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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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SSEA-4+ TICs Undergo Mesenchymal Differentiation to Generate SSEA-4− Cells.(a) Heatmap showing expression levels of the signature gene sets that indicate the mesenchymal differentiation status of W0823-derived SSEA-4+ TICs or SSEA-4− osteosarcoma cells. (b) Western blotting assays for the levels of PPAR-γ and RUNX2 in sorted SSEA-4+ or SSEA-4− osteosarcoma cells growing in vivo. The cropped blots were run under the same experimental conditions. The full-length blots are included in Supplementary Figure 8. (c) Co-immunofluorescent staining and inspection of cytospun Well5-xenograft cells (left panel) or frozen sectioned (right panel) L1031-derived xenografts. The antibody against SSEA-4 was labeled with Alexa555 (Red) and the antibody against OCN was FITC-labeled (green). (d) SSEA-4+ cell frequency decreased in Well5 or MG63 cells undergoing osteogenic or adipogenic differentiation, as measured by flow cytometry. (e) Tumorigenic xenograft-forming rates of Well5 cells after prior treatment with DMSO or differentiation inducers for 3–5 days as in (d) (P = 0.002 for each induction versus control). (f) SSEA-4+ cell frequency measured for Well5 or MG63 cells under different culture conditions. The percentages indicate cell densities. One hundred %-D2 or 100%-D4 indicates an additional culture for 2 days or 4 days post-confluence. Results are shown as mean ± SD, n = 3. (g) TIC frequency measurement of Well5 cells obtained from different culture conditions as in (f). One hundred %-D4 versus 50%: P < 0.001. 100% versus 50%: P = 0.034.
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f3: SSEA-4+ TICs Undergo Mesenchymal Differentiation to Generate SSEA-4− Cells.(a) Heatmap showing expression levels of the signature gene sets that indicate the mesenchymal differentiation status of W0823-derived SSEA-4+ TICs or SSEA-4− osteosarcoma cells. (b) Western blotting assays for the levels of PPAR-γ and RUNX2 in sorted SSEA-4+ or SSEA-4− osteosarcoma cells growing in vivo. The cropped blots were run under the same experimental conditions. The full-length blots are included in Supplementary Figure 8. (c) Co-immunofluorescent staining and inspection of cytospun Well5-xenograft cells (left panel) or frozen sectioned (right panel) L1031-derived xenografts. The antibody against SSEA-4 was labeled with Alexa555 (Red) and the antibody against OCN was FITC-labeled (green). (d) SSEA-4+ cell frequency decreased in Well5 or MG63 cells undergoing osteogenic or adipogenic differentiation, as measured by flow cytometry. (e) Tumorigenic xenograft-forming rates of Well5 cells after prior treatment with DMSO or differentiation inducers for 3–5 days as in (d) (P = 0.002 for each induction versus control). (f) SSEA-4+ cell frequency measured for Well5 or MG63 cells under different culture conditions. The percentages indicate cell densities. One hundred %-D2 or 100%-D4 indicates an additional culture for 2 days or 4 days post-confluence. Results are shown as mean ± SD, n = 3. (g) TIC frequency measurement of Well5 cells obtained from different culture conditions as in (f). One hundred %-D4 versus 50%: P < 0.001. 100% versus 50%: P = 0.034.

Mentions: The formation of osteoids observed in cases of high-grade osteosarcoma indicates that even highly dedifferentiated immature SSEA-4+ TICs might undergo spontaneous osteogenic maturation, although at a significantly reduced rate. A comparison of the mRNA expression profiles of SSEA-4+ cells and SSEA-4− cells freshly isolated from the W0823-derived P2 xenografts showed that 1491 of a total of 54614 probe-sets (2.7%) were differentially expressed (fold changes > 1.5, P < 0.05) (also see the database deposit). Interestingly, in accordance with the assumption that SSEA-4+ osteosarcoma cells differentiate to produce SSEA-4− cells, analyses of pathways revealed significant upregulation of several mesenchymal differentiation signature genes, such as RUNX2, BMPR2, COL3A1, IBSP and ADIPOQ, in SSEA-4− cells (Fig. 3a and Supplementary Fig. 3a)3334. Especially, Western blotting showed that freshly-isolated SSEA-4− cells expressed elevated levels of the osteogenic transcription factor RUNX2 (Fig. 3b), which is indicative of osteoblastic commitment from MSCs or osteosarcoma TICs16. Additionally, the immunofluorescent inspection of Well5- or L1031-derived xenografts showed that some SSEA-4− cells also expressed osteocalcin (OCNhi), a late-stage maturation marker of osteoblasts, but no SSEA-4+ cells did (Fig. 3c).


