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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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Terminal Differentiation Induction by mTOR-inactivation Decreases SSEA-4+ TICs in vivo.(a) SSEA-4+ cells (1–50 × 105) from the different resources as indicated were subcutaneously inoculated into NOD/SCID mice. The oral administration of PBS (filled box) or 5 mg/kg RAD001 (filled circle) commenced 2 days later. Tumor volumes are shown as the means ± SDs, n = 3–5. (b–c) Representative images of whole-body (b) or lung metastasis (c) bioluminescence 4 weeks following subcutaneous (b) or tail vein injection (c) of 1 × 105 PBS- or RAD001-treated SSEA-4+ Well5 cells (as in (a)) into NOD/SCID mice. (d) Two representative tissue samples retrieved from the PBS-treated and RAD001-treated groups, respectively. (e) Immunohistochemical staining of p-S6, SSEA-4, or OCN in xenografts retrieved from the PBS-or RAD001-treated group, as in (a and b). Scale bars represent 100 μm. HE: hematoxylin-eosin. (f) Secondary tumorigenic xenograft formation of tumor tissue or cells after post-PBS or -RAD001 treatment, as in (a and b) (upper panel). Secondary tumorigenic xenografting rates (n/n) are summarized in the bottom table (P < 0.01).
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f6: Terminal Differentiation Induction by mTOR-inactivation Decreases SSEA-4+ TICs in vivo.(a) SSEA-4+ cells (1–50 × 105) from the different resources as indicated were subcutaneously inoculated into NOD/SCID mice. The oral administration of PBS (filled box) or 5 mg/kg RAD001 (filled circle) commenced 2 days later. Tumor volumes are shown as the means ± SDs, n = 3–5. (b–c) Representative images of whole-body (b) or lung metastasis (c) bioluminescence 4 weeks following subcutaneous (b) or tail vein injection (c) of 1 × 105 PBS- or RAD001-treated SSEA-4+ Well5 cells (as in (a)) into NOD/SCID mice. (d) Two representative tissue samples retrieved from the PBS-treated and RAD001-treated groups, respectively. (e) Immunohistochemical staining of p-S6, SSEA-4, or OCN in xenografts retrieved from the PBS-or RAD001-treated group, as in (a and b). Scale bars represent 100 μm. HE: hematoxylin-eosin. (f) Secondary tumorigenic xenograft formation of tumor tissue or cells after post-PBS or -RAD001 treatment, as in (a and b) (upper panel). Secondary tumorigenic xenografting rates (n/n) are summarized in the bottom table (P < 0.01).

Mentions: mTORC1 activity is elevated by p53 deficiency through attenuation of AMPK activity and potentially by various types of other osteosarcoma-related oncogenic events4042434445. Nevertheless, we argued that mTORC1 activity is also subject to negative regulation by various types of environmental cues, such as the lack of nutrients that significantly decreased mTORC1 activity by activating AMPK in p53 or/and Rb-deficient osteosarcoma cells (Supplementary Fig. 5c–f). Since previous studies using mTOR inhibitors showed differential inhibitory effects on the in vivo growth of different osteosarcoma cell lines23242526, we examined whether mTOR inactivation alone would cause loss of tumorigenicity in SSEA-4+ TICs and their progeny by enforcing terminal differentiation in vivo. Interestingly, following the subcutaneous or intravenous inoculation of NOD/SCID mice with 1–50 × 105 SSEA-4+ osteosarcoma cells freshly- isolated from 3 different xenografts, RAD001 gavage for about 4–8 weeks significantly inhibited the formation and/or growth of subcutaneous or lung xenografts in these animals (Fig. 6a–c). Notably, pathological studies showed that compared to bulky PBS-treated tumorigenic xenografts, the tiny residual xenografts recovered from the RAD001-treated group were mostly the accumulation of a cellular collagenous masses akin to osteoid structures. These masses were only sparsely infiltrated by SSEA-4− p-S6low OCNhi mature osteogenic cells (Fig. 6d, e). To test whether tumorigenicity was decreased by RAD001 treatment, we then re-inoculated the retrieved xenograft tissues or isolated tumor cells into secondary recipients without further intervention. Strikingly, no tumorigenic engraftments were detected after 7 inoculations of RAD001-treated xenografts, whereas all 9 comparable inoculations from PBS-treated xenografts generated the palpable tumors (Fig. 6f). This demonstrates a strong inhibitory effect of mTOR inactivation on the propagation of SSEA-4+ TICs through the enforcement of terminal maturation. On the other hand, although systemic chemotherapy with Methotrexate (MTX) also partially inhibited the growth of xenografts, abundant SSEA-4+ p-S6+OCN− immature TICs were detected (Supplementary Fig. 6a, b), and these cells generated the tumorigenic xenografts upon the secondary inoculation (data not shown).


