mTORC1 Maintains the Tumorigenicity of SSEA-4+ High-Grade Osteosarcoma

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.

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 1 tumor cells represent a subset of tumor-initiating cells (TICs) whose pool size is maintained by mTORC1 activity. mTORC1 supports not only SSEA-4 1 cell self-renewal through S6K but also the regeneration of SSEA-4 1 TICs by SSEA-4 2 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 2 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 1 TICs and their progeny.
O steosarcoma represents a type of highly aggressive bone tumor prevalent in adolescents and is characterized by composite genetic defects. Early observations pointed to genetic defects in the Rb or p53 pathway as driving events behind tumorigenesis 1,2 . Accordingly, recent studies have indicated that in all osteosarcoma cases, the p53 pathway is functionally defective 3 . More than 70% of sporadic osteosarcoma cases harbor genetic abnormalities involving the Rb pathway 4 . Although the targeted deletion of p53 but not Rb alone caused murine osteosarcomas to develop, the simultaneous deletion of p53 and Rb significantly accelerated tumorigenesis [4][5][6] , indicating that inactivation of these two pathways cooperates to drive malignant transformation [7][8][9][10] .
The dedifferentiation process essentially underlies the genesis of osteosarcoma that is marked with mesenchymal immaturity, as the targeted-deletion of p53 or both p53 and Rb in committed or mature osteoblasts rather than in mesenchymal stem cells (MSCs) results in osteosarcoma in mouse models 4,[11][12][13] . In agreement with this, recent studies demonstrate that p53 or Rb deficiency activates the dedifferentiation potential of many types of somatic cells 10,14 . Since a prior cell-cycle exit is required for immature osteosarcoma cells to undergo the terminal maturation that likely eliminates their tumorigenicity 15,16 , and p53 and Rb pathways share the critical activity of restraining cell-cycle progression, defects in the p53 or/and Rb pathway(s) may confer dedifferentiation potential to osteosarcoma cells largely by reinstituting their entry into the cell cycle from the post-mitotic state 17 .
On the other hand, cases of osteosarcoma can be highly heterogeneous in terms of their clinical prognosis. With the introduction of combined systemic chemotherapy plus surgery, approximately 60-70% of newly-diagnosed osteosarcoma patients actually achieve long-term survival 18 , whereas the remaining cases are chemo-resistant and prone to metastasize, thus constituting a high-grade subgroup 19,20 . Few studies have addressed this clinical heterogeneity 21 . A recent study did report that the level of p-S6, an indictor of mTOR activity, positively correlated with poor prognosis in osteosarcoma 22 . In parallel, a phase I-II clinical trial of mTOR inhibitors on a panel of pediatric tumors revealed a plausible therapeutic benefit in a few advanced osteosarcoma cases [23][24][25] . More recently, use of a combination of multi-kinase inhibitors restrained the growth of osteosarcoma cell lines in vitro and in vivo, which was accompanied by a reduction in the activities of mTORC1 and mTORC2 26 . Nevertheless, the precise role mTOR plays in the regulation of the malignant stemness of osteosarcoma cells remains unclear.
Interestingly, all osteosarcoma cases, including high-grade ones, are pathologically characterized by the spontaneous formation of osteoid, an unmineralized pre-bone structure produced largely by mature osteocytes. This seems to indicate that putative osteosarcoma tumor-initiating cells (TICs) retain the potential to undergo terminal osteogenic maturation in vivo. In this study, we explore which cellular mechanisms control terminal differentiation versus the maintenance of malignant immaturity associated with high-grade osteosarcoma, which may shed potential light on the design of new therapeutic regimens for this currently-incurable malignancy.

Results
SSEA-4 1 osteosarcoma cells represent a subset of xenografting-TICs. To identify putative TICs for osteosarcoma, we subcutaneously inoculated minced human osteosarcoma tissue (typically # 3 3 3 3 1.5 mm 3 ) 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 3 10 6 huCD44-labeled P1 cells per recipient, only the four P1 xenografts containing SSEA-4 1 cells (.1%) but not others generated P2 xenografts (Fig. 1a, b). Likewise, as assayed by immunohistochemical staining, only SSEA-4 1 cells-containing primary samples generated P1 xenografts, with SSEA-4 1 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 1 cells showed that the expression of p53 or/and Rb was absent in most SSEA-4 1 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.
