Rapamycin induces feedback activation of Akt signaling ... - Nature

Oncogene (2007) 26, 1932–1940

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ORIGINAL ARTICLE

Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism X Wan, B Harkavy, N Shen, P Grohar and LJ Helman Molecular Oncology Section, Pediatric Oncology Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, MD, USA

Rapamycin and several analogs, such as CCI-779 and RAD001, are currently undergoing clinical evaluation as anticancer agents. In this study, we show that inhibition of mammalian target of rapamycin (mTOR) signaling by rapamycin leads to an increase of Akt phosphorylation in Rh30 and RD human rhabdomyosarcoma cell lines and xenografts, and insulin-like growth factor (IGF)II-treated C2C12 mouse myoblasts and IGF-IIoverexpressing Chinese hamster ovary cells. RNA interference-mediated knockdown of S6K1 also results in an increase of Akt phosphorylation. These data suggest that mTOR/S6K1 inhibition either by rapamycin or small interfering RNA (siRNA) triggers a negative feedback loop, resulting in the activation of Akt signaling. We next sought to investigate the mechanism of this negative feedback regulation from mTOR to Akt. Suppression of insulin receptor substrate (IRS)-1 and tuberous sclerosis complex-1 by siRNAs failed to abrogate rapamycininduced upregulation of Akt phosphorylation in both Rh30 and RD cells. However, pretreatment with h7C10 antibody directed against insulin-like growth factor-1 receptor (IGF-1R) led to a blockade of rapamycin-induced Akt activation. Combined mTOR and IGF-1R inhibition with rapamycin and h7C10 antibody, respectively, resulted in additive inhibition of cell growth and survival. These data suggest that rapamycin mediates Akt activation through an IGF-1R-dependent mechanism. Thus, combining an mTOR inhibitor and an IGF-1R antibody/inhibitor may be an appropriate strategy to enhance mTOR-targeted anticancer therapy. Oncogene (2007) 26, 1932–1940. doi:10.1038/sj.onc.1209990; published online 25 September 2006 Keywords: mTOR; rapamycin; Akt; IGF-1R; rhabdomyosarcoma

Correspondence: Dr X Wan, Molecular Oncology Section, Pediatric Oncology Branch, Bldg. 10, Rm CRC-1W-3750, NCI, National Institutes of Health, Bethesda, MD 20892-1928, USA. E-mail: [email protected] Received 19 May 2006; revised 20 July 2006; accepted 8 August 2006; published online 25 September 2006

Introduction Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood related to the myogenic lineage, accounting for nearly 250 cases of childhood cancer in United States each year (Pappo et al., 1995; Dagher and Helman, 1999; Womer and Pressey, 2000). Based on histopathologic features, two subtypes, alveolar (ARMS) and embryonal (ERMS), were identified and associated with distinct clinical characteristics and genetic alterations. ARMS carries a characteristic chromosomal translocation involving the DNA-binding domain of the Pax3 or Pax7 gene to the transactivation domain of the FKHR gene resulting in t(2;13) or t(1;13), respectively (Merlino and Helman, 1999). In contrast to the specific translocations found in ARMS, ERMS is characterized by loss of heterozygosity at chromosome 11p15.5. Our previous studies have demonstrated that insulin-like growth factor (IGF)-II) is overexpressed and functions as an autocrine factor in RMS (Minniti et al., 1995; Wan and Helman, 2003), and that IGF-IImediated cell survival is associated with activation of the mammalian target of rapamycin (mTOR) pathway (Wan and Helman, 2002). The target of rapamycin, TOR (mTOR in mammals) is an evolutionarily conserved serine/threonine protein kinase that integrates nutrient- and growth factorderived signals to supply a required input for cell growth and proliferation, a process whereby cells accumulate mass and increase in cell size and divide (Schmelzle and Hall, 2000; Fingar et al., 2002). In mammals, TOR is best known to regulate translation through the ribosomal S6 kinases (S6K1 and S6K2) and the eucaryotic initiation factor 4E (eIF-4E)-binding protein 1 (4E-BP1). mTOR activity leads to phosphorylation and activation of S6K1 and to 4E-BP1 phosphorylation and release from the cap-dependent translation initiation factor eIF-4E, which play fundamental roles in ribosome biogenesis and cap-dependent translation, respectively. Thus, mTOR and its downstream effectors have emerged as novel targets for cancer therapeutics (Huang and Houghton, 2003). Inhibition of mTOR by the immunosuppressant rapamycin results in reduced cell size and cell proliferation (Abraham and Wiederrecht, 1996). Rapamycin has limited water solubility and stability in solution, and a variety of analogs such as CCI-779and RAD001are

