Current Development of Mtor Inhibitors As Anticancer Agents

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Current development of mTOR inhibitors as anticancer agents

Sandrine Faivre*, Guido Kroemer‡ and Eric Raymond* Abstract | Mammalian target of rapamycin (mTOR) is a kinase that functions as a master switch between catabolic and anabolic metabolism and as such is a target for the design of anticancer agents. The most established mTOR inhibitors — rapamycin and its derivatives — showed long-lasting objective tumour responses in clinical trials, with CCI-779 being a first- in-class mTOR inhibitor that improved the survival of patients with advanced renal cell carcinoma. This heralded the beginning of extensive clinical programmes to further evaluate mTOR inhibitors in several tumour types. Here we review the clinical development of this drug class and look at future prospects for incorporating these agents into multitarget or multimodality strategies against cancer.

Signal transduction in cancer cells frequently involves the The PI3K/AKT/mTOR signalling pathway Receptor tyrosine kinases (RTKs). A family of conditional or constitutive activation of receptor tyrosine The PI3K/AKT/mTOR pathway has a cardinal role transmembrane receptors kinases (RTKs) that trigger multiple cytoplasmic kinases, in cancer cell metabolism (for a review, see REF. 8). that are physiologically which are often serine/threonine kinases. Such cellular Activation of various RTKs leads to autophosphorylation activated by the extracellular signalling pathways can operate independently, in para- of the intracellular portion of these receptors, which in binding of growth factor(s) and which initiate intracellular llel and/or through interconnections to promote cancer turn serves as a ‘docking station’ for selected intracell- signalling, ultimately leading development. Three major signalling pathways that have ular proteins. In particular, phosphorylated tyrosine to many cellular responses been identified as important in cancer include the phos- residues of the RTK interact with p85, the regulatory such as proliferation. phatidylinositol 3-kinase (PI3K)/AKT kinase cascade1,2, subunit of PI3K. PI3K is a heterodimer consisting of Abnormalities of these the protein kinase C (PKC) family3,4 and the mitogen-acti- the p85 regulatory subunit and a p110 catalytic subunit, receptors are often reported 5 γ in human malignancies. vated protein kinase (MAPK)/Ras signalling cascades . which can transfer the -phosphate group from ATP to

Mammalian target of rapamycin (mTOR, also known phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), Translational research as FRAP, RAFT1 and RAP1) has been identified as a key thereby generating phosphatidylinositol-3,4,5-trisphos- Research aimed at using kinase acting downstream of the activation of PI3K6. phate (PtdIns(3,4,5)P ) and ADP. The binding of p85 to biological tools for clinical 3 applications. Cumulative evidence supports the hypothesis that mTOR phosphotyrosine residues of the RTK serves to bring acts as a master switch of cellular catabolism and anabo- PI3K into proximity to its substrate PtdIns(4,5)P2 in the lism, thereby determining whether cells — and in par- plasma membrane and probably also induces the allos- ticular tumour cells — grow and proliferate. In addition, teric activation of PI3K. RTKs can also indirectly activate mTOR has been found to have profound effects on the PI3K by causing activation of Ras, which in turn binds regulation of apoptotic cell death, which is mainly dictated to and activates the p110 subunit of PI3K. To negatively by the cellular context and downstream targets including regulate PI3K, cells contain phosphatase and tensin p53, B-cell lymphoma 2 (BCL2), BCL2-antagonist of cell homologue deleted on chromosome 10 (PTEN) and 7 *Service Inter Hospitalier de death (BAD), p21, p27 and c-MYC . other phosphatases that dephosphorylate PtdIns(3,4,5)P3 Cancérologie (SIHC), Beaujon Rapamycin and rapamycin derivatives that specifically back to PtdIns(4,5)P2. A reduction in PTEN expression University Hospital, 100 block mTOR have been developed during the past 5 years indirectly stimulates PI3K activity (and PtdIns(3,4,5)P3 Boulevard du General Leclerc, as potential anticancer agents. In this review we focus on concentrations), thereby contributing to oncogenesis 92118 Clichy Cedex, France. ‡CNRS-UMR 8125, Institute the role of the PI3K/AKT/mTOR signalling pathway in in humans. PtdIns(3,4,5)P3 serves as a ligand to recruit Gustave-Roussy, 39 rue cancer, and the effects of drugs that inhibit mTOR on AKT to the plasma membrane through direct interac- Camille-Desmoulins 94805 the function of cancer cells and tumour angiogenesis. tions with the pleckstrin homology (PH) domain of AKT. Villejuif Cedex, France. We further emphasize the role of translational research on Once at the inner leaflet of the plasma membrane, AKT Correspondence to E.R. e-mail: the determination of the biologically active dose and the becomes phosphorylated by the serine/threonine kinase [email protected] identification of tumour types that are likely to respond, phosphatidylinositol 3-dependent kinase 1 (PDK1), doi:10.1038/nrd2062 or be resistant, to mTOR inhibition. resulting in AKT activation. Activated ATK, itself a

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serine/threonine kinase, promotes cell proliferation, Downstream effects of mTOR. Following phosphory- growth and survival and other processes involved in can- lation, mTOR modulates two distinct downstream sig- cer development by phosphorylating various intracellular nalling pathways that control the translation of specific proteins. Of particular interest among the AKT targets subsets of mRNAs including S6K1 and 4EBP1. Activation is the downstream effector mTOR. Interestingly, several of either PI3K and/or AKT, and/or loss of PTEN sup- genetic syndromes with well-characterized somatic gene pressor function, is necessary and sufficient to induce mutations affecting PTEN or the PI3K/AKT/mTOR the phosphorylation of both S6K1 and 4EBP1 through pathway have enhanced our understanding of human mTOR33–35. Thus, rapamycin-derivatives block the carcinogenesis9–13. In such genetic syndromes, the use phosphorylation of S6K1 and 4EBP1 in cells expressing of mTOR inhibitors could control disease progression activated PI3K or AKT or lacking PTEN36,37. The process and/or exert antitumour effects (TABLE 1). by which mTOR transmits signals depends on its interaction with Raptor, an evolutionarily conserved protein General functions of mTOR of 150 kDa that forms a complex with mTOR and also Activation of mTOR. The TOR family of proteins has binds to both 4EBP1 and S6K1. Although Raptor itself pleiotropic functions, and participates in the regulation is not a kinase, it is required for the mTOR-mediated of the initiation of mRNA transcription and protein phosphorylation of 4EBP1 and S6K1 (REFS 38,39). translation in response to intracellular concentrations of amino acids and other essential nutrients, in 4EBP1. 4EBP1 is a small protein that represses the ini- the organization of the actin cytoskeleton, membrane tiation of protein translation through its association trafficking, protein degradation, PKC signalling with eIF4E, the mRNA cap-binding subunit of the eIF4F and ribosome biogenesis14,15. Of note, there are two complex40–42. Overexpression of eIF4E alone is sufficient mTOR-containing complexes: a rapamycin-sensitive to induce cell transformation43,44. The binding of 4EBPs to complex (also called mTOR complex 1, mTORC1), eIF4E depends on the phosphorylation status of 4EBP1. which is defined by its interaction with the acces- A recent review by Houghton et al.45 has summarized sory protein Raptor (regulatory-associated protein of the interaction of mTOR with translation proteins. In mTOR); and a rapamycin-insensitive complex (also quiescent cells and under growth-factor-deprived condi- called mTOR complex 2, mTORC2), which is defined tions, unphosphorylated 4EBP1 binds tightly to eIF4E, by its interaction with RICTOR (rapamycin-insensi- inhibiting initiation of protein translation. In response to tive companion of mTOR). In contrast to mTORC1, proliferative stimuli triggered by hormones, growth fac- which phosphorylates the well-characterized mTOR tors, mitogens, cytokines and G-protein-coupled agonists, effectors S6 kinase 1 (S6K1, also known as p70S6K) and 4EBP1 becomes phosphorylated at several serine/threo- eukaryotic initiation factor 4E (eIF4E)-binding protein nine sites through the action of mTOR and other kinases, 1 (4EBP1, which is encoded by the gene phosphor- promoting the dissociation of eIF4E from 4EBP1. Free ylated heat- and acid-stable protein regulated insulin 1 eIF4E can then bind to eIF4G (a large scaffolding protein), (PHAS1)), mTORC2 controls the actin cytoskeleton as eIF4A (an ATP-dependent RNA helicase), and eIF4B, well as AKT/PKB16,17. forming the multisubunit eIF4F complex and facilitat- mTOR regulates essential signal transduction ing cap-dependent protein translation46–48. This cascade pathways and is involved in coupling growth stimuli of events induces an increase in translation of mRNAs to cell-cycle progression. In response to growth-induc- with regulatory elements in the 5′-untranslated terminal ing signals, quiescent cells increase the translation of a regions (5′-UTR), including mRNAs that encode c-MYC, subset of mRNAs, the protein products of which are cyclin D1 and ornithine decarboxylase. By contrast, required for progression through the G1 phase of the growth-factor deprivation or treatment with rapamycin cell cycle. PI3K and AKT are the key elements of the results in dephosphorylation of 4EBP1, increased eIF4E upstream pathway that links the ligation of growth binding and a concomitant impairment of the initiation of factor receptors to the phosphorylation and activation the translation of mRNAs with 5′−UTRs that is required state of mTOR18,19. With regard to the role of the PI3K/ for the G1-to-S phase transition of the cell cycle. AKT/mTOR pathway in the genesis and proliferation There is abundant experimental evidence indicating of cancer cells, elements of the PI3K/AKT/mTOR that mTOR is directly responsible for 4EBP1 phospho- pathway have been demonstrated to be activated by rylation and the activation of eIF4E induced by various the erythroblastic leukaemia viral oncogene homo- mitogenic stimuli. For example, the phosphorylation logue (ERB) family of surface receptors, the insulin- of 4EBP1 in insulin-treated cells has been shown to be like growth factor receptors (IGFRs), and oncogenic effectively blocked by mTOR inhibitors49–52. In fact, a low Ras20–23. Overexpression of insulin-like growth-factor cellular ratio of 4EBP1 to eIF4E can cause resistance to 1 receptor (IGF1R) and its ligand, insulin growth- mTOR inhibitors53. Furthermore, sites of 4EBP1 that are factor 1 (IGF1), commonly occurs in several cancers24–28. phosphorylated by mTOR are identical to those induced Additionally, several elements of the PI3K/AKT/mTOR by insulin treatment, and are rapidly dephosphorylated pathway have been demonstrated to be constitution- following exposure to mTOR inhibitors54–56. Some obser- ally activated in malignancies29–31. The hyperactivation vations indicate that mTOR might also act indirectly as an of PI3K/AKT/mTOR signalling elements in PTEN- inhibitor of a protein serine/threonine phosphatase, which deficient malignancies suggests that cancers often functions to dephosphorylate 4EBP1 when conditions depend on this pathway for growth and sustenance32. are appropriate for the G1-to-S phase transition57,58.

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Table 1 | Tumour-prone syndromes that might benefit from treatment with mTOR inhibitors Gene Clinical presentation Syndrome Aetiology PTEN Hamartomatous tumour Cowden disease PTEN is a negative regulator syndromes Cowden syndrome of PI3K Cowden syndrome-like phenotype Bannayan–Zonana syndrome Bannayan–Riley–Ruvalcaba syndrome Lhermitte–Duclos disease Endometrial carcinoma Malignancies Prostate carcinoma Malignant melanoma TSC1 Hamartomas in multiple Tuberous sclerosis complex TSC1 is part of a heterodimer organs (with TSC2) that negatively regulates mTOR TSC2 Abnormal proliferation of Lymphangioleiomyomatosis TSC2 is part of a heterodimer smooth muscle-like cells in (with TSC1) that negatively the lung regulates mTOR NF1 Benign and malignant Neurofibromatosis 1 Loss of NF1 cause AKT peripheral nerve sheath activation via PI3K and Ras tumours AMPK Cardiomyopathy Familial hypertrophic cardiomyopathy On ATP depletion, AMPK can activate TSC2 LKB1 Gastrointestinal Peutz–Jeghers syndrome LKB1 (STK11) phosphorylated hamartomas and activated AMPK on ATP depletion AMPK, AMP-dependent protein kinase; STK11, serine/threonine kinase 11; mTOR, mammalian target of rapamycin; NF1, neurofibromin 1; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; TSC1, tuberous sclerosis 1.