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)

Terminal Differentiation Induction by mTOR-inactivation Decreases SSEA-4+ TICs in vivo.(a) SSEA-4+ cells (1–50 × 105) from the different resources as indicated were subcutaneously inoculated into NOD/SCID mice. The oral administration of PBS (filled box) or 5 mg/kg RAD001 (filled circle) commenced 2 days later. Tumor volumes are shown as the means ± SDs, n = 3–5. (b–c) Representative images of whole-body (b) or lung metastasis (c) bioluminescence 4 weeks following subcutaneous (b) or tail vein injection (c) of 1 × 105 PBS- or RAD001-treated SSEA-4+ Well5 cells (as in (a)) into NOD/SCID mice. (d) Two representative tissue samples retrieved from the PBS-treated and RAD001-treated groups, respectively. (e) Immunohistochemical staining of p-S6, SSEA-4, or OCN in xenografts retrieved from the PBS-or RAD001-treated group, as in (a and b). Scale bars represent 100 μm. HE: hematoxylin-eosin. (f) Secondary tumorigenic xenograft formation of tumor tissue or cells after post-PBS or -RAD001 treatment, as in (a and b) (upper panel). Secondary tumorigenic xenografting rates (n/n) are summarized in the bottom table (P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Terminal Differentiation Induction by mTOR-inactivation Decreases SSEA-4+ TICs in vivo.(a) SSEA-4+ cells (1–50 × 105) from the different resources as indicated were subcutaneously inoculated into NOD/SCID mice. The oral administration of PBS (filled box) or 5 mg/kg RAD001 (filled circle) commenced 2 days later. Tumor volumes are shown as the means ± SDs, n = 3–5. (b–c) Representative images of whole-body (b) or lung metastasis (c) bioluminescence 4 weeks following subcutaneous (b) or tail vein injection (c) of 1 × 105 PBS- or RAD001-treated SSEA-4+ Well5 cells (as in (a)) into NOD/SCID mice. (d) Two representative tissue samples retrieved from the PBS-treated and RAD001-treated groups, respectively. (e) Immunohistochemical staining of p-S6, SSEA-4, or OCN in xenografts retrieved from the PBS-or RAD001-treated group, as in (a and b). Scale bars represent 100 μm. HE: hematoxylin-eosin. (f) Secondary tumorigenic xenograft formation of tumor tissue or cells after post-PBS or -RAD001 treatment, as in (a and b) (upper panel). Secondary tumorigenic xenografting rates (n/n) are summarized in the bottom table (P < 0.01).
Mentions: mTORC1 activity is elevated by p53 deficiency through attenuation of AMPK activity and potentially by various types of other osteosarcoma-related oncogenic events4042434445. Nevertheless, we argued that mTORC1 activity is also subject to negative regulation by various types of environmental cues, such as the lack of nutrients that significantly decreased mTORC1 activity by activating AMPK in p53 or/and Rb-deficient osteosarcoma cells (Supplementary Fig. 5c–f). Since previous studies using mTOR inhibitors showed differential inhibitory effects on the in vivo growth of different osteosarcoma cell lines23242526, we examined whether mTOR inactivation alone would cause loss of tumorigenicity in SSEA-4+ TICs and their progeny by enforcing terminal differentiation in vivo. Interestingly, following the subcutaneous or intravenous inoculation of NOD/SCID mice with 1–50 × 105 SSEA-4+ osteosarcoma cells freshly- isolated from 3 different xenografts, RAD001 gavage for about 4–8 weeks significantly inhibited the formation and/or growth of subcutaneous or lung xenografts in these animals (Fig. 6a–c). Notably, pathological studies showed that compared to bulky PBS-treated tumorigenic xenografts, the tiny residual xenografts recovered from the RAD001-treated group were mostly the accumulation of a cellular collagenous masses akin to osteoid structures. These masses were only sparsely infiltrated by SSEA-4− p-S6low OCNhi mature osteogenic cells (Fig. 6d, e). To test whether tumorigenicity was decreased by RAD001 treatment, we then re-inoculated the retrieved xenograft tissues or isolated tumor cells into secondary recipients without further intervention. Strikingly, no tumorigenic engraftments were detected after 7 inoculations of RAD001-treated xenografts, whereas all 9 comparable inoculations from PBS-treated xenografts generated the palpable tumors (Fig. 6f). This demonstrates a strong inhibitory effect of mTOR inactivation on the propagation of SSEA-4+ TICs through the enforcement of terminal maturation. On the other hand, although systemic chemotherapy with Methotrexate (MTX) also partially inhibited the growth of xenografts, abundant SSEA-4+ p-S6+OCN− immature TICs were detected (Supplementary Fig. 6a, b), and these cells generated the tumorigenic xenografts upon the secondary inoculation (data not shown).

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