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currently being studied in the clinic for the treatment of a variety of tumor types (Hidalgo and Rowinsky, 2000; Huang and Houghton, 2003). The serine/threonine protein kinase Akt, a downstream effector of phosphatidylinositol-3 kinase (PI3K), is one of the most frequently hyperactivated protein kinases in human cancer. Hyperactivation of Akt is associated with resistance to apoptosis, increased cell growth, cell proliferation and cell energy metabolism (Hay, 2005). Akt can potentially phosphorylate over 9000 proteins in mammalian cells (Lawlor and Alessi, 2001). However, it remains to be determined which downstream targets are most critical for tumorigenesis. mTOR has emerged as a critical downstream effector of Akt. The mechanism by which Akt activates mTOR is not fully understood. Recent new findings of the tuberous sclerosis complex (TSC)-2) and Ras homolog enriched in brain (Rheb) provide an evolutionarily conserved link between the growth factor-regulated PI3K/Akt and the nutrient-sensitive mTOR pathways (Fingar and Blenis, 2004). Akt-activated mTOR signaling appears to be through direct phosphorylation and inhibition of TSC-2 (Inoki et al., 2002; Potter et al., 2002). TSC-1 and TSC-2 form a heterodimer with GTPase that inhibits the activity of Rheb, a small GTPase required for mTOR activation (Hay and Sonenberg, 2004). Recently, a negative feedback loop from mTOR and S6K1 to PI3K and Akt has been found not only in Drosophila (Radimerski et al., 2002; Stocker et al., 2004), but also in mammalian cells when studying insulin signaling (Tremblay and Marette, 2001; Harrington et al., 2004). However, the underlying mechanism of this negative feedback regulation and its physiological implications are not well understood. In this study, mTOR inhibition by rapamycin led to an increase in Akt phosphorylation on serine 473 in Rh30 and RD RMS cell lines and xenografts, and IGF-II-treated C2C12 mouse myoblasts and IGF-IIoverexpressing Chinese hamster ovary (CHO) cells. The rapamycin-mediated Akt activation is prevented by the PI3K inhibitor LY294002 and the antibody, h7C10, to insulin-like growth factor 1 receptor (IGF-1R). These data indicate that mTOR inhibition by rapamycin induces negative feedback activation of Akt that is IGF-1R/PI3K-dependent.

Results Upregulation of Akt phosphorylation by rapamycin is associated with suppression of mTOR signaling in RMS cell lines and in tumors of mice bearing RMS xenografts. To evaluate the relationship between suppression of mTOR signaling and status of Akt phosphorylation, we exposed Rh30 and RD cells to different concentrations of rapamycin ranging from 1.0 to 100 nM for 3 h in serum or serum-free medium, and then examined phosphorylation of Akt and S6 in both cell lines. As

