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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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mTORC1 Activity Maintains SSEA-4+ TIC Frequency.(a) MG63 or Well5 cells were treated with negative control DMSO, Wnt-β catenin inhibitor indomethacin, Notch inhibitor γ-secretase inhibitor, Hedgehog inhibitor cyclopamine, PI3K-AKT inhibitor LY294002, mTOR inhibitor RAD001, p38 MAPK inhibitor SB203580, JAK-STAT inhibitor AG490, or PKC inhibitor GO6976 for 24 hours and the frequency of SSEA-4+ cells was measured by flow cytometry. Results are expressed as the mean ± SD (**P < 0.01). (b) Western blotting assays for phosphorylated levels of mTORC1 pathway components S6K or/and S6 as well as the β-catenin level in SSEA-4+ or SSEA-4− cells freshly sorted from osteosarcoma xenografts. The cropped blots were run under the same experimental conditions. The full-length blots can be found in Supplementary Figure 8. (c) Immunofluorescent co-staining of SSEA-4 and p-S6 in cytospun MG63 and Well5 cells. (d–e) The effects of Dox-inducible Raptor or Rictor knockdown (d) or S6K knockdown (e) on the frequency of SSEA-4+ cells in MG63 cell culture. Results are expressed as mean ± SD (*P < 0.05, **P < 0.01). NC: control shRNA; shRaptor: shRNA for Raptor; shRictor: shRNA for Rictor; sh-S6K-1 and sh-S6K-2: shRNAs for S6K. (f) p-S6 level is positively correlated with SSEA-4 staining intensity among 98 human osteosarcoma samples (P = 0.000155). Representative immunohistochemical staining of p-S6 in one SSEA-4− and SSEA-43+ sample is shown on the left panel. Scale bars represent 100 μm. (g) MG63 cells were treated with DMSO or RAD001 for 3 days, then stained with the fluorescent antibodies against SSEA-4, OCN and RUNX2, and viewed microscopically. (h) ALP activity and OCN mRNA levels were measured in MG63 cells with or without Raptor knockdown (**P < 0.01, ***P < 0.001).
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f4: mTORC1 Activity Maintains SSEA-4+ TIC Frequency.(a) MG63 or Well5 cells were treated with negative control DMSO, Wnt-β catenin inhibitor indomethacin, Notch inhibitor γ-secretase inhibitor, Hedgehog inhibitor cyclopamine, PI3K-AKT inhibitor LY294002, mTOR inhibitor RAD001, p38 MAPK inhibitor SB203580, JAK-STAT inhibitor AG490, or PKC inhibitor GO6976 for 24 hours and the frequency of SSEA-4+ cells was measured by flow cytometry. Results are expressed as the mean ± SD (**P < 0.01). (b) Western blotting assays for phosphorylated levels of mTORC1 pathway components S6K or/and S6 as well as the β-catenin level in SSEA-4+ or SSEA-4− cells freshly sorted from osteosarcoma xenografts. The cropped blots were run under the same experimental conditions. The full-length blots can be found in Supplementary Figure 8. (c) Immunofluorescent co-staining of SSEA-4 and p-S6 in cytospun MG63 and Well5 cells. (d–e) The effects of Dox-inducible Raptor or Rictor knockdown (d) or S6K knockdown (e) on the frequency of SSEA-4+ cells in MG63 cell culture. Results are expressed as mean ± SD (*P < 0.05, **P < 0.01). NC: control shRNA; shRaptor: shRNA for Raptor; shRictor: shRNA for Rictor; sh-S6K-1 and sh-S6K-2: shRNAs for S6K. (f) p-S6 level is positively correlated with SSEA-4 staining intensity among 98 human osteosarcoma samples (P = 0.000155). Representative immunohistochemical staining of p-S6 in one SSEA-4− and SSEA-43+ sample is shown on the left panel. Scale bars represent 100 μm. (g) MG63 cells were treated with DMSO or RAD001 for 3 days, then stained with the fluorescent antibodies against SSEA-4, OCN and RUNX2, and viewed microscopically. (h) ALP activity and OCN mRNA levels were measured in MG63 cells with or without Raptor knockdown (**P < 0.01, ***P < 0.001).

