Rapamycin Inhibition of the Akt/mTOR Pathway Blocks Select Stages of VEGF-A164–Driven Angiogenesis, in Part by Blocking S6Kinase
Objective— We evaluated the stages of VEGF-A164 driven angiogenesis that are inhibited by therapeutic doses of rapamycin and the potential role of S6K1 in that response.
Methods and Results— We assessed the effects of rapamycin on the several stages of angiogensis and lymphangiogenesis induced with an adenovirus expressing VEGF-A164 (Ad-VEGF-A164) in the ears of adult nude mice. Rapamycin (0.5 mg/kg/d) effectively inhibited mTOR and downstream S6K1 signaling and partially inhibited Akt signaling, likely through effects on TORC2. The earliest stages of angiogenesis, including mother vessel formation and increased vascular permeability, were strikingly inhibited by rapamycin, as was subsequent formation of daughter glomeruloid microvasular proliferations. However, later stage formation of vascular malformations and lymphangiogenesis were unaffected. Retrovirally delivered isoforms and shRNAs demonstrated that S6K1 signaling plays an important role in early VEGF-A164-angiogenesis.
Conclusions— Rapamycin potently inhibited early and mid stages of VEGF-A164–driven angiogenesis, but not late-stage angiogenesis or lymphangiogenesis. Rapamycin decreased phosphorylation of both Akt and S6, suggesting that both the TORC1 and TORC2 pathways are impacted. Inhibition of S6K1 signaling downstream of mTOR is a major component of the antiangiogenesis action of rapamycin.
The success of Avastin in treating vascular endothelial growth factor–A (VEGF-A)–driven cancers and macular edema has demonstrated the potential clinical importance of antiangiogenesis as a therapeutic approach.1 Nonetheless, much remains to be learned about the steps and mechanisms by which VEGF-A induces the formation of new blood vessels and lymphatics.2,3
Rapamycin, an inhibitor of “mammalian target of rapamycin” (mTOR), is a natural macrolide antibiotic derived from Streptomyces hygroscopicus. It has been used for some years for immunosuppressing patients after organ transplantion,4 and recently it has shown potential in cancer therapy.5 In addition to its likely role in inhibiting tumor cell growth, rapamycin has also been found to be antiangiogenic,6 and we have shown that its effects on the tumor vasculature contributed importantly to its anticancer efficacy in a spontaneous mouse model of breast cancer.7
The PI3K/Akt/mTOR pathway plays a central role in cell growth and metabolism, and rapamycin and related inhibitors have multiple effects on this pathway. Recently we demonstrated that, at low doses, rapamycin inhibits not only mTOR and downstream signaling, but also the Akt pathway upstream of mTOR.8 This was puzzling but perhaps can be explained by the finding that mTOR is distributed in 2 discrete protein complexes, TORC1 and TORC2, only 1 of which, TORC1, binds rapamycin. In addition to mTOR, the TORC2 complex contains rictor, mLST8 and SIN1, and regulates Akt phosphorylation at S473.9–12 Some divergences in Akt downstream signaling have recently been found to depend on differences in the composition of TORC1 and TORC2. For example, although usually present in both TORC1 and TORC2, mLST8 is required for phosphorylation of the Akt-FOXO3 pathway, but not for the Akt-GSK3β (S9) or mTOR-S6K1 (S240/244) pathways.13 Although TORC2 does not bind rapamycin, it has been thought that prolonged rapamycin treatment causes mTOR to be sequestered in TORC1 (its rapamycin binding complex), thus reducing the availability of mTOR for TORC2 assembly.10 However, rapamycin also induces an early transient rise in Akt signaling, perhaps via derepression of IRS1, insulin receptor substrate 1, which potentiates PI3K signaling.14
Studying a spontaneous mouse model of breast cancer, we recently reported that different cells and tissues responded with different levels of sensitivity to the inhibitory effects of rapamycin on the Akt pathway.7 Further, we found that, in general, tumor cells are more likely to maintain elevated Akt phosphorylation during prolonged rapamycin treatment, whereas Akt S473 phosphorylation was strongly inhibited in stromal cells.7 However, because endothelial cells are particularly sensitive to rapamycin-mediated p-Akt S473 downregulation, we were not able to separate the effects of rapamycin on p-Akt S473 from its effects on mTOR downstream signaling, and particularly its effects on S6K1.