S6K1. Another important downstream target of mTOR effects65. mTOR inhibitors increase the turnover of cyclin is the serine/threonine kinase S6K1. Activated mTOR D1 at both the mRNA and protein levels66. A decrease phosphorylates S6K1 on Thr389, which, in turn, phos- in the translation of cyclin D1 mRNA due to inhibi- phorylates the 40S ribosomal protein S6K159. mTOR might tion of 4EBP1 results in a deficiency of active cyclin- therefore function as a rapamycin-activated phosphatase. dependent kinase 4 (CDK4)–cyclin D1 complexes, The phosphorylation of S6K1 leads to the recruitment of which are required for phosphorylation of the protein the 40S ribosomal subunit into actively translating polys- retinoblastoma (pRb). mTOR inhibition also blocks omes, thereby enhancing the translation of mRNAs with the elimination of the CDK inhibitor p27, thereby a 5′-terminal oligopolypyrimidine (5′-TOP), including prolonging its half-life and facilitating the formation of mRNAs that encode ribosomal proteins, elongation factors complexes between p27 and CDK/cyclin complexes67–69. and insulin growth factor 2 (REFS 60–62). Importantly, S6K1 mTOR inhibition also results in the upregulation of p27 might also be activated by TOR-insensitive signalling path- at the mRNA and protein levels, and inhibits cyclin-A- ways involving PDK1, MAPK and stress-activated protein dependent kinase activity in exponentially growing cells. kinase (SAPK). Reportedly, S6K1 can also phosphorylate Combined together, these cellular effects, along with the the eIF4G and eIF4B units of the eIF4F complex. translational inhibition of other mRNAs, might explain At least three phosphorylation sites have been iden- the profound inhibition of the G1-to-S transition that tified in S6K1, and all of them are blocked by mTOR mTOR inhibitors cause in sensitive cells. Of note, how- inhibitors. The phosphorylation of Thr389 is particu- ever, is that cells derived from p27-knockout mice are larly important because substitution of this residue with only partially resistant to mTOR inhibitors, suggesting alanine blocks the activation of the kinase domain63. that mTOR is also involved in cell-cycle progression There is also evidence indicating that Thr389 is directly through p27-independent mechanisms70,71. phosphorylated by mTOR. Alternatively, or in addition, Recent evidence indicates that mTOR also modulates mTOR might repress a serine/threonine phosphatase that the transcription of DNA to RNA. Inhibition of mTOR dephosphorylates rapamycin-sensitive sites on S6K1. might therefore block the function of polymerases (Pol) If this is correct, the de-repression of this phosphatase by I and III in yeast and mammalian cells, thereby reducing the binding of the FKBP12-RAP complex to mTOR might the transcription of rRNAs and tRNAs, respectively72,73. explain why S6K1 undergoes rapid dephosphorylation Inhibition of mTOR activity results in the inhibition when cells are treated with mTOR inhibitors following of rRNA synthesis and might involve the tumour sup- stimulation with insulin and other growth factors64. pressor pRb, which represses both Pol I and Pol III74. In addition, mTOR inhibition might also inhibit pRb phos- Other molecular interactions of mTOR. In addition to phorylation by modulating the stability and expression its well-characterized inhibitory effects on the activation of cyclin D1 and p27, which regulate CDKs upstream of of S6K1 and 4EBP1, mTOR inhibitors block cell-cycle pRb. mTOR might therefore regulate protein synthesis at progression and therefore mediate antiproliferative both transcriptional and translational levels.

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pathway can also be activated via exogenous oncogenes, Receptor tyrosine kinase including overexpressed or mutated tyrosine kinase recep- Ras (ErbR, PDGFR/KIT, IGFR) tors such as human epidermal growth factor receptors Activation 1–4 (HER1–4), platelet-derived growth factor receptor (PDGFR)/KIT and IGFR, and Ras directly binds the p110 subunit of PI3K75, the latter interaction being important p110 PTEN for membrane anchoring. Downstream of mTOR, the PI3K overexpression and/or amplification of S6K1 or eIF4E p85 could also contribute to oncogenesis. Interestingly, no mutation of mTOR itself has been described. However, there is another rationale for mTOR activation in cancer. Activated p53 acts as a negative regulator of mTOR — for instance, in conditions of glucose deprivation76. p53 func- AKT tion is often lost in cancer, and so this might favour the TSC1/2 constitutive activation of mTOR. The role of these signal transduction proteins in carcinogenesis has been exten- RHEB sively studied in a number of cellular and animal models, mTOR suggesting that activation of the PI3K/AKT/mTOR pathway alone is not sufficient to induce cancer, but rather 4EBP1 requires a secondary oncogenic event to induce cellular transformation. S6K1 eIF4E PI3K/AKT/mTOR activation affects many tumour types, as specified in FIG. 1. RTK activation is frequent Protein Dysfunction/effect Tumour type in malignancies. For instance, activation of PI3K is mediated by K-Ras mutations in certain gastrointestinal K-Ras Mutation resulting in activation Pancreatic, gastric, colon cancers, and more particularly in pancreatic, gastric and Receptor tyrosine kinases Receptor activation Many tumour types colon cancer. Loss of PTEN function via gene mutation, p110 Gene amplification Head and neck, ovarian deletion or promoter methylation has been reported in a Gene mutation Gastrointestinal, brain more selected subset of tumours, including endometrial p85 Gene mutation Colon, ovarian cancer, glioblastoma, prostate, ovarian, thyroid carcinoma, PTEN Gene mutation, deletion or promoter Endometrial, glioblastoma, methylation (loss of function) thyroid, HCC, Cowden syndrome and less frequently in hepatocellular carcinoma, breast, AKT Gene amplification Breast, ovarian, colon lung, renal cell carcinoma and melanoma. Tumours Protein overexpression Ovarian, breast associated with PTEN inactivation are particularly sus- TSC1/2 Gene mutation TSC syndrome ceptible to the therapeutic effects of mTOR inhibitors. 4EBP1 and eIF4E Gene amplification Breast In addition to rare genetic syndromes associated with Protein overexpression Squamous cell, adenocarcinoma PTEN mutation, such as Cowden syndrome, PTEN S6K1 Gene amplification Breast, ovarian inactivation corresponding to genetic or epigenetic alterations can result in the loss of protein expression Figure 1 | Dysregulation of the PI3K/AKT/mTOR signalling pathway in human cancer. Dysregulation of the PI3K/AKT/mTOR pathway can result from exogenous or in several sporadic tumours, such as endometrial endogenous activation. Exogenous factors include activation by Ras, mostly restricted carcinoma, glioblastoma and thyroid carcinoma. In to gastrointestinal malignancies, whereas receptor tyrosine kinase activation has been TABLE 2, we attempt to classify major dysfunctions reported in a broad variety of haematological and solid tumours. Endogenous factors in selected tumour types that might be relevant for include either kinase activation resulting from gene mutation/amplification or PTEN loss therapeutic intervention using mTOR inhibitors. of function. The tumour types that are most frequently affected are shown in the table. eIF4E, eukaryotic initiation factor 4E; 4EBP1, eIF4E-binding protein 1; HCC, hepatocellular Genetic alterations of PTEN. PTEN is found mutated in carcinoma; IGFR, insulin-like growth factor receptor; mTOR, mammalian target of as many as 36–66% of endometrial carcinomas, which is rapamycin; PDGFR, platelet-derived growth factor receptor; PI3K, phosphatidylinositol one of the highest incidences observed among analysed 3-kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; RHEB, tumours77,78. This feature is associated with high levels Ras homologue enriched in brain; TSC, tuberous sclerosis 1. of phospho-AKT (p-AKT represents the active form of AKT) and BAD (p-BAD loses its apoptogenic activity), which improves tumour cell survival69. Among 99 The PI3K/AKT/mTOR pathway in human cancer patients with advanced endometrial carcinoma analysed A plethora of distinct mechanisms can result in the con- for PTEN, p-AKT and Ki-67 expression in tissue speci- stitutive activation of the PI3K/AKT/mTOR pathway in mens, patients with PTEN-positive and p-AKT-negative Glioblastoma High-grade brain malignancy cancer cells (FIG. 1). Cell-intrinsic processes resulting in expression had a higher survival rate than patients in arising from astrocytes with mTOR activation involve loss of PTEN function, mutation all other groups. Multivariate analysis revealed that the abnormal cellular proliferation or amplification of the PI3K p110 catalytic unit, mutation combination of PTEN/ and AKT expression was an and increased tumour of the PI3K p85 regulatory unit, amplification of either independent prognostic factor for survival. Negative angiogenesis. This cancer is usually refractory to of the AKT isoenzymes AKT1 and 2, and inactivation or p-AKT expression was related to a decrease in Ki-67 chemotherapy and has a mutations of AKT-associated mTOR-regulatory proteins that could at least in part explain the better progno- very poor prognosis. such as tuberous sclerosis 1 (TSC1) or TSC2. The mTOR sis. Interestingly, these molecular events are observed

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Table 2 | Molecular events resulting in the activation of the PI3K/AKT/mTOR pathway in human tumours Tumour types Molecular events Comments PTEN PI3K AKT Endometrial Early mutation (30–50%) Increased PI3K activity Early activation (66% in Rapamycin analogues for the carcinoma hyperplasia) treatment of platinum-refractory endometrial carcinoma Glioblastoma Loss of expression (30–40%) Increased PI3K activity Increased expression or EGFR has a role in PI3K activation activation Head and neck No alteration in most cases Overexpression associated with Increased activation Early genetic alteration of PI3Kα cancer lymph node metastasis: (57–81%) Role of PI3K in transition of • EGFR-independent dysplasia to carcinoma • Involved in angiogenic switch Bad prognostic value of PI3Kα • Involved in cisplatin resistance Colon cancer Mutation uncommon in Activity not altered in most cases Increased expression or Early genetic alteration induced sporadic colon cancer; methyl- Low incidence of PI3KCA activation by K-Ras mutations ation of PTEN promoter in mutation (13.6%) Increase tumorigenic potential MSI-H sporadic colon cancer Pancreatic Infrequent loss of Increased activity caused by Ras Increased expression or Early event in carcinogenesis cancer function (PTEN mutation mutation activation in sporadic endocrine pancreatic tumours) Gastric cancer Loss of function infrequent Infrequent upregulation or Increased expression or p-AKT might have a role in (11%) mutation (4.3–10.6%) of PI3KCA activation (28.9%) cisplatin resistance Concurrent K-Ras mutation Hepato- Mutation Increased PI3K activity Increased expression or PI3K pathway might contribute to carcinoma Promoter inactivation activation the angiogenic switch Ovarian cancer Rare cases of mutation Gene amplification Increased expression or Somatic mutation (12%) activation of AKT2 (gene amplification) S6K1 gene amplification Thyroid Deletion (20–60%) Increased PI3K activity Increased expression or Higher percentage of PTEN loss in carcinoma activation aggressive phenotypes Role of EGR1 transcription factor in silencing PTEN Breast cancer Loss of PTEN in 30% of PI3KCA mutation (18–26%), Increased expression or PTEN loss is a bad prognosis in sporadic tumours of which mostly if PTEN functional activation of AKT high-grade tumour with distant 60% have a methylated S6K1 gene amplification metastases and poorer disease- promoter free survival Frequent in Cowden disease Correlated with nodal involvement and resistance to tamoxifen Prostate Low expression in advanced Increased PI3K activity Increased expression or Long-term androgen deprivation cancer tumours activation of AKT reinforces the PI3K/AKT pathway Lung cancer Mutation rare Increased PI3K activity Increased expression Role of EGFR in PTEN silencing Reduced protein expression or activation of AKT, PI3K/AKT activated by MAPK (24–74%), presumably by activation of S6K1 promoter silencing EGFR, epidermal growth factor receptor; EGR1, early growth response 1; MAPK, mitogen-activated protein kinase; MSI-H, microsatellite instability-high; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10.

already in pre-malignant endometrial hyperplasia, p-mTOR and p-S6K1. Expression of the mutant epi- which can be successfully treated with oral progester- dermal growth factor receptor VIII was also tightly one in 30–50% of cases. Among non-responders to pro- correlated with phosphorylation of these effectors, gesterone, increased or persistent p-mTOR expression demonstrating an additional route to PI3K pathway was observed. Although mTOR phosphorylation does activation in glioblastomas in vivo80. Among 92 patients not imply its activation, studies are ongoing to evaluate with glioma, the levels of p-PI3K, p-AKT and p-S6K1 the role of mTOR inhibitors in progesterone-refractory all correlated inversely with the levels of cleaved caspase endometrial hyperplasia79. 3, indicating that PI3K pathway activation suppresses In 30–40% of glioblastomas, PTEN has been found to apoptosis. Importantly, activation of the PI3K/AKT/ be inactivated by mutation, homozygous deletion or loss mTOR axis was associated with increasing tumour grade of expression. In previously untreated patients, loss of and reduced patient survival81. PTEN gene inactiva- PTEN was associated with significant increase in p-AKT, tion or loss has been correlated with poor prognosis in

