Dysregulation of mTOR function via physiologic or mutational activation of upstream pathways is a common event in tumors from many lineages (2
). We speculated that tumor cells with activated mTOR display a phenotype functionally equivalent to insulin-resistant diabetes with an exaggerated down-regulation of upstream signaling molecules like IRS-1 (4
). We report here that, in a variety of tumor cell lines, the mTOR inhibitory drug rapamycin up-regulates IRS-1 protein levels and induces Akt phosphorylation, protein kinase activity, and downstream signaling. In parallel clinical studies with the rapamycin derivative RAD001 (Novartis Pharma), the intensity of immunohistochemical staining of tumor biopsies for p-Akt was significantly elevated after 4 weeks of drug treatment. These data suggest that the induction of Akt activity observed in tumor cells in tissue culture is biologically relevant and may be important clinically.
Rapamycin and rapamycin-like molecules inhibit mTOR efficiently in patients, are useful as immunosuppressants, and suppress S6 kinase activity in normal and tumor cells in vitro
and in vivo
). Pharmacologic inhibition of mTOR has been shown to potently inhibit tumor cells with activation of PI3K/Akt signaling due either to PTEN loss, expression of Akt, or growth factor activation (16
). In this regard, rapamycin derivatives are effective in in vivo
models, as recently shown in myr-Akt-driven transgenic models of prostatic neoplasia (17
). The Akt activation induced by rapamycin in tumor cells, however, is likely to reduce its antitumor effects, by activating pathways that attenuate its effects on proliferation and apoptosis. In tumors in which Akt activation is induced, rapamycin will not effectively inhibit PI3K/Akt kinase signaling except insofar as it is mediated through mTOR. We show here that IGF-I overcomes the rapamycin-induced inhibition of MCF-7 proliferation in serum-free medium. This result is consistent with those of Houghton and coworkers, who showed that induction of apoptosis by rapamycin via ASK-1 activation occurs in serum-free but not serum-containing medium (22
). We find that inhibition of induction of Akt activation with agents that block IGF-I signaling enhances cell cycle arrest and apoptosis induction by rapamycin.
Despite the results from model systems, the clinical antitumor activity of mTOR inhibitor analogues has been modest at best. Our demonstration that rapamycin can induce Akt phosphorylation in tumors implies that its potential antitumor activity is attenuated by release of feedback inhibition of growth signaling pathways. The results also suggest a new model for the development of effective combinatorial anticancer therapy. Combined inhibition of constitutively activated oncoproteins and of normal pathways that are down-regulated by oncoprotein-inhibition (and thus up-regulated by oncoprotein-targeted drugs) may be much more effective than either alone. For the specific case discussed in this article, the work provides a rationale for tailored combination therapy with an mTOR inhibitor and an inhibitor of the growth factor receptor, such as IGF-IR, that normally drives PI3K activity in that tumor. Considering our results in which LY294002 abrogates rapamycininduced Akt kinase activity, and the report by Sun et al. detailing the combined efficacy of LY294002 and rapamycin in non-small cell lung cancer cell lines, mTOR inhibitors and PI3K inhibitors might also be a promising combination therapy (15
We have shown that rapamycin induces Akt activity and that abrogating this induction could enhance the antitumor effects of rapamycin in vitro
, but the mechanism of rapamycin-induced Akt activity remains unclear. Our finding that rapamycin treatment induces IRS-1 expression suggests that rapamycin’s inhibition of p70/S6K results in increased IGF-IR/IRS-1/PI3K signaling to Akt. Previous reports have shown that p70/S6K mediates phosphorylation of IRS-1 inhibitory serine sites (S312 and/or S636/639) which lead to IRS-1 degradation (8
). Thus, suppression of p70 activity by rapamycin may prevent inhibitory IRS-1 phosphorylation, thereby stabilizing IRS-1. An increase in IRS-1 adapter protein levels may induce Akt activity by augmenting IGF-IR signaling to PI3K/Akt. The inhibition of pAkt induction with LY294002 implies that the phenomenon is PI3K-dependent and supports this mechanism. However, LY294002 inhibits several PI3K-like kinases, including mTOR, and has been reported to inhibit the activity of rictor-mTOR, recently described as the Akt S473 kinase/PDK2 (25
). It is possible that rapamycin, by some unknown mechanism, induces rictor-mTOR activity, resulting in increased S473 Akt levels. These possibilities are currently under investigation.