Actually, SSEA-4 1 cells were also present within W0823-derived Well5 cells passaged in vitro and in osteosarcoma cell lines MG63 and U2OS cells, but not Saos-2 cells ( Supplementary Fig. 1f, g and data not shown) 29 . Both Well5 and MG63 cells possessed mesenchymal multipotency showing bi-differentiation potential towards osteogenic and adipocytic lineages ( Supplementary Fig. 1h), indicating that SSEA-4 1 TICs remain at an immature stage before osteoblastic commitment. Tumorigenic xenograft-forming or tumorsphere-forming assays of Well5 or MG63 cells confirmed that tumorigenicity was much more enriched in the SSEA-4 1 cell fraction than in SSEA-4 2 cells (Fig. 1e, f). Nevertheless, ISP-1, an inhibitor of SSEA-4 synthesis, did not reduce the tumorsphere-forming potential of MG63 cells ( Supplementary Fig. 1i), indicating that SSEA-4 itself represents a biomarker rather than a functional regulator of malignant stemness.
Frequency of SSEA-4 1 TICs predicts prognosis. SSEA-4 1 osteosarcoma cells were readily detectable only in a small fraction (8/21) of primary osteosarcoma specimens (Fig. 1c, d), which prompted us to test whether the osteosarcoma cases containing SSEA-4 1 TICs represent a subtype of osteosarcoma distinct from the majority of SSEA-4 neg cases. To address this, we performed a retrospective analysis of a cohort of osteosarcoma cases collected over .10 years. Remarkably, the frequency of SSEA-4 1 TICs alone, as indicated by immunohistochemical staining (arbitrarily determined as -, 11, 21, or 31; see Supplementary Fig. 2a) before or after the first round of chemotherapy, correlated inversely with the overall-survival probabilities of patients (Fig. 2a). Strikingly, .90% of SSEA-4 neg cases but no SSEA-4 21-31 cases survived more than 10 years after diagnosis. Notably, transcription factor Oct3/4, the expression of which marks MSCs and was reported in certain cultivated osteosarcoma cells to associate with a gain of tumorigenicity and probably reprogramming events 28,30,31 , was consistently and abundantly expressed in bulk tissue cells of SSEA-4 12-3 primary specimens (n 5 26) wherein SSEA-4 1 TICs constituted a small subfraction of Oct3/4 1 osteosarcoma cells (Fig. 2b, c). In striking contrast, no Oct3/4 expression was detected in eight SSEA-4 neg cases examined (Fig. 2b, Supplementary Fig. 2b, c), thus indicating that SSEA-4 1 TICs but not TICs from other subtypes of osteosarcoma have been reprogrammed to a highly immature status comparable to MSCs or even ES cells in certain molecular programs 32 .
Mesenchymal differentiation of SSEA-4 1 TICs parallels a gradual loss of tumorigenicity. The formation of osteoids observed in cases of high-grade osteosarcoma indicates that even highly dedifferentiated immature SSEA-4 1 TICs might undergo spontaneous osteogenic maturation, although at a significantly reduced rate. A comparison of the mRNA expression profiles of SSEA-4 1 cells and SSEA-4 2 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 1 osteosarcoma cells differentiate to produce SSEA-4 2 cells, analyses of pathways revealed significant upregulation of several mesenchymal differentiation signature genes, such as RUNX2, BMPR2, COL3A1, IBSP and ADIPOQ, in SSEA-4 2 cells ( Fig. 3a and Supplementary Fig. 3a) 33,34 . Especially, Western blotting showed that freshly-isolated SSEA-4 2 cells expressed elevated levels of the osteogenic transcription factor RUNX2 (Fig. 3b), which is indicative of osteoblastic commitment from MSCs or osteosarcoma TICs 16 . Additionally, the immunofluorescent inspection of Well5-or L1031-derived xenografts showed that some SSEA-4 2 cells also expressed osteocalcin (OCN hi ), a late-stage maturation marker of osteoblasts, but no SSEA-4 1 cells did (Fig. 3c).