shown in Figure 1a, increased Akt phosphorylation on Ser473 and decreased S6 phosphorylation, a downstream target of S6K1, were detected in both cell lines in response to rapamycin treatment at all tested doses either in serum or serum-free medium. Although 1 nM rapamycin completely eliminated the phosphorylation of S6, this dose is not sufficient to inhibit phosphorylation of mTOR and 4E-BP1 (data not shown). Thus, we also did a time course to examine the effects of rapamycin at 100 nM on Akt and S6 phosphorylation in the presence of serum. Inhibition of S6 phosphorylation and activation of Akt phosphorylation were seen 1 h after 100 nM rapamycin treatment (Figure 1b). These results indicate that suppression of S6 phosphorylation and activation of Akt are rapid and concurrent in RMS cells treated with 100 nM rapamycin. However, exposure of cells to 100 nM rapamycin failed to alter Akt and S6 protein expression (Figure 1a and b). In addition, we do not see any effect on non-targeted kinase such as mitogen-activated protein kinase for this dose (data not shown). To further determine whether rapamycin also induces an increase in Akt phosphorylation in vivo, we performed in vivo experiments to examine the effect of CCI-779, an analog of rapamycin that has been shown activity against a wide range of cancer in preclinical models with low toxicity and stability in solution, allowing parenteral administration of drug to tumorbearing mice. As shown in Figure 1c, CCI-779 inhibited mTOR signaling, indicated by a reduction of S6 phosphorylation in both xenografts after 24 h of treatment. Concurrently, Akt phosphorylation on Ser473 increased after 24 h of treatment in the RD xenograft and 48 h of treatment in the Rh30 xenograft. The discrepancy of CCI-779-mediated inhibition of S6 phosphorylation and increase of Akt phosphorylation in Rh30 xenograft may be explained by the fact that S6 is a direct target of rapamycin, whereas rapamycin-induced Akt activation is mediated via a indirect feedback mechanism. Taken together, these data suggest that mTOR inhibition by rapamycin triggers a negative feedback mechanism resulting in the activation of Akt signaling. PI3K is involved in the rapamycin-mediated increase of Akt phosphorylation As Akt is a major downstream regulator of PI3K, we sought to determine whether the rapamycin-induced increase of Akt phosphorylation was dependent on PI3K. Thus, we tested the effect of a PI3K inhibitor, LY294002, on the rapamycin-mediated feedback activation of Akt signaling. As shown in Figure 1d, pretreatment of Rh30 and RD cells with LY294002 resulted in a reduction of rapamycin-induced increase of Akt phosphorylation. Compared to LY294002, U0126, a mitogen-induced extracellular kinase (MEK) inhibitor, failed to affect the rapamycin-mediated activation of Akt. These data suggest that the rapamycin-induced increase of Akt phosphorylation is PI3K dependent, but independent of the MEK pathway. Oncogene


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Figure 1 Inhibition of mTOR signaling by rapamycin leads to an increase of Akt phosphorylation in Rh30 and RD human RMS cell lines and xenografts. (a) Rh30 and RD cells were treated with rapamycin at the indicated dose for 3 h in complete Rosewell Park Memorial Institute medium (RPMI) or serum-free medium and then lysed in lysis buffer for Western blot analysis phosphorylation and expression of Akt and S6. (b) Rh30 and RD cells were treated with 100 nM rapamycin at indicated hours in the complete RPMI medium. (c) Mice bearing Rh30 and RD xenografts were treated with CCI-779 at 20 mg/kg/IP from 24 to 96 h. The phosphorylation status of Akt and S6 were determined by Western blot analysis of these tumor samples. (d) PI3K inhibitor, LY294002, blocks rapamycin-mediated activation of Akt. Rh30 and RD cells were pretreated with either 10 mM LY294002 or10 mM U0126 for 1 h, then cotreated with 10 nM rapamycin for 3 h in complete RPMI medium and then lysed in lysis buffer for Western blot analysis of Akt phosphorylation and expression. Blotting with antibody against actin was used to confirm equal loading of all proteins.

Suppression of S6K1 by siRNA leads to an increase of Akt phosphorylation Recent studies have reported that mTOR activation by insulin initiates a feedback inhibition of PI3K/Akt signaling through S6K1 activation (Radimerski et al., 2002; Harrington et al., 2004; Manning, 2004). To verify whether S6K1, a major downstream target of mTOR, is involved in the feedback regulation of Akt activation, we eliminated S6K1 expression by using small interfering RNA (siRNA). As shown in Figure 2, siRNA against S6K1 in both Rh30 and RD cells led to a significant inhibition of S6K1 expression, and concurrently also resulted in elevated Akt phosphorylation. These data support a direct role of S6K1 in feedback inhibition of Akt. Rapamycin-induced Akt activation is dependent on signaling of IGFs IGFs are overexpressed in a wide variety of human tumors. Our previous data have demonstrated that Oncogene

