S 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 (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 Benzocaine 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]. 4-IBP 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 and 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 24786787 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 osteosarcom.S 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 (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 and 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 24786787 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 osteosarcom.
