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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

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).
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f1: 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).

Mentions: We previously demonstrated that WWOX levels are reduced in most OS cells12. Indeed, both HOS and LM-7 metastatic OS cells express very low or undetectable levels of endogenous WWOX (Fig. 1A). To study effect of WWOX expression on metastatic traits in these cells, we used lentiviral vectors to generate stable clones of HOS and LM7 cells expressing WWOX or empty vector (EV) and then measured their cell migration and invasion characteristics in vitro. Overexpression of WWOX was validated by immunoblotting using anti-WWOX antibody (Fig. 1 upper panel). To examine effect of WWOX on cell migration, we performed a wound-healing assay and found that HOS-WWOX cells have low cell motility compared with HOS-EV cells when grown in a serum-free media (Fig. 1B,C). Next, we determined WWOX action on invasion using a Boyden chamber assay and found that HOS-WWOX cells exhibited reduced invasiveness relative to EV-expressing cells (Fig. 1D). Similar traits were observed when LM7-WWOX stable clones were compared with LM7-EV cells (Fig. 1 lower panel). These data suggest that WWOX restoration can attenuate metastatic characteristics in vitro in metastatic OS cell lines.


Tumor Suppressor WWOX inhibits osteosarcoma metastasis by modulating RUNX2 function.

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

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

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

f1: 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).
Mentions: We previously demonstrated that WWOX levels are reduced in most OS cells12. Indeed, both HOS and LM-7 metastatic OS cells express very low or undetectable levels of endogenous WWOX (Fig. 1A). To study effect of WWOX expression on metastatic traits in these cells, we used lentiviral vectors to generate stable clones of HOS and LM7 cells expressing WWOX or empty vector (EV) and then measured their cell migration and invasion characteristics in vitro. Overexpression of WWOX was validated by immunoblotting using anti-WWOX antibody (Fig. 1 upper panel). To examine effect of WWOX on cell migration, we performed a wound-healing assay and found that HOS-WWOX cells have low cell motility compared with HOS-EV cells when grown in a serum-free media (Fig. 1B,C). Next, we determined WWOX action on invasion using a Boyden chamber assay and found that HOS-WWOX cells exhibited reduced invasiveness relative to EV-expressing cells (Fig. 1D). Similar traits were observed when LM7-WWOX stable clones were compared with LM7-EV cells (Fig. 1 lower panel). These data suggest that WWOX restoration can attenuate metastatic characteristics in vitro in metastatic OS cell lines.

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