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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 Labels Xenografting-TICs Present in a Minority of Human Osteosarcoma Cases.(a) The frequency of SSEA-4+ cells in P1 xenografts correlates with xenograft-formation potential in secondary recipients. (b) Live 7-AAD− and murine CD45+/MHC-I+-excluded osteosarcoma xenograft cells were analyzed for expression of the indicated antigens by flow cytometry. (c) Primary osteosarcoma samples that generate both P1 and P2 xenografts show higher SSEA-4-staining intensity than those that produced only P1 xenografts. Primary specimens that produced no P1 xenografts stained negatively for SSEA-4. Immunohistochemical staining signals for SSEA-4 are indicated by arrows. Scale bar represents 100 μm. (d) Engrafting efficiencies of SSEA-4+ or SSEA-4− cells freshly isolated from three individual tumorigenic xenografts (P2 to P5). (e) Xenografting efficiency of SSEA-4+ or SSEA-4− osteosarcoma cells isolated from in vitro-cultivated Well5 cells (P < 0.05). (f) Sphere-forming rate of SSEA-4+ or SSEA-4− cells isolated from in vitro cultivated osteosarcoma MG63 cells (P < 0.05). Microscopic inspection of representative tumor-sphere or cellular debris is shown in the upper panel.
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f1: SSEA-4 Labels Xenografting-TICs Present in a Minority of Human Osteosarcoma Cases.(a) The frequency of SSEA-4+ cells in P1 xenografts correlates with xenograft-formation potential in secondary recipients. (b) Live 7-AAD− and murine CD45+/MHC-I+-excluded osteosarcoma xenograft cells were analyzed for expression of the indicated antigens by flow cytometry. (c) Primary osteosarcoma samples that generate both P1 and P2 xenografts show higher SSEA-4-staining intensity than those that produced only P1 xenografts. Primary specimens that produced no P1 xenografts stained negatively for SSEA-4. Immunohistochemical staining signals for SSEA-4 are indicated by arrows. Scale bar represents 100 μm. (d) Engrafting efficiencies of SSEA-4+ or SSEA-4− cells freshly isolated from three individual tumorigenic xenografts (P2 to P5). (e) Xenografting efficiency of SSEA-4+ or SSEA-4− osteosarcoma cells isolated from in vitro-cultivated Well5 cells (P < 0.05). (f) Sphere-forming rate of SSEA-4+ or SSEA-4− cells isolated from in vitro cultivated osteosarcoma MG63 cells (P < 0.05). Microscopic inspection of representative tumor-sphere or cellular debris is shown in the upper panel.

Mentions: To identify putative TICs for osteosarcoma, we subcutaneously inoculated minced human osteosarcoma tissue (typically ≤ 3 × 3 × 1.5 mm3) into NOD/SCID mice. Eight of 21 primary specimens generated passage 1 (P1) xenografts wherein the retention of osteosarcoma identity was evidenced by the presence of osteoid formations (Supplementary Fig. 1a). Interestingly, upon the secondary inoculation of 1–10 × 106 huCD44-labeled P1 cells per recipient, only the four P1 xenografts containing SSEA-4+ cells (>1%) but not others generated P2 xenografts (Fig. 1a, b). Likewise, as assayed by immunohistochemical staining, only SSEA-4+ cells-containing primary samples generated P1 xenografts, with SSEA-4+ cells being more frequently detected in those also producing P2 xenografts (Fig. 1c and data not shown). Immunofluorescent co-staining of tissue sections and FISH assays on sorted SSEA-4+ cells showed that the expression of p53 or/and Rb was absent in most SSEA-4+ cells, thus discriminating them from nonmalignant stromal cells (Supplementary Fig. 1b). As this secondary xenografting rate was likely determined by the frequency of TIC within the P1 xenografts, we assumed that SSEA-4 could be used as a xenografting-TIC-enriching biomarker.


