (DNLPR5, Frzb, WIF-1, DKK3) on NRP-1 expression in OS cell lines (LM7, 143B, and 143.98.2) Results: In order to develop a new therapeutic target for OS, the most common primary malignant bone tumor, we examined the expression of NRP-1 and vascular endothelial growth factor (VEGF) in 8 established OS cell lines, as well as NHOst. Both VEGF and NRP-1 (a co-receptor of VEGF) were up-regulated in most OS cell lines compared with normal osteoblast, suggesting that VEGF and NRP-1 may be responsible for the excessive blood vessel formation in OS. Given our previous report that Wnt inhibitors Frzb, WIF1, DKK3, and DNLRP5, inhibited tumor growth and metastasis of OS, we hypothesize that one of the potential mechanisms may be through inhibition of tumor angiogenesis. When HUVEC assay was examined, DKK3, Frzb, WIF-1 condition medium significantly inhibited tube formation compared with control, suggesting that these Wnt inhibitors suppressed angiogenesis. Further microarray assay, real time PCR and western blots indicated that these Wnt inhibitors (DKK3, Frzb, WIF-1 and DNLRP5) dramatically down-regulated the expression of NRP-1, but VEGF level remained unchanged. These results show that inhibition of angiogenesis by Wnt signaling may be mediated by down-regulation of NRP-1 instead of VEGF. Discussion and Conclusions: Our study suggests that the Wnt-signaling reduces tumor growth and metastasis by inhibiting angiogenesis and by down-regulating NRP-1 expression in OS. We are further studying the role of NRP-1 in osteosarcoma by knockdown of NRP1 expression in OS cells to assess tumor proliferation, metastases and angiogenesis. This may represent a novel therapeutic strategy for osteosarcoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3963.
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