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Knockdown of IRS-1 and TSC-2 by siRNAs failed to block rapamycin-induced upregulation of Akt phosphorylation Recent studies of the mTOR signaling pathway have discovered that the TSC-1/-2 complex is an upstream regulator of mTOR, providing an evolutionarily conserved link between the growth factor-regulated PI3K/ Akt and nutrient-sensitive mTOR pathways (Inoki et al., 2002; Potter et al., 2002). Additional studies have implicated insulin receptor substrate-1 (IRS)-1 as an intermediary in the mTOR-PI3K/Akt feedback loop (Radimerski et al., 2002; Harrington et al., 2004). To determine the mechanism by which rapamycin induces Akt activation, we knocked down IRS-1 and TSC-2 expression by siRNAs in both RMS cell lines. As shown in Figure 4a, phosphorylated and unphosphorylated IRS-1 was detected in the untreated cells. Following treatment with rapamycin, phosphorylated IRS-1 was decreased, whereas the unphosphorylated IRS-1 was increased in both RMS cell lines. To further determine the effect of rapamycin on the inhibition of IRS-1 phosphorylation, we tested IRS-1 phosphorylation in these cells by using a specific antibody against phosphoIRS1 (Ser636/639). Treatment of cells with rapamycin resulted in a significant inhibition of IRS-1 phosphorylation (Figure 4a). Suppression of IRS-1 by siRNA led to a reduction of Akt and S6 phosphorylation, but failed to abrogate rapamycin-induced upregulation of Akt.

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IGF-II is overexpressed in RMS cell lines (Minniti et al., 1995; Wan and Helman, 2003). To determine whether rapamycin-induced feedback activation of Akt in RMS cells is associated with IGF signaling, we examined the ability of rapamycin to induce activation of Akt in IGF-II-treated and -nontreated C2C12 mouse myoblasts as well as CHO cell line with tetracycline-regulatable IGF-II expression. Our previous published data demonstrated that C2C12 cells do not express detectable levels of IGF-II (Wan and Helman, 2003). Rapamycin-induced increase of Akt phosphorylation was only seen in the IGF-II-treated C2C12 cells (Figure 3a), but was not seen in the IGF-IInon-treated C2C12 cells (Figure 3b). The phosphorylation of S6K1 and Akt are very strong and easy to detect in IGF-II-stimulated C2C12 cells, but their levels are much lower in IGF-II-non-stimulated cells, which required substantially longer exposure times. In the CHO model, cells were grown with/without tetracycline (10 mg/ml) for 48 h to turn off/on IGF-II expression (Figure 3c) and then treated with rapamycin or LY294002 for 1 h. As shown in Figure 3d, rapamycininduced increase of Akt phosphorylation was observed in CHO cells with high IGF-II expression (Tet), but not in CHO cells without IGF-II expression ( þ Tet) (Figure 3e). These results suggest that rapamycininduced feedback activation of Akt is dependent on IGF signaling.

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Figure 3 Rapamycin inhibits the mTOR signaling and increases Akt phosphorylation in IGF-II-treated C2C12 mouse myoblast cells and IGF-II-overexpressing CHO cells. (a) C2C12 cells were cotreated with IGF-II and either rapamycin or LY294002 and wortmannin for 1 h in complete Dulbecco’s modified Eagle’s medium (DMEM) and then lysed in lysis buffer for Western blot analysis of Akt and S6K1 phosphorylation and expression. (b) C2C12 cells were treated with rapamycin or LY294002 for 1 h in complete DMEM medium (c) Northern blot analysis of IGF-II mRNA expression from control CHO AA8 and tetracycline-regulated IGF-II-overexpressing CHO cells growing in complete a-MEM medium without tetracycline or with10 mg/ml of tetracycline at indicated hours. (d and e) Tetracycline-regulated IFG-II-overexpressing CHO cells were grown without tetracycline or with 10 mg/ml of tetracycline for 48 h and then treated with rapamycin or wortmannin for 1 h. These cells were lysed in lysis buffer and then subjected to Western blot analysis of Akt phosphorylation. Oncogene


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Figure 4 Suppression of IRS-1 and TSC-2 by siRNAs failed to block rapamycin-mediated activation of Akt. Rh30 and RD cells plated in six-well plates were transfected with 100 nmol/l control, or IRS-1 or TSC-2 siRNA using LipofectAMINE 2000 reagent. After 3 days, cells were treated without or with 10 nM rapamycin for 3 h in the complete RPMI medium and then lysed in lysis buffer and subjected to Western blot analysis.