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levels of EGR1 are associated with an increased risk Box 1 | Protective use of mTOR inhibitors in neurodegeneration of recurrence and poor survival after surgical resection of Inhibitors of mammalian target of rapamycin (mTOR) might have other roles than tumours in patients with early stage NSCLC88. controlling proliferation of cancer cells. As part of multiple effects of mTOR inhibition, In cervical cancer, PTEN mutations seem uncommon89 there is genetic evidence that invalidation of the mTOR pathway can increase the at early stages but might increase in frequency in advanced lifespan of nematodes and fruit flies, presumably by mimicking the effects of caloric disease, particularly in tumours exposed to radiation restriction121. Whether chronic mTOR inhibition prolongs lifespan in mammals, however, therapy90. Conversely, loss of function of PTEN, either has yet to be tested. Interestingly, inhibition of mTOR has been proposed as a strategy for stimulating the autophagic removal of neurotoxic proteins carrying polyglutamine by loss of heterozygosity or promoter methylation, was extensions, such as mutated huntingtin. Accordingly, CCI-779 improved performance in more frequently reported from the early stages of carcino- behavioural tasks and decreased aggregate formation in a mouse model of Huntington’s genesis, including dysplasia to invasive carcinoma91. Loss disease122. Furthermore, Alzheimer’s disease is accompanied by a marked activation of of PTEN function in patients with cervical carcinoma is the mTOR pathway, correlating with the levels of the protein tau, which participates in associated with a poor outcome. the formation of neurofibrillary tangles123. In addition, enhanced expression of mTOR has been found in multi-nucleated giant cells (also called syncytia) that develop in human PI3K/AKT pathway abnormalities. Inactivation of 124 immunodeficiency virus (HIV)-associated encephalitis . In an in vitro model of HIV1- PTEN, as measured by immunohistochemistry, was elicited syncytium formation, mTOR was able to participate in pathological cell death by found in about one-third (28%) of 236 breast cancer phosphorylating p53 on serine 15, thereby causing activation of this lethal transcription specimens92. The reduced expression of PTEN protein factor125. Inhibition of mTOR can therefore inhibit syncytial cell death in vitro, and it might also be possible to use mTOR inhibitors to palliate HIV-associated encephalitis. correlated with lymph-node metastases and poor prog- Further research is underway to better characterize the role of mTOR in diseases nosis. In another study including 100 breast cancers, associated with cell loss and the effects of mTOR to prevent neurodegeneration. downregulation of PTEN expression was predictive of the failure of tamoxifen treatment93. In addition to loss of PTEN, activating PI3K mutations on the catalytic high-grade astrocytomas. In particular, the PI3K/AKT subunit were identified in 26% of 342 breast cancers. pathway might be involved in glioma cell migration and Interestingly, PI3K mutations mostly occurred when stimulated by the urokinase-type plasminogen activator PTEN function was retained. In addition, PI3K muta- in glioma cells82. tions were associated with expression of oestrogen Cowden disease (also known as multiple harmartoma (ER) and progesterone receptors (PR), lymph-node syndrome) is an autosomal-dominant cancer syndrome metastasis, and HER2/neu overexpression. Although associated with a high risk of breast and thyroid can- PI3K mutations correlate with ER/PR-overexpression, cer caused by germline mutations in PTEN. Although PTEN loss correlates with the loss of either/or ER/PR, somatic mutations of the PTEN gene are rare in sporadic suggesting that breast cancer might follow two dis- thyroid carcinoma, loss of heterozygosity at 10q23 (which tinct patterns of carcinogenesis94. In yet another study, includes the PTEN gene) is present in 20–60% of thy- among 402 ERα-positive breast carcinomas taken from roid malignancies, with a higher percentage in the more patients treated with tamoxifen, high p-AKT levels were aggressive histotypes. For example, a screening for PTEN predictive of reduced overall survival95. These data sup- mRNA expression in 87 sporadic thyroid tumours includ- port in vitro evidence that AKT mediates tamoxifen ing 14 anaplastic carcinomas, 37 follicular carcinomas, 21 resistance. atypical adenomas and 15 ordinary adenomas showed a Allelic imbalance and mutations of the PTEN gene complete loss of PTEN mRNA expression in six of the have only been found in 9% of ovarian cancers96. tumours, including four anaplastic carcinomas. PTEN However, somatic missense mutations affecting the cata- transcriptional silencing is therefore probably involved lytic unit of PI3K have been reported in 24 of 198 (12%) in the carcinogenesis of highly malignant or late-stage of ovarian cancers97. Increased expression or activation thyroid cancers, whereas it seems to be of minor impor- of AKT2 due to gene amplification, together with S6K1 tance in differentiated follicular thyroid tumours83. gene amplification, has also been observed24. In prostate cancer, the probability of PTEN loss and Epigenetic alteration of PTEN. It has previously been PI3K/AKT pathway activation increases in advanced demonstrated that PTEN promoter activity is increased stages98,99. Androgen independence of prostate cancer by overexpression of the transcription factor early has been associated with the activation of either the growth response protein 1 (EGR1)84. Preclinical stud- PI3K or the MAPK pathways100, suggesting a rationale ies using thyroid tumour cell lines revealed that the for novel therapeutic approaches. absence of EGR1 mRNA is associated with the silencing Conflicting data have been published on the role of PTEN gene expression that occurs during thyroid of PTEN loss in squamous head and neck carcinoma. cell transformation85. A similar scenario applies to non- Although one study reported PTEN loss of heterozygos- small-cell lung cancer (NSCLC). Most NSCLC cell lines ity in 41% of the cases101, another analysis of 21 cases demonstrate PI3K/AKT/mTOR activation yet harbour found no PTEN loss or mutation102. The most frequent a wild-type PTEN gene86. In tumour specimens from abnormality is the overexpression or amplification of the patients, about 24–74% of cases showed no PTEN pro- catalytic unit of PI3K, which can be found both in the tein expression87, presumably because of the silencing early stage of carcinogenesis (dysplasia) and in advanced of the PTEN promoter gene by the transcription factor head and neck cancer. Activation of the PI3K pathway EGR1. A recent study suggests that EGR1 gene expres- is correlated with lymph node metastases, enhanced sion is predictive of PTEN concentrations and that low angiogenesis and resistance to cisplatin103.

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agent was given a low priority and was halted in 1982, Rapamycin derivatives resuming in 1988 to be postponed again until the end of the 1990s following the discovery of CCI-779, a novel G1–S cell-cycle transition Effects on apoptosis soluble rapamycin derivative formulated for intravenous infusion that had a safe toxicological profile in animals. In the meantime, rapamycin became an immuno- 4EBP1mTOR BAD suppressive agent, which stimulated the exploration of the mechanism of action of this agent. Rapamycin inhibits T-cell proliferation induced by cross-linking p34cdc2 of the T-cell receptor or antigenic peptides pre- Cyclin D1 Cyclin E BCL2 sented by major histocompatibility complex (MHC) molecules. Rapamycin also inhibits proliferative responses induced by several cytokines, including p27 p53 interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-6, IGF, PDGF and colony-stimulating factors (CSFs). The preclini- Figure 2 | Effect of mTOR inhibitors on cancer cells. Cellular effects of mTOR activation cal development of rapamycin as an immunosuppres- include the facilitation of G1–S cell cycle transition and inhibition of apoptosis through sor has been extensively reviewed113. Rapamycin can the interaction with key molecules of cell-cycle control (4EBP1, cyclin D1 and p27) and exert its immunosuppressive effect in synergy with apoptosis (BAD, BCL2 and p53). These processes are reversed by mTOR inhibitors. cyclosporine114. The combination of rapamycin and 4EBP1, eukaryotic initiation factor 4E-binding protein 1; BAD, BCL2-antagonist of cell cyclosporine A enhanced rejection prevention in renal death; BCL2, B-cell lymphoma 2; mTOR, mammalian target of rapamycin. transplantation, and enabled the use of lower doses of cyclosporine, minimizing its toxicity115,116. Rapamycin was approved in the USA in 1999 (and 2000 in Europe) In gastrointestinal tumours, the role of PTEN seems for the prevention of acute rejection in combination to be limited, with infrequent mutations. Sporadic PTEN with cyclosporine and steroids. Interestingly, rapamy- loss of function has been described in colon, gastric and cin, unlike cyclosporine, does not increase the risk of pancreatic carcinoma104–109. In colon and pancreatic malignancy but rather decreases the risk of post-trans- tumours, K-Ras-induced PI3K/AKT activation was plant lymphoproliferative disorders117. Apart from its reported as an early event in carcinogenesis. In hepato- immunosuppressive capacity, rapamycin was also cellular carcinoma, studies were mainly performed using recently shown to be capable of preventing coronary human cancer cell lines, with no available data from artery re-stenosis118. High doses of rapamycin block patient specimens. These preclinical studies indicated the proliferative responses to cytokines in vascular that the function of the PTEN gene might be impaired smooth-muscle cells after mechanical injury, such as by point mutations or promoter inactivation110,111. balloon angioplasty119. Growth, migration and differentiation of vascular smooth-muscle cells are also two Rapamycin: a prototype for mTOR inhibition major features of neo-intimal proliferation after vascu- So far, four mTOR inhibitors are available for clinical tri- lar injury. The proposed mechanisms of inhibition of als: the prototype rapamycin and three rapamycin deriv- vascular smooth-muscle cell proliferation were related atives, CCI-779 (temsirolimus), RAD001 (everolimus) to cell-cycle blockage and/or the inhibition of PDGF- and AP23573. Each of these inhibitors forms a complex induced migration120. Recent studies have suggested with the intracellular immunophilin FKBP12; the result- applications of rapamycin derivatives in the treatment ing complex interacts with and inhibits mTOR. No other of neurodegenerative diseases121–125 (BOX 1). proteins have been identified as rapamycin targets, Recently, rapamycin has been shown to inhibit the and the requirement for a cofactor makes the mTOR– growth of several murine and human cancer cell lines in rapamycin interaction very specific. Indeed, treatment a concentration-dependent manner, both in tissue cul- of mice embryos with rapamycin induces exactly the ture and in xenograft models including B16 melanoma, same developmental defect as the mTOR knockout. This P388 leukaemia, MiaPaCa-2 and Panc-1 human pancre- accurate phenocopy suggests that rapamycin is a truly atic carcinomas126,127. In the 60-tumour cell-line screen monospecific mTOR inhibitor. by the US NCI, the spectrum of activity of rapamycin Rapamycin (also named sirolimus) is a macrocy- was different from that of other anticancer agents in clic lactone produced by Streptomyces hygroscopicus. leukaemia, ovarian, breast, central nervous system, and Rapamycin was initially developed as an antifungal small-cell lung cancer cell lines. Furthermore, rapamycin drug directed against Candida albicans, Cryptococcus alone induces p53-independent apoptosis in childhood neoformans and Aspergillus fumigatus112. It is a white rhabdomyosarcoma and sensitized cells to apoptosis crystalline solid that is insoluble in aqueous solutions, induced by cisplatin in HL-60 promyelocytic leukae- but soluble in organic solvents. When rapamycin was mias and ovarian SKOV3 carcinoma cell lines128,129. evaluated by the Developmental Therapeutic Branch of Conversely, rapamycin inhibits taxol-induced apoptosis the National Cancer Institute (NCI), it was identified as in human B-cell lines, probably through prevention of a non-cytotoxic agent that had cytostatic activity against BCL2 inactivation130. Rapamycin also inhibits hybridoma several human cancers in vitro and in vivo. However, the cell death in bioreactors, thereby increasing the produc- development programme of rapamycin as an anticancer tion of monoclonal antibody131. In addition, rapamycin

