Antiangiogenic therapy with mammalian target of rapamycin inhibitor RAD001 (Everolimus) increases radiosensitivity in solid cancer.

PURPOSE: Radiotherapy exerts direct antivascular effects in tumors and also induces a proangiogenic stress response in tumor cells via the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. Therefore, the combination of radiotherapy and antiangiogenic therapy with mTOR inhibitor RAD001 (Everolimus) might exert additive/synergistic effects on tumor growth. EXPERIMENTAL DESIGN: Effects of radiation combined with mTOR inhibitor RAD001 were studied on proliferation of murine colon cancer CT-26, human pancreatic cancer L3.6pl, and human umbilical vascular endothelial cells in vitro. In vivo tumor growth of subcutaneous colon cancer CT 26 and orthotopic pancreatic cancer L3.6pl was assessed after fractionated radiotherapy (5 x 2 or 5 x 4 Gy) with or without the addition of the mTOR inhibitor RAD001. RAD001 (1.5 mg/kg/d) was administered until the end of experiments beginning before or after radiotherapy. RESULTS: A single dose of 2 Gy reduced in vitro proliferation of L3.6pl (-16%), CT-26 (-70%), and human umbilical vascular endothelial cells (HUVEC; -72%). The mTOR inhibitor RAD001 (10 ng/mL) suppressed proliferation of HUVEC (-83%), L3.6pl (-8%), and CT-26 (-82%). Combination of even low concentrations of 0.01 ng/mL RAD001 and 0.25 Gy radiation significantly reduced proliferation of HUVECs (-57%), whereas additive effects of RAD001 and radiation on tumor cells were seen only at the highest concentrations tested. In vivo, RAD001 introduced before radiotherapy (5 x 2 Gy) improved tumor growth control in mice (L3.6pl: 326 mm(3) versus 1144 mm(3); CT-26: 210 mm(3) versus 636 mm(3); P < 0.05 versus control). RAD001 turned out to possess a dose-modifying effect on radiotherapy. CONCLUSION: Endothelial cells seem to be most sensitive to combination of mTOR inhibition and radiotherapy. Additive tumor growth delay using the mTOR inhibitor RAD001 and radiotherapy in vivo therefore might rely on combined antiangiogenic and antivascular effects.     

mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt

Stimulation of the insulin and insulin-like growth factor I (IGF-I) receptor activates the phosphoinositide-3-kinase/Akt/mTOR pathway causing pleiotropic cellular effects including an mTOR-dependent loss in insulin receptor substrate-1 expression leading to feedback down-regulation of signaling through the pathway. In model systems, tumors exhibiting mutational activation of phosphoinositide-3-kinase/Akt kinase, a common event in cancers, are hypersensitive to mTOR inhibitors, including rapamycin. Despite the activity in model systems, in patients, mTOR inhibitors exhibit more modest antitumor activity. We now show that mTOR inhibition induces insulin receptor substrate-1 expression and abrogates feedback inhibition of the pathway, resulting in Akt activation both in cancer cell lines and in patient tumors treated with the rapamycin derivative, RAD001. IGF-I receptor inhibition prevents rapamycin-induced Akt activation and sensitizes tumor cells to inhibition of mTOR. In contrast, IGF-I reverses the antiproliferative effects of rapamycin in serum-free medium. The data suggest that feedback down-regulation of receptor tyrosine kinase signaling is a frequent event in tumor cells with constitutive mTOR activation. Reversal of this feedback loop by rapamycin may attenuate its therapeutic effects, whereas combination therapy that ablates mTOR function and prevents Akt activation may have improved antitumor activity.     

Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors

The mammalian target of rapamycin (mTOR) is a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway and a central modulator of cell proliferation in malignant gliomas. Therefore, the targeting of mTOR signaling is considered a promising therapy for malignant gliomas. However, the mechanisms underlying the cytotoxic effects of a selective mTOR inhibitor, rapamycin, on malignant glioma cells are poorly understood. The purpose of this study was thus to elucidate how rapamycin exerts its cytotoxic effects on malignant glioma cells. We showed that rapamycin induced autophagy but not apoptosis in rapamycin-sensitive malignant glioma U87-MG and T98G cells by inhibiting the function of mTOR. In contrast, in rapamycin-resistant U373-MG cells, the inhibitory effect of rapamycin was minor, although the phosphorylation of p70S6 kinase, a molecule downstream of mTOR, was remarkably inhibited. Interestingly, a PI3K inhibitor, LY294002, and an Akt inhibitor, UCN-01 (7-hydroxystaurosporine), both synergistically sensitized U87-MG and T98G cells as well as U373-MG cells to rapamycin by stimulating the induction of autophagy. Enforced expression of active Akt in tumor cells suppressed the combined effects of LY294002 or UCN-01, whereas dominant-negative Akt expression was sufficient to increase the sensitivity of tumor cells to rapamycin. These results indicate that rapamycin exerts its antitumor effect on malignant glioma cells by inducing autophagy and suggest that in malignant glioma cells a disruption of the PI3K/Akt signaling pathway could greatly enhance the effectiveness of mTOR inhibitors.     

Intracellular signaling in tumor and endothelial cells: The expected and, yet again, the unexpected

In this issue of Cancer Cell, Phung and coworkers demonstrate that sustained endothelial activation of Akt by expression of constitutively activated Akt1 (myrAkt1) leads to blood vessels that essentially recapitulate the complex structural and functional abnormalities of tumor vessels. The authors provide evidence that rapamycin inhibition of PI3K/Akt/mTOR signaling in endothelial cells (ECs), by either reducing Akt activity or blocking mTOR, reverses the pathologic effects associated with excess VEGF signaling in the tumor vasculature. However, unexpected findings following mTOR inhibition in vivo highlight the seemingly paradoxical and complex effects of rapamycin on various cell types within the tumor microenvironment.     

Therapeutic targets: MTOR and related pathways

The mammalian target of rapamycin (mTOR), a protein kinase of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, has a central role in controlling malignant cellular growth. As a result, mTOR is viewed as an important target for anticancer drug development. Inhibitors of mTOR currently under evaluation in cancer clinical trials are rapamycin (also known as sirolimus, Wyeth) and derivatives temsirolimus (CCI-779, Wyeth), everolimus, (RAD001, Novartis Pharma AG), and AP23573 (Ariad Pharmaceuticals). Preclinical studies suggest that sensitivity to mTOR inhibitors may correlate with activation of the PI3K pathway and/or with aberrant expression of cell cycle regulatory or anti-apoptotic proteins. Clinical trial results show that mTOR inhibitors are well tolerated and may induce prolonged stable disease and tumor regressions in cancer patients. Future research should evaluate optimal, schedule, patient selection, and combination strategies for this novel class of agents.     

A specific antagonist of the p110delta catalytic component of phosphatidylinositol 3'-kinase, IC486068, enhances radiation-induced tumor vascular destruction

The phosphatidylinositol 3'-kinase (PI3k)/protein kinase B (PKB/Akt) signal transduction pathway plays a critical role in mediating endothelial cell survival and function during oxidative stress. The role of the PI3k/Akt signaling pathway in promoting cell viability was studied in vascular endothelial cells treated with ionizing radiation. Western blot analysis showed that Akt was rapidly phosphorylated in response to radiation in primary culture endothelial cells (human umbilical vascular endothelial cells) in the absence of serum or growth factors. PI3k consists of p85 and p110 subunits, which play a central upstream role in Akt activation in response to exogenous stimuli. The delta isoform of the p110 subunit is expressed in endothelial cells. We studied the effects of the p110delta specific inhibitor IC486068, which abrogated radiation-induced phosphorylation of Akt. IC486068 enhanced radiation-induced apoptosis in endothelial cells and reduced cell migration and tubule formation of endothelial cells in Matrigel following irradiation. In vivo tumor growth delay was studied in mice with Lewis lung carcinoma and GL261 hind limb tumors. Mice were treated with daily i.p. injections (25 mg/kg) of IC486068 during 6 days of radiation treatment (18 Gy). Combined treatment with IC486068 and radiation significantly reduced tumor volume as compared with either treatment alone. Reduction in vasculature was confirmed using the dorsal skinfold vascular window model. The vascular length density was measured by use of the tumor vascular window model and showed IC486068 significantly enhanced radiation-induced destruction of tumor vasculature as compared with either treatment alone. IC486068 enhances radiation-induced endothelial cytotoxicity, resulting in tumor vascular destruction and tumor control when combined with fractionated radiotherapy in murine tumor models. These findings suggest that p110delta is a therapeutic target to enhance radiation-induced tumor control.     

