BackgroundAberrations in the Wnt pathway have been reported to be involved in the metastasis of prostate cancer (PCa) to bone. We investigated the effect and underlying mechanism of a naturally-occurring Wnt inhibitor, WIF1, on the growth and cellular invasiveness of a bone metastatic PCa cell line, PC3.ResultsThe WIF1 gene promoter was hypermethylated and its expression down-regulated in the majority (7 of 8) of PCa cell lines. Restoration of WIF1 expression in PC-3 cells resulted in a decreased cell motility and invasiveness via up-regulation of epithelial markers (E-cadherin, Keratin-8 and-18), down-regulation of mesenchymal markers (N-cadherin, Fibronectin and Vimentin) and decreased activity of MMP-2 and -9. PC3 cells transfected with WIF1 consistently demonstrated reduced expression of Epithelial-to-Mesenchymal Transition (EMT) transcription factors, Slug and Twist, and a change in morphology from mesenchymal to epithelial. Moreover, WIF1 expression significantly reduced tumor growth by approximately 63% in a xenograft mouse model. This was accompanied by an increased expression of E-cadherin and Keratin-18 and a decreased expression of vimentin in tumor tissues.ConclusionThese data suggest that WIF1 regulates tumor invasion through EMT process and thus, may play an important role in controlling metastatic disease in PCa patients. Blocking Wnt signaling in PCa by WIF1 may represent a novel strategy in the future to reduce metastatic disease burden in PCa patients.

Osteosarcoma is the most common primary bone malignancy, with a high propensity for local invasion, early metastasis and relapse. While the molecular mechanisms behind osteosarcoma development and metastasis have not yet been fully elucidated, research has highlighted an important role for Wnt signaling. Several Wnt ligands, receptors and coreceptors are highly expressed in osteosarcoma cell lines, while Wnt inhibitors are downregulated. As a result, research has begun to identify mechanisms with which to inhibit Wnt signaling. The use of Wnt pathway inhibitors and the targeting of c-Met, a Wnt regulated proto-oncogene, may be two possible mechanisms for treatment of osteosarcoma. In addition, as the Wnt signaling pathway is a regulator of stem cells, reagents that function as Wnt inhibitors are currently under investigation as inhibitors of cancer stem cell proliferation. Research involving the Wnt signaling pathway and cancer stem cells holds promise for novel treatment options in the future.

BackgroundNeuropilin 2 (NRP2) isa multi-functional co-receptor to many receptors, including VEGF receptor, c-Met and others. NRP2 has recently been implicated in tumor angiogenesis, growth, and metastasis of many other cancers. However, its role in osteosarcoma remains poorly understood.ResultsNRP2 was overexpressed in osteosarcoma cell lines and tissues, and associated with poor survival of osteosarcoma patients. Knockdown of NRP2 expression by short-hairpin (Sh) RNA resulted in reduced tumor growth, metastasis, and blood vessel formation of osteosarcoma. Knockdown of NRP2 expression by ShRNA also inhibited the recruitment of HUVEC cells to osteosarcoma cells. Inhibition of Wnt signaling by overexpression of secreted Wnt antagonists soluble LRP5, Frzb, and WIF1 markedly down-regulated mRNA and protein expression of NRP2 in osteosarcoma cell lines.ConclusionsRegulation of NRP2 receptor expression may represent a novel approach for treatment of osteosarcoma through retarding osteosarcoma growth, metastasis and blood vessel formation. In addition, down-regulation of NRP2 expression can be achieved by expression of secreted Wnt antagonists.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0359-4) contains supplementary material, which is available to authorized users.

Osteosarcoma (OS) is the most common primary bone malignancy with a high propensity for local invasion and distant metastasis. Despite current multidisciplinary treatments, there has not been a drastic change in overall prognosis within the past 2 decades. Dickkopf-3 protein (Dkk-3/REIC) has been known to inhibit canonical Wnt/β-catenin pathway, and its expression has been shown to be downregulated in OS cell lines. Using in vivo and in vitro studies, we demonstrated that Dkk-3-transfected 143B cells inhibited tumorigenesis and metastasis in an orthotopic xenograft model of OS. Inoculation of Dkk-3-transfected 143B cell lines into nude mice showed significant decreased tumor growth and less metastatic pulmonary nodules (88.7%) compared to the control vector. In vitro experiments examining cellular motility and viability demonstrated less anchorage-independent growth and decreased cellular motility for Dkk-3-transfected 143B and SaOS2 cell lines compared to the control vector. Downstream expressions of Met, MAPK, ALK, and S1004A were also downregulated in Dkk-3-transfected SaOS2 cells, suggesting the ability of Dkk-3 to inhibit tumorigenic potential of OS. Together, these data suggest that Dkk-3 has a negative impact on the progression of osteosarcoma. Reexpressing Dkk-3 in Dkk-3-deficient OS tumors may prove to be of benefit as a preventive or therapeutic strategy.

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.