Similarly, inhibition of TSC-2 by siRNA also failed to block rapamycin-induced activation of Akt (Figure 4b). These data suggest that mTOR inhibition-mediated Akt activation in RMS cell lines is not mediated through IRS-1 and TSC-2 signaling.

the mechanism by which mTOR inhibition-induced Akt activation is dependent on IGFs/IGF-1R signaling.

Discussion Akt activation induced by mTOR inhibition is IGF-1R dependent Our above data have shown that rapamycin-induced Akt activation is dependent on IGF signaling. We therefore hypothesized that blockade of IGF-1R would prevent rapamycin-mediated Akt activation. As shown in Figure 5, 3 h of pretreatment with 1 mg/ml h7C10, a human monoclonal antibody against IGF-1R (Goetsch et al., 2005), resulted in abrogation of rapamycininduced Akt phosphorylation in both cell lines (Figure 5a). Pretreatment with 10 mg/ml h7C10 displayed an even more significant effect on the complete blockade of Akt activation induced by rapamycin (Figure 5b). Furthermore, treatment of cells with rapamycin at 10 nM in combination with IGF-1R antibody h7C10 at 10 mg/ml exhibited a greater inhibition of cell growth and survival than either alone (Figure 5c). However, treating these cells with 1 nM rapamycin could affect neither cell proliferation nor enhancement of the effect of h7C10 on the inhibition of cell growth (data not shown). These data suggest that Oncogene

The PI3K/Akt/mTOR pathway plays an important role in the biology of human cancers. Components of this pathway are frequently deregulated in a wide range of tumors, making them an attractive target for cancer therapy. Inhibition of mTOR by rapamycin and its analogs is a promising anticancer strategy. However, our recent studies found that treatment with rapamycin failed to completely inhibit RMS cell growth (unpublished data), indicating that these cells are, at least partially, resistant to mTOR inhibition. Thus, it is necessary to understand the mechanism by which cells are resistant to mTOR inhibition. In this study, inhibition of mTOR signaling by rapamycin led to upregulation of Akt phosphorylation in Rh30 and RD human RMS cell lines and xenografts, IGF-II-treated C2C12 mouse myoblasts and IGF-II-overexpressing CHO cells. Our previous studies have demonstrated that IGF-II is overexpressed and functions as an autocrine factor in RMS (Minniti et al., 1995; Wan and Helman, 2003). Thus, it is expected that even in serum-free condition, rapamycin still induces Akt


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Figure 5 Blockade of IGF-1R by h7C10 antibody prevents rapamycin-mediated Akt activation and enhances the effect of rapamycin on the inhibition of cell growth and survival. (a and b) Rh30 and RD cells were pretreated with 1 or 10 mg/ml of h7C10, or 1 mg/ml of MOPC-21 as control for 4 h and then cotreated with rapamycin for 3 h in complete RPMI medium. These cells were lysed in lysis buffer and then subjected to Western blot analysis. (c) Rh30 and RD cells were pretreated with 1 or 10 mg/ml of h7C10, or 1 mg/ml of MOPC21 for 4 h and then cotreated with rapamycin for 48 h in complete RPMI medium. Cell growth and survival was determined by MTT assay. Data are mean7s.e. (n ¼ 8).

phosphorylation in both Rh30 and RD cell lines. However, rapamycin treatment of C2C12 and CHO cells, with very low endogenous levels of IGF-II expression, failed to induce an increase of Akt phosphorylation in the absence of exogenous IGF stimulation. These data suggest that mTOR inhibition by rapamycin triggers a feedback mechanism resulting in the activation of Akt signaling, and that this feedback activation of Akt is dependent on IGFs/IGF-1R signaling. Our results are consistent with recent reports that rapamycin induces Akt activation in human nonsmall-cell lung cancer and breast cancer cell lines (Sun et al., 2005; O’Reilly et al., 2006). Akt is frequently hyperactivated in a variety of human cancers, which is associated with resistance to apoptosis. Thus, mTOR inhibition-induced feedback activation of Akt may contribute to the resistance of cancer cells to rapamycin and its analogs, thus attenuating their potential antitumor activity. Recent studies have shown that insulin-induced mTOR activation initiates a feedback inhibition of PI3K/Akt through S6K1 and subsequent phosphorylation of IRS-1 (Radimerski et al., 2002; Harrington et al., 2004; Manning, 2004). A kinase-dead version of S6K1