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Rapamycin proper control of the activated PI3K/AKT/mTOR signal- derivatives ling pathway and can be viewed as a ‘gain of function’ rather than a trivial inhibitory effect on protein function. Interestingly, molecular interactions between rapamycin, HIF1α PDGFR KIT FKBP12 and mTOR can persist for about 72 hours even after a short exposure to rapamycin, blocking mTOR PDGFR IGFR function for several days. As a result, S6K1 undergoes PI3K/AKT/mTOR dephosphorylation, which results in a reduction of protein synthesis, linked to reduced cell size and motility. The VEGFR ERB rapamycin-triggered dephosphorylation of 4EBP1 results in a reduced translation capacity of mRNAs encoding c-MYC, cyclin D1 and ornithine decarboxylase, increased eIF4E binding, and a concomitant impairment of the initiation of the translation of mRNAs with 5′-UTRs that are required for the transition in the G1/S phase of the cell cycle. Overall, effects of rapamycin consistently induce a decrease in cyclin D1 expression and lead to an increase Anabolism in p27 that will lead to late G1/S cell-cycle blockage. These Proliferation rapamycin-mediated metabolic and cell-cycle effects slow Migration Endothelial Tumour down the proliferation of several human cancer cell lines cells cells in a dose-dependent manner. In a relatively limited number of tumour models, Figure 3 | The effect of modulation of the PI3K/AKT/mTOR pathway with rapamycin was shown to induce cancer cell death either rapamycin derivatives on endothelial and tumour cells. Key molecular factors by inducing apoptosis or autophagy. The molecular that activate the PI3K/AKT/mTOR pathway in either endothelial or cancer cells are mechanisms leading to apoptosis in cancer cells have shown. In addition to effects reported in cancer cells, mTOR inhibitors might also not yet been fully deciphered. One link between mTOR act as anti-angiogenic agents by intercepting the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) signalling cascades. inhibition and apoptosis induction might be provided by ERB, erythroblastic leukaemia viral oncogene homologue; HIF1α, hypoxia-inducible the downstream target S6K1, which can phosphorylate factor-α; IGFR, insulin growth factor receptor; mTOR, mammalian target of the pro-apoptotic molecule BAD on Ser136, a reaction rapamycin; PI3K, phosphatidylinositol 3-kinase. that disrupts BAD’s binding to the mitochondrial death inhibitors BCL-XL and BCL2, thereby inactivating BAD133,134. In this scenario, rapamycin-mediated S6K1 inhibits the oncogenic transformation of human cells inactivation would indirectly cause BAD activation. In induced by either PI3K or AKT, and inhibits metastatic addition, two recent studies have suggested that mem- tumour growth and tumour vascularization in vivo in bers of the BCL2 family could be downstream media- suitable mouse models132. tors of the IGF1-stimulated, PI3K-dependent survival of On the basis of these preclinical results, studies with cells135,136. Furthermore, several growth factors that acti- rapamycin as an anticancer drug have been launched, vate the PI3K and S6K1 pathways were recently shown and rapamycin derivatives with improved pharma- to increase expression of BCL2, thereby promoting cell cokinetics and reduced immunosuppressive effects have survival in myeloid progenitor cells123. been developed. Unlike rapamycin, the three rapamycin The contribution of BCL2 to chemotherapy resistance derivatives do not mediate any manifest immunosup- has been investigated extensively in cultured cells, animal pression when administered intermittently in clinical models and clinical studies. In studies with patients suf- settings1. CCI-779 is administered either orally or intra- fering from ovarian carcinoma, high levels and/or aber- venously, RAD001 is available as an oral formulation and rant patterns of BCL2 expression have been correlated AP23573 is either given orally or intravenously. with resistance to commonly used anticancer agents137. This also applies to rapamycin-like agents. Ovarian Properties of tumour growth inhibition cancer cells that are resistant to mTOR inhibitors over- Direct exposure of cancer cells to rapamycin and rapamy- express BCL2 and an antisense BCL2 oligonucleotide cin derivatives results in several effects that depend on can sensitize ovarian cancer cells to rapamycin and specific cellular characteristics and drug concentra- RAD001138. Furthermore, one of the most potent mTOR tion. In addition, mTOR inhibitors can target tumour activators is a high level of nutrients, and in particular growth indirectly, by interacting with the maintenance amino acids. As mTOR acts as an endogenous suppressor of endothelial cells and pericytes that are required for of autophagy, nutrient depletion (which inhibits mTOR) tumour angiogenesis. results in the activation of autophagy, allowing the cell to catabolize macromolecules and meet its energy demands Effects of mTOR inhibitors in cancer cells. In cancer cells with low molecular mass metabolites139. Rapamycin acti- possessing an activated PI3K/AKT/mTOR pathway, vates autophagic processes that might, at least in some rapamycin and its derivatives block the binding of raptor instances, participate in the cytostatic or cytotoxic effects to mTOR, which is required for downstream phosphor- of rapamycin on tumour cells140. Surprisingly, cell death ylation of 4EBP1 and S6K1 (FIG. 2). This effect restores the induced by rapamycin and its derivatives does not seem

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Table 3 | Current status of mTOR inhibitors in clinical development Compounds Development status Dose and schedule Main clinical results Refs (number of patients) CCI-779 Phase I 7.5–220 mg per m2 per week Recommended doses <220 mg per m2 148 (temsirolimus) (n = 24) Dose-limiting toxicity: mucositis, depression, intravenous and thrombocytopaenia and hyperlipaemia oral formulation 0.75–19.1 mg per m2 per day x Maximum tolerated dose: 19.1 mg per m² 149 5 days every 2 weeks (n = 24) Dose-limiting toxicity: thrombocytopaenia and mucositis Phase II*: endometrial 250 mg per week (n = 16) Response rate: 31% 152 carcinoma Rate of stable disease: 63% Phase II: renal cell carcinoma 25 versus 75 versus 250 mg Response rate: 7% with a 2–3 fold survival 155 per week (n = 111) improvement for intermediate/poor prognosis patients versus historical series Phase III: renal cell carcinoma 25 mg per week (n = 626) Significantly longer survival in CCI-779 arm (10.9 156 months) compared with interferon-α (7.3 months) Phase II: breast cancer 75 versus 250 mg per week Response rate: 9.2% (recommended dose: 75 mg 158 (n = 109) per week) No clear dose–effect relationship Phase II: glioblastoma 250 mg per week Time to progression: 2.3 months 153,154 (total n = 65) Partial response reported Phase II: mantle cell lymphoma 250 mg per week (n = 43) Response rate: 38% 159 Time to progression: 6.5 months RAD001 Phase I Single agent: 5–20 mg per Recommended dose: 20 mg per week 150 (everolimus) oral week Dose-limiting toxicity: thrombocytopaenia and formulation gastritis Phase I/II in patients with Combination: 2.5–5 mg per Recommended dose: 2.5 mg per day 163 Gleevec-refractory gastro- day with imatinib at 600 mg Partial response in 2 patients intestinal stromal tumours per day Tumour stabilization in 8 patients AP23573 Phase I 6.25–100 mg per day weekly Antitumour activity in sarcoma 151 intravenous and oral formulation Phase II in advanced sarcomas 3–28 mg per day x 5 days Objective responses in 4 patients, decrease 164 every 2 weeks in PET uptake in 8 patients and symptomatic 12.5 mg per day x 5 days every improvement in 13 patients 2 weeks *On the basis of pharmacokinetic data, all Phase II trials were conducted using flat dosing. PET, positron emission tomography.

to be dose-dependent. Altogether, it seems that rapamy- cells or pericytes142. Cellular proliferation, survival and cin can induce growth arrest and death of tumour cells migration required for vascular sprouting, and endothe- through various pathways. lial cell differentiation leading to tubule formation are primarily driven by VEGF/VEGFR activation that can Effects of mTOR inhibitors on tumour angiogenesis. in turn trigger the PI3K/AKT/mTOR pathway. One of Tumour angiogenesis relies on an intricate interplay the major stimuli of cancer angiogenesis is hypoxia, between tumour cells, endothelial cells and surround- which activates hypoxia-inducible transcription factors ing mesenchymal cells (pericytes in microvessels and (HIFs), which in turn induce the expression of VEGF, vascular smooth-muscle cells in large vessels) to acti- VEGFR, bFGF, PDGF and ANG2 (REF. 143). mTOR vate endothelial cell proliferation, to recruit migrating can facilitate the translation of HIF1α mRNA, thereby endothelial cells and pericytes and to form new vessels/ enhancing vascular growth factor expression144, a proc- capillaries through vascular remodelling and matura- ess that is influenced by PTEN. In normal vessels, HIF1α tion141. At the molecular level, tumour angiogenesis is transiently expressed as a result of the action of the depends on shear stress and coordinated interactions HIF-prolyl hydroxylase that targets HIF1α to a ubiquitin between endothelial vascular growth factors such as ligase complex containing von Hippel–Lindau (pVHL), vascular endothelial growth factor (VEGF), angiopoi- which marks it for destruction by the proteasome. In etin 1 (ANG1), ANG2, basic fibroblast growth factor tumour cells, a number of factors stabilize HIF1α and (bFGF), PDGF-B, ephrin-B2 and members of the translocate HIF1α into the nucleus. For instance, BCL2 tumour growth factor-β (TGFβ) superfamily; intrac- and hypoxia synergize to induce HIF1α and VEGF ellular signalling molecules including NOTCH1 and expression in melanoma cells145. In addition, loss-of- COUP-TFII; and intercellular contacts via connexins function mutations of VHL, as they frequently occur and vascular cell-adhesion molecule 1 (VCAM1)130. in renal cell carcinoma, can cause HIF1α stabilization, Interestingly, all the above factors can activate the thereby inducing PDGF and VEGF overexpression and PI3K/AKT/mTOR pathway in cancer cells, endothelial sustained tumour angiogenesis in humans133.

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continuous dosing until tumour progression148–151. Dose- Box 2 | Chemical structure of rapamycin and rapamycin derivatives limiting toxicities were consistent between the three com- The rapamycin derivatives pounds and consisted of reversible mucositis, asthenia used as inhibitors of OH (weakness and fatigue) and thrombocytopaenia, and mammalian target of were mainly observed with the five-times daily dosing rapamycin (mTOR) share Binding site OCH3 Binding site schedule. Severe psychiatric disorders at very high doses the same molecular to FKBP12 to mTOR were also reported in the weekly schedule of CCI-779 scaffold but with substitution of the lactonic O O OH (REF. 146). No significant immunosuppression was N macrocycle, making the observed for CCI-779, RAD001 and AP23573, and at compound either suitable O O O the recommended dose the most prevalent toxic effects O H3CO for intravenous (CCI-779 HO were reversible cutaneous side effects, such as herpes and AP23573) or oral O OCH3 lesions, aseptic acne-like rash, maculopapular rash and (RAD001) formulation. nail disorders, and were observed in about 75% of the Rapamycin derivatives patients. The longer-term side effects of rapamycin have the same binding sites derivatives are not yet known, but should be carefully for FK506-binding protein 12 (FKBP12) and mTOR. The semi-synthetic modifications of assessed because it is speculated that long-term treat- rapamycin derivatives produced only few structural changes, mostly located at the C40 ment with weekly CCI-779 might cause nonspecific hydroxyl group outside FKBP12- and mTOR-binding domains. All these derivatives are therefore likely to share similar properties to rapamycin. Modifications in the rapamycin pneumonia. At the recommended doses, these com- biosynthetic gene cluster using conjugate methods for DNA transfer to the rapamycin- pounds displayed a side-effect profile that renders them producing organism Streptomyces hygroscopicus NRRL5491 could provide access to potentially amenable to combination with cytotoxic novel rapalogues. CCI-779 can be considered as a prodrug that is bioconverted into agents or other targeted therapies. sirolimus, whereas RAD001 and AP23573 are not metabolized in this way. The weekly administration schedule that showed the lowest toxic side effects was selected for further clinical evaluation of rapamycin derivatives in Phase II and III It is possible that the anticancer effects of mTOR inhib- trials. Early on in Phase I studies of rapamycin-deriva- itors involve anti-angiogenic processes mediated by effects tives, antitumour activity included an objective response on pericytes and endothelial cells rather than on cancer in renal (FIG. 4), breast and non-small cell lung cancer, as cells themselves131. As outlined above, mTOR inhibition well as several cases of minor responses and prolonged can block angiogenesis by inhibition of HIF1α transla- disease stabilization. These responses were observed tion as well as by intercepting the VEGF/VEGFR and/or over a broad range of dose levels. Phase II trials were PDGF/PDGFR signalling cascades (FIG. 3). Chemical continued using weekly oral and intravenous schedules inactivation of mTOR in hypoxia-activated endothelial in tumours types that had shown promising results in cells and pericytes can induce a G0–G1 cell-cycle block Phase I trials or in tumours likely to be driven by the (associated with reduced cyclin D1 expression and p27 PI3K/AKT/mTOR pathway and/or the loss of PTEN. accumulation) rather than apoptosis146. In mice bearing One such example is a recent Phase II trial investigat- human tumour xenografts, RAD001 significantly reduced ing CCI-779 that was conducted in recurrent or meta- tumour angiogenesis by blocking tumour growth. If these static endometrial cancer, based on the fact that this type data can be extrapolated to the human system, then mTOR of tumour frequently loses PTEN. In the first part of the inhibitors should be particularly efficient in inhibiting the study, 18 patients received CCI-779 at the dose of 250 mg angiogenesis of tumours that bear VHL mutations and/or per week for a median duration of treatment of 6 months. constitutively activate the VEGFR and PDGFR tyrosine Five out of the 16 patients who were suitable for evaluat- kinase signalling pathways, such as renal cell carcinoma. ing efficacy showed partial responses (31%) and ten out The assumption that VHL has a role in sensitivity/resist- of 16 patients (63%) had stable disease, with only one ance to mTOR inhibitors has been supported by recent patient showing progressive disease. These preliminary work showing that CCI-779 preferentially inhibits VHL- results suggest that monotherapy with CCI-779 could be null renal cell carcinomas, which provides a rationale for an option for the treatment of endometrial carcinoma, a prospective biomarker-driven clinical trials in patients disease for which no standard of care currently exists152. with kidney cancer147. It remains to be seen whether Two Phase II studies have been performed on patients Mucositis mTOR inhibitors are particularly efficient when combined with recurrent glioblastoma treated with CCI-779 at the Inflammation and/or ulceration 153,154 of mouth mucosa. with anti-angiogenic drugs targeting VEGF (bevacizu- initial dose of 250 mg per week . The results of both mab (Avastin; Genentech)), PDGFR (imatinib mesylate studies are consistent, showing limited antitumour activ- Thrombocytopaenia (Gleevec; Novartis)) or VEGFR (sunitinib malate (Sutent; ity as measured by a computerized tomography (CT) Low level of platelets in Pfizer) and sorafenib (Nexavar; Bayer)). scan and/or magnetic resonance imaging (MRI) with a circulating blood. median time to progression of disease of 2.3 months. Objective response Clinical development of rapamycin-derivatives Reports of objective responses in this disease were Shrinkage of at least 50% of So far, most data exist for the clinical development of the anecdotal and mainly based on non-validated functional malignant target lesions rapamycin derivatives CCI-779 (temsirolimus), RAD001 neuroimaging methods. So, therapy with CCI-779 as a (according to World Health (everolimus) and AP23573 (TABLE 3, BOX 2). These single-agent for recurrent glioblastoma seems to have Organization criteria) after administration of anticancer compounds were studied in the clinic using three main only limited activity. However, it might be possible to treatment as compared with oral and intravenous schedules: five-times daily dosing combine CCI-779 with other treatment modalities such baseline measurement. every two weeks, once-weekly dosing or daily oral as radiotherapy and temozolomide chemotherapy. It is