The current study had 2 goals: (1) Define the steps in VEGF-A driven angiogenesis and lymphangiogenesis that are rapamycin-sensitive, and (2) determine whether S6K1 signaling plays a key role in the angiogeneic response to VEGF-A.
Materials and Methods
Animals and Materials
Four- to 6-week-old female athymic Nu/Nu mice (NCI, Bethesda, Md) were used. Animal protocols were approved by the BIDMC IACUC. A nonreplicating adenoviral vector (Ad-VEGF-A164) was engineered to express the predominant (164 aa) murine isoform of VEGF-A.3 Recombinant human VEGF-A165 was from R&D Systems (Minneapolis, Minn), and rapamycin was from Alexis Corporation (San Diego, Calif). Antibodies were purchased as follows: anti-AKT, antiphospho-AKT(Ser-473), antiphospho-p70S6K(Thr-389), anti-S6, antiphospho-S6 (Ser-240/244) and anti-HA-Tag from Cell Signaling Technology; antip70S6K1 from Santa Cruz Biotechnology; anti–β-Actin from Sigma-Aldrich Inc; anti-CD31 from BD Pharmingen; and anti-Lyve1 from Upstate.
Analysis of Mouse Ear Vasculature
5×106 pfu of Ad-VEGF-A164 or LacZ were injected into mouse dorsal ear skin with a 30-gauge needle in 10 μL and treated with rapamycin i.p. as indicated. Ears were photographed as described (n=12 mice).3,15 Plasma leakage was measured with a double tracer method (6 ear sites, 3 mice per group).3 Ear lymphatics were demonstrated by perfusion with colloidal carbon.15 Lymphangiogenesis was quantified by measuring the fraction of total ear sections tissue occupied by lymphatics in Giemsa-stained 1 μm sections15 with NIH Image analysis software.
Immunohistochemistry for CD31, pAkt, and pS6 were performed as previously described on paraformaldehyde fixed tissue.16 For immunofluorescence, frozen sections were fixed in 4% paraformaldehyde, pH 7.4, for 5 minutes at room temperature, washed in PBS, blocked in 5% goat serum-0.1% Triton X-100 in PBS for 1 hour at room temperature. Sections were then incubated with primary antibodies to mouse anti-CD31 (1:100 dilution), and rabbit anti-lyve-1 polyclonal secondary antibodies were diluted 1:200 in 5% goat serum in PBS.
Primary human umbilical vein endothelial cells (HUVECs) were from Clonetics. They were grown on plates coated with 30 μg/mL Vitrogen (Cohesion) in EGM-MV Bullet Kit at 37°C, 5% CO2. Passage 4 to 6 HUVECs at ≈80% confluence were washed with PBS and serum-starved in 0.1% FBS in endothelial cell basic medium for 18 hours. Rapamycin was added for 1 hour before VEGF-A165 stimulation. 293T cells and NIH 3T3 cells were grown in Dulbecco modified Eagle medium with 10% fetal calf serum (FCS), 2 mmol/L glutamine, penicillin (100 U/mL), and streptomycin (100 μg/mL).
Retroviral Constructs and Cell Infection
A plasmid containing a constitutively active rapamycin-insensitive form of S6K1 (HA-S6K1-F5A-E3893A) was kindly provided by Dr John Blenis (Dept. of Cell Biology, Harvard Medical School).17 It was subcloned into a pCMBP retroviral vector after digestion with EcoRI restriction; ends were blunted and digested with SalI. After purification through LMP agarose, it was cloned into the pCMBP vector that was digested with BamHI, ends blunted and digested with XhoI. Retrovirus was generated using 293T cells, and HUVECs were infected as described.18 Briefly, 2 μg of target gene plasmid, 1.5 μg of pMD gag pol, and 0.5 μg of pMD G were mixed, and transfected into 293T cells. Medium was changed after 16 hours. The medium containing retrovirus was collected 48 hours after transfection and used for infection into HUVECs after adding 10 μg/mL Polybrene. HUVECs were used for experiments 48 hours after infection.