Compensatory activation of Akt in response to mTOR and Raf inhibitors – A rationale for dual-targeted therapy approaches in neuroendocrine tumor disease

Several studies have established a link between aberrant PI(3)K–Akt–mTOR- and Ras–Raf–MEK–Erk1/2 signaling and neuroendocrine tumor disease. In this study, we comparatively investigate the antitumor potential of novel small-molecule inhibitors targeting mTOR (RAD001), mTOR/PI(3)K (NVP-BEZ235) and Raf (Raf265) on human NET cell lines of heterogeneous origin. All inhibitors induced potent antitumor effects which involved the induction of apoptosis and G0/G1 arrest. However, the dual mTOR/PI(3)K inhibitor NVP-BEZ235 was more efficient compared to the single mTOR inhibitor RAD001. Consistently, NVP-BEZ235 prevented the negative feedback activation of Akt as observed after treatment with RAD001. Raf265 inhibited Erk1/2 phosphorylation but strongly induced Akt phosphorylation and VEGF secretion, suggesting the existence of a compensatory feedback loop on PI3K-Akt signaling. Finally, combined treatment with RAD001 or NVP-BEZ235 and Raf265 was more efficient than single treatment with either kinase inhibitor. Together, our data provide a rationale for dual targeting of PI(3)K–Akt–mTOR- and Ras–Raf–MEK–Erk1/2 signaling in NET disease.     

The impact of tumor microenvironment on cancer treatment and its modulation by direct and indirect antivascular strategies

Tumor cells exploit their microenvironment by growth factors and cytokines such as vascular endothelial growth factor (VEGF) to stimulate abnormal vessel formation that is leaky and tortuous, causing irregular blood flow. The combination of poor perfusion, raised interstitial fluid pressure and areas of vascular collapse leads to hypoxia within tumor. The latter activates factors such as hypoxia inducible factor 1 (HIF-1) that serve to make cancer cells more aggressive and also markedly influences the response of malignant tumors to conventional irradiation and chemotherapy. Accumulating data now suggest that blockade of oncogenic signaling, for example by PI3K/Akt/mTOR inhibitors, might consist a promising strategy since these agents do not only possess antitumor effects but can also alter tumor vasculature and oxygenation to improve the response to radiation and chemotherapy. In many cases, these changes are related to downregulation of HIF-1α and VEGF. Here, we review the pathophysiology of tumor microenvironment (TME) and how it adversely affects cancer treatment. The complex interaction of tumor vasculature and radiotherapy is examined together the preclinical evidence supporting a proinvasive/metastatic role for ionising radiation. We will discuss the expanding role of oncogenic signaling, especially PI3K/Akt/mTOR, on tumor angiogenesis. Special emphasis will be paid to the potential of different oncogenic pathways blockade and other indirect antivascular strategies to alter the TME for the benefit of cancer treatment, as an alternative to the classical angiogenetic treatment.     

