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Tumor Suppressor WWOX inhibits osteosarcoma metastasis by modulating RUNX2 function.

Del Mare S, Aqeilan RI - Sci Rep (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Lautenberg Center for Immunology and Cancer Research, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem, Israel 91220.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f2: 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.

Mentions: The presence of metastasis at diagnosis is the most important predictor of disease-free survival with a 5-year survival rate of only 20% for OS patients with metastasis compared to 65% for patients with localized disease4. Since our findings indicated that WWOX significantly diminish migration and invasion potential of OS cells in vitro, we next set to examine whether WWOX expression inhibits seeding of metastasis in vivo. In order to assess whether WWOX could affect the metastatic potential of OS cell lines, we labeled manipulated HOS or LM-7 cells with GFP, injected them intratibially (IT) and followed formation of primary tumors and examined for lung metastasis, the predominate site of OS metastasis. By three weeks of IT injections, HOS EV cells formed large tumors that mice had to be removed from the experiment (Fig. 2A). By contrast, mice injected with WWOX-expressing HOS cells displayed significantly smaller tumors as compared to HOS-EV cells (0.5 cm3 compared to 2.0 cm3, respectively) (Fig. 2A) To compare metastatic lesions, HOS-WWOX injected mice were sacrificed at ~5-weeks allowing primary tumors to reach comparable size to HOS-EV bearing mice. As shown in Fig. 2B,C, higher incidence of pulmonary metastasis was observed in matched sized tumor bearing-mice inoculated with HOS-EV cells as compared to HOS-WWOX cells. In fact, all HOS-EV IT-injected mice developed macrometastasis whereas 2 out of 5 (40%) mice injected with HOS-WWOX developed only micrometastasis (Fig. 2C). These data suggest that WWOX overexpression leads to an attenuation of the metastatic seeding potential of HOS cells when injected orthotopically. LM-7 cells did not form tumors in the IT model in same time period and therefore WWOX effect on metastatsis using this model could not be assessed.


Tumor Suppressor WWOX inhibits osteosarcoma metastasis by modulating RUNX2 function.

Del Mare S, Aqeilan RI - Sci Rep (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4542681&req=5

f2: 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.
Mentions: The presence of metastasis at diagnosis is the most important predictor of disease-free survival with a 5-year survival rate of only 20% for OS patients with metastasis compared to 65% for patients with localized disease4. Since our findings indicated that WWOX significantly diminish migration and invasion potential of OS cells in vitro, we next set to examine whether WWOX expression inhibits seeding of metastasis in vivo. In order to assess whether WWOX could affect the metastatic potential of OS cell lines, we labeled manipulated HOS or LM-7 cells with GFP, injected them intratibially (IT) and followed formation of primary tumors and examined for lung metastasis, the predominate site of OS metastasis. By three weeks of IT injections, HOS EV cells formed large tumors that mice had to be removed from the experiment (Fig. 2A). By contrast, mice injected with WWOX-expressing HOS cells displayed significantly smaller tumors as compared to HOS-EV cells (0.5 cm3 compared to 2.0 cm3, respectively) (Fig. 2A) To compare metastatic lesions, HOS-WWOX injected mice were sacrificed at ~5-weeks allowing primary tumors to reach comparable size to HOS-EV bearing mice. As shown in Fig. 2B,C, higher incidence of pulmonary metastasis was observed in matched sized tumor bearing-mice inoculated with HOS-EV cells as compared to HOS-WWOX cells. In fact, all HOS-EV IT-injected mice developed macrometastasis whereas 2 out of 5 (40%) mice injected with HOS-WWOX developed only micrometastasis (Fig. 2C). These data suggest that WWOX overexpression leads to an attenuation of the metastatic seeding potential of HOS cells when injected orthotopically. LM-7 cells did not form tumors in the IT model in same time period and therefore WWOX effect on metastatsis using this model could not be assessed.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Lautenberg Center for Immunology and Cancer Research, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem, Israel 91220.

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.

No MeSH data available.


Related in: MedlinePlus