could abrogate this feedbacck loop and activate Akt (Shah et al., 2004). In our study, suppression of S6K1 by siRNA led to an increase of Akt phosphorylation (Figure 2). These data suggest that mTOR activation and feedback PI3K/Akt inhibition is at least partially mediated by S6K1. To determine whether IRS-1 is related to the feedback mechanism of Akt activation, we knocked down IRS-1 expression by siRNA. In controlsiRNA-treated cells, rapamycin inhibits phosphorylated IRS-1, and subsequently increases unphosphorylated levels of IRS-1, and increases Akt phosphorylation (lanes 3 and 4 of Figure 4a). However, in IRS-1 knockdown cells, rapamycin still induces Akt activation (lanes 1 and 2 of Figure 4a). These data suggest that mTOR/S6K1 inhibition-mediated feedback activation of Akt is through an IRS-1-independent mechanism in RMS cells. This apparent discrepancy between the roles of IRS-1 in Akt activation in our model may be explained by the importance of cell context. So far, four members of IRS family (IRS-1, IRS-2, IRS-3 and IRS-4) have been identified which differ in their subcellular distribution and interaction with Src homology 2 (SH2) domain of signaling proteins like PI3K (Sun et al., 1991; White, 1998). These differences may Oncogene


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contribute to IRS subtype specificity in their signaling potential. Indeed, IRS-1 and IRS-2 mediate PI3K activation to different extents in distinct tissues (Kido et al., 2000). Thus, it remains possible that other members of IRS family may be involved in the feedback regulation of Akt in RMS cells, and further experiments will be required to specifically address this. The discoveries of proteins TSC-1 and TSC-2 and Rheb provide novel functional links between the PI3K/ Akt pathway and the mTOR pathway. Genetic, biochemical and biologic analysis have highlighted their roles as negative regulators of mTOR signaling. The best-characterized cellular function of TSC-2, serving as a substrate of Akt, is to inhibit the phosphorylation of S6K1 (Goncharova et al., 2002; Inoki et al., 2002). Our data show that suppression of TSC-2 by siRNA resulted in a reduction of Akt phosphorylation (Figure 4b). This could be explained by suppression of TSC-2 leading to release of mTOR/S6K1 inhibition, subsequently increasing the negative feedback regulation of Akt and reducing Akt activation. However, suppression of TSC-2 by siRNA failed to abrogate the rapamycininduced increase of Akt activation. These results suggest that the main route for Akt signaling to mTOR/S6K1 is via Akt-dependent phosphorylation and inactivation of TSC-2 (Goncharova et al., 2002; Inoki et al., 2002), whereas mTOR/S6K1 inhibition-induced feedback regulation of Akt activation is independent of TSC-2. The PI3K/Akt pathway is regulated by upstream receptor tyrosine kinases, specifically the IGF-1R that mediates IGF-1 and IGF-II signaling. Interaction of IGF-1 and -II with IGF-1R plays a critical role in tumorigenesis, proliferation and increased resistance to chemotherapy. Thus, not surprisingly, a wide variety of tumors show increased expression levels of IGFs and IGF-1R, and enhanced activity of IGF-1R, which have been correlated with advanced cancer disease stage, reduced survival and development of metastases (LeRoith et al., 1995; LeRoith and Helman, 2004). Our data demonstrate that mTOR inhibition mediates Akt activation through IGFs/IGF-1R signaling. Blockade of IGFs signaling using h7C10, an antibody against IGF-1R, completely abrogated rapamycin-induced increase of Akt phosphorylation on both Ser473 and Thr308 sides. Our previous data has shown that blockade of IGF-1R signaling by aIR3, a neutralizing antibody against IGF-1R a chain, downregulates Akt phosphorylation on Ser473 but not on Thr308 (Wan and Helman, 2003). The mechanism by which h7C10 affects both Ser473 and Thr308 phosphorylation of Akt, whereas aIR3 only affects Ser473 phosphorylation of Akt is not clear. h7C10 has displayed significant effects on blocking IGF-1R b chain and inhibiting phosphorylation of both b chain and IRS-1, whereas aIR3 failed to inhibit phosphorylation of b chain and IRS-1 (Goetsch et al., 2005). The difference of h7C10 and aIR3 may be related to different effects of h7C10 and aIR3 on the inhibition of Thr308 phosphorylation of Akt. The specific mechanism remains to be further elucidated. Our data in Figure 1d show that LY294002, a PI3K inhibitor, blocks Akt activation induced by Oncogene

