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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 Supports the De-differentiate Potential of Early SSEA-4− Progeny.(a) SSEA-4− cells were sorted at different time points during MG63 cells' differentiation to osteocytes and individually inoculated into fresh medium for measuring their clonal efficiency or into special medium for measuring their tumorsphere-forming potential (*P < 0.05, ***P < 0.001). (b) Enforced activation of AKT-mTOR pathway increases SSEA-4+ cell frequency in MG63 cells undergoing mesenchymal differentiation. Transduced cells were plated at 60% confluence and the expression of Myr-AKT was induced by Dox; SSEA-4+ cell frequency was measured 48 hours later. Results are shown as means ± SDs. The cropped blots were run under the same experimental conditions. The full-length blots can be seen in Supplementary Figure 8. (c) mTOR inhibitor relieves the AKT activation-caused differentiation arrest of MG63 cells. Data for ALP activity and OCN mRNA levels are presented as means ± SDs (*P < 0.05, **P < 0.01, ***P < 0.001). The cropped blots were run under the same experimental conditions. The full-length blots can be seen in the Supplementary Figure 8. (d) Tumorsphere-forming rates of single SSEA-4− MG63 cells expressing vector or Myr-AKT (as in b or c) are shown on the upper panel (***P < 0.001). The enhancing effect of Myr-AKT induction on the tumorsphere-forming potential of SSEA-4− cells was abolished by RAD001 (bottom panel).
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f5: mTORC1 Supports the De-differentiate Potential of Early SSEA-4− Progeny.(a) SSEA-4− cells were sorted at different time points during MG63 cells' differentiation to osteocytes and individually inoculated into fresh medium for measuring their clonal efficiency or into special medium for measuring their tumorsphere-forming potential (*P < 0.05, ***P < 0.001). (b) Enforced activation of AKT-mTOR pathway increases SSEA-4+ cell frequency in MG63 cells undergoing mesenchymal differentiation. Transduced cells were plated at 60% confluence and the expression of Myr-AKT was induced by Dox; SSEA-4+ cell frequency was measured 48 hours later. Results are shown as means ± SDs. The cropped blots were run under the same experimental conditions. The full-length blots can be seen in Supplementary Figure 8. (c) mTOR inhibitor relieves the AKT activation-caused differentiation arrest of MG63 cells. Data for ALP activity and OCN mRNA levels are presented as means ± SDs (*P < 0.05, **P < 0.01, ***P < 0.001). The cropped blots were run under the same experimental conditions. The full-length blots can be seen in the Supplementary Figure 8. (d) Tumorsphere-forming rates of single SSEA-4− MG63 cells expressing vector or Myr-AKT (as in b or c) are shown on the upper panel (***P < 0.001). The enhancing effect of Myr-AKT induction on the tumorsphere-forming potential of SSEA-4− cells was abolished by RAD001 (bottom panel).

Mentions: Notably, a partial retention of tumorigenicity by SSEA-4− osteosarcoma cells was actually detectable by xenografting or tumorsphere-forming assay (Fig. 1d–f). Most of these cells were still highly immature osteosarcoma cells as marked by Oct3/4 expression (Fig. 2b, c). As suggested by recent studies in osteosarcoma and other types of aggressive tumors373839, we suspected that a dedifferentiation potential existed within the progeny of SSEA-4+ TICs, which contributed to the retention of tumorigenicity. To test this, we retrieved single SSEA-4− MG63 or Well5 cells from the ordinary passage (cellular confluence was <80%) and inoculated them into 96-well plates with nutrient-replenished medium. Indeed, the colony-forming rate of SSEA-4− cells was comparable to that of SSEA-4+ cells, although the formation of colonies by SSEA-4− cells usually took longer times (≈21 days versus 10 days) (Supplementary Fig. 5a), thus excluding the possibility that colony formation by SSEA-4− cells was the result of contamination by SSEA-4+ cells. Interestingly, SSEA-4+ cells were consistently detected within colonies generated by SSEA-4− cells (Supplementary Fig. 5b), indicating that the dedifferentiation of SSEA-4− cells explained their colony-forming ability. Nevertheless, it was interesting to note that when mesenchymal differentiation inducers were added to the culture, the clonal recovery rate and the tumorsphere-forming rate among live SSEA-4− cells progressively decreased along with induced osteogenic differentiation (Fig. 5a), suggesting that dedifferentiation potential was inversely correlated to the maturation status of heterogeneous SSEA-4− 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 Supports the De-differentiate Potential of Early SSEA-4− Progeny.