Next, we examined whether the induced mesenchymal differentiation of SSEA-4 1 TICs in vitro would reduce tumorigenicity. As expected, the SSEA-4 1 cell frequency declined when MG63 cells, www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 9604 | DOI: 10.1038/srep09604 U-2OS cells or Well5 cells were exposed to osteogenic or adipocytic differentiation inducers ( Fig. 3d and Supplementary Fig. 3b) before any obvious apoptosis-induction was detectable ( Supplementary Fig.  3c). As assayed by xenografting experiments, the tumorigenicity within the live Well5 cells was significantly decreased in conjunction with this process (Fig. 3e). Analogous to this, the frequency of SSEA-4 1 cells declined along with the elongated post-confluence cultures in Well5 and MG63 cells (Fig. 3f), which, as previously reported 35 , induced osteogenic or adipocytic differentiation in osteosarcoma cells ( Supplementary Fig. 3d). Again, a parallel gradual reduction in xenografting-TIC frequency was observed for Well5 cells ( Fig. 3g and Supplementary Fig. 3e). Taken together, these results indicate that tumorigenicity was inversely associated with the differentiation status of osteosarcoma cells.   mTORC1 activity maintains the immaturity of SSEA-4 1 TICs. We next explored which molecular mechanisms maintain the immaturity of SSEA-4 1 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-b catenin, Notch and AKT-mTOR axes, exhibited significant differences when comparing SSEA-4 1 to SSEA-4 2 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, b-catenin and Notch inhibitors) quickly reduced the SSEA-4 1 cell frequency (Fig. 4a). A detailed analysis of dose-or timedependent inhibition of SSEA-4 1 cell frequency by RAD001 or LY294002 indicated that the SSEA-4 1 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-AKT S473 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 1 cells than in SSEA-4 2 cells asfreshly isolated from Well5-or L1031-derived xenografts, whereas there was no difference in the level of b-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 1 TICs autonomously. To further examine whether mTORC2 was involved at all in this process 36 , 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 1 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 prognosis 22 , 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 1 TICs in human osteosarcoma cells.
As expected, mTORC1 inactivation by RAD001 drove MG63 and Well5 SSEA-4 1 cells into osteogenic differentiation as evident by the upregulation of RUNX2 and OCN (Fig. 4g). Surprisingly, some of these osteosarcoma cells actually reached the terminal osteocytic stage as evident by positive Alizarin Red S staining ( Supplementary  Fig. 4h). Likewise, Raptor knockdown but not Rictor knockdown resulted in the upregulation of both OCN mRNA and ALP-another osteogenic maturation marker ( Fig. 4h and Supplementary Fig. 4i). Nevertheless, the upregulation of RUNX2 (Supplementary Fig. 4g) but not of OCN mRNA levels or ALP activity could be caused by S6K knockdown (Supplementary Fig. 4j), thus indicating that SSEA-4 2 osteosarcoma cells are a heterogeneous population lying at discrete differentiation stages, and that S6K inhibition alone promoted only an osteogenic commitment of SSEA-4 1 TICs but not further maturation.
Partially differentiated SSEA-4 2 osteosarcoma cells dedifferentiate to regenerate SSEA-4 1 TICs. Notably, a partial retention of tumorigenicity by SSEA-4 2 osteosarcoma cells was actually detectable by xenografting or tumorsphere-forming assay (Fig. 1df). 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 tumors 37-39 , we suspected that a dedifferentiation potential existed within the progeny of SSEA-4 1 TICs, which contributed to the retention of tumorigenicity. To test this, we retrieved single SSEA-4 2 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 2 cells was comparable to that of SSEA-4 1 cells, although the formation of colonies by SSEA-4 2 cells usually took longer times (<21 days versus 10 days) ( Supplementary Fig. 5a), thus excluding the possibility that colony formation by SSEA-4 2 cells was the result of contamination by SSEA-4 1 cells. Interestingly, SSEA-4 1 cells were consistently detected within colonies generated by SSEA-4 2 cells ( Supplementary Fig. 5b), indicating that the dedifferentiation of SSEA-4 2 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 2 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 2 osteosarcoma cells.