For S6K1-siRNA studies, we used recombinant pSUPER retro vector system (OligoEngine). The OligoEngine Workstation designed 2 gene-specific inserts with starting sites from 545 GCAGTTAGAAAGAGAGGGA (S6K1 siRNA #1) and from 1075 AACTTCTGGCTCGAAAGGT (S6K1 siRNA #2), which were separated by a 9-nucleotide noncomplementary spacer (tctcttgaa) from the reverse complement of the same 19-nucleotide sequence. Nontargeting pSuper-control was constructed using a sequence AAGAGGGAGGGAGACATAT. These oligos were inserted into the pSUPER.retro.circular.vectors after digestion with BglII and HindIII. To make a siRNA pool, 1 μg of each pSUPER vector (containing oligo 545 or 1075 inserts), 1.5 μg of pMD gag pol, and 0.5 μg of pMD G were mixed and transfected into 293T cells to produce retroviruses as described above.
In Vivo Retrovirus Studies
For preparation of retroviruses, culture medium from virus-producing 293T cells was passed through a 0.45 μm syringe filter and centrifuged at 16 500 RPM, 4°C, for 2 hours, the pellet was resuspended in PBS and titered in NIH3T3 cells. For in vivo studies, mouse ears were injected with 5×106 pfu Ad-VEGF-A164 and 2 days later with 10 μL each of retrovirus (5×108 pfu) in Polybrene (20 μg/mL).
Quantitative data are expressed as mean±SD or SEM. The unpaired Student t test or Mann–Whitney test were used for statistical analysis.
Initial experiments tested whether rapamycin would inhibit the angiogenic response induced by an adenoviral vector expressing VEGF-A164 (Ad-VEGF-A164).3 This model has been extensively characterized and reproducibly induces, in sequence, new blood vessels of the several types that form in tumors and in healing wounds. Greatly enlarged, thin-walled, pericyte-poor, hyperpermeable mother vessels (MVs) develop initially (1 to 5 days); subsequently these evolve into several types of daughter vessels, particularly glomeruloid microvascular proliferations (GMP) and vascular malformations (VMs). Ad-VEGF-A164 also initiates lymphangiogenesis.15 Hence, by selecting defined time windows for rapamycin treatment, we hoped to assess the impact of mTOR inhibition on successive stages of angiogenesis and on lymphangiogenesis.
Effects of Rapamycin on the Earliest Stage of VEGF-Induced Angiogenesis (1 to 5 Days)
Intradermal injection of 5×106 pfu of Ad-VEGF-A164 into nude mouse ears induced the expected robust angiogenic response. Treatment with rapamycin, beginning 24 hours before Ad-VEGF-A164 injection, inhibited angiogenesis in a dose-dependent manner (Figure 1A). Ad-lacZ served as a negative control. To document the efficacy of rapamycin on mTOR inhibition, we collected 8-mm punch biopsies of the ear reaction sites on day 5 for immunoblotting to assess expression and phosphorylation of S6 and Akt (Figure 1B). Rapamycin potently inhibited phosphorylation of S6 (downstream of S6K) in a dose-dependent manner. As previously shown,8 p-Akt (S473) was somewhat reduced.
We recently made a careful study of the doses of rapamycin in mice that achieve the clinically recommended blood levels of rapamycin used for treating human transplant recipients.7 We found that the relatively low dose of 0.5 mg/kg/d resulted in the 10 to 20 ng/mL trough plasma levels of rapamycin desired clinically. This low dose of rapamycin partially inhibited angiogenesis (Figure 1A), and reduced plasma leakage by ≈60% (Figure 1C), an impressive feat in that Ad-VEGF-A164 generates high tumor-like concentrations of VEGF-A164 (up to 120 ng/8 mm punch site at 24 hours).3 Associated tissue edema, assessed by ear thickness, was also strikingly reduced, as were the number of new MVs that formed (Figure 1D, left panel).
Immunohistochemistry with antibodies specific for the phosphorylated forms of S6 (S240/244) and Akt (S473) were used to assess the effects of rapamycin on cell signaling. MV staining for p-S6 was strikingly reduced, whereas p-Akt staining was less affected (Figure 1D, right panels). Of interest, lymphatic vessels in mice injected with Ad-VEGF-A164 alone did not stain strongly for p-Akt, even though the endothelial cells lining these vessels were actively proliferating15 (Figure 1D).
