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Tumor Suppressor WWOX Inhibits Osteosarcoma Metastasis by Modulating RUNX2 Function

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Tumor Suppressor WWOX Inhibits Osteosarcoma Metastasis by Modulating RUNX2 Function

Sara Del Mare et al. Sci Rep.

Abstract

Osteosarcoma (OS) is among the most frequently occurring primary bone tumors, primarily affecting adolescents and young adults. This malignant osteoid forming tumor is characterized by its metastatic potential, mainly to lungs. We recently demonstrated that WW domain-containing oxidoreductase (WWOX) is frequently inactivated in human OS and that WWOX restoration in WWOX-negative OS cells suppresses tumorigenicity. Of note, WWOX levels are reduced in paired OS samples of post-treatment metastastectomies as compared to pre-treatment biopsies suggesting that decreased WWOX levels are associated with a more aggressive phenotype at the metastatic site. Nevertheless, little is known about WWOX function in OS metastasis. Here, we investigated the role of tumor suppressor WWOX in suppressing pulmonary OS metastasis both in vitro and in vivo. We demonstrated that ectopic expression of WWOX in OS cells, HOS and LM-7, inhibits OS invasion and cell migration in vitro. Furthermore, WWOX expression reduced tumor burden in vivo and inhibited metastases' seeding and colonization. Mechanistically, WWOX function is associated with reduced levels of RUNX2 metastatic target genes implicated in adhesion and motility. Our results suggest that WWOX plays a critical role in determining the aggressive phenotype of OS, and its expression could be an attractive therapeutic target to combat this devastating adolescent disease.

Figures

Figure 1
Figure 1. Restoration of WWOX in OS cells suppresses migration and invasion in vitro.
(A) Western blot analysis of WWOX expression in HOS (upper panel) and LM7 (lower panel)- EV and WWOX cells showing restoration of WWOX. GAPDH served as loading control. Images shown were cropped to indicate relevant lanes. (B) Wound healing assay, reduced migration of HOS and LM7 WWOX-expressing compared to control cells. Wound closures were photographed at 0, 9 and 24 hrs after scratch. Cells were grown serum-free media. The scale lines represent scratch. (C) Quantification of the % of wound closure in WWOX-manipulated cell lines from (B). Wound size at different time points was measured using the software ImageJ in three spots of the wound on each triplicate. Results are expressed as mean ± SD. *p < 0.01 as compared with HOS/LM7-EV cells. (D) Matrigel invasion assay. HOS and LM7 stable WWOX clone exhibits markedly reduced Matrigel invasion properties (P = 0.003).
Figure 2
Figure 2. WWOX-overexpressing HOS cells display an impaired metastatic potential when injected intratibially.
(A) HOS EV derived tumors grow much faster than HOS WWOX tumors. Measurements of tumor volumes were performed every week. (B,C) HOS-EV-GFP and HOS-WWOX-GFP cells were injected into the tibia (IT) of NOD/SCID mice (5 mice per group). Lung metastases were visualized by fluorescent imaging after 5 weeks from injection. (B) Representative images showing macrometastasis in HOS cells. (C) Quantification of micro (less than or equal to 2 mm) and macro (greater than 2 mm)–metastasis, as assessed by counting using fluorescent stereoscope.
Figure 3
Figure 3. WWOX expression inhibited OS metastatic potential when injected intravenously.
(A,B) HOS-EV-GFP and HOS-WWOX-GFP (A,B) and LM7 -EV-GFP and LM7-GFP-WWOX cells (C,D) were injected into the lateral tail vein (IV) of NOD/SCID mice (5 per group). The formation of metastasis in the lungs was detected after 8 weeks from the injection. Images obtained by fluorescent microscopy displaying macrometastasis in HOS and LM7 cells are shown. Quantification of micro and macro –metastasis (as in Fig. 2C) is shown in (B,D).
Figure 4
Figure 4. High levels of WWOX in OS cells are associated with decreased expression of RUNX2 target genes.
(A,B) HOS and LM7 control and WWOX-restored cells were subjected to real-time PCR for genes known to be involved in OS metastasis. (C,D) RUNX2 target genes are significantly downregulated upon WWOX restoration in both HOS and LM7 cell lines. Relative fold induction is shown. Results are expressed as mean ± SEM. *p < 0.05 as compared with HOS/LM7-EV cells.

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References

    1. McKenna W. G. et al. Combined modality treatment of adult soft tissue sarcomas of the head and neck. International journal of radiation oncology, biology, physics 13, 1127–1133 (1987). - PubMed
    1. Sissons H. A. The WHO classification of bone tumors. Recent results in cancer research. Fortschritte der Krebsforschung. Progres dans les recherches sur le cancer. 104–108 (1976). - PubMed
    1. Kager L. et al. Primary metastatic osteosarcoma: presentation and outcome of patients treated on neoadjuvant Cooperative Osteosarcoma Study Group protocols. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 21, 2011–2018, 10.1200/JCO.2003.08.132 (2003). - DOI - PubMed
    1. Kaste S. C., Pratt C. B., Cain A. M., Jones-Wallace D. J. & Rao B. N. Metastases detected at the time of diagnosis of primary pediatric extremity osteosarcoma at diagnosis: imaging features. Cancer 86, 1602–1608 (1999). - PubMed
    1. Bacci G. et al. High grade osteosarcoma of the extremities with lung metastases at presentation: treatment with neoadjuvant chemotherapy and simultaneous resection of primary and metastatic lesions. Journal of surgical oncology 98, 415–420, 10.1002/jso.21140 (2008). - DOI - PubMed

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