mTORC1 induces a bias toward dedifferentiation rather than terminal differentiation in SSEA-4 2 osteosarcoma cells. In parallel to this flexible tumorigenicity observed in osteosarcoma cells, it is well known that the mTORC1 activity is reversibly modulated by the AKT and AMPK pathways 40,41 . As expected, Dox-induced expression of Myr-AKT activated mTORC1, which in turn prevented the loss of the SSEA-4 1 cells during the elongated period of culturing (Fig. 5b). Parallel measurements of ALP and OCN indicated that the induced terminal differentiation of MG63 cells was partially arrested (Fig. 5c), a result that was then reversed by RAD001, which preferentially targeted the mTORC1 complex (Fig. 5c). Conversely, glucose depletion for 5 days, which is known to inactivate mTORC1 via AMPK activation, induced terminal osteoblastic differentiation in MG63 cells ( Supplementary Fig. 5c, d), and accordingly decreased SSEA-4 1 cell frequency and tumorsphere-forming rate in these cells (Supplementary Fig. 5e, f). Notably, an inducible Myr-AKT activity endowed SSEA-4 2 cells with a measurable gain in tumorsphere-forming potential, which was abolished by RAD001 (Fig. 5d). Taken together, these findings indicate that mTORC1 maintains the SSEA-4 1 TIC pool not only by supporting their selfrenewal but also by biasing the dedifferentiation of certain SSEA-4 2 progeny at the expense of their terminal maturation ( Supplementary  Fig. 5g). In line with this, in vitro pretreatment with RAD001 significantly decreased the tumorigenic potential of MG63 cells and Well5 cells ( Supplementary Fig. 5h, i), which was mimicked by Dox-induced Raptor knockdown but not by Rictor knockdown after transient treatment ( Supplementary Fig. 5j). mTOR inactivation causes the terminal differentiation of apical SSEA-4 1 TICs in vivo. mTORC1 activity is elevated by p53 deficiency through attenuation of AMPK activity and potentially by various types of other osteosarcoma-related oncogenic events 40,[42][43][44][45] . 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 lines 23-26 , we examined whether mTOR inactivation alone would cause loss of tumorigenicity in SSEA-4 1 TICs and their progeny by enforcing terminal differentiation in vivo. Interestingly, following the subcutaneous or intravenous inoculation of NOD/SCID mice with 1-50 3 10 5 SSEA-4 1 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 2 p-S6 low OCN hi mature osteogenic cells (Fig. 6d, e). To test whether tumorigenicity was decreased by RAD001 treatment, we then reinoculated 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 1 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 1 p-S6 1 OCN 2 immature TICs were detected ( Supplementary Fig. 6a, b), and these cells generated the tumorigenic xenografts upon the secondary inoculation (data not shown). mTORC1 inactivation derepresses p27 resulting in terminal differentiation of p53/Rb-deficient osteosarcoma cells. As S6K inactivation promoted only a partial osteogenic differentiation of SSEA-4 1 TICs (Supplementary Fig. 4j), we wanted to investigate the downstream mechanisms of mTORC1 in the regulation of dedifferentiation versus terminal maturation of partially differentiated SSEA-4 2 osteosarcoma cells, as this marks a critical point for retention or elimination of tumorigenicity. In this regard, previous studies indicated that cell-cycle exit or reentry controlled by p27 level/activity is a prerequisite for the terminal differentiation or malignant transformation of osteoblasts, respectively 15,16 . Especially, p27 levels in high-grade human osteosarcoma were found to be greatly reduced compared to such levels in low-grade osteosarcoma 16 . Interestingly, among different osteogenic differentiation models tested, we noticed a common molecular occurrence: a reduction in mTORC1 activity was accompanied selectively by a progressive upregulation of the cell cycle inhibitor p27 (Supplementary Fig.  5c, Supplementary Fig. 7a, b and data not shown), while neither p53 nor Rb expression was upregulated in Rb-null Well5 cells nor in p53null MG63 cells during induced mesenchymal differentiation (Supplementary Fig. 7c). Indeed, p27 levels in SSEA-4 1 TICs- initiated xenografts were significantly upregulated by RAD001 (Supplementary Fig. 7d). In line with this, previously studies have demonstrated that p27 repression represents a key mechanism whereby mTOR activity supports cellular proliferation [46][47][48] . We confirmed that RAD001 elevated the levels of p27 mRNA in parallel with OCN mRNA levels ( Supplementary Fig. 7e).
Indeed, consistent with a progressive elevation in p27 but not in p21 levels ( Fig. 7a and supplementary Fig. 7f), RAD001 induced a pre-S phase cell cycle arrest in MG63, U-2OS and Well5 cells prior to inducing apoptosis ( Fig. 7b and Supplementary Fig. 7g-h). These results indicated that, analogous to a well-documented role for p21 upregulation in driving the differentiation of certain types of osteosarcoma cell lines 49 , p27 upregulation represents a critical molecular event following mTOR inactivation in SSEA-4 1 TIC-derived progeny, especially for their late-stage differentiation. Consistent with this observation, while Raptor knockdown upregulated both p27 and p21, Rictor knockdown upregulated p21 but not p27 in p53-deficient MG63 cells (Supplementary Fig. 7i). Knockdown of Raptor resulted  in obvious cell-cycle arrest ( Supplementary Fig. 7j), while such arrest was not seen in Rictor knockdown.