Effects of Rapamycin on MV Evolution Into Daughter Vessels and on Lymphangiogenesis (Days 5 to 12)
By 5 days after injection of Ad-VEGF-A164, tissue levels of VEGF-A164 have fallen ≈ 10-fold from those at 1 day and new MV formation has ceased.2 Over the course of the following week, MVs that had already formed differentiated into daughter glomeruloid microvascular proliferations (GMP) and vascular malformations (VMs). In addition, a robust lymphangiogenic response developed, leading to the formation of large numbers of abnormally enlarged “giant” lymphatics.15 To assess the effects of rapamycin on this stage of VEGF-A164–driven angiogenesis and lymphangiogenesis, we began treatment on day 5 after Ad-VEGF-A164 administration and continued it daily until harvesting ears on day 12. Rapamycin dramatically reduced overall vascularity by day 12 (Figure 2A) and strikingly reduced the numbers and size of GMP (Figure 2B and 2C), but did not noticeably affect VM formation. GMP stained strongly for both p-Akt and p-S6 in mice receiving Ad-VEGF-A164 alone, whereas in rapamycin-treated mice, the few poorly formed GMP that developed stained weakly for p-Akt and even more weakly for p-S6.
To assess the effects of rapamycin on VEGF-A164–induced lymphangiogenesis, we performed intravital perfusion of the lymphatics in mouse ears with colloidal carbon. Figure 3A illustrates the ears shown in Figure 2A following such perfusion; rapamycin at a dose of 0.5 mg/kg/d had little or no effect on the new VEGF-A164–induced lymphatic network. Consistently, immunofluorescent double staining (Figure 3B) demonstrated that rapamycin-treated ears had many fewer CD31-stained blood vessels but not fewer Lyve-1–positive lymphatic vessels. Quantification of the lymphatic response is shown in Figure 3C.
Effects of Rapamycin on Late Stages of VEGF-Induced Angiogenesis (Days 35 to 49)
VEGF-A-164–driven angiogenesis culminates in the formation of VMs, essentially MVs that have acquired an irregular coat of smooth muscle cells.2 Unlike MVs and GMP, VMs, once formed, persist indefinitely (>1 year); the giant lymphatics induced by Ad-VEGF-A164 also persist indefinitely. To test the effects of rapamycin on these vessels, we began treatment at 35 days after injection of Ad-VEGF-A164 and continued it daily until day 49. Because we have not observed any complications of rapamycin treatment in otherwise mature and normal vascular beds, we were surprised to find macroscopic evidence of vascular changes at this late stage (Figure 4A, day 49, note intense, focal redness). Histology revealed that VM formation was not diminished in rapamycin-treated mice. However, a number of VMs in rapamycin-treated mice had thrombosed and were filled with platelet- and fibrin-rich blood clots (Figure 4B). As at earlier times, lymphatics in ears injected with Ad-VEGF-A164 (here containing colloidal carbon following intralymphatic injection) did not stain detectably with antibodies against p-Akt and p-S6 (Figure 4B).
Finally, we performed Western blots to assess p-Akt and p-S6 activity over the entire time course of our experiments. As shown in Figure 4C, the intensity of both p-Akt and p-S6 bands declined gradually over time from peak values at day 5 in the ears of untreated mice. In rapamycin treated mice, p-S6 bands were strikingly reduced, in comparison with comparable controls, at all time points; p-Akt bands were also reduced, but to a lesser extent.