rapamycin. These findings suggest that mTOR/S6K1 inhibition triggers a negative feedback mechanism, resulting in the activation of Akt signaling via IGF-1R/ PI3K signaling to Akt. A recent study has found that the mTOR–rictor complex can directly phosphorylate Akt at Ser473 (Sarbassov et al., 2005). However, this complex is not sensitive to rapamycin (Sarbassov et al., 2004). It remains possible that mTOR inhibition induces Akt activation by other unknown mechanisms, such as indirect activation of mTOR–rictor by rapamycin, which require further elucidation. Our data also show that IGF-1R antibody in combination with rapamycin displays additive inhibition of cell growth and survival versus either alone, suggesting that combining therapy with mTOR and IGF-1R inhibitors may be an appropriate strategy to enhance mTOR-targeted anticancer therapy.

Materials and methods Cell lines Rh30 ARMS cells, RD ERMS cells and the mouse C2C12 myoblast cells have been described previously (Wan and Helman, 2003). Tetracycline-regulated IGF-II-overexpressing CHO and control CHO-AA8 cells were generated as described previously (Zhang et al., 1999), and were grown without tetracycline or with 10 mg/ml of tetracycline for 48 h. Reagents and antibodies Rapamycin, LY294002, wortmannin and U0126 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). CCI-779 was obtained from Developmental Therapeutics Program, National Cancer Institute (Bethesda, MD, USA) and Wyeth Laboratories (Philadelphia, PA, USA). Antibodies to phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, phospho-S6K1 (Thr389), S6K1, phospho-S6 (Ser235/236), S6 and phospho-IRS-1 (Ser636/639 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-IRS-1 antibody was purchased from Upstate (Lake Placid, NY, USA). Anti-TSC-2 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-actin antibody was purchased from Abcam Inc. (Cambridge, MA, USA). Anti-IGF-1R antibody (h7C10) was obtained from Merck (Whitehouse Station, NJ, USA) and isotype-matched control antibody (MOPC-21) was purchased from Sigma (St Louis, MO, USA). Western blot analysis Western blot analysis was performed as published previously (Wan et al., 2005). RNA interference The siRNAs directed against IRS-1 and TSC-2 were purchased from Upstate (Lake Placid, NY, USA) and Cell Signaling Technology Inc. (Beverly, MA, USA), respectively. The S6K1 siRNA (target sequence: 50 -AACAGUGGAGGAGAAC UAUUU-30 ) and a negative control siRNA (nonspecific target sequence: 50 -AATAGCGACTAAACACATCAA-30 ) were purchased from Dharmacon Research Inc. (Lafayette, CO, USA). We applied siRNA duplexes at a final concentration of 100 nM using LipofectAmine 2000 Reagent (Invitrogen, Carlsbad, CA, USA). Lysates were made 72 h post-transfection.


1939 Northern blot analysis Northern blot analysis of IGF-II expression was performed as described previously (Wan and Helman, 2002). MTT assay Rh30 and RD cells were harvested and seeded in 96-well plates 1 104 (1 104 cells/well). These cells were pretreated for 4 h with either 1 or 10 mg/ml of a monoclonal antibody against the IGF-1R (h7C10) (Merck, Whitehouse Station, NJ, USA) or 1 mg/ml of an isotype-matched control antibody (MOPC-21) (Sigma Chemical Co., St Louis, MO, USA), and then treated with 10 nM rapamycin for 48 h. Cell survival was determined using MTT assay according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Statistics All values are presented as mean7s.e. (n ¼ 8). Comparisons between two groups were performed using a two-tailed Student’s t-test. The criterion for statistical significance is Po0.05.

Acknowledgements This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. We thank Merck Inc. for providing us with the h7C10 antibody.

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