Ll type in relation with FHL2 interaction with other proteins, causing either get Peptide M repression or activation of target genes [13]. The present finding that FHL2 protein level is high in osteosarcoma tumors and correlates with osteosarcoma aggressiveness in human osteosarcoma supports a positive role of FHL2 in bone tumor development. To investigate the specific role of FHL2 in osteosarcoma tumor development, we used K7M2 murine 
osteosarcoma cells that express high FHL2 levels in basal conditions. We found that silencing FHL2 though transduction with a lentivirus encoding a specific shRNA that efficiently reduced FHL2 levels in these cells, reduced cell proliferation and repressed the oncogene c-Myc, supporting a role of FHL2 in osteosarcoma cell growth. This is consistent with the recent observation that FHL2 deficiencyFHL2 Silencing Reduces Tumorigenesis and Metastasis in vivoBased on the above evidence that FHL2 silencing reduces mouse osteosarcoma cell migration and invasiveness in vitro, we hypothesized that this effect may impact osteosarcoma tumorigenesis in vivo. To investigate this hypothesis, shControl- and shFHL2-transduced K7M2 cells were injected in thigh muscle of BALB/c mice. As expected, injected K7M2 cells developed large tumors which were detectable after 6 weeks (Fig. 5A). We found that FHL2 silencing strikingly reduced tumor size compared to control cells (Fig. 5A). Quantification of the tumor samples confirmed that FHL2 silencing reduced tumor volume by about 2fold compared to control tumors (Fig. 5B), which is consistent with the anticancer activity of FHL2 silencing that we found in vitro.FHL2 Silencing Reduces Osteosarcoma TumorigenesisFigure 3. FHL2 silencing decreases osteosarcoma cell growth. After treatment with Wnt3a CM (A) or FGF-2 18055761 (0.50 ng/ml) (B) for 3 days, DNA replication was evaluated by BrdU incorporation in shControl and shFHL2-transduced K7M2 cells. Apoptosis was induced by serum deprivation and effector caspases activity was evaluated at 48 h in cells treated with or without Wnt3a CM (C). a: p,0.05 vs untreated, b:p,0.05 vs shControl. TUNEL analysis was performed in basal and serum deprivation conditions at 72 h (D). *: P,0.05 vs the indicated group or shControl cells. doi:10.1371/journal.pone.0055034.greduces intestinal tumorigenesis in Apc mutant mice [32]. Several mechanisms such as the androgen receptor [35], partners of the MAPK pathway [36,37,38], Smad proteins [39] and cyclin D1 [40] have been reported to be involved in the control of cell replication and the response to mitogenic stimulation by FHL2. We buy 16960-16-0 focused here on Wnt/b-catenin signaling since FHL2 interacts with this pathway in several cell types [14,15,41] including in osteoblast progenitor cells [19]. Additionally, Wnt/b-catenin signaling was reported to be dysregulated in osteosarcoma [7,8,9,11]. We found that FHL2 silencing in murine osteosarcoma cells led to reduce b-catenin transcription as well as the expressionof c-myc, a target of Wnt/b-catenin signaling which controls cell replication, and concomitantly reduced the expression of Axin2 and WISP-1 which are direct Wnt/b-catenin-target genes [3]. Although FHL2 silencing abrogated the positive effect of Wnt3a on osteosarcoma cell growth, this effect might be explained by overall reduced proliferation since FHL2 silencing also abrogated FGF-2-induced cell proliferation. We also found that FHL2 silencing slightly reduced osteosarcoma cell apoptosis in vitro. To date, both pro-apoptotic a.Ll type in relation with FHL2 interaction with other 
proteins, causing either repression or activation of target genes [13]. The present finding that FHL2 protein level is high in osteosarcoma tumors and correlates with osteosarcoma aggressiveness in human osteosarcoma supports a positive role of FHL2 in bone tumor development. To investigate the specific role of FHL2 in osteosarcoma tumor development, we used K7M2 murine osteosarcoma cells that express high FHL2 levels in basal conditions. We found that silencing FHL2 though transduction with a lentivirus encoding a specific shRNA that efficiently reduced FHL2 levels in these cells, reduced cell proliferation and repressed the oncogene c-Myc, supporting a role of FHL2 in osteosarcoma cell growth. This is consistent with the recent observation that FHL2 deficiencyFHL2 Silencing Reduces Tumorigenesis and Metastasis in vivoBased on the above evidence that FHL2 silencing reduces mouse osteosarcoma cell migration and invasiveness in vitro, we hypothesized that this effect may impact osteosarcoma tumorigenesis in vivo. To investigate this hypothesis, shControl- and shFHL2-transduced K7M2 cells were injected in thigh muscle of BALB/c mice. As expected, injected K7M2 cells developed large tumors which were detectable after 6 weeks (Fig. 5A). We found that FHL2 silencing strikingly reduced tumor size compared to control cells (Fig. 5A). Quantification of the tumor samples confirmed that FHL2 silencing reduced tumor volume by about 2fold compared to control tumors (Fig. 5B), which is consistent with the anticancer activity of FHL2 silencing that we found in vitro.FHL2 Silencing Reduces Osteosarcoma TumorigenesisFigure 3. FHL2 silencing decreases osteosarcoma cell growth. After treatment with Wnt3a CM (A) or FGF-2 18055761 (0.50 ng/ml) (B) for 3 days, DNA replication was evaluated by BrdU incorporation in shControl and shFHL2-transduced K7M2 cells. Apoptosis was induced by serum deprivation and effector caspases activity was evaluated at 48 h in cells treated with or without Wnt3a CM (C). a: p,0.05 vs untreated, b:p,0.05 vs shControl. TUNEL analysis was performed in basal and serum deprivation conditions at 72 h (D). *: P,0.05 vs the indicated group or shControl cells. doi:10.1371/journal.pone.0055034.greduces intestinal tumorigenesis in Apc mutant mice [32]. Several mechanisms such as the androgen receptor [35], partners of the MAPK pathway [36,37,38], Smad proteins [39] and cyclin D1 [40] have been reported to be involved in the control of cell replication and the response to mitogenic stimulation by FHL2. We focused here on Wnt/b-catenin signaling since FHL2 interacts with this pathway in several cell types [14,15,41] including in osteoblast progenitor cells [19]. Additionally, Wnt/b-catenin signaling was reported to be dysregulated in osteosarcoma [7,8,9,11]. We found that FHL2 silencing in murine osteosarcoma cells led to reduce b-catenin transcription as well as the expressionof c-myc, a target of Wnt/b-catenin signaling which controls cell replication, and concomitantly reduced the expression of Axin2 and WISP-1 which are direct Wnt/b-catenin-target genes [3]. Although FHL2 silencing abrogated the positive effect of Wnt3a on osteosarcoma cell growth, this effect might be explained by overall reduced proliferation since FHL2 silencing also abrogated FGF-2-induced cell proliferation. We also found that FHL2 silencing slightly reduced osteosarcoma cell apoptosis in vitro. To date, both pro-apoptotic a.