Effects of Constitutively Active S6K1 and S6K1-Specific shRNAs on Ad-VEGF-A164–Induced Angiogenesis
Rapamycin strongly inhibits S6K1 phosphorylation of S6 but also has a minor effect on Akt phosphorylation. Therefore, to test the effects of S6K1 inhibition independent of concomitant changes on Akt signaling, we prepared retroviruses that expressed either a constitutively active S6K1 or S6K1-specific shRNAs and determined their effects on Ad-VEGF-A164–induced angiogenesis. We first tested these retroviruses on HUVECs (Figure 5A). Constitutively activated S6K1 expression was monitored by its HA tag and by increased phosphorylation of S6K1 at threonine 389, the rapamycin-sensitive residue (Figure 5A). As expected, rapamycin did not inhibit phosphorylation of SK61 at T389 in cells expressing the constitutively active S6K1 because this isoform is mTOR independent. These cells also did not exhibit any change in p-Akt expression. HUVECs were also infected with retroviruses expressing S6K1-specific shRNAs. When silenced with either of 2 independent shRNAs, or by the 2 shRNAs pooled, total and phosphorylated S6K1 levels were strikingly reduced (Figure 5B). Total Akt was not changed but p-AktS473 may have increased slightly. A similar finding has been reported previously and was proposed to reflect derepression of IRS-1.14
We next injected these retroviruses into the ears of mice that had been injected 2 days previously with Ad-VEGF-A164. We chose this time because endothelial cells have begun to divide rapidly at this time and retroviruses only stably integrate into proliferating cells. As shown in Figure 5C, ears injected with retroviruses expressing constitutively active S6K1 showed an enhanced angiogenic response at day 5, above that induced by Ad-VEGF-A164 alone; this response was slightly reduced by rapamycin, likely because not all endothelial cells are infected by the retrovirus. Ears injected with an S6K1 silencing retrovirus exhibited a reduced angiogenic response (Figure 5C).
Supplemental Figure IA (available online at http://atvb.ahajournals.org) shows the histology of these ears at 5 days. Large numbers of typical MVs were found in mice injected with Ad-VEGF-A164 and also in those injected with a retrovirus expressing constitutively active S6K. Rapamycin had little inhibitory effect on MV formation in the presence of the constitutively active S6K1, compared to its dramatic inhibition of MV formation in ears injected with Ad-VEGF-A164 alone (compare with Figure 1A and 1D). However, injecting Ad-VEGF-A164 infected ears with an S6K1 silencing retrovirus substantially reduced the number and size of MVs that formed (supplemental Figure IA, right panel). Immunohistochemistry demonstrated strong p-S6 reactivity in the great majority of MV endothelial cells induced by Ad-VEGF-A164 alone and also in those also injected with a retrovirus expressing active S6K1; in the latter mice, some endothelial cells, presumably those not infected with the retrovirus expressing activated S6K1, exhibited reduced staining as the result of rapamycin repression. Injection of the retrovirus expressing the shRNA silencing S6K1 showed greatly reduced MV p-S6 and slightly increased p-Akt staining. Together the histology demonstrates that S6K1 has a critical role in VEGF-A164-induced MV induction.
This conclusion was further substantiated by immunoblots of whole ear reaction site lysates (supplemental Figure IB). S6K1 and p-S6 were increased in the Ad-VEGF-A164 injection sites of ears that were also injected with retrovirus-expressing S6K1, but p-Akt and total Akt were unchanged. Rapamycin treatment reduced p-S6 in these ears and to a lesser extent p-Akt. When Ad-VEGF-A164 injection sites were injected 2 days later with retroviruses expressing either of 2 shRNAs that silence S6K1, or with a combination of the 2 shRNAs, p-S6 was strikingly reduced whereas p-Akt was modestly increased.