Two hits are better than one: targeting both phosphatidylinositol 3-kinase and mammalian target of rapamycin as a therapeutic strategy for acute leukemia treatment

Phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) are two key components of the PI3K/Akt/mTOR signaling pathway. This signal transduction cascade regulates a wide range of physiological cell processes, that include differentiation, proliferation, apoptosis, autophagy, metabolism, motility, and exocytosis. However, constitutively active PI3K/Akt/mTOR signaling characterizes many types of tumors where it negatively influences response to therapeutic treatments. Hence, targeting PI3K/Akt/mTOR signaling with small molecule inhibitors may improve cancer patient outcome. The PI3K/Akt/mTOR signaling cascade is overactive in acute leukemias, where it correlates with enhanced drug-resistance and poor prognosis. The catalytic sites of PI3K and mTOR share a high degree of sequence homology. This feature has allowed the synthesis of ATP-competitive compounds targeting the catalytic site of both kinases. In preclinical models, dual PI3K/mTOR inhibitors displayed a much stronger cytotoxicity against acute leukemia cells than either PI3K inhibitors or allosteric mTOR inhibitors, such as rapamycin. At variance with rapamycin, dual PI3K/mTOR inhibitors targeted both mTOR complex 1 and mTOR complex 2, and inhibited the rapamycin-resistant phosphorylation of eukaryotic initiation factor 4E-binding protein 1, resulting in a marked inhibition of oncogenic protein translation. Therefore, they strongly reduced cell proliferation and induced an important apoptotic response. Here, we reviewed the evidence documenting that dual PI3K/mTOR inhibitors may represent a promising option for future targeted therapies of acute leukemia patients.     

Mammalian target of rapamycin: a new molecular target for breast cancer

The mammalian target of rapamycin (mTOR), a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signaling pathway that mediates cell survival and proliferation, is a prime strategic target for anticancer therapeutic development. By targeting mTOR, the immunosuppressant and antiproliferative agent rapamycin inhibits signals required for cell cycle progression, cell growth, and proliferation. Both rapamycin and novel rapamycin analogues with more favorable pharmaceutical properties, such as CCI-779, RAD 001, and AP23573, are highly specific inhibitors of mTOR. In essence, these agents gain function by binding to the immunophilin FK506 binding protein 12 and the resultant complex inhibits the activity of mTOR. Because mTOR activates both the 40S ribosomal protein S6 kinase (p70s6k) and the eukaryotic initiation factor 4E-binding protein-1, rapamycin-like compounds block the actions of these downstream signaling elements, which results in cell cycle arrest in the G1 phase. Rapamycin and its analogues also prevent cyclin-dependent kinase (CDK) activation, inhibit retinoblastoma protein phosphorylation, and accelerate the turnover of cyclin D1, leading to a deficiency of active CDK4/cyclin D1 complexes, all of which potentially contribute to the prominent inhibitory effects of rapamycin at the G1/S boundary of the cell cycle. Rapamycin and rapamycin analogues have demonstrated impressive growth-inhibitory effects against a broad range of human cancers, including breast cancer, in preclinical and early clinical evaluations. In breast cancer cells, PI3K/Akt and mTOR pathways seem to be critical for the proliferative responses mediated by the epidermal growth factor receptor, the insulin growth factor receptor, and the estrogen receptor. Furthermore, these pathways may be constitutively activated in cancers with many types of aberrations, including those with loss of PTEN suppressor gene function. Therefore, the development of inhibitors of mTOR and related pathways is a rational therapeutic strategy for breast and other malignancies that possess a wide range of aberrant molecular constituents. This review will summarize the principal mechanisms of action of rapamycin and rapamycin derivatives, as well as the potential utility of these agents as anticancer therapeutic agents with an emphasis on breast cancer. The preliminary results of early clinical evaluations with rapamycin analogues and the unique developmental challenges that lie ahead will also be discussed.     

Inhibition of mammalian target of rapamycin or apoptotic pathway induces autophagy and radiosensitizes PTEN null prostate cancer cells