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 Labels Xenografting-TICs Present in a Minority of Human Osteosarcoma Cases.(a) The frequency of SSEA-4+ cells in P1 xenografts correlates with xenograft-formation potential in secondary recipients. (b) Live 7-AAD− and murine CD45+/MHC-I+-excluded osteosarcoma xenograft cells were analyzed for expression of the indicated antigens by flow cytometry. (c) Primary osteosarcoma samples that generate both P1 and P2 xenografts show higher SSEA-4-staining intensity than those that produced only P1 xenografts. Primary specimens that produced no P1 xenografts stained negatively for SSEA-4. Immunohistochemical staining signals for SSEA-4 are indicated by arrows. Scale bar represents 100 μm. (d) Engrafting efficiencies of SSEA-4+ or SSEA-4− cells freshly isolated from three individual tumorigenic xenografts (P2 to P5). (e) Xenografting efficiency of SSEA-4+ or SSEA-4− osteosarcoma cells isolated from in vitro-cultivated Well5 cells (P < 0.05). (f) Sphere-forming rate of SSEA-4+ or SSEA-4− cells isolated from in vitro cultivated osteosarcoma MG63 cells (P < 0.05). Microscopic inspection of representative tumor-sphere or cellular debris is shown in the upper panel.
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Related In: Results  -  Collection

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f1: SSEA-4 Labels Xenografting-TICs Present in a Minority of Human Osteosarcoma Cases.(a) The frequency of SSEA-4+ cells in P1 xenografts correlates with xenograft-formation potential in secondary recipients. (b) Live 7-AAD− and murine CD45+/MHC-I+-excluded osteosarcoma xenograft cells were analyzed for expression of the indicated antigens by flow cytometry. (c) Primary osteosarcoma samples that generate both P1 and P2 xenografts show higher SSEA-4-staining intensity than those that produced only P1 xenografts. Primary specimens that produced no P1 xenografts stained negatively for SSEA-4. Immunohistochemical staining signals for SSEA-4 are indicated by arrows. Scale bar represents 100 μm. (d) Engrafting efficiencies of SSEA-4+ or SSEA-4− cells freshly isolated from three individual tumorigenic xenografts (P2 to P5). (e) Xenografting efficiency of SSEA-4+ or SSEA-4− osteosarcoma cells isolated from in vitro-cultivated Well5 cells (P < 0.05). (f) Sphere-forming rate of SSEA-4+ or SSEA-4− cells isolated from in vitro cultivated osteosarcoma MG63 cells (P < 0.05). Microscopic inspection of representative tumor-sphere or cellular debris is shown in the upper panel.
Mentions: To identify putative TICs for osteosarcoma, we subcutaneously inoculated minced human osteosarcoma tissue (typically ≤ 3 × 3 × 1.5 mm3) into NOD/SCID mice. Eight of 21 primary specimens generated passage 1 (P1) xenografts wherein the retention of osteosarcoma identity was evidenced by the presence of osteoid formations (Supplementary Fig. 1a). Interestingly, upon the secondary inoculation of 1–10 × 106 huCD44-labeled P1 cells per recipient, only the four P1 xenografts containing SSEA-4+ cells (>1%) but not others generated P2 xenografts (Fig. 1a, b). Likewise, as assayed by immunohistochemical staining, only SSEA-4+ cells-containing primary samples generated P1 xenografts, with SSEA-4+ cells being more frequently detected in those also producing P2 xenografts (Fig. 1c and data not shown). Immunofluorescent co-staining of tissue sections and FISH assays on sorted SSEA-4+ cells showed that the expression of p53 or/and Rb was absent in most SSEA-4+ cells, thus discriminating them from nonmalignant stromal cells (Supplementary Fig. 1b). As this secondary xenografting rate was likely determined by the frequency of TIC within the P1 xenografts, we assumed that SSEA-4 could be used as a xenografting-TIC-enriching biomarker.

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