Mentions: We next explored which molecular mechanisms maintain the immaturity of SSEA-4+ TICs. Actually, the pathway enrichment analysis described above (Fig. 3a) revealed that the functional states of multiple signal transduction and/or transcription pathways, including the Jak-Stat, the Wnt-β catenin, Notch and AKT-mTOR axes, exhibited significant differences when comparing SSEA-4+ to SSEA-4− osteosarcoma cells (Supplementary Fig. 4a,b). To screen for the most critical one, we applied individual pathway inhibitors to ordinary cultures of Well5 and MG63 cells. The results showed that the mTOR inhibitor RAD001 but not others (including Jak, β-catenin and Notch inhibitors) quickly reduced the SSEA-4+ cell frequency (Fig. 4a). A detailed analysis of dose- or time-dependent inhibition of SSEA-4+ cell frequency by RAD001 or LY294002 indicated that the SSEA-4+ cell decrease was consistently associated with a reduction in mTORC1 (as indicated by p-S6K or p-S6 level) but not mTORC2 activity (as indicated by p-AKTS473 level) (Supplementary Fig. 4c,d)36. Consistent with this, mTORC1 activity, as indicated by the p-S6K or p-S6 level, was higher in SSEA-4+ cells than in SSEA-4− cells asfreshly isolated from Well5- or L1031-derived xenografts, whereas there was no difference in the level of β-catenin (Fig. 4b). Moreover, the expression of SSEA-4 and p-S6 signals overlapped to a notable extent at the single-cell level (Fig. 4c), indicating that mTORC1 regulates SSEA-4+ TICs autonomously. To further examine whether mTORC2 was involved at all in this process36, we knocked down the Raptor, Rictor or S6K in MG63 cells individually using a Dox-inducible shRNA expression system (Supplementary Fig. 4e). Interestingly, the knockdown of mTORC1-related Raptor or S6K but not of mTORC2-related Rictor significantly decreased the frequency of SSEA-4+ cells (Fig. 4d, e and Supplementary Fig. 4f, g). Notably, in line with a previous study indicating that p-S6K levels in human osteosarcomas reliably predicted the prognosis22, the staining intensity of p-S6 and that of SSEA-4 were found to be positively correlated among 98 primary osteosarcoma specimens examined (Fig. 4f). This suggested a general contribution of elevated mTORC1 activity to the maintenance of SSEA-4+ TICs in human osteosarcoma cells.


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)

mTORC1 Activity Maintains SSEA-4+ TIC Frequency.(a) MG63 or Well5 cells were treated with negative control DMSO, Wnt-β catenin inhibitor indomethacin, Notch inhibitor γ-secretase inhibitor, Hedgehog inhibitor cyclopamine, PI3K-AKT inhibitor LY294002, mTOR inhibitor RAD001, p38 MAPK inhibitor SB203580, JAK-STAT inhibitor AG490, or PKC inhibitor GO6976 for 24 hours and the frequency of SSEA-4+ cells was measured by flow cytometry. Results are expressed as the mean ± SD (**P < 0.01). (b) Western blotting assays for phosphorylated levels of mTORC1 pathway components S6K or/and S6 as well as the β-catenin level in SSEA-4+ or SSEA-4− cells freshly sorted from osteosarcoma xenografts. The cropped blots were run under the same experimental conditions. The full-length blots can be found in Supplementary Figure 8. (c) Immunofluorescent co-staining of SSEA-4 and p-S6 in cytospun MG63 and Well5 cells. (d–e) The effects of Dox-inducible Raptor or Rictor knockdown (d) or S6K knockdown (e) on the frequency of SSEA-4+ cells in MG63 cell culture. Results are expressed as mean ± SD (*P < 0.05, **P < 0.01). NC: control shRNA; shRaptor: shRNA for Raptor; shRictor: shRNA for Rictor; sh-S6K-1 and sh-S6K-2: shRNAs for S6K. (f) p-S6 level is positively correlated with SSEA-4 staining intensity among 98 human osteosarcoma samples (P = 0.000155). Representative immunohistochemical staining of p-S6 in one SSEA-4− and SSEA-43+ sample is shown on the left panel. Scale bars represent 100 μm. (g) MG63 cells were treated with DMSO or RAD001 for 3 days, then stained with the fluorescent antibodies against SSEA-4, OCN and RUNX2, and viewed microscopically. (h) ALP activity and OCN mRNA levels were measured in MG63 cells with or without Raptor knockdown (**P < 0.01, ***P < 0.001).