Discussion
A previous study demonstrated that the Oct3/4 promoter is active in certain types of osteosarcoma TICs 50 , while the reason for the obvious discrepancy in the prognoses of clinical cases was not addressed. In this study, we show that SSEA-4 1 cells represent a particular subset of TICs in human osteosarcomas. These SSEA-4 1 cells represent a small fraction of Oct3/4 1 tumor cells, whose abundance is the hallmark of high-risk cases but not those with good prognosis following conventional therapy. The co-purification of SSEA-4 and Oct3/4 exclusively in high-grade cases supports the notion that a reprogramming process may accompany the creation of TICs for high-grade osteosarcoma cases but not for low-grade cases. During such reprogramming, cells at least partially approach the level of MSCs or even show certain ES cell-like features 30,32,51 .
Theoretically, factors associated with the prognosis of any type of cancer ought to be ones that play a critical role in the regulation of tumorigenesis. In this regard, it was previously observed that a reduced p27 level or an elevated level of p-S6K level was found in association with high-grade rather than low-grade osteosarcoma samples 16,22 . In accordance with this, multiple independent studies have already demonstrated that mTORC1 activity represses p27 expression in a variety of tumor types [46][47][48] . In this study, we demonstrated that SSEA-4 1 TIC frequency is associated with mTORC1 activity to predict prognosis in osteosarcoma. Taken together, these observations suggest that a possible link exists between mTORC1 activity and p27 levels in the regulation of the SSEA-4 1 TIC pool.
The question, then, is how mTORC1-p27 regulates the pool of SSEA-4 1 TICs. Previous in vitro studies that used different cell models resulted in differing conclusions regarding the role of mTORC1 or/and mTORC2 in controlling stemness versus maturation of osteosarcoma cells 52,53 . Recent studies indicate that aggressive types of malignancies are characterized by tumor cells that possess dedifferentiation potential 37,38 . In the case of osteosarcoma, since the dedifferentiation of p53-or/and Rb-deficient mature osteoblasts underlies their malignant transformation 4,11-13 , it is not surprising to find that SSEA-4 2 osteosarcoma cells retain the capacity to dedifferentiate, thus regenerating SSEA-4 1 TICs.
There are redundant cell-cycle regulators that potentially drive osteoblasts out of the cell-cycle, which in turn hinders the dedifferentiation potential of osteosarcoma cells 15,16 . The genetic inactivation of the Rb pathway, including the deletion of CDK inhibitors p16 INK4A and the pocket protein Rb, as well as amplification of CDK4, is part of the reason why osteosarcoma cells are able to avoid permanent cellcycle exit. The repression of p21 expression, as implicated by the genetic inactivation of p53 and p14 ARF as well as the amplification of MDM2 (p14 ARF -MDM2-p53-p21) or the functional activation of USP1-IDs 49 , contributes to tumorigenesis by blocking osteosarcoma cells from cell-cycle exit. However, it seems that these abnormalities taken together still do not prevent the possible upregulation of p27, which has been shown to be sufficient to drive Rb-deficient osteoblasts out of the cell-cycle 54 . In this study, we show that mTORC1 not only supports SSEA-4 1 TIC expansion via activating S6K, but also determines the retention of the tumorigenicity of p53/Rb-deficient osteosarcoma cells by repressing p27. Moreover, it is highly possible that p27 may also play an additional role in promoting the early or intermediate differentiation phase of SSEA-4 1 TICs, for example, by facilitating Runx2 function through maintenance of residual Rb in its hypophosphorylated form, or other unknown pathways 16 .
It is interesting to consider what factors regulate the increases and decreases of mTORC1 activity in osteosarcoma cells growing in vivo. Previous work has indicated that the deficiency of Rb or p53 activities may increase mTORC1 activity in osteosarcoma cells via compromising AMPK activity or elevating mTOR expression 43,44 . Nevertheless, we argue that a variety of environmental cues as well as other osteosarcoma-related genetic mutations would influence mTOR activity 13,21 . We showed that the glucose-withdrawal activated AMPK to inhibit mTORC1 activity in p53-deficient MG63 cells. On the other hand, our data also support previous findings that growth factor-stimulated mTORC1 activation promotes TIC activity in osteosarcomas resulting in metastasis 40 . Nevertheless, our results do not exclude an additional contribution by the inactivation of mTORC2, probably secondary to mTORC1 inactivation 55 in the restriction of tumorigenicity in high-grade osteosarcoma 26 . When considered in their broader context, our observations indicate that mTOR-targeting represents a promising way to improve the treatment of high-grade osteosarcoma, especially those characterized by the presence of highly immature SSEA-4 1 TICs.

