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mTORC1 maintains the tumorigenicity of SSEA-4(+) high-grade osteosarcoma.

Zhang W, Ding ML, Zhang JN, Qiu JR, Shen YH, Ding XY, Deng LF, Zhang WB, Zhu J - Sci Rep (2015)

Bottom Line: Here we show that in certain high-grade osteosarcomas, immature SSEA-4(+) tumor cells represent a subset of tumor-initiating cells (TICs) whose pool size is maintained by mTORC1 activity. mTORC1 supports not only SSEA-4(+) cell self-renewal through S6K but also the regeneration of SSEA-4(+) TICs by SSEA-4(-) osteosarcoma cell dedifferentiation.Mechanistically, active mTORC1 is required to prevent a likely upregulation of the cell-cycle inhibitor p27 independently of p53 or Rb activation, which otherwise effectively drives the terminal differentiation of SSEA-4(-) osteosarcoma cells at the expense of dedifferentiation.Thus, mTORC1 is shown to critically regulate the retention of tumorigenicity versus differentiation in discrete differentiation phases in SSEA-4(+) TICs and their progeny.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology and Collaborative Innovation Center of Hematology, Rui-Jin Hospital affiliated to Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, People's Republic of China [2] Collaborative Innovation Center of Systems Biomedicine, Shanghai 200025, People's Republic of China.

ABSTRACT
Inactivation of p53 and/or Rb pathways restrains osteoblasts from cell-cycle exit and terminal differentiation, which underpins osteosarcoma formation coupled with dedifferentiation. Recently, the level of p-S6K was shown to independently predict the prognosis for osteosarcomas, while the reason behind this is not understood. Here we show that in certain high-grade osteosarcomas, immature SSEA-4(+) tumor cells represent a subset of tumor-initiating cells (TICs) whose pool size is maintained by mTORC1 activity. mTORC1 supports not only SSEA-4(+) cell self-renewal through S6K but also the regeneration of SSEA-4(+) TICs by SSEA-4(-) osteosarcoma cell dedifferentiation. Mechanistically, active mTORC1 is required to prevent a likely upregulation of the cell-cycle inhibitor p27 independently of p53 or Rb activation, which otherwise effectively drives the terminal differentiation of SSEA-4(-) osteosarcoma cells at the expense of dedifferentiation. Thus, mTORC1 is shown to critically regulate the retention of tumorigenicity versus differentiation in discrete differentiation phases in SSEA-4(+) TICs and their progeny.

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p27-initiated Cell-cycle Exit Contributes to mTOR Inactivation-induced Terminal Osteogenic Differentiation of SSEA-4+ TICs.(a) Western blotting assays for p27 and p21 in MG63 and Well5 cells treated with DMSO or RAD001 for varying lengths of time. The cropped blots were run under the same experimental conditions. The full-length blots can be viewed in Supplementary Figure 8. (b) Cell cycle status of RAD001-treated MG63 and Well5 cells. Data are presented as means ± SDs (*P < 0.05, **P < 0.01, ***P < 0.001). (c) Inducible expression of exogenous p27 elevates ALP activity and OCN mRNA levels in MG63 cells. Left panel, Western blot assay of p27-overexpressing MG63 cells. Data are presented as the means ± SDs (**P < 0.01). (d) Western blot assay on RAD001-treated MG63 and Well5 cells with or without p27 knockdown. si-CTL: control siRNA; si-p27: p27 siRNA mixture. The cropped blots were run under the same experimental conditions. The full-length blots can be viewed in Supplementary Figure 8. (e–f) S-phase distribution (e) and terminal osteogenic maturation (f) of RAD001-treated MG63 and Well5 cells with or without p27 knockdown were evaluated by flow cytometry and Alizarin Red-S staining, respectively. (g) Dox-induced p27 knockdown enhanced the tumorsphere-forming ability of LZJ1-derived total or SSEA-4− osteosarcoma cells. (h) Monitoring of the in vivo growth of LZJ1-derived osteosarcoma cells which were treated with either RAD001 or RAD001 plus p27 knockdown. (i) Immunohistochemical staining of p27, SSEA-4, OCN or ALP in xenografts recovered from RAD001 or RAD001 plus p27 knockdown groups. Scale bars represent 100 μm. HE: hematoxylin-eosin.
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f7: p27-initiated Cell-cycle Exit Contributes to mTOR Inactivation-induced Terminal Osteogenic Differentiation of SSEA-4+ TICs.(a) Western blotting assays for p27 and p21 in MG63 and Well5 cells treated with DMSO or RAD001 for varying lengths of time. The cropped blots were run under the same experimental conditions. The full-length blots can be viewed in Supplementary Figure 8. (b) Cell cycle status of RAD001-treated MG63 and Well5 cells. Data are presented as means ± SDs (*P < 0.05, **P < 0.01, ***P < 0.001). (c) Inducible expression of exogenous p27 elevates ALP activity and OCN mRNA levels in MG63 cells. Left panel, Western blot assay of p27-overexpressing MG63 cells. Data are presented as the means ± SDs (**P < 0.01). (d) Western blot assay on RAD001-treated MG63 and Well5 cells with or without p27 knockdown. si-CTL: control siRNA; si-p27: p27 siRNA mixture. The cropped blots were run under the same experimental conditions. The full-length blots can be viewed in Supplementary Figure 8. (e–f) S-phase distribution (e) and terminal osteogenic maturation (f) of RAD001-treated MG63 and Well5 cells with or without p27 knockdown were evaluated by flow cytometry and Alizarin Red-S staining, respectively. (g) Dox-induced p27 knockdown enhanced the tumorsphere-forming ability of LZJ1-derived total or SSEA-4− osteosarcoma cells. (h) Monitoring of the in vivo growth of LZJ1-derived osteosarcoma cells which were treated with either RAD001 or RAD001 plus p27 knockdown. (i) Immunohistochemical staining of p27, SSEA-4, OCN or ALP in xenografts recovered from RAD001 or RAD001 plus p27 knockdown groups. Scale bars represent 100 μm. HE: hematoxylin-eosin.