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a Before treatment On the basis of these data, requests for registration for CCI-779 have been filed in the USA and Europe for first-line treatment of patients with advanced/metastatic renal cell carcinoma. Promising preliminary results showing antitumour activity have also been reported Lung metastases with RAD001 in patients with advanced renal cell carcinoma157. Although no translational research was associated with those trials, consistent results showing antitumour effects of rapamycin derivatives in patients with renal cell carcinoma suggest an important role b After treatment for mTOR in either renal cell carcinogenesis and/or tumour-associated angiogenesis. CCI-779 has also shown some efficacy in breast cancer. In a study of 109 patients with advanced or metastatic Tumour size breast cancer and extensive treatment histories, patients reduction were randomized to receive 75 or 250 mg of CCI-779 158 Modiﬁcation per week . Efficacy was similar for both doses with a of tumour rate of objective response of only 9.2%. However, given density the favourable safety profile, it might be worthwhile to combine CCI-779 with other agents in future clinical Figure 4 | Antitumour effects of mTOR inhibitors in patients with cancer. studies of advanced breast cancer. Antitumour activity, including objective response and tumour stabilization, has been Preclinical studies revealed that mTOR inhibitors could reported consistently in patients with renal cell carcinoma in several trials. The downregulate cyclin D1 in mantle cell lymphoma, a disease computerized tomography scans in this figure show lung metastases in a patient driven by a chromosomal translocation t(11;14)(q13;q32) with advanced renal cell carcinoma before (a) and after (b) treatment with an mTOR that places the cyclin D1 gene under the influence of the inhibitor. The reduction in tumour size and modification of tumour density might reflect a direct effect on cancer cells and on tumour angiogenesis, respectively. immunoglobulin heavy chain enhancer region, resulting mTOR, mammalian target of rapamycin. in cyclin D1 overexpression. A Phase II trial in 35 patients with mantle cell lymphoma that had relapsed after chemotherapy and rituximab treatment indicated that CCI-779 significant that patients receiving concomitant cyto- treatment resulted in a remarkable overall response rate chrome P450 (CYP450) enzyme-inducing anti-epileptic of 38%, with a median duration of responses of 6 months. drugs such as phenobarbital and carbamazepine toler- This suggests that mTOR inhibitors can mediate sustained ated higher doses of CCI-779 than patients who did antilymphoma effects159. Interestingly, the effect seems to not receive such a co-treatment and who required dose be similar in those patients who received 250 mg and 25 reduction to 170 mg per week. mg weekly, suggesting no dose–activity relationships in CCI-779 was also investigated in a large dose-rand- this disease160. omized Phase II study of patients with advanced renal cell The most promising results with mTOR inhibitors, carcinomas that were classified in three groups according so far, have been obtained in renal cell carcinoma, to Motzer’s criteria (good, intermediate and poor prog- mantle cell lymphoma and endometrial cancers. nosis)155. As a single agent, CCI-779 displayed a relatively This is particularly significant because these patients low objective response rate of 7%, with 26% additional had relapsed after therapy or had not responded to minor responses, and was associated with better survival standard therapies. In other tumour types, including in patients with intermediate or poor prognosis who glioblastoma, breast cancer and neuroendocrine carci- experienced a two- or threefold increase in survival (22.5 noma161,162, objective response rates with CCI-779 were months for the intermediate group, 8.2 months for the consistently lower than 10%. Preliminary results with group with poor prognosis) compared with historical RAD001 and AP23573 given as single agents in patients controls treated with interferon-α (IFNα) (13.8 and 4.9 with gastrointestinal sarcoma and different pathological months for the intermediate and poor prognosis groups, subtypes of advanced sarcoma (including osteosarcoma, respectively). Those promising results subsequently led leiomyosarcoma and liposarcoma) also yielded a low to the initiation of a large multicentre randomized Phase rate of objective responses163,164. This low response rate III trial comparing IFNα given either alone or with CCI- in many tumour types remains a major hurdle to the 779, or single agent weekly intravenous administration development of rapamycin derivatives. of 25 mg CCI-779 as a first-line treatment in a total of 626 high-risk patients with advanced/metastatic renal Biomarkers for mTOR inhibitor development Sarcoma cell carcinoma. This trial was presented in June 2006 at Since 2000, major efforts have been undertaken to Malignant tumour arising from the annual meeting of the American Society of Clinical identify surrogate markers for assessing the efficacy of the bone (osteosarcoma) or the Oncology156, and showed that patients treated with CCI- mTOR inhibitors, either in skin, in peripheral blood soft tissues with high risk of 779 had a statistically significant longer median survival mononuclear cells (PBMCs) and/or in the tumour lung metastasis. Patients with (FIG. 5) soft tissue sarcomas are (10.9 months) than those receiving IFNα (7.3 months). itself . Progress in this field has been disappoint- frequently poor responders to The combination of CCI-779 with IFNα did not improve ing, but this is at least in part because there is great classical chemotherapy. survival in those patients. variation in the reproducibility and robustness of using

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Translational research

Human cancer Human tumour Peripheral Human skin biopsy Human imaging and cell in culture xenografts blood tumour biopsies mononuclear cells Activation of p-AKT expression p-AKT expression ND p-AKT expression p-AKT expression PI3K/AKT/mTOR PTEN mutation, PTEN mutation, in biopsy (tumour cell deletion, silencing deletion, silencing proliferation) Anabolism/ S6K1 S6K1 S6K1 S6K1 S6K1 in tumour biopsy catabolism 4EBP1 4EBP1 4EBP1 18FdG positron emission (tumour starvation) tomography (PET-scan) Cell cycle control G1–S arrest Delayed tumour ND ND Tumour stabilization (cytostatic effects) ↓ cyclin D1 growth (CT-scan) ↓ cyclin D1 ↓ p27 (mantle cell lymphoma) Apoptosis Induction of BCL2 expression ND ND Unknown (cytotoxic effects) apoptosis BCL2 expression Angiogenesis ↑ VEGF Imaging of tumour NA ND Vascular density in biopsy (anti-angiogenic ↑ HIF1α angiogenesis Vascularization in CT scan, effects) MRI and high frequency Doppler ultrasound Figure 5 | Bench-to-bedside translational research using molecular markers to assess biologically active doses of mTOR inhibitors. This figure shows a process by which molecular markers related to tumour proliferation, metabolism, cell cycle, apoptosis and tumour angiogenesis were identified in preclinical trials, including experiments in cultured human cancer cells and tumour xenografts. These markers were further evaluated in several clinical trials using peripheral blood mononuclear cells and skin biopsies to help determine the biologically active doses of mTOR inhibitors. Direct access to tumour biopsies confirmed the usefulness of a few molecular markers. The difficulties of this approach reside in the limited possibilities to perform iterative tumour biopsies, heterogeneity of sample tumour biopsies, lack of accuracy and reproducibility of bioassays and the small number of patients entering most translational research programmes. Additional assessments using imaging techniques provided further information on the effects of mTOR inhibitors in cancer cells and tumour angiogenesis. Furthermore, predictive factors of tumour sensitivity/ resistance remain an ongoing conundrum. 4EBP1, eukaryotic initiation factor 4E-binding protein 1; BCL2, B-cell lymphoma 2; HIF1α, hypoxia-inducible transcription factor 1α; mTOR, mammalian target of rapamycin; NA, not applicable; ND, not determined; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; VEGF, vascular endothelial growth factor.

biomarkers to study the efficacy of kinase inhibitors. the other study, p-AKT was paradoxically found to be Preclinical assays have allowed the identification of activated after treatment in tumour specimens159. molecular markers such as S6K1 that were further Intratumoural inhibition of mTOR signalling was used in clinical trials165. These studies, summarized also observed in Phase I trials of RAD001. This inhibi- in TABLE 4, assessed the capacity of rapamycin deriva- tion could be detected as a reduction of p-S6K1 (–92.5 to tives to inhibit the phosphorylation of downstream –100% of initial values) and p-4EBP1 (–5.9 to –63.8% of elements of mTOR, including S6K1 and 4EBP1. Of initial values) in tumour biopsies performed early after note, these studies often included a small number of administration of RAD001. Intriguingly, an increase in patients, treated across various doses levels in Phase p-AKT (+22.2 to 63.1% of initial values) was observed I or Phase II randomized trials and are therefore only at the highest RAD001 dose168. RAD001 also induced a exploratory in nature. Two studies linked to clinical significant reduction of p-S6K1 in PBMCs 148. trials of CCI-779 revealed that CCI-779 reduces the In patients with recurrent glioblastoma, AP23573 phosphorylation of components of the PI3K/AKT/ reached its potential target in the brain, since a major mTOR pathway, particularly p-S6K1, both in PBMCs inhibition of p-S6K1 was reported in 6 out of 8 brain and in tumour biopsies166,167. One study included an tumour biopsy specimens169. Pharmacodynamic studies untreated control from healthy volunteers that showed with AP23573 explored the reduction of p-4EBP1 in 22 limited intrapatient variability over time, contrasting patients treated at various dose levels over several Phase with marked interpatient variability, emphasizing the I trials using the weekly schedule and the five-times daily need to use each patient as his/her own control160. In dosing schedule. A rapid and major reduction of p-4EBP1

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Table 4 | Translational research linked to clinical trials with mTOR inhibitors Compound (doses) Molecular markers evaluated Main results Refs (number of patients) CCI-779 (temsirolimus) 25 mg per week p53, eIF4G, p-AKT Significant inhibition in p-S6K1 post-dosing 166,167 p-mTOR, p-S6K1, CD34 in paired Minor inhibition in p-mTOR, p-eIFEG and CD34 tumour biopsies, pre- and 2 weeks post-dosing post-treatment (n = 13) Activation in p-AKT post-dosing 25, 75 or 250 mg per p-S6K1 in PBMC (n = 9) Inhibition of p-S6K1 24 h post-dosing, reaching week a maximum at day 8 No correlation with dose RAD001 (everolimus) 20–70 mg per week p-S6K1, p-4EBP1, p-AKT in Major inhibition of p-S6K1 at all dose levels and 168 or 5–10 mg per d x 5 tumour biopsies (n = 33) schedules after dosing every 2 weeks Dose-related inhibition of p-4EBP1 Activation p-AKT for 10 mg daily and ≥50 mg weekly 5–30 mg per week p-S6K1 in PBMC (n = 18) Significant inhibition of p-S6K1 in PBMCs, 150 major and durable at doses ≥20 mg AP23573 12.5–15 mg 4 times p-4EBP1 in PBMC Major inhibition of p-4EBP1 in PBMCs up to 6 169 daily before surgery p-S6K1 in surgical resection (n days = 8) Major inhibition (–50 to 100%) of p-S6K1 in tumour brain tissue after resection 3–28 mg per d x 5 p-4EBP1 in PBMC (n = 22) Rapid and major (>70%) decrease of p-4EBP1, 170 days every 2 weeks up to 7 days for weekly schedule and up to 10 and 6.25–100 mg per days for daily x 5 schedule week 3–28 mg per d x 5 p-S6K1 in skin biopsies (n = 23) p-S6K1 inhibition (≥50%) in 20 out of 23 patients 171 every 2 weeks (87%) in one or more time-points after dosing, sustained for at least 10 days post-dosing Not correlated with dose eIF4G, eukaryotic initiation factor 4G; 4EBP1, eIF4E-binding protein 1; mTOR, mammalian target of rapamycin; PBMCs, peripheral blood mononuclear cells; SKG1, S6 kinase 1.

