Aberrant Wnt signaling plays a major role in multiple cancers, including OS (7
). Therefore, inhibition of Wnt effects in OS may represent major therapeutic potentials. At present, little is known about the functional role of naturally occurring, secreted Wnt antagonists, including WIF-1 in sarcoma. In this study, we showed that the expression of WIF-1 was commonly down-regulated in OS cell lines and human tumor tissues. By re-expressing WIF-1 in OS cell line 143B, we demonstrated inhibition of anchorage independent growth and cellular motility. Furthermore, the marked inhibitory effect of WIF-1 on both tumor growth and metastasis was demonstrated in animal models. These findings strongly suggest a tumor suppressive role for WIF-1 in human OS.
In this study, WIF-1 promoter was found to be hypermethylated in multiple OS cell lines, which closely related to the down-regulation of WIF-1 mRNA expression in these cell lines when compared to normal osteoblasts. In addition, immunohistochemical staining of 50 paraffin-embedded OS patient tissue samples with a WIF-1 antibody showed reduced WIF-1 expression in the majority (76%) of the samples. These results suggest WIF-1 may function as a common tumor suppressor in OS. However, our study has been restricted to a small sample size of paraffin embedded tissues obtained through the Cooperative Human Tissue Network and the Children’s Oncology Group. To determine whether WIF-1 expression has prognostic value for clinical OS, we have obtained IRB approval for collecting surgical specimens and clinicopathologic data to establish an OS patient follow-up cohort. In addition, the exact mechanism by which WIF-1 is hypermethylated in OS cell lines is not very clear at this moment. Further studies are also ongoing to examine the mechanisms of WIF-1 inactivation by promoter hypermethylation in OS.
WIF-1 silencing due to promoter hypermethylation has also been shown in many other cancers including colorectal, prostate, bladder, melanoma, lung cancers, etc. (26
). Restoring WIF-1 expression in these cancer cells to study its biological function has been done by several groups (26
). The commonly described effect of WIF-1 on these cancer cells is the inhibition of cell growth (26
). In this study, we showed the inhibitory effect of WIF-1 expression in OS on cell motility, anchorage-independent growth in soft agar, and in vivo
tumor growth in nude mice of 143B cells. These results are consistent with reported findings in melanoma, esophageal adenocarcinoma, and bladder cancer (31
). However, we are unable to show the inhibitory effect of WIF-1 on anchorage-dependent growth in cell cultures (data not shown) as reported in other cancers (31
). During our investigation, a publication by Kansara et al. (34
) showed that recombinant WIF-1 protein can significantly decrease 143B cell proliferation at concentrations up to 2μg/ml. We also recently reported that recombinant WIF-1 protein achieved similar anti-proliferative effect in bladder cancer cell lines (33
). It is possible that high concentration of WIF-1 is needed to achieve its anti-proliferative effect or apoptosis and that gene transfection may not as effective as recombinant protein as a therapeutic agent. The mechanisms of cell motility and anchorage independence remain unclear for 143B cell line and likely are the result of several context dependent processes. We are currently investigating these mechanisms through detailed microarray analysis. However, difference in cell motility did not appear to involve changes in the epithelial to mesenchymal transition (EMT), given E-cadherin level did not change significantly in 143B cells after WIF-1 transfection (data not shown).
The complexity of Wnt extracellular and membrane components which consist of at least 19 Wnts, 10 Frizzled receptors, and co-receptors LRP5 and 6 suggest the existence of multiple Wnt-receptor interactions for initiation of Wnt signaling in OS. At this moment, little is known about the exact Wnt-receptor interactions for OS development and progression. It is reasonable to speculate that the difference between our results and others in the effect of WIF-1 anchorage-dependent growth of 143B cells may be due to context-dependent effect of WIF-1 on Wnt activity and cell growth inhibition. Further studies are necessary to determine which Wnt-receptor interactions are predominantly affected by WIF-1 in OS.
Patients with OS frequently present with hematogenous metastasis to the lungs (35
). Despite multi-modality treatment including surgery and chemotherapy, five-year survival of patients with relapsed OS remains approximately 20% (37
). At present, there is no targeted anti-metastatic therapy available for OS. To our best knowledge, we are first to report that WIF-1 markedly inhibits tumor metastasis in an orthotopic animal model of OS. This model of intra-tibial injection of 143B cells leading to 100% lung metastasis (20
) closely recapitulates the process of OS tumor metastasis in human. Therefore, our results suggest the potential for developing WIF-1 as a targeted anti-metastatic agent for clinical use in OS cases with aberrant Wnt signaling.
Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that can degrade the ECM and facilitate cellular invasion and migration (41
). High MMP-9 expression was observed in pre-treatment OS tumor samples and in a majority of metastatic lesions, leading to speculation that MMP-9 expression is associated with the micrometastatic behavior of OS (42
). Membrane-type metalloproteinase (MT1-MMP), also known as MMP-14, has been shown to also play a critical role in metastasis (43
). MMP-9 and 14 are transcriptional targets of Wnt signaling (23
) and have been correlated with poor disease-free survival in OS (44
). We demonstrated that WIF-1 overexpression reduced MMP-9 and MMP-14 protein expression in 143B cells. Taken together, these results suggest that WIF-1 exhibits its markedly anti-tumor metastasis effect in OS through multiple complex mechanisms. Further studies are in progress to dissect pathways modulated by WIF-1 for metastasis in OS.
Based on its significant anti-tumor and anti-metastasis effects in vivo in animal models of OS, WIF-1 represents a promising target for developing therapeutic and preventive strategies against metastatic OS with aberrant Wnt signaling. At this point, the interaction of WIF-1 with key Wnt components at the membrane and extracellular levels needs further delineation. In addition, study of WIF-1 regulated pathways for OS tumor growth and metastasis may be helpful for identification of biomarkers and targets for WIF-1 deficient OS as WIF-1 expression is commonly down-regulated in this disease.