(a) SSEA-4− cells were sorted at different time points during MG63 cells' differentiation to osteocytes and individually inoculated into fresh medium for measuring their clonal efficiency or into special medium for measuring their tumorsphere-forming potential (*P < 0.05, ***P < 0.001). (b) Enforced activation of AKT-mTOR pathway increases SSEA-4+ cell frequency in MG63 cells undergoing mesenchymal differentiation. Transduced cells were plated at 60% confluence and the expression of Myr-AKT was induced by Dox; SSEA-4+ cell frequency was measured 48 hours later. Results are shown as means ± SDs. The cropped blots were run under the same experimental conditions. The full-length blots can be seen in Supplementary Figure 8. (c) mTOR inhibitor relieves the AKT activation-caused differentiation arrest of MG63 cells. Data for ALP activity and OCN mRNA levels are presented as means ± SDs (*P < 0.05, **P < 0.01, ***P < 0.001). The cropped blots were run under the same experimental conditions. The full-length blots can be seen in the Supplementary Figure 8. (d) Tumorsphere-forming rates of single SSEA-4− MG63 cells expressing vector or Myr-AKT (as in b or c) are shown on the upper panel (***P < 0.001). The enhancing effect of Myr-AKT induction on the tumorsphere-forming potential of SSEA-4− cells was abolished by RAD001 (bottom panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: mTORC1 Supports the De-differentiate Potential of Early SSEA-4− Progeny.(a) SSEA-4− cells were sorted at different time points during MG63 cells' differentiation to osteocytes and individually inoculated into fresh medium for measuring their clonal efficiency or into special medium for measuring their tumorsphere-forming potential (*P < 0.05, ***P < 0.001). (b) Enforced activation of AKT-mTOR pathway increases SSEA-4+ cell frequency in MG63 cells undergoing mesenchymal differentiation. Transduced cells were plated at 60% confluence and the expression of Myr-AKT was induced by Dox; SSEA-4+ cell frequency was measured 48 hours later. Results are shown as means ± SDs. The cropped blots were run under the same experimental conditions. The full-length blots can be seen in Supplementary Figure 8. (c) mTOR inhibitor relieves the AKT activation-caused differentiation arrest of MG63 cells. Data for ALP activity and OCN mRNA levels are presented as means ± SDs (*P < 0.05, **P < 0.01, ***P < 0.001). The cropped blots were run under the same experimental conditions. The full-length blots can be seen in the Supplementary Figure 8. (d) Tumorsphere-forming rates of single SSEA-4− MG63 cells expressing vector or Myr-AKT (as in b or c) are shown on the upper panel (***P < 0.001). The enhancing effect of Myr-AKT induction on the tumorsphere-forming potential of SSEA-4− cells was abolished by RAD001 (bottom panel).
Mentions: Notably, a partial retention of tumorigenicity by SSEA-4− osteosarcoma cells was actually detectable by xenografting or tumorsphere-forming assay (Fig. 1d–f). Most of these cells were still highly immature osteosarcoma cells as marked by Oct3/4 expression (Fig. 2b, c). As suggested by recent studies in osteosarcoma and other types of aggressive tumors373839, we suspected that a dedifferentiation potential existed within the progeny of SSEA-4+ TICs, which contributed to the retention of tumorigenicity. To test this, we retrieved single SSEA-4− MG63 or Well5 cells from the ordinary passage (cellular confluence was <80%) and inoculated them into 96-well plates with nutrient-replenished medium. Indeed, the colony-forming rate of SSEA-4− cells was comparable to that of SSEA-4+ cells, although the formation of colonies by SSEA-4− cells usually took longer times (≈21 days versus 10 days) (Supplementary Fig. 5a), thus excluding the possibility that colony formation by SSEA-4− cells was the result of contamination by SSEA-4+ cells. Interestingly, SSEA-4+ cells were consistently detected within colonies generated by SSEA-4− cells (Supplementary Fig. 5b), indicating that the dedifferentiation of SSEA-4− cells explained their colony-forming ability. Nevertheless, it was interesting to note that when mesenchymal differentiation inducers were added to the culture, the clonal recovery rate and the tumorsphere-forming rate among live SSEA-4− cells progressively decreased along with induced osteogenic differentiation (Fig. 5a), suggesting that dedifferentiation potential was inversely correlated to the maturation status of heterogeneous SSEA-4− 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