mTOR and its downstream effectors, eIF4E and S6K, have been implicated in cellular transformation, although their contribution to glial transformation remains undefined. In this work we show that growth of glioma cells in soft agar, a stringent assay for transformation, is blocked by down regulation of mTORC1 and that signaling through S6K, but not eIF4E, maintains glial transformation. We also show that in vivo suppression of S6K results in reduced intracranial glioma growth. These findings indicate that the S6K arm may have special significance in glial transformation.
Our data suggest that mTORC2 function is less significant in mTOR-dependent anchorage-independent growth for a few reasons. Rapamycin has been reported to have alternate effects on Akt phosphorylation; prolonged rapamycin exposure has been shown to inhibit assembly of mTORC2, thereby inhibiting Akt (28
), and mTOR inhibition has also been described to induce insulin receptor substrate-1 (IRS-1), leading to Akt activation (26
). In the human glioma cell lines U251 and U373, we found that suppression of mTOR resulted in a significant increase in phosphorylated Akt Ser473 as compared to cells expressing scramble control. Despite the increase in phosphorylated Akt Ser473, mTOR knockdown nonetheless significantly compromised these cells’ anchorage independent growth. These data suggest that in gliomas, mTORC2 is not the dominant arm supporting mTOR-dependent transformation, although it is possible that mTORC2 has effects on tumorigenesis that are Akt independent.
Our data also suggest that S6K and eIF4E have distinct roles in gliomagenesis. While prior findings have shown that eIF4E transforms rat fibroblasts, we found that eIF4E expression in U373 glioma cells and HRasV12
- and HRasV12
/Akt- transformed human astrocytes failed to restore anchorage-independent growth in the setting of mTOR inhibition. Silencing 4EBP1 also failed to rescue anchorage-independent growth from rapamycin-mediated suppression. eIF4E overexpression, however, increased colony formation in HRasV12
/Akt- transformed human astrocytes, suggesting that eIF4E plays a positive role in transformation, and it is possible that eIF4E’s effects on transformation require other mTOR-dependent pathways such as S6K1. Another reason we cannot fully exclude a role by eIF4E in glial transformation is that superphysiologic levels of eIF4E beyond the two to fourfold increases generated in this study may be required for transformation. Malignant gliomas have been described immunohistochemically to express more eIF4E as compared to normal neuroglial cells (29
), although the degree of eIF4E overexpression in malignant gliomas remains undefined.
Although S6K has not been shown to be a n oncoprotein, in the human gliomas assessed in this study, S6K appeared to be a key factor in maintaining anchorage-independent growth. The actions of S6K demonstrated in human astrocytes may indicate that S6K has differing roles in various tissue types: for example, S6K1 deletion blocks growth factor stimulated hypertrophy in muscle but not in neurons (30
). S6K has numerous downstream targets, among them mRNAs with 5′ TOP sequences, the protein products of which are starting to be understood. The critical targets of S6K are not well defined, although the present data clearly suggest that these targets may be distinct from those influenced by eIF4E, and may represent better therapeutic targets. It should be noted that while the mTOR-S6K pathway appears to be critical for the growth of cells in soft agar, transformation of glial cells requires a series of events (p53 inactivation, Rb inactivation, telomerase reactivation, Ras activation) to which the mTOR-S6K pathway is merely a contributor (22
). This observation is consistent with the finding that supplying S6K to rapamycin-treated cells only partially rescues growth. The present findings suggest that S6K, but not eIF4E, plays a key, although not sufficient, role in glial transformation.
In addition to our data, which shows an important role for S6K1 in supporting gliomagenesis in vitro
and in vivo
, recently published data describe ribosomal S6Kinase 2 (RSK2) as supporting anchorage independent growth induced by tumor promoting agents such as Epidermal Growth Factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (32
). RSK2, a homologue of S6K1, is similarly activated by mitogens and inhibited by rapamycin (33
). Although both RSK2 and S6K1 phosphorylate S6 in vivo
, these kinases do not appear to be functionally redundant for a few reasons. S6K1 knockout mice have a small-body phenotype, despite the finding that mouse embryo fibroblasts from these animals show normal S6 phosphorylation in vivo
, suggesting that RSK2 does not completely duplicate S6K1 functions (34
). Comparisons of amino acid sequences and localization between the two S6 Kinases also suggest distinct functional differences (33
). It remains possible that S6K1 and RSK2 support tumor growth through similar mechanisms, and further studies defining the transformation-promoting effects common or specific to these kinases are needed.
Defining the role of the mTOR-S6K pathway in glial transformation may have an impact on the design and implementation of glioma therapies. Current targeted therapies are based on our knowledge of pathways thought to be critical for tumorigenesis and proliferation. This rationale has led to the clinical testing of signaling inhibitors such as Tarceva and CCI-779. Despite this mechanistic approach to drug development, these agents have shown only modest effects, and combinatorial strategies that inhibit multiple kinases (for example PI3K or Akt in combination with mTOR) show more promise than strategies employing single kinase inhibition (36
). In the case of Akt/mTOR combinatorial therapy, the fact that mTOR inhibition can induce Akt activation through IRS-1 may explain why targeting the same pathway at multiple sites is associated with better efficacy. Concerns have been raised that Akt activation with mTORC1 inhibition could represent a mechanism for drug resistance and sustained tumor growth, although in our model, Akt activation did not rescue tumor growth from mTORC1 inhibition. Our observation that the mTOR-S6K pathway plays a key role in glial transformation suggests that targeting the Akt-mTOR-S6K pathway at a more distal point may be as effective as dual inhibition at a more proximal point. Selective S6K inhibitors are not at present available at the clinical or pre-clinical level, although the present studies suggest that such agents, alone or in combination with other agents, might be rational choices for glioma therapy, and perhaps other tumors dependent on mTOR-S6K signaling for maintenance of the transformed phenotype.