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f4: mTORC1 Activity Maintains SSEA-4+ TIC Frequency.(a) MG63 or Well5 cells were treated with negative control DMSO, Wnt-β catenin inhibitor indomethacin, Notch inhibitor γ-secretase inhibitor, Hedgehog inhibitor cyclopamine, PI3K-AKT inhibitor LY294002, mTOR inhibitor RAD001, p38 MAPK inhibitor SB203580, JAK-STAT inhibitor AG490, or PKC inhibitor GO6976 for 24 hours and the frequency of SSEA-4+ cells was measured by flow cytometry. Results are expressed as the mean ± SD (**P < 0.01). (b) Western blotting assays for phosphorylated levels of mTORC1 pathway components S6K or/and S6 as well as the β-catenin level in SSEA-4+ or SSEA-4− cells freshly sorted from osteosarcoma xenografts. The cropped blots were run under the same experimental conditions. The full-length blots can be found in Supplementary Figure 8. (c) Immunofluorescent co-staining of SSEA-4 and p-S6 in cytospun MG63 and Well5 cells. (d–e) The effects of Dox-inducible Raptor or Rictor knockdown (d) or S6K knockdown (e) on the frequency of SSEA-4+ cells in MG63 cell culture. Results are expressed as mean ± SD (*P < 0.05, **P < 0.01). NC: control shRNA; shRaptor: shRNA for Raptor; shRictor: shRNA for Rictor; sh-S6K-1 and sh-S6K-2: shRNAs for S6K. (f) p-S6 level is positively correlated with SSEA-4 staining intensity among 98 human osteosarcoma samples (P = 0.000155). Representative immunohistochemical staining of p-S6 in one SSEA-4− and SSEA-43+ sample is shown on the left panel. Scale bars represent 100 μm. (g) MG63 cells were treated with DMSO or RAD001 for 3 days, then stained with the fluorescent antibodies against SSEA-4, OCN and RUNX2, and viewed microscopically. (h) ALP activity and OCN mRNA levels were measured in MG63 cells with or without Raptor knockdown (**P < 0.01, ***P < 0.001).
Mentions: We next explored which molecular mechanisms maintain the immaturity of SSEA-4+ TICs. Actually, the pathway enrichment analysis described above (Fig. 3a) revealed that the functional states of multiple signal transduction and/or transcription pathways, including the Jak-Stat, the Wnt-β catenin, Notch and AKT-mTOR axes, exhibited significant differences when comparing SSEA-4+ to SSEA-4− osteosarcoma cells (Supplementary Fig. 4a,b). To screen for the most critical one, we applied individual pathway inhibitors to ordinary cultures of Well5 and MG63 cells. The results showed that the mTOR inhibitor RAD001 but not others (including Jak, β-catenin and Notch inhibitors) quickly reduced the SSEA-4+ cell frequency (Fig. 4a). A detailed analysis of dose- or time-dependent inhibition of SSEA-4+ cell frequency by RAD001 or LY294002 indicated that the SSEA-4+ cell decrease was consistently associated with a reduction in mTORC1 (as indicated by p-S6K or p-S6 level) but not mTORC2 activity (as indicated by p-AKTS473 level) (Supplementary Fig. 4c,d)36. Consistent with this, mTORC1 activity, as indicated by the p-S6K or p-S6 level, was higher in SSEA-4+ cells than in SSEA-4− cells asfreshly isolated from Well5- or L1031-derived xenografts, whereas there was no difference in the level of β-catenin (Fig. 4b). Moreover, the expression of SSEA-4 and p-S6 signals overlapped to a notable extent at the single-cell level (Fig. 4c), indicating that mTORC1 regulates SSEA-4+ TICs autonomously. To further examine whether mTORC2 was involved at all in this process36, we knocked down the Raptor, Rictor or S6K in MG63 cells individually using a Dox-inducible shRNA expression system (Supplementary Fig. 4e). Interestingly, the knockdown of mTORC1-related Raptor or S6K but not of mTORC2-related Rictor significantly decreased the frequency of SSEA-4+ cells (Fig. 4d, e and Supplementary Fig. 4f, g). Notably, in line with a previous study indicating that p-S6K levels in human osteosarcomas reliably predicted the prognosis22, the staining intensity of p-S6 and that of SSEA-4 were found to be positively correlated among 98 primary osteosarcoma specimens examined (Fig. 4f). This suggested a general contribution of elevated mTORC1 activity to the maintenance of SSEA-4+ TICs in human osteosarcoma cells.

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