In this study we examined the effects of rapamycin on VEGF-A164–induced angiogenesis and lymphangiogensis. Previous studies using tumor models were limited by their inability to dissect apart the effects of rapamycin on tumor versus stromal cells. The adenoviral model offers the added advantage that different types of vessels form sequentially in a temporally defined manner; thus, the angiogenic response can be studied at discrete early, mid, and late stages, each involving different types of blood vessels. Using this model, we made several novel observations. First, the Akt and S6K1 signaling pathways are differentially upregulated during this response. At early times, MVs highly expressed both Akt and S6K1 signaling. At middle times, it was proliferating GMP that were active, and at later stages, VMs. However, VEGF-A164–induced lymphatics exhibited relatively little Akt and S6 phosphorylation. The inhibitory effects of rapamycin consistently matched the stages of highest p-Akt expression, and VEGF-A164–induced lymphatics were insensitive to rapamycin. The VEGF-A164 levels induced in our adenovirus model are high and formation of new vessels could not be completely suppressed by low dose (0.5 mg/kg/d) rapamcyin, compared with higher doses (Figure 1A). Nonetheless, the 0.5 mg/kg/d dose provides plasma levels of rapamycin that are clinically relevant. Therefore, they allowed us to determine which VEGF-A164–driven vessel subtypes are most sensitive to rapamycin and are useful for translating our findings to patients receiving rapamycin treatment. In studies of the antiangiogenic activity of rapamycin, several laboratories have demonstrated that rapamycin has anticancer efficacy.8,19,20 Moreover, a relatively tumor-specific thrombosis of blood vessels has been correlated to changes in endothelial expression of tissue factor.19
Using the Ad-VEGF-A164 model combined with retrovirally-delivered dominant active and loss-of-function S6K1 isoforms, we were able to demonstrate the importance of S6K1 signaling at least in the early and midstages of VEGF-A164–driven angiogenesis and separated that requirement from overall Akt signaling. As Akt has multiple downstream targets that lead to pathways other than mTOR and S6K, this finding clarifies the importance of S6K1 downstream of Akt signaling in the overall angiogenic response. Earlier studies with rapamycin could not definitively make this conclusion because of the feedback on Akt. Given that we did not observe a reduction in p-Akt in cells overexpressing the dominant active S6K1 (Figure 5A), or by immunohistochemistry (supplemental Figure IA), or in whole lysates (supplemental Figure IB), we conclude that the observed reduction in angiogenesis when we silence S6K1 is not via feedback inhibition of p-Akt, as we find with rapamycin, and supports the conclusion that S6K1 itself is essential for VEGF-A164–driven angiogenesis. If anything, as we also observed in cell culture, there is a slight increase in p-Akt in the ear model when S6K1 is silenced. Although we did not test silencing of S6K1 at later stages of VEGF-A164–driven angiogenesis, extrapolating our findings suggests that later stages that are rapamycin-sensitive, such as GMP formation, will have a similar dependency on S6K. We also predict that vessels with low expression of phosphorylated S6, such as lymphatics and VMs, will have less sensitivity to S6K1 inhibition, as they do to rapamycin treatment.
The late stage of VEGF-A164–induced angiogenesis (35 to 49 days) was confounding in that local increases in p-Akt were observed in response to rapamycin, but in the absence of altered p-S6 expression. This suggests that rapamycin is inducing a different response in these vessels and may have a role in inducing the thrombosis observed in VMs. Taken together, it is clear that the vascular response to rapamycin is heterogeneous and that different vessel subtypes are differentially affected. The thrombosis observed in VMs could be of clinical concern if in fact rapamycin induces vascular thrombosis. However, further investigation will be necessary to assess whether VM thrombosis within tumors is a risk or, possibly, a benefit to cancer patients.
A final surprise was the lack of p-Akt and p-S6 expression in lymphatics. This is in contrast to reports in other models and may reflect a difference between the lymphangiogenesis induced by VEGF-A164 through VEGFR2 versus that induced by other stimuli such as VEGF-C through VEGFR3.21–23 Future studies are required to determine the explanation for the difference in signaling in our Ad-VEGF-A–induced lymphatics and those induced by other means. A recent report showed that although VEGF isoforms that only bind VEGFR2 are capable of inducing lymphangiogenesis, they cannot rescue loss of lymphatics because of inhibition of VEGFR3, further supporting the notion that VEGF-A164–driven lymphangiogenesis may be wholly independent of VEGFR3 signaling.24 This also suggests that the new lymphatics induced by VEGF-A164 can remain functional in reducing edema and clearing waste products even while VEGF-A164–driven angiogenesis is inhibited by rapamycin.
Thanks to H. Zeng and D. Zhao for help in retroviral cloning of shRNA and transduction of HUVEC.
Sources of Funding
This work was supported by P01 CA092644 (to H.F.D. and L.E.B.), R01 CA131064 & HL1049 (to L.E.B.), and RO1 HL 64402 and a contract from the National Foundation for Cancer Research (to H.F.D.).
H.F.D. and L.E.B. contributed equally this study.
Received September 13, 2008; revision accepted May 6, 2009.
Motzer RJ, Escudier B, Oudard S, Hutson TE, Porta C, Bracarda S, Grunwald V, Thompson JA, Figlin RA, Hollaender N, Urbanowitz G, Berg WJ, Kay A, Lebwohl D, Ravaud A. Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled phase III trial. Lancet. 2008; 372: 449–456.