was observed, lasting for more than 7 days in both sched- with S6K1 and AKT. It remains unclear how realistic it ules in PBMCs170. In skin biopsies, AP23573 induced a will be to conduct large systematic studies of genomes and significant reduction (>85%) of p-S6K1 in most patients, proteomes for the identification of markers and predic- confirming that the drug efficiently penetrates into the tors of clinical outcome and, moreover, whether it will be skin. However, the degree of p-S6K1 inhibition did not academia, industry or a collaborative effort that makes this correlate with the dose nor with the clinical response, sug- endeavour feasible. Smaller studies might be more incisive gesting that useful biomarkers for predicting therapeutic and have real value to those working in the field. There are responses to mTOR inhibitors are still elusive171. also problems associated with using humans in studies In a limited number of pilot trials using RAD001 and of biomarkers, such as limited availability of tissue from AP23573, 18F-2-fluoro-2-deoxy-d-glucose positron emis- biopsy, and therefore the search for new surrogate markers sion tomography (PET) was used to monitor the glucose is of paramount importance. At this stage, it only can be uptake of tumours following administration of mTOR speculated that downstream effectors tied to the cyto- inhibitors. In those preliminary studies, some tumours static and cytotoxic activities of mTOR inhibitors, such as exhibited a decrease in glucose uptake that was not con- cyclin D1, as well as markers of apoptosis, autophagy or sistently associated with objective responses determined angiogenesis, might constitute such molecular markers. by radiological methods172. One of the main pitfalls of this approach is that rapamycin can inhibit insulin and IGF1 Identifying rapamycin sensitivity and resistance signalling, thereby reducing the uptake of glucose, perhaps The identification of tumour types that respond to mTOR independently of the cytostatic/cytotoxic effects. inhibitors remains a major issue for the development of In conclusion, it seems that immunoblot analyses of p- rapamycin derivatives. mTOR is ubiquitously expressed S6K1 and p-4EBP1 can be used to monitor the biological and therefore the sensitivity or resistance of specific effects of biologically active doses of mTOR inhibitors in tissues to rapamycin cannot be predicted solely on the normal cells taken from blood, skin and, whenever possible, basis of whether the target protein can be detected in tumour tissues, although it might be necessary to develop the tumour tissue. As it stands, elucidating the activa- more accurate molecular markers. One problem is the dif- tion status of the PI3K/AKT/mTOR signalling pathway ficulty associated with using 4EBP1 as a marker compared seems to be the only means of identifying tumour types

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Another hypothesis lies in the paradoxical activation of Box 3 | Molecular patterns of mTOR inhibitors in patients the AKT pathway on addition of rapamycin derivatives. Translational and clinical data suggest that sensitive tumour types that have adequate On the one hand, mTOR and S6K1 activation can cause signalling parameters and functional apoptosis pathways might not require high doses of phosphorylation and degradation of insulin-receptor inhibitors of mammalian target of rapamycin (mTOR) to trigger apoptosis. This matches substrate 1 (IRS1); inhibition of mTOR will therefore the clinical situation, in which sporadic objective responses were observed when mTOR prevent the degradation of IRS1, which is an important inhibitors were used at doses well below the maximum tolerated dose. However, mediator of insulin-receptor-dependent activation sensitive tumour types might only represent a limited number of tumours. In most cases, 175 cancer cells might only be marginally sensitive to mTOR inhibitors because of redundant of PI3K . On the other hand, only mTOR–RICTOR signal transduction pathways or lack of functional apoptosis signalling pathways. In this (which is not inhibited by rapamycin derivatives), but not particular situation, high doses might be required to achieve a proliferative arrest. mTOR—raptor, can phosphorylate AKT on Ser473, and Clinical observations of prolonged tumour stabilization with mTOR inhibitors might be this mTOR activity might be enhanced in the presence of attributable to this cytostatic effect. Future studies should aim at identifying biological rapamycin derivatives176. In theory, it might be interesting parameters that are predictive of whether mTOR inhibitors will behave as apoptosis to combine rapamycin-like agents with AKT inhibitors177 inducers as single agents or would need to be combined with more potent inducers of or yet-to-be-developed mTOR–RICTOR inhibitors178. apoptosis to trigger cell death. Some progress with making associations between level of Indeed, it has been shown that malignant gliomas sensitivity to mTOR inhibitors and molecular patterns in the cell has already been made: can be killed by the synergistic action of rapamycin Sensitive tumours (shown by objective response — for example, endometrial and PI3K or AKT inhibitors179,180. and mantle cell lymphoma) But what is the current state of knowledge about • PTEN (phosphatase and tensin homologue deleted on chromosome 10) inactivated the mechanisms of rapamycin resistance? Apart from • PI3K (phosphatidylinositol 3-kinase)/AKT/mTOR activated pharmacological parameters that can affect the anti- • Cyclin D1 overexpressed tumour activity of anticancer agents including rapamycin- • Apoptosis pathways functional derivatives (such as individual pharmacokinetic profiles, • No dose-dependent induction of apoptosis tumour tissue distribution, and ATP-binding cassette- transporter protein expression in cancer cells), down- Marginally sensitive tumours (shown by tumour stabilization or lack of stream effectors of cell-cycle progression and apoptosis progression in the clinic — for example, renal cell carcinoma and breast cancer) might account for rapamycin-resistance (BOX 3). In • Redundant signal transduction (crosstalk with mitogen-activated protein kinase pathway) tumours with activated AKT and functional apop- • Non-functional apoptosis pathways (B-cell lymphoma 2 (BCL2), BCL2-antagonist of cell tosis, relatively low doses of rapamycin-derivatives death expression) might be sufficient to induce cell death, which could • Dose-dependent cell-cycle inhibition observed explain both why antitumour activity in the clinic was not dose-dependent and why sporadic but sustained objective responses with rapamycin derivatives were potentially sensitive to rapamycin derivatives. Tumour observed over a broad range of doses. Conversely, in profiling using molecular biology or immunohistochem- those tumours that are marginally sensitive or resistant istry has been carried out in a limited number of Phase I/ to mTOR inhibitors, rapamycin-derivatives might have II trials. Molecular markers of mTOR pathway activation been given at insufficient doses. Alternatively, combi- investigated were loss of PTEN expression, and phos- nations with other anticancer drugs will be required phorylation of AKT, S6K1 and 4EBP1. In most cases in to circumvent rapamycin-resistance. Illustrating this which PTEN expression was reduced, AKT, S6K1 and/or possibility, a recent study showed that rapamycin-resist- 4EBP1 were phosphorylated, demonstrating that loss of ant ovarian cancer cells with a functional PI3K/AKT PTEN is associated with activation of the PI3K/AKT/ pathway expressed high levels of the apoptosis-inhibi- mTOR signalling pathway. In a recent study carried out tory protein BCL2 and that antisense oligonucleotides in patients with renal cell carcinoma, resistance to the designed to downregulate BCL2 re-established the rapamycin-derivative CCI-779 was associated with low apoptotic response to RAD001 (REF. 137). It remains levels of p-AKT and p-S6K1. Conversely, loss of PTEN to be seen, however, whether the expression levels of or activation of AKT, S6K1 or 4EBP1 were significantly BCL2 and its homologues (such as BCL-XL, BCL-w associated with objective responses173. Taken together, and myeloid cell leukaemia sequence 1 (MCL1)) are these data strongly suggest that there is a cohort of predictive of therapeutic responses to mTOR inhibitors. tumours with an activated PI3K/AKT/mTOR signalling It would be worthwhile to perform a large systematic pathway that does not respond to mTOR inhibitors. As study that includes systematic genome-wide and pro- it stands, tumour profiling by immunohistochemistry teomic approaches to identify optimal predictors of the might be more useful for predicting tumour resistance clinical outcome of mTOR inhibition. than tumour sensitivity. Therefore one recommendation To summarize, current data are insufficient to for future studies would be to exclude patients whose authoratively predict tumour types that are sensitive tumour biopsies have low or negative p-AKT levels from to rapamycin. However, the available data allow us to trials with mTOR inhibitors. Paired tissue biopsies before better characterize tumours that might not respond to and after treatment with rapamycin-derivatives revealed rapamycin, such as those with non-activated AKT or that non-responders frequently developed an increase high BCL2 levels. Future clinical studies should attempt in the level of p-AKT174. The increased phosphoryla- to prospectively validate molecular markers of rapamycin tion of AKT might reflect a mechanism of rapamycin resistance for a more accurate identification of tumours resistance that should be explored in further detail. that could benefit from mTOR inhibition.

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EGF, PDGF, causes a rapid decline in the cell-cycle inhibitor p21, VEGF, IGF which is required for DNA repair183. Rapamycin can enhance apoptosis that is induced by the drug paclitaxel Receptor R R tyrosine kinase by yet another pathway that involves glycogen synthase β184 Cell kinase 3 . membrane A loss-of-function of PTEN might account for the resistance of breast cancer cells against trastuzumab Farnesyl (Herceptin; Genentech), a monoclonal antibody transferase EGFR: Geﬁtinib, Erlotinib, inhibitor Tyrosine GW5720165 that binds HER2/neu and demonstrated significant Zarnestra kinase PDGFR: Imatinib and SCH66336 inhibitor antitumour activity in a fraction of patients. Patients SU11248 with PTEN-deficient breast cancer are usually poorer Ras VEGFR: SU11248, PTK787, AEE788, Bay 43-9006 responders to trastuzumab therapy than those with RAF normal PTEN185. In addition, preclinical studies dem- inhibitor Cytoplasm Bay 43-9006 RAF PI3K onstrated that blocking the PI3K signalling pathway PTEN prevented loss of PTEN and associated trastuzumab AKT resistance. Other studies have shown that resistance to MEK gefitinib (Iressa; AstraZeneca), a small tyrosine kinase mTOR inhibitor of EGFR, might be linked to increased AKT inhibitor mTOR phosphorylation and reduced PTEN expression in Erk non-small-cell lung adenocarcinoma cells186. Taken CCI-779, RAD001 together, these data strongly support the idea that Rapamycin, AP23573 mTOR inhibitors could be clinically used to overcome the chemoresistance against agents targeting HER2/neu Gene Cell survival Proliferation expression and EGFR. Fortunately, recent Phase I studies showed (anti-apoptosis) no pharmacokinetic interactions between gefitinib and Chemotherapy/ RAD001 (REF. 187), paving the way for further evaluation radiotherapy sensitivity Angiogenesis Nucleus of combined therapies. Figure 6 | Complexity of the multitargeted approach to cancer therapy. Anticancer In breast carcinoma, high AKT activity was associated therapy targeting multiple signalling pathways is an emerging paradigm that requires with poor prognosis as well as resistance to the growth extensive knowledge of the activation status of receptor tyrosine kinases and cyto- inhibitory effects of the anti-oestrogen tamoxifen. In vitro, plasmic serine/threonine kinases in cancer cells. Targeted agents blocking the PI3K/ CCI-779 can restore sensitivity to tamoxifen, suggesting AKT/mTOR pathway along with alternative Ras/Raf/MEK signal transduction that AKT-induced tamoxifen resistance is mediated in pathways could be used together to generate a synergistic effect and maximize the part by mTOR pathway signalling188. On the basis of these efficacy of each individual novel anticancer agent at controlling cell proliferation, data, clinical studies are evaluating the effects of rapamy- invasion and angiogenesis, while hopefully reducing resistance to existing cin derivatives in hormone refractory breast cancers as chemotherapy/radiotherapy regimens. EGF, endothelial growth factor; IGF, insulin- well as in combination with hormone therapy. like growth factor; MEK, mitogen-activated protein kinase/extracellular signal- Resistance to imatinib mesylate in advanced gas- related kinase; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; PDGF, platelet-derived growth factor; PTEN, phosphatase and tensin trointestinal stromal tumours might involve alterna- homologue deleted on chromosome 10; VEGF, vascular endothelial growth factor. tive oncogenic signals, including the activation of the PI3K/AKT/mTOR pathway. Synergistic interactions between imatinib mesylate and RAD001 were observed in human gastrointestinal tumour cell lines resistant to Multitargeting approaches imatinib mesylate, which supported the development Combination therapies are being developed that combine of a Phase I combination trial in patients refractory to mTOR inhibitors with additional compounds, based on imatinib mesylate. Unfortunately, pharmacokinetic the rationale that simultaneous inhibition of multiple interactions were observed between RAD001 and imat- signalling pathways should reduce the likelihood of inib mesylate as a result of them both being metabo- resistance. Accordingly, several clinical trials are actively lized by the same CYP450 mechanism. Nevertheless, investigating the potential benefits of combining mTOR preliminary evidence of objective response rates sup- inhibitors with hormonal therapy, chemotherapy or other ports further evaluation of this combination in Phase targeted therapies (FIG. 6). II studies156. Combinations of rapamycin derivatives with gemcit- abine and 5-fluorouracil (5-FU) led to unacceptable toxic Conclusions side effects such as thrombocytopaenia and mucositis. The proof of principle that mTOR inhibitors can improve Cisplatin-resistance of squamous cell carcinomas can cancer patient survival has been recently obtained from a involve AKT signalling as well as the Raf/MEK/ERK sig- large randomized trial in advanced poor prognostic renal nalling cascade, providing a rationale for the combination cell carcinoma. Recent knowledges on the status of PTEN of cisplatin with mTOR inhibitors181,182. Reportedly, and PI3K/AKT/mTOR-linked pathways might help in RAD001 can sensitize tumour cells to apoptosis induc- the selection of other tumour types that will respond to tion by the DNA-damaging agent cisplatin. This effect mTOR inhibitors. Examples of successful translation of might be due to a general inhibition of translation that this basic knowledge on mTOR-associated pathways to