The phosphatidylinositol 3-kinase/Akt pathway plays a critical role in oncogenesis, and dysregulation of this pathway through loss of PTEN suppression is a particularly common phenomenon in aggressive prostate cancers. The mammalian target of rapamycin (mTOR) is a downstream signaling kinase in this pathway, exerting prosurvival influence on cells through the activation of factors involved in protein synthesis. The mTOR inhibitor rapamycin and its derivatives are cytotoxic to a number of cell lines. Recently, mTOR inhibition has also been shown to radiosensitize endothelial and breast cancer cells in vitro. Because radiation is an important modality in the treatment of prostate cancer, we tested the ability of the mTOR inhibitor RAD001 (everolimus) to enhance the cytotoxic effects of radiation on two prostate cancer cell lines, PC-3 and DU145. We found that both cell lines became more vulnerable to irradiation after treatment with RAD001, with the PTEN-deficient PC-3 cell line showing the greater sensitivity. This increased susceptibility to radiation is associated with induction of autophagy. Furthermore, we show that blocking apoptosis with caspase inhibition and Bax/Bak small interfering RNA in these cell lines enhances radiation-induced mortality and induces autophagy. Together, these data highlight the emerging importance of mTOR as a molecular target for therapeutic intervention, and lend support to the idea that nonapoptotic modes of cell death may play a crucial role in improving tumor cell kill.     

Overcoming mTOR inhibition-induced paradoxical activation of survival signaling pathways enhances mTOR inhibitors' anticancer efficacy

The mammalian target of rapamycin (mTOR) has emerged as an important cancer therapeutic target. Several mTOR inhibitors are currently being tested in cancer clinical trials. Both PI3K/Akt and MEK/ERK signaling regulate mTOR axis. However, inhibition of mTOR activates Akt survival signaling, which in turn attenuates mTOR inhibitors' anticancer efficacy. We are interested in developing strategies for enhancing mTOR-targeted cancer therapy. In this study, we report that mTOR inhibition also induced activations of the MEK/ERK signaling pathway in some cancer cell lines after a prolonged treatment. The combination of rapamycin with the MEK inhibitor U0126 significantly enhanced growth inhibitory effects of cancer cells, suggesting that MEK/ERK activation may counteract mTOR inhibitors' anticancer efficacy. Similarly, the combination of an mTOR inhibitor with the EGF receptor inhibitor erlotinib synergistically inhibited the growth of both human cancer cells in cell cultures and xenografts in nude mice. Moreover, the presence of erlotinib suppressed rapamycin-induced phosphorylation of Akt, ERK and eIF4E as well, implying that erlotinib can suppress mTOR inhibition-induced feedback activation of several survival signaling pathways including Akt, ERK and eIF4E. Thus, we suggest a therapeutic strategy for enhancing mTOR-targeted cancer therapy by preventing mTOR inhibition-induced feedback activation of several survival mechanisms.     

Longitudinal inhibition of PI3K/Akt/mTOR signaling by LY294002 and rapamycin induces growth arrest of adult T-cell leukemia cells

This study found that phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling was activated in human T-cell lymphotropic virus type I (HTLV-1)-infected leukemia cells. Rapamycin (1-100 nM, 48h), the inhibitor of mTOR and its analog RAD001 (1-100 nM, 48 h)-induced growth inhibition and G0/G1 cell cycle arrest of these cells in association with de-phosphorylation of p70S6K and 4E-BP-1, although IC50 was not achieved. Paradoxically, rapamycin-stimulated phosphorylation of Akt at Ser473. Blockade of Akt signaling by the PI3K inhibitor LY294002 (1-20 microM, 48 h) also resulted in the growth inhibition and G0/G1 cell cycle arrest of HTLV-1-infected cells, with IC50 ranging from 5 to 20muM, and it caused de-phosphorylation of p70S6K and 4E-BP-1. Of note, when rapamycin was combined with LY294002, rapamycin-induced phosphorylation of Akt was blocked, and the ability of rapamycin to induce growth arrest of HTLV-1-infected T-cells and suppress the p-p70S6K and p-4E-BP-1 proteins was potentiated. Moreover, both LY294002 and rapamycin down-regulated the levels of c-Myc and cyclin D1 proteins in these cells, and their combination further decreased levels of these cell cycle-regulating proteins. Taken together, longitudinal inhibition of PI3K/Akt/mTOR signaling represents a promising treatment strategy for individuals with adult T-cell leukemia.     

PTEN loss does not predict for response to RAD001 (Everolimus) in a glioblastoma orthotopic xenograft test panel.