Mentions: Indeed, consistent with a progressive elevation in p27 but not in p21 levels (Fig. 7a and supplementary Fig. 7f), RAD001 induced a pre-S phase cell cycle arrest in MG63, U-2OS and Well5 cells prior to inducing apoptosis (Fig. 7b and Supplementary Fig. 7g–h). These results indicated that, analogous to a well-documented role for p21 upregulation in driving the differentiation of certain types of osteosarcoma cell lines49, p27 upregulation represents a critical molecular event following mTOR inactivation in SSEA-4+ TIC-derived progeny, especially for their late-stage differentiation. Consistent with this observation, while Raptor knockdown upregulated both p27 and p21, Rictor knockdown upregulated p21 but not p27 in p53-deficient MG63 cells (Supplementary Fig. 7i). Knockdown of Raptor resulted in obvious cell-cycle arrest (Supplementary Fig. 7j), while such arrest was not seen in Rictor knockdown.


mTORC1 maintains the tumorigenicity of SSEA-4(+) high-grade osteosarcoma.

Zhang W, Ding ML, Zhang JN, Qiu JR, Shen YH, Ding XY, Deng LF, Zhang WB, Zhu J - Sci Rep (2015)

p27-initiated Cell-cycle Exit Contributes to mTOR Inactivation-induced Terminal Osteogenic Differentiation of SSEA-4+ TICs.(a) Western blotting assays for p27 and p21 in MG63 and Well5 cells treated with DMSO or RAD001 for varying lengths of time. The cropped blots were run under the same experimental conditions. The full-length blots can be viewed in Supplementary Figure 8. (b) Cell cycle status of RAD001-treated MG63 and Well5 cells. Data are presented as means ± SDs (*P < 0.05, **P < 0.01, ***P < 0.001). (c) Inducible expression of exogenous p27 elevates ALP activity and OCN mRNA levels in MG63 cells. Left panel, Western blot assay of p27-overexpressing MG63 cells. Data are presented as the means ± SDs (**P < 0.01). (d) Western blot assay on RAD001-treated MG63 and Well5 cells with or without p27 knockdown. si-CTL: control siRNA; si-p27: p27 siRNA mixture. The cropped blots were run under the same experimental conditions. The full-length blots can be viewed in Supplementary Figure 8. (e–f) S-phase distribution (e) and terminal osteogenic maturation (f) of RAD001-treated MG63 and Well5 cells with or without p27 knockdown were evaluated by flow cytometry and Alizarin Red-S staining, respectively. (g) Dox-induced p27 knockdown enhanced the tumorsphere-forming ability of LZJ1-derived total or SSEA-4− osteosarcoma cells. (h) Monitoring of the in vivo growth of LZJ1-derived osteosarcoma cells which were treated with either RAD001 or RAD001 plus p27 knockdown. (i) Immunohistochemical staining of p27, SSEA-4, OCN or ALP in xenografts recovered from RAD001 or RAD001 plus p27 knockdown groups. Scale bars represent 100 μm. HE: hematoxylin-eosin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4389812&req=5