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the clinic include endometrial cancer and mantle cell lym- or sensitivity to these compounds. Moreover, it seems that phoma, two neoplastic diseases that frequently respond to it would be useful to immediately apply existing knowl- mTOR inhibitors. Translational studies investigating bio- edge that mTOR inhibition can restore sensitivity to some logical parameters, including the inhibition of p-S6K1 or existing chemotherapeutic agents such as tamoxifen, p-4EBP1 revealed that these markers might guide optimal trastuzumab and gefitinib in breast cancer, or to imatinib application schemes at the pharmacokinetic level. Because mesylate in gastrointestinal stromal tumours in the clinic. most tumour types still do not respond to single agent Translational studies widely performed during current therapy with rapamycin derivatives, it will be important and future Phase II trials, together with functional imag- to continue the search for factors predictive of resistance ing techniques, will certainly help to achieve this aim.

1. Vignot, S., Faivre, S., Aguirre, D. & Raymond, E. 24. Hankinson, S. E. et al. Circulating concentrations of insulin requires insulin receptor substrate 1 and mTOR-targeted therapy of cancer with rapamycin insulin-like growth factor–I and risk of breast cancer. phosphatidyl inositol 3 kinase. Mol. Cell Biol. 16, derivatives. Ann. Oncol. 16, 525–537 (2005). Lancet 351, 1393–1396 (1998). 5991–6001 (1996). 2. Larue, L. & Bellacosa, A. Epithelial-mesenchymal 25. Smith, G. D., Gunnell, D. & Holly, J. Cancer and 47. Rosenwald, I. B. et al. Eukaryotic translation initiation transition in development and cancer: role of insulin-like growth factor-I. A potential mechanism factor 4E regulates expression of cyclin D1 at phosphatidylinositol 3′ kinase/AKT pathways. linking the environment with cancer risk. BMJ 321, transcriptional and post-transcriptional levels. J. Biol. Oncogene 24, 7443–7454 (2005). 847–848 (2000). Chem. 270, 21176–21180 (1995). 3. Gschwendt, M. Protein kinase Cδ. Eur. J. Biochem. 26. Pollack, M. Insulin like growth factor physiology and 48. Rousseau, D. et al. Translation initiation of ornithine 259, 555–564 (1999). cancer risk. Eur. J. Cancer 36, 1224–1228 (2000). decarboxylase and nuceocytoplasmic transport of 4. Steinberg, S. F. Distinctive activation mechanisms and 27. Yu, H. & Rohan, T. Role of the insulin-like growth factor cyclin D1 mRNA are increased in cells overexpressing functions for protein kinase Cδ. Biochem. J. 384, family in cancer development and progression. J. Natl eukaryotic initiation factor 4E. Proc. Natl Acad. Sci. 449–459 (2004). Cancer Inst. 92, 1472–1489 (2000). USA 93, 1065–1070 (1996). 5. Fang, J. Y. & Richardson, B. C. The MAPK signalling 28. Resnicoff, M. & Baserga, R. The role of the insulin-like 49. Berretta, L. et al. Rapamycin blocks the phos- pathways and colorectal cancer. Lancet Oncol. 6, growth factor–I receptor in transformation and phorylation of 4E-BP1 and inhibits cap-dependent 322–327 (2005). apoptosis. Ann. NY Acad. Sci. 842, 76–81 (1998). initiation of translation. EBMO J. 15, 658–664 6. Hay, N. The Akt-mTOR tango and its relevance to 29. Cheng, J. Q. et al. Akt2, a putative oncogene encoding (1996). cancer. Cancer Cell 8, 179–183 (2005). a member of a subfamily of serine/threonine kinases, 50. Graves, L. M. et al. c-AMP –and rapamycin-sensitive 7. Castedo, M., Ferri, K. F. & Kroemer, G. Mammalian is amplified in human ovarian carcinomas. Proc. Natl regulation of the association of eukaryotic initiation target of rapamycin (mTOR): pro- and anti-apoptotic. Acad. Sci. USA 89, 9267–9271 (1992). factor 4E and the translational regulator PHAS-I in Cell Death Differ. 9, 99–100 (2002). 30. Cheng, J. Q. et al. Amplification of AKT 2 in human aortic smooth muscle cells. Proc. Natl Acad. Sci. USA 8. Vivanco, I. & Sawyers, C. L. The phosphatidylinositol pancreatic cells and inhibition of Akt 2 expression and 92, 7222–7226 (1995). 3-kinase AKT pathway in human cancer. Nature Rev. tumorigenicity by antisense RNA. Proc. Natl Acad. Sci. 51. Lin, T. A. et al. Control of PHAS-I by insulin in 3T3-L1 Cancer 2, 489–501 (2002). USA 93, 3636–3641 (1996). adipocytes. Synthesis, degradation, and An important review with most references that 31. Bellacosa, A. et al. Molecular alterations of the AKT2 phosphorylation by a rapamycin — sensitive and emphasize PI3K/AKT functions and implications in oncogene in ovarian and breast carcinomas. Int. J. mitogen-activated protein kinase-independent malignant tumours. Cancer 64, 280–285 (1995). pathway. J. Biol. Chem. 270, 18531–18538 (1995). 9. Inoki, K., Corradetti, M. N. & Guan, K. L. 32. Neshat, M. S. et al. Enhanced sensitivity of PTEN- 52. Dilling, B. D. et al. 4E-binding proteins, the Dysregulation of the TSC–mTOR pathway in human deficient tumors to inhibition of FRAP/mTOR. Proc. suppressors of eukaryotic initiation factor 4E, are disease. Nature Genet. 37, 19–24 (2005). Natl Acad. Sci. USA 98, 10314–10319 (2001). down-regulated in cells with acquired or intrinsic 10. Johannessen, C. M. et al. The NF1 tumor suppressor This paper describes the crucial role of PTEN and resistance to rapamycin. J. Biol. Chem. 277, critically regulates TSC2 and mTOR. Proc. Natl Acad. PTEN deletions as well as the effects of mTOR 13907–13917 (2002). Sci. USA. 102, 8573–8578 (2005). inhibitors using in vivo models. 53. Von Manteuffel, S. et al. 4E-Bp1 phosphorylation is 11. Luo, Z., Saha, A. K., Xiang, X. & Ruderman, N. B. 33. Brunn, G. J. et al. Phosphorylation of the translational mediated by the FRAP-p70s6k pathway and is AMPK, the metabolic syndrome and cancer. Trends repressor PHAS-I by the mammalian target of independent of mitogen-activated protein kinase. Pharmacol. Sci. 26, 69–76 (2005). rapamycin. Science 277, 99–101 (1997). Proc. Natl Acad. Sci. USA 93, 4076–4080 (1996). 12. Shaw, R. J. et al. The LKB1 tumor suppressor 34. Hara, K. et al. Regulation of eIF-4E BP1 54. Brunn, G. J. et al. Three mammalian target of negatively regulates mTOR signaling. Cancer Cell 6, phosphorylation by mTOR. J. Biol. Chem. 272, rapamycin phosphorylates sites having a (Ser/Thr)-Pro 91–99 (2004). 26457–26463 (1997). motif and is activated by antibodies to a region near 13. Lee, L. et al. Efficacy of a rapamycin analog (CCI-779) 35. Chung, J. et al. PDGF- and insulin-dependent its COOH terminus. J. Biol. Chem. 272, 32547– and IFN-γ in tuberous sclerosis mouse models. Genes pp70S6k activation mediated by phosphatidylinositol- 32550 (1997). Chromosomes Cancer 42, 213–227 (2005). 3-OH kinase. Nature 370, 71–75 (1994). 55. Fadden, P. Jr. Identification of phosphorylation sites in 14. Thomas, G. & Hall, M. N. TOR signaling and control of 36. Gingras, A. C. et al. 4E-BP1, a repressor of mRNA the translational regulator, PHAS-I, that are controlled control of cell growth. Curr. Opin. Cell Biol. 9, 782– translation, is phosphorylated and inactivated by the by insulin and rapamycin in rat adipocytes. J. Biol. 787 (1997). Akt(PKB) signaling pathway. Genes Dev. 12, 502–513 Chem. 272, 10240–10247 (1997). 15. Schmelzle, T. & Hall, M. N. TOR, a central controller of (1998). 56. Herbert, P. T., Tee, A. R. & Proud, C. G. The cell growth. Cell 103, 253–262 (2000). 37. Brunn, G. J. et al. Phosphorylation of the translational extracellular signal-regulated kinase pathway 16. Guertin, D. A. & Sabatini, D. M. An expanding role for repressor PHAS-I by the mammalian target of regulates the phosphorylation of 4E-BP1 at multiple mTOR in cancer. Trends Mol. Med. 11, 353–361 rapamycin. Science 277, 99–101 (1997). sites. J. Biol. Chem. 277, 11591–11596 (2002). (2005). 38. Hara, K. et al. Raptor, a binding partner of target of 57. Di Como, C. J. & Arndt, K. T. Nutrients, via TOR 17. Martin, D. E. & Hall, M. N. The expanding TOR rapamycin (TOR), mediates TOR actions. Cell 110, proteins, stimulate the association of Tap42 with type signaling network. Curr. Opin. Cell Biol. 17, 158–166 177–189 (2002). 2A phosphatases. Genes Dev. 10, 1904–1916 (2005). 39. Kim, D.-H. et al. MTOR interacts with raptor to form a (1996). 18. Scott, P. H. et al. Evidence of insulin-stimulated nutriement –sensitive complex that signals to the cell 58. Murata, K., Wu, J. & Brautigan, D. L. B cell receptor- phosphorylation and activation of the mammalian growth machinery. Cell 110, 163–175 (2002). associated protein α4 displays rapamycin-sensitive target of rapamycin mediated by a protein kinase B 40. Pain, V. M. Initiation of protein synthesis in eukaryotic binding directly to the catalytic subunit of protein signaling pathway. Proc. Natl Acad. Sci. USA 95, cells. Eur. J. Biochem. 236, 747–771 (1996). phosphatase 2A. Proc. Natl Acad. Sci. USA 148, 7772–7777 (1998). 41. Proud, C. G. & Denton, R M. Molecular mechanism for 71–82 (1997). 19. Nave, B T. et al. Mammalian target of rapamycin is a the control of translation by insulin. Biochem. J. 328, 59. Park, I.-H. et al. Regulation of ribosomal S6 kinase 2 direct target for protein kinase B: identification of a 329–341 (1997). by mammalian target of Rapamycin. J. Biol. Chem. convergence point for opposing effects of insulin and 42. Sonenberg, N. & Gingras, A. C. The m RNA 5′ cap- 277, 31423–31429 (2002). amino-acid deficiency on protein translation. Biochem. binding protein eIF4E and control of cell growth. Curr. 60. Seufferlein, T. & Rozengurt, E. Rapamycin inhibits J. 344, 427–431 (1999). Opin. Cell Biol. 10, 268–275 (1998). constitutive p70s6k phosphorylation, cell 20. Fry, M. J. Phosphoinositide 3-kinase signaling in 43. Smith, M. R. et al. Translation initiation factors induce proliferation, and colony formation in small cell lung breast cancer: how big a role might it play? Breast DNA synthesis and transform NIH 3T3 cells. New Biol. cancer cells. Cancer Res. 56, 3895–3897 (1996). Cancer Res. 3, 304–312 (2001). 2, 648–654 (1990). 61. Brown EJ, Beal PA, Keith CT et al. Control of p70s6 21. Hu, Q. et al. Ras-dependent induction of cellular 44. Rousseau, D. et al. The eIF4E-binding protein 1 and 2 kinase by kinase activity of FRAP in vivo. Nature 377, responses by constitutively active phosphatidylinositol- are negative regulators of cell growth. Oncogene 13, 441–446 (1995). 3 kinase. Science 268, 100–102 (1995). 2415–2420 (1996). 62. Burnett, P. E. et al. RAFT 1 phosphorylation of the 22. Lee, A. V. & Yee, D. Insulin-like growth factors and 45. Easton, J. B., Kurmasheva, R. T. & Houghton, P. J. translational regulators p70 s6 kinase and 4E-BP1. breast cancer. Biomed. Pharmacother. 49, 415–421 IRS-1: auditing the effectiveness of mTOR inhibitors. Proc. Natl Acad. Sci. USA 95, 1432–1437 (1998). (1995). Cancer Cell 9, 153–155 (2006). 63. Dennis, P. B. et al. The principal rapamycin-sensitive 23. Scheid, M. P. & Woodgett, J. R. Phosphatidylinositol 46. Mendez, R. et al. Stimulation of protein synthesis, p70s6k phosphorylation sites T229 and T389 are 3′ kinase signaling in mammary tumorigenesis. J. eukaryotic translation initiation factor 4E differentially regulated by rapamycin-insensitive kinase- Mammary Gland Biol. Neoplasia 6, 83–99 (2001). phosphorylation and PHAS-I phosphorylation by kinases. Mol. Cell Biol. 16, 6242–6251 (1996).