PURPOSE: Hyperactivation of the phosphatidylinositol 3-kinase/Akt signaling through disruption of PTEN function is common in glioblastoma multiforme, and these genetic changes are predicted to enhance sensitivity to mammalian target of rapamycin (mTOR) inhibitors such as RAD001 (everolimus). EXPERIMENTAL DESIGN: To test whether PTEN loss could be used as a predictive marker for mTOR inhibitor sensitivity, the response of 17 serially transplantable glioblastoma multiforme xenografts was evaluated in an orthotopic therapy evaluation model. Of these 17 xenograft lines, 7 have either genomic deletion or mutation of PTEN. RESULTS: Consistent with activation of Akt signaling, there was a good correlation between loss of PTEN function and elevated levels of Akt phosphorylation. However, of the 7 lines with disrupted PTEN function, only 1 tumor line (GBM10) was significantly sensitive to RAD001 therapy (25% prolongation in median survival), whereas 1 of 10 xenograft lines with wild-type PTEN was significantly sensitive to RAD001 (GS22; 34% prolongation in survival). Relative to placebo, 5 days of RAD001 treatment was associated with a marked 66% reduction in the MIB1 proliferation index in the sensitive GBM10 line (deleted PTEN) compared with a 25% and 7% reduction in MIB1 labeling index in the insensitive GBM14 (mutant PTEN) and GBM15 (wild-type PTEN) lines, respectively. Consistent with a cytostatic antitumor effect, bioluminescent imaging of luciferase-transduced intracranial GBM10 xenografts showed slowed tumor growth without significant tumor regression during RAD001 therapy. CONCLUSION: These data suggest that loss of PTEN function is insufficient to adequately predict responsiveness to mTOR inhibitors in glioblastoma multiforme.     

Vascular responses to radiotherapy and androgen-deprivation therapy in experimental prostate cancer

Background

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated in tumor cells and promotes tumor cell survival after radiation-induced DNA damage. Because the pathway may not be completely inhibited after blockade of PI3K itself, due to feedback through mammalian target of rapamycin (mTOR), more effective inhibition might be expected by targeting both PI3K and mTOR inhibition.

Materials and methods

We investigated the effect of two dual PI3K/mTOR (both mTORC1 and mTORC2) inhibitors, NVP-BEZ235 and NVP-BGT226, on SQ20B laryngeal and FaDu hypopharyngeal cancer cells characterised by EGFR overexpression, on T24 bladder tumor cell lines with H-Ras mutation and on endothelial cells. Analysis of target protein phosphorylation, clonogenic survival, number of residual ??H2AX foci, cell cycle and apoptosis after radiation was performed in both tumor and endothelial cells. In vitro angiogenesis assays were conducted as well.

Results

Both compounds effectively inhibited phosphorylation of Akt, mTOR and S6 target proteins and reduced clonogenic survival in irradiated tumor cells. Persistence of DNA damage, as evidenced by increased number of ??H2AX foci, was detected after irradiation in the presence of PI3K/mTOR inhibition, together with enhanced G2 cell cycle delay. Treatment with one of the inhibitors, NVP-BEZ235, also resulted in decreased clonogenicity after irradiation of tumor cells under hypoxic conditions. In addition, NVP-BEZ235 blocked VEGF- and IR-induced Akt phosphorylation and increased radiation killing in human umbilical venous endothelial cells (HUVEC) and human dermal microvascular dermal cells (HDMVC). NVP-BEZ235 inhibited VEGF-induced cell migration and capillary tube formation in vitro and enhanced the antivascular effect of irradiation. Treatment with NVP-BEZ235 moderately increased apoptosis in SQ20B and HUVEC cells but not in FaDu cells, and increased necrosis in both tumor and endothelial all cells tumor.

Conclusions

The results of this study demonstrate that PI3K/mTOR inhibitors can enhance radiation-induced killing in tumor and endothelial cells and may be of benefit when combined with radiotherapy.     