f7: p27-initiated Cell-cycle Exit Contributes to mTOR Inactivation-induced Terminal Osteogenic Differentiation of SSEA-4+ TICs.(a) Western blotting assays for p27 and p21 in MG63 and Well5 cells treated with DMSO or RAD001 for varying lengths of time. The cropped blots were run under the same experimental conditions. The full-length blots can be viewed in Supplementary Figure 8. (b) Cell cycle status of RAD001-treated MG63 and Well5 cells. Data are presented as means ± SDs (*P < 0.05, **P < 0.01, ***P < 0.001). (c) Inducible expression of exogenous p27 elevates ALP activity and OCN mRNA levels in MG63 cells. Left panel, Western blot assay of p27-overexpressing MG63 cells. Data are presented as the means ± SDs (**P < 0.01). (d) Western blot assay on RAD001-treated MG63 and Well5 cells with or without p27 knockdown. si-CTL: control siRNA; si-p27: p27 siRNA mixture. The cropped blots were run under the same experimental conditions. The full-length blots can be viewed in Supplementary Figure 8. (e–f) S-phase distribution (e) and terminal osteogenic maturation (f) of RAD001-treated MG63 and Well5 cells with or without p27 knockdown were evaluated by flow cytometry and Alizarin Red-S staining, respectively. (g) Dox-induced p27 knockdown enhanced the tumorsphere-forming ability of LZJ1-derived total or SSEA-4− osteosarcoma cells. (h) Monitoring of the in vivo growth of LZJ1-derived osteosarcoma cells which were treated with either RAD001 or RAD001 plus p27 knockdown. (i) Immunohistochemical staining of p27, SSEA-4, OCN or ALP in xenografts recovered from RAD001 or RAD001 plus p27 knockdown groups. Scale bars represent 100 μm. HE: hematoxylin-eosin.
Mentions: Indeed, consistent with a progressive elevation in p27 but not in p21 levels (Fig. 7a and supplementary Fig. 7f), RAD001 induced a pre-S phase cell cycle arrest in MG63, U-2OS and Well5 cells prior to inducing apoptosis (Fig. 7b and Supplementary Fig. 7g–h). These results indicated that, analogous to a well-documented role for p21 upregulation in driving the differentiation of certain types of osteosarcoma cell lines49, p27 upregulation represents a critical molecular event following mTOR inactivation in SSEA-4+ TIC-derived progeny, especially for their late-stage differentiation. Consistent with this observation, while Raptor knockdown upregulated both p27 and p21, Rictor knockdown upregulated p21 but not p27 in p53-deficient MG63 cells (Supplementary Fig. 7i). Knockdown of Raptor resulted in obvious cell-cycle arrest (Supplementary Fig. 7j), while such arrest was not seen in Rictor knockdown.

Bottom Line: Here we show that in certain high-grade osteosarcomas, immature SSEA-4(+) tumor cells represent a subset of tumor-initiating cells (TICs) whose pool size is maintained by mTORC1 activity. mTORC1 supports not only SSEA-4(+) cell self-renewal through S6K but also the regeneration of SSEA-4(+) TICs by SSEA-4(-) osteosarcoma cell dedifferentiation.Mechanistically, active mTORC1 is required to prevent a likely upregulation of the cell-cycle inhibitor p27 independently of p53 or Rb activation, which otherwise effectively drives the terminal differentiation of SSEA-4(-) osteosarcoma cells at the expense of dedifferentiation.Thus, mTORC1 is shown to critically regulate the retention of tumorigenicity versus differentiation in discrete differentiation phases in SSEA-4(+) TICs and their progeny.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology and Collaborative Innovation Center of Hematology, Rui-Jin Hospital affiliated to Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, People's Republic of China [2] Collaborative Innovation Center of Systems Biomedicine, Shanghai 200025, People's Republic of China.

ABSTRACT
Inactivation of p53 and/or Rb pathways restrains osteoblasts from cell-cycle exit and terminal differentiation, which underpins osteosarcoma formation coupled with dedifferentiation. Recently, the level of p-S6K was shown to independently predict the prognosis for osteosarcomas, while the reason behind this is not understood. Here we show that in certain high-grade osteosarcomas, immature SSEA-4(+) tumor cells represent a subset of tumor-initiating cells (TICs) whose pool size is maintained by mTORC1 activity. mTORC1 supports not only SSEA-4(+) cell self-renewal through S6K but also the regeneration of SSEA-4(+) TICs by SSEA-4(-) osteosarcoma cell dedifferentiation. Mechanistically, active mTORC1 is required to prevent a likely upregulation of the cell-cycle inhibitor p27 independently of p53 or Rb activation, which otherwise effectively drives the terminal differentiation of SSEA-4(-) osteosarcoma cells at the expense of dedifferentiation. Thus, mTORC1 is shown to critically regulate the retention of tumorigenicity versus differentiation in discrete differentiation phases in SSEA-4(+) TICs and their progeny.

Show MeSH
Related in: MedlinePlus