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)

SSEA-4+ TICs Undergo Mesenchymal Differentiation to Generate SSEA-4− Cells.(a) Heatmap showing expression levels of the signature gene sets that indicate the mesenchymal differentiation status of W0823-derived SSEA-4+ TICs or SSEA-4− osteosarcoma cells. (b) Western blotting assays for the levels of PPAR-γ and RUNX2 in sorted SSEA-4+ or SSEA-4− osteosarcoma cells growing in vivo. The cropped blots were run under the same experimental conditions. The full-length blots are included in Supplementary Figure 8. (c) Co-immunofluorescent staining and inspection of cytospun Well5-xenograft cells (left panel) or frozen sectioned (right panel) L1031-derived xenografts. The antibody against SSEA-4 was labeled with Alexa555 (Red) and the antibody against OCN was FITC-labeled (green). (d) SSEA-4+ cell frequency decreased in Well5 or MG63 cells undergoing osteogenic or adipogenic differentiation, as measured by flow cytometry. (e) Tumorigenic xenograft-forming rates of Well5 cells after prior treatment with DMSO or differentiation inducers for 3–5 days as in (d) (P = 0.002 for each induction versus control). (f) SSEA-4+ cell frequency measured for Well5 or MG63 cells under different culture conditions. The percentages indicate cell densities. One hundred %-D2 or 100%-D4 indicates an additional culture for 2 days or 4 days post-confluence. Results are shown as mean ± SD, n = 3. (g) TIC frequency measurement of Well5 cells obtained from different culture conditions as in (f). One hundred %-D4 versus 50%: P < 0.001. 100% versus 50%: P = 0.034.
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Related In: Results  -  Collection

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Show All Figures
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f3: SSEA-4+ TICs Undergo Mesenchymal Differentiation to Generate SSEA-4− Cells.(a) Heatmap showing expression levels of the signature gene sets that indicate the mesenchymal differentiation status of W0823-derived SSEA-4+ TICs or SSEA-4− osteosarcoma cells. (b) Western blotting assays for the levels of PPAR-γ and RUNX2 in sorted SSEA-4+ or SSEA-4− osteosarcoma cells growing in vivo. The cropped blots were run under the same experimental conditions. The full-length blots are included in Supplementary Figure 8. (c) Co-immunofluorescent staining and inspection of cytospun Well5-xenograft cells (left panel) or frozen sectioned (right panel) L1031-derived xenografts. The antibody against SSEA-4 was labeled with Alexa555 (Red) and the antibody against OCN was FITC-labeled (green). (d) SSEA-4+ cell frequency decreased in Well5 or MG63 cells undergoing osteogenic or adipogenic differentiation, as measured by flow cytometry. (e) Tumorigenic xenograft-forming rates of Well5 cells after prior treatment with DMSO or differentiation inducers for 3–5 days as in (d) (P = 0.002 for each induction versus control). (f) SSEA-4+ cell frequency measured for Well5 or MG63 cells under different culture conditions. The percentages indicate cell densities. One hundred %-D2 or 100%-D4 indicates an additional culture for 2 days or 4 days post-confluence. Results are shown as mean ± SD, n = 3. (g) TIC frequency measurement of Well5 cells obtained from different culture conditions as in (f). One hundred %-D4 versus 50%: P < 0.001. 100% versus 50%: P = 0.034.
Mentions: The formation of osteoids observed in cases of high-grade osteosarcoma indicates that even highly dedifferentiated immature SSEA-4+ TICs might undergo spontaneous osteogenic maturation, although at a significantly reduced rate. A comparison of the mRNA expression profiles of SSEA-4+ cells and SSEA-4− cells freshly isolated from the W0823-derived P2 xenografts showed that 1491 of a total of 54614 probe-sets (2.7%) were differentially expressed (fold changes > 1.5, P < 0.05) (also see the database deposit). Interestingly, in accordance with the assumption that SSEA-4+ osteosarcoma cells differentiate to produce SSEA-4− cells, analyses of pathways revealed significant upregulation of several mesenchymal differentiation signature genes, such as RUNX2, BMPR2, COL3A1, IBSP and ADIPOQ, in SSEA-4− cells (Fig. 3a and Supplementary Fig. 3a)3334. Especially, Western blotting showed that freshly-isolated SSEA-4− cells expressed elevated levels of the osteogenic transcription factor RUNX2 (Fig. 3b), which is indicative of osteoblastic commitment from MSCs or osteosarcoma TICs16. Additionally, the immunofluorescent inspection of Well5- or L1031-derived xenografts showed that some SSEA-4− cells also expressed osteocalcin (OCNhi), a late-stage maturation marker of osteoblasts, but no SSEA-4+ cells did (Fig. 3c).

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