Guba M, von Breitenbuch P, Steinbauer M, Koehl G, Flegel S, Hornung M, Bruns CJ, Zuelke C, Farkas S, Anthuber M, Jauch KW, Geissler EK. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med. 2002; 8: 128–135.
Phung TL, Eyiah-Mensah G, O'Donnell RK, Bieniek R, Shechter S, Walsh K, Kuperwasser C, Benjamin LE. Endothelial Akt signaling is rate-limiting for rapamycin inhibition of mouse mammary tumor progression. Cancer Res. 2007; 67: 5070–5075.
Phung TL, Ziv K, Dabydeen D, Eyiah-Mensah G, Riveros M, Perruzzi C, Sun J, Monahan-Earley RA, Shiojima I, Nagy JA, Lin MI, Walsh K, Dvorak AM, Briscoe DM, Neeman M, Sessa WC, Dvorak HF, Benjamin LE. Pathological angiogenesis is induced by sustained Akt signaling and inhibited by rapamycin. Cancer Cell. 2006; 10: 159–170.
Sarbassov dos D, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science. 2005; 307: 1098–1101.
Yang Q, Inoki K, Ikenoue T, Guan KL. Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity. Genes Dev. 2006; 20: 2820–2832.
Guertin DA, Stevens DM, Thoreen CC, Burds AA, Kalaany NY, Moffat J, Brown M, Fitzgerald KJ, Sabatini DM. Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1. Dev Cell. 2006; 11: 859–871.
O'Reilly KE, Rojo F, She QB, Solit D, Mills GB, Smith D, Lane H, Hofmann F, Hicklin DJ, Ludwig DL, Baselga J, Rosen N. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res. 2006; 66: 1500–1508.
Nagy JA, Vasile E, Feng D, Sundberg C, Brown LF, Detmar MJ, Lawitts JA, Benjamin L, Tan X, Manseau EJ, Dvorak AM, Dvorak HF. Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis. J Exp Med. 2002; 196: 1497–1506.
Xue Q, Hopkins B, Perruzzi C, Udayakumar D, Sherris D, Benjamin LE. Palomid 529, a novel small-molecule drug, is a TORC1/TORC2 inhibitor that reduces tumor growth, tumor angiogenesis, and vascular permeability. Cancer Res. 2008; 68: 9551–9557.
Schalm SS, Tee AR, Blenis J. Characterization of a conserved C-terminal motif (RSPRR) in ribosomal protein S6 kinase 1 required for its mammalian target of rapamycin-dependent regulation. J Biol Chem. 2005; 280: 11101–11106.
Zeng H, Zhao D, Yang S, Datta K, Mukhopadhyay D. Heterotrimeric G alpha q/G alpha 11 proteins function upstream of vascular endothelial growth factor (VEGF) receptor-2 (KDR) phosphorylation in vascular permeability factor/VEGF signaling. J Biol Chem. 2003; 278: 20738–20745.
Guba M, Yezhelyev M, Eichhorn ME, Schmid G, Ischenko I, Papyan A, Graeb C, Seeliger H, Geissler EK, Jauch KW, Bruns CJ. Rapamycin induces tumor-specific thrombosis via tissue factor in the presence of VEGF. Blood. 2005; 105: 4463–4469.
Stephan S, Datta K, Wang E, Li J, Brekken RA, Parangi S, Thorpe PE, Mukhopadhyay D. Effect of rapamycin alone and in combination with antiangiogenesis therapy in an orthotopic model of human pancreatic cancer. Clin Cancer Res. 2004; 10: 6993–7000.
Salameh A, Galvagni F, Bardelli M, Bussolino F, Oliviero S. Direct recruitment of CRK and GRB2 to VEGFR-3 induces proliferation, migration, and survival of endothelial cells through the activation of ERK, AKT, and JNK pathways. Blood. 2005; 106: 3423–3431.
Wirzenius M, Tammela T, Uutela M, He Y, Odorisio T, Zambruno G, Nagy JA, Dvorak HF, Yla-Herttuala S, Shibuya M, Alitalo K. Distinct vascular endothelial growth factor signals for lymphatic vessel enlargement and sprouting. J Exp Med. 2007; 204: 1431–1440.