Methods
Patients. All of the patients included in this study were admitted to Shanghai Rui-Jin Hospital during the last 12 years. All patients received systemic chemotherapy according to the IOR/OS-N4 protocol, plus surgery 18 . All patients provided written informed consent for the use of their tumor samples under a protocol approved by the ethics committee of Shanghai Rui-Jin Hospital.
Human Osteosarcoma Xeno-transplantation. Human primary osteosarcoma samples were obtained in accordance with the ethical guidelines established by Shanghai Rui-Jin Hospital. Fresh osteosarcoma samples were dissected into small pieces (,3 3 3 3 1.5 mm 3 ) within an hour after surgery. The pieces were then implanted subcutaneously into the two flanks of 6 to 8 week-old female NOD/SCID mice through a 3-mm incision, which was then wrapped twice with a 5-0 suture. All animal experiments were carried out in accordance with the approved guidelines provided by the Laboratory Animal Resource Center of Shanghai Jiao Tong University School of Medicine.
For the isolation and further passage of osteosarcoma cells from the xenografts, tumor tissues were dissected into small pieces (2 3 2 3 1 mm 3 ) and then incubated with Collagenase Type IV (Invitrogen) in IMDM medium (250 units/ml) at 37uC for 1 hour. Single cell suspensions were obtained by filtering the collagenase-treated samples through a 70-mm nylon cell strainer (BD Falcon, Bedford, USA). One thousand to 1 million unsorted, sorted SSEA-4 1 or SSEA-4 2 osteosarcoma cells suspended in PBS were then injected into the distal ends of femurs or subcutaneously into the flanks of 6 to 8 week-old female NOD/SCID mice. Tumor volumes were estimated by following an established formula (tumor volume 5 p/6 3 0.5 3 length 3 width 2 ).
Flow Cytometric Analysis and Cell Sorting. Cells were suspended in FACS buffer (PBS containing 1% FBS, with or without 0.1% Na 3 N) and subsequently stained with fluorochrome-conjugated antibodies. Flow cytometric data were collected using an LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) or a MoFlo flowsorter (Beckman coulter, Fullerton, CA, USA), and were analyzed with either FlowJo software (TreeStar, Ashland, OR, USA) or Summit software (Beckman coulter). Cells were sorted by MoFlo into DMEM supplemented with 2% FBS.
Gene Expression Profiling. Total RNA was extracted from sorted SSEA-4 1 and SSEA-4 2 cells using Trizol (Invitrogen). Three biological replicates were performed and all samples were subjected to strict quality control. Gene expression profiling was conducted by Shanghai Biotechnology Corporation using Affymetrix U133 plus 2.0 arrays (Affymetrix, Santa Clara, CA). All data were analyzed according to the manufacturer's protocol. Raw data generated from Affymetrix CEL files were normalized by RMA background correction; values were log 2 transformed. Comparison of the data sets by t test showed that 1491 of the total of 54614 probe-sets (2.7%) were differentially expressed (fold changes . 1.5, P , 0.05). For the www.nature.com/scientificreports enrichment of P values of each GO term, we used Fisher's exact test to calculate P values and R package stats to calculate FDR (q value) by BH method (www.r-project. org). All microarray data have been deposited with Gene Expression Omnibus (GEO) under accession number GSE58209.
Tissue Microarrays and Immunohistochemical Analyses. The osteosarcoma tissue microarray was provided by the Department of Pathology of Rui-Jin Hospital. Two representative cores of each paraffin-embedded tissue sample (1 mm in diameter) were selected for immunohistochemical staining, which was performed on 5 mmthick paraffin-embedded sections. All experimental protocols for osteosarcoma samples were approved by the ethics committee of Shanghai Rui-Jin Hospital.
Statistical Analysis. Data are presented as arithmetic means 6 SD (standard deviations). Kaplan-Meier survival analysis, student's t tests or x 2 tests were used to calculate P values where appropriate. P , 0.05 was considered to be significant.