Combination of PI3K/mTOR inhibitors: antitumor activity and molecular correlates

The phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR pathway is a major target for cancer therapy. As a strategy to induce the maximal inhibition of this pathway in cancer cells, we combined allosteric mTOR inhibitors (rapamycin and RAD001) with a dual PI3K/mTOR kinase inhibitor (PI-103). Both in vitro and in vivo, the combination exhibited more activity than single agents in human ovarian and prostate cancer cells that harbor alterations in the pathway. At the molecular level, combined inhibition of mTOR prevented the rebound activation of Akt that is seen after treatment with rapamycin and its analogues and caused more sustained inhibition of Akt phosphorylation. Furthermore, the combination strongly inhibited the expression of PI3K/Akt/mTOR downstream proteins. In particular, it showed greater activity than the single agents in inhibiting the phosphorylation of 4EBP1, both in vitro and in vivo, resulting in selective inhibition of CAP-dependent translation. A proteomic approach was used to confirm the identification of c-Myc as the key regulator for the reduction in downstream proteins affected by the combined inhibition of mTOR. In conclusion, the combination of a catalytic and an allosteric inhibitor of mTOR shows greater activity, without a concomitant increase in toxicity, than either drug alone, and this may have therapeutic implications for inhibiting this pathway in the clinical setting.     

Mechanisms of mammalian target of rapamycin inhibition in sarcoma: present and future

The mammalian target of rapamycin (mTOR) is a protein kinase that plays a pivotal role in the control of cell growth and development. A part of the PI3K/Akt pathway, mTOR responds to growth factor stimuli as well as nutrient availability by variations in downstream phosphorylation. Increasing knowledge of the upstream regulators and downstream targets of mTOR has led to the development of anticancer drugs that suppress protein synthesis and metabolism. Rapamycin (sirolimus) and three rapamycin analogues are currently being evaluated in clinical trials: temsirolimus (CCI-779, Wyeth), everolimus (RAD001, Novartis Pharma AG), and AP23573 (Ariad Pharmaceuticals Inc.). This review will highlight the role of these inhibitors in the treatment of sarcoma.     

Mechanisms of mammalian target of rapamycin inhibition in sarcoma: present and future

The mammalian target of rapamycin (mTOR) is a protein kinase that plays a pivotal role in the control of cell growth and development. A part of the PI3K/Akt pathway, mTOR responds to growth factor stimuli as well as nutrient availability by variations in downstream phosphorylation. Increasing knowledge of the upstream regulators and downstream targets of mTOR has led to the development of anticancer drugs that suppress protein synthesis and metabolism. Rapamycin (sirolimus) and three rapamycin analogues are currently being evaluated in clinical trials: temsirolimus (CCI-779, Wyeth), everolimus (RAD001, Novartis Pharma AG), and AP23573 (Ariad Pharmaceuticals Inc.). This review will highlight the role of these inhibitors in the treatment of sarcoma.     

PKB/Akt mediates radiosensitization by the signaling inhibitor LY294002 in human malignant gliomas

The phosphoinositide 3-kinase (PI3-kinase) signaling pathway is frequently aberrantly activated in glioblastoma multiforme (GM) by mutation or loss of the 3 phospholipid phosphatase PTEN. PTEN abnormalities result in inappropriate signaling to downstream molecules including protein kinase B (PKB/Akt), and mammalian target of rapamycin (mTOR). PI3-kinase activation increases resistance to radiation-induced cell death; conversely, PI3-kinase inhibition enhances the sensitivity of tumors to radiation. The effects of LY294002, a biochemical inhibitor of PI3-kinase, on the response to radiation were examined in the PTEN mutant glioma cell line U251 MG. Low doses of LY294002 sensitized U251 MG to clinically relevant doses of radiation. In contrast to LY294002, rapamycin, an inhibitor of mTOR, did not result in radiosensitization. We demonstrate that among multiple known targets of LY294002, PI3-kinase is the most likely molecule responsible for LY294002-induced radiosensitization. Furthermore, using a myristoylated PKB/Akt construct, we identified PKB/Akt as the downstream molecule that mediates the synergistic cytotoxicity between LY294002 and radiation. Thus PI3-kinase dysregulation may contribute to the notable radioresistance of GM tumors and inhibition of PKB/Akt offers an excellent target to enhance radiosensitivity.