Nd anti-apoptotic effects of FHL2 have been reported [42,43]. This dual effect is likely to be related to the cellular context, namely the molecular interactions between FHLFHL2 Silencing Reduces Osteosarcoma TumorigenesisFigure 4. FHL2 silencing decreases bone tumor cell migration and invasion. Migration of shControl and shFHL2-transduced K7M2 cells was evaluated by Boyden’s chamber (A) and wounding assays (C) and migrating cell number was evaluated (B, D). K7M2 cell invasion was evaluated by the Matrigel invasion assay (E, F). *: P,0.05 vs shControl-transduced cells. doi:10.1371/journal.pone.0055034.gand specific partners [13]. In the get NT-157 present study, FHL2 silencing may have inhibited cell proliferation independently of cell death since FHL2 was found to regulate tumor cell growth through the control of G1/S transition during cell cycle rather than apoptosis [43]. One possibility is that the depletion of FHL2 may have resulted in cells becoming more quiescent, thus avoiding cell cyclerelated initiation of apoptosis. The observed anti-apoptotic effect of FHL2 silencing in osteosarcoma cells may be linked in part to the observed decrease in Wnt5a, since this protein exerts antiapoptotic activity in cells of the osteoblast lineage [44]. FHL2 is known to interact with Foxo1 [45] and Foxo1 was shown to increase osteoblast apoptosis in vivo [46]. We found that FHL2 silencing increased Foxo1 expression in osteosarcoma cells, suggesting a possible implication of Foxo1 in the anti-apoptotic effect of FHL2 silencing in osteosarcoma cells. Despite our finding that FHL2 silencing reduced osteosarcoma cell apoptosis in vitro and in vivo, we found that the overall effect of FHL2 silencingin vivo is to suppress tumor growth, indicating that FHL2 acts mostly as an oncoprotein in osteosarcoma cells. Osteosarcoma tumorigenesis is often associated with tumor cell invasion leading to metastasis and reduced patient’s survival [1,26]. Few experimental studies suggest that FHL2 may play a role in cancer cell invasion and migration in some soft tissue cancers [32,47,48]. However, nothing is known on the role of FHL2 in osteosarcoma cell metastasis capacity. Strikingly, we found that FHL2 silencing reduced osteosarcoma cell invasion and migration in vitro and metastatic development in vivo. These results provide the first evidence that FHL2 is involved in the invasiveness capacity of osteosarcoma cells and that silencing FHL2 reduces osteosarcoma tumorigenesis in mice. One mechanism underlying the anti-oncogenic effect of FHL2 silencing could be the 
decreased expression of the Wnt family members Wnt5a and Wnt10b that we observed in vitro and in vivo, because these proteins confer cell invasiveness, metastasis and reduced survival in osteosarcomas [22,23,24] and thereby JWH-133 contribute to tumorigenesis [49,50]. InFHL2 Silencing Reduces Osteosarcoma TumorigenesisFigure 5. FHL2 silencing decreases bone tumor growth in vivo. shRNA control and shFHL2-transduced murine K7M2 cells were injected in BALB/c mice and tumor size (A) and volume (B) were determined at 6 weeks (n = 9 per group). Cell proliferation and apoptosis in tumors was determined by histological analysis using Ki67 (C, D) and TUNEL staining (arrows), respectively (E, F). Wnt5a and Wnt10b mRNA expression was evaluated in the tumors by q-PCR analysis 
(G). *: P,0.05 vs shControl cells. doi:10.1371/journal.pone.0055034.gaddition to involve Wnt proteins, the anti-oncogenic effect of FHL2 silencing may involve dec.Nd anti-apoptotic effects of FHL2 have been reported [42,43]. This dual effect is likely to be related to the cellular context, namely the molecular interactions between FHLFHL2 Silencing Reduces Osteosarcoma TumorigenesisFigure 4. FHL2 silencing decreases bone tumor cell migration and invasion. Migration of shControl and shFHL2-transduced K7M2 cells was evaluated by Boyden’s chamber (A) and wounding assays (C) and migrating cell number was evaluated (B, D). K7M2 cell invasion was evaluated by the Matrigel invasion assay (E, F). *: P,0.05 vs shControl-transduced cells. doi:10.1371/journal.pone.0055034.gand specific partners [13]. In the present study, FHL2 silencing may have inhibited cell proliferation independently of cell death since FHL2 was found to regulate tumor cell growth through the control of G1/S transition during cell cycle rather than apoptosis [43]. One possibility is that the depletion of FHL2 may have resulted in cells becoming more quiescent, thus avoiding cell cyclerelated initiation of apoptosis. The observed anti-apoptotic effect of FHL2 silencing in osteosarcoma cells may be linked in part to the observed decrease in Wnt5a, since this protein exerts antiapoptotic activity in cells of the osteoblast lineage [44]. FHL2 is known to interact with Foxo1 [45] and Foxo1 was shown to increase osteoblast apoptosis in vivo [46]. We found that FHL2 silencing increased Foxo1 expression in osteosarcoma cells, suggesting a possible implication of Foxo1 in the anti-apoptotic effect of FHL2 silencing in osteosarcoma cells. Despite our finding that FHL2 silencing reduced osteosarcoma cell apoptosis in vitro and in vivo, we found that the overall effect of FHL2 silencingin vivo is to suppress tumor growth, indicating that FHL2 acts mostly as an oncoprotein in osteosarcoma cells. Osteosarcoma tumorigenesis is often associated with tumor cell invasion leading to metastasis and reduced patient’s survival [1,26]. Few experimental studies suggest that FHL2 may play a role in cancer cell invasion and migration in some soft tissue cancers [32,47,48]. However, nothing is known on the role of FHL2 in osteosarcoma cell metastasis capacity. Strikingly, we found that FHL2 silencing reduced osteosarcoma cell invasion and migration in vitro and metastatic development in vivo. These results provide the first evidence that FHL2 is involved in the invasiveness capacity of osteosarcoma cells and that silencing FHL2 reduces osteosarcoma tumorigenesis in mice. One mechanism underlying the anti-oncogenic effect of FHL2 silencing could be the decreased expression of the Wnt family members Wnt5a and Wnt10b that we observed in vitro and in vivo, because these proteins confer cell invasiveness, metastasis and reduced survival in osteosarcomas [22,23,24] and thereby contribute to tumorigenesis [49,50]. InFHL2 Silencing Reduces Osteosarcoma TumorigenesisFigure 5. FHL2 silencing decreases bone tumor growth in vivo. shRNA control and shFHL2-transduced murine K7M2 cells were injected in BALB/c mice and tumor size (A) and volume (B) were determined at 6 weeks (n = 9 per group). Cell proliferation and apoptosis in tumors was determined by histological analysis using Ki67 (C, D) and TUNEL staining (arrows), respectively (E, F). Wnt5a and Wnt10b mRNA expression was evaluated in the tumors by q-PCR analysis (G). *: P,0.05 vs shControl cells. doi:10.1371/journal.pone.0055034.gaddition to involve Wnt proteins, the anti-oncogenic effect of FHL2 silencing may involve dec.