REVIEWS

64. Begum, N. & Ragolia, L. cAMP counter-regulates 89. Su, T. H. et al. Mutation analysis of the putative tumor 115. Kahan, B. D. Optimization of cyclosporine therapy. insulin-mediated protein phosphatase-2A inactivation suppressor gene PTEN/MMAC1 in cervical cancer. Transplant. Proc. 25, 5–9 (1993). in rat skeletal muscle cells. J. Biol. Chem. 271, Gynecol. Oncol. 76, 193–199 (2000). 116. Trepanier, D. J. et al. Rapamycin: distribution, 31166–31171 (1996). 90. Harima, Y. et al. Mutation of the PTEN gene in pharmacokinetics and therapeutic range 65. Nourse, J. et al. Interleukin-2-mediated elimination of advanced cervical cancer correlated with tumor investigations: an update. Clin. Biochem. 31, the p27Kip1 cyclin-dependent kinase inhibitor progression and poor outcome after radiotherapy. Int. 345–351 (1998). prevented by rapamycin. Nature 372, 570–573 J. Oncol. 18, 493–497 (2001). 117. Kauffman, H. M., Cherikh, W. S., Cheng, Y., Hanto, D. (1994). 91. Cheung, T. H. et al. Epigenetic and genetic alternation W. & Kahan, B. D. Maintenance immunosuppression 66. Grewe., M. et al. Regulation of cell growth and cyclin of PTEN in cervical neoplasm. Gynecol. Oncol. 93, with target-of-rapamycin inhibitors is associated with a D1 expression by the constitutively active FRAP- 621–627 (2004). reduced incidence of de novo malignancies. p70s6K pathway in human pancreatic cancer cells. 92. Tsutsui, S. et al. Reduced expression of PTEN protein Transplantation 80, 883–889 (2005). Cancer Res. 59, 3581–3587 (1999). and its prognostic implications in invasive ductal 118. Gallo, R. et al. Inhibition of intimal thickening after 67. Kawamata, S. et al. The upregulation of p27Kip1 by carcinoma of the breast. Oncology 68, 398–404 balloon angioplasty in porcine coronary arteries by rapamycin results in G1 arrest in exponentially (2005). targeting regulators of the cell cycle. Circulation 99, growing T-cell lines. Blood 91, 561–569 (1998). 93. Shoman, N. et al. Reduced PTEN expression predicts 2164–2170 (1999). 68. Hashemolhosseini, S. et al. Rapamycin inhibition of relapse in patients with breast carcinoma treated by 119. Sousa, J. E. et al. Use of rapamycin-impregnated the G1 to S transition is mediated by effects on cyclin tamoxifen. Mod. Pathol. 18, 250–259 (2005). stents in coronary arteries. Transplant. Proc. 35, D1 mRNA and protein stability. J. Biol. Chem. 273, 94. Saal, L. H. et al. PIK3CA mutations correlate with 165S–170S (2003). 14424–14429 (1998). hormone receptors, node metastasis, and ERBB2, and 120. Mohacsi, P. J. et al. Different inhibitory effects of 69. Morice, W. G. et al. Rapamycin inhibition of are mutually exclusive with PTEN loss in human breast immunosuppressive drugs on human and rat aortic interleukin-2-dependent p33cdk2 and p34cdc2 carcinoma. Cancer Res. 65, 2554–2559 (2005). smooth muscle and endothelial cell proliferation kinase activation in T lymphocytes. J. Biol. Chem. 95. Kirkegaard, T. et al. AKT activation predicts outcome stimulated by platelet-derived growth factor or 268, 22737–22745 (1993). in breast cancer patients treated with tamoxifen. endothelial cell growth factor. J. Heart Lung 70. Luo, Y. et al. Rapamycin resistance tied to defective J. Pathol. 207, 139–146 (2005). Transplant. 16, 484–492 (1997). regulation of p27Kip1. Mol. Cell. Biol. 16, 96. Saito, M. et al. Allelic imbalance and mutations of the 121. Martin, D. E. & Hall, M. N. The expanding TOR 6744–6751 (1996). PTEN gene in ovarian cancer. Int. J. Cancer 85, signaling network. Curr. Opin. Cell. Biol. 17, 158–166 71. Rosenwald, I. B. et al. Eukaryotic translation initiation 160–165 (2000). (2005). factor 4E regulates expression of cyclin D1 at 97. Levine, D. A. et al. Frequent mutation of the PIK3CA 122. Ravikumar, B. et al. Inhibition of mTOR induces transcriptional and post-transcriptional levels. J. Biol. gene in ovarian and breast cancers. Clin. Cancer Res. autophagy and reduces toxicity of polyglutamine Chem. 270, 21176–21180 (1995). 11, 2875–2878 (2005). expansions in fly and mouse models of Huntington 72. Mahajan, P. B. Modulation of transcription of rRNA 98. Koksal, I. T. et al. The assessment of PTEN tumor disease. Nature Genet. 36, 585–595 (2004). genes by rapamycin. Int. J. Immunopharmacol. 16, suppressor gene in combination with Gleason scoring 123. Li, X., Alafuzoff, I., Soininen, H., Winblad, B. & Pei, J. 711–721 (1994). and serum PSA to evaluate progression of prostate J. Levels of mTOR and its downstream targets 4E- 73. Leicht, M. et al. Okadaic acid induces cellular carcinoma. Urol. Oncol. 22, 307–312 (2004). BP1, eEF2, and eEF2 kinase in relationships with tau hypertrophy in AKR-2B fibroblasts: involvment of the 99. Pfeil, K. et al. Long-term androgen-ablation causes in Alzheimer's disease brain. FEBS J. 272, 4211– p70S6 kinase in the onset of protein and rRNA increased resistance to PI3K/Akt pathway inhibition in 4220 (2005). synthesis. Cell Growth Differ. 7, 1199–1209 (1996). prostate cancer cells. Prostate 58, 259–268 (2004). 124. Nardacci, R. et al. Characterization of cell death 74. White, R. J. Regulation of RNA polymerases I and III by 100. Edwards, J., Krishna, N. S., Witton, C. J. & pathways in human immunodeficiency virus- the retinoblastoma protein: a mechanism for growth Bartlett, J. M. Gene amplifications associated with the associated encephalitis. Am. J. Pathol. 167, 695–704 control? Trends Biochem. Sci. 22, 77–80 (1997). development of hormone-resistant prostate cancer. (2005). 75. Rodriguez-Viciana, P. Phosphatidylinositol-3-OH Clin. Cancer Res. 9, 5271–5281 (2003). 125. Castedo, M. et al. Sequential involvement of Cdk1, kinase as a direct target of Ras. Nature 370, 527– 101. Okami, K. et al. Analysis of PTEN/MMAC1 alterations mTOR and p53 in apoptosis induced by the HIV-1 532 (1994). in aerodigestive tract tumours. Cancer Res. 58, envelope. EMBO J. 21, 4070–4080 (2002). 76. Feng, Z., Zhang, H., Levine, A. J. & Jin, S. The 509–511 (1998). 126. Busca, R. et al. Inhibition of the phosphatidylinositol coordinate regulation of the p53 and mTOR pathways 102. Kubo, Y., Urano, Y., Hida, Y. & Arase, S. Lack of 3-kinase/p70(S6)-kinase pathway induces B16 in cells. Proc. Natl Acad. Sci. USA 102, 8204–8209 somatic mutation in the PTEN gene in squamous cell melanoma cell differentiation. J. Biol. Chem. 271, (2005). carcinoma of human skin. J. Dermatol. Sci. 19, 31824–31830 (1996). 77. Kanamori, Y. et al. Correlation between loss of PTEN 199–201 (1999). 127. Grewe, M. et al. Regulation of cell growth and cyclin expression and Akt phosphorylation in endometrial 103. Aoki, K. et al. Cisplatin activates survival signals in D1 expression by the constitutively active FRAP– carcinoma. Clin. Cancer Res. 7, 892–895 (2001). UM-SCC-23 squamous cell carcinoma and these signal p70s6K pathway in human pancreatic cancer cells. 78. Uegaki, K. et al. PTEN-positive and phosphorylated- pathways are amplified in cisplatin-resistant squamous Cancer Res. 59, 3581–3587 (1999). Akt-negative expression is a predictor of survival for cell carcinoma. Oncol. Rep. 11, 375–379 (2004). 128. Hosoi, H. et al. Rapamycin causes poorly reversible patients with advanced endometrial carcinoma. 104. Khaleghpour, K., Li, Y., Banville, D., Yu, Z. & Shen, S. inhibition of mTOR and induces p53-independent Oncol. Rep. 14, 389–392 (2005). H. Involvement of the PI 3-kinase signaling pathway in apoptosis in human rhabdomyosarcoma cells. Cancer 79. Soliman, P. T. et al. mTOR inhibition as a potential progression of colon adenocarcinoma. Carcinogenesis Res. 59, 886–894 (1999). treatment for progesterone-refractory endometrial 25, 241–248 (2004). 129. Shi, Y. et al. Rapamycin enhances apoptosis and hyperplasia. Proc. Am. Soc. Clin. Oncol. 23, 474s, 105. Velho, S. et al. The prevalence of PIK3CA mutations increases sensitivity to cisplatin in vitro. Cancer Res. A5080 (2005). in gastric and colon cancer. Eur. J. Cancer 41, 55, 1982–1988 (1995). 80. Choe, G. et al. Analysis of the phosphatidylinositol 1649–1654 (2005). 130. Calastretti, A. et al., Damaged microtubules can 3-kinase signaling pathway in glioblastoma patients in 106. Agbunag, C. & Bar-Sagi, D. Oncogenic K-ras drives inactivate BCL-2 by means of the mTOR kinase. vivo. Cancer Res. 63, 2742–2746 (2003). cell cycle progression and phenotypic conversion of Oncogene 20, 6172–6180 (2001). 81. Chakravarti, A. et al. The prognostic significance of primary pancreatic duct epithelial cells. Cancer Res. 131. Balcarcel, R. R. & Stephanopoulos, G. Rapamycin phosphatidylinositol 3-kinase pathway activation in 64, 5659–5663 (2004). reduces hybridoma cell death and enhances human gliomas. J. Clin. Oncol. 22, 1926–1933 107. Altomare, D. A. et al. Frequent activation of AKT2 monoclonal antibody production. Biotechnol. Bioeng. (2004). kinase in human pancreatic carcinomas. J. Cell 76,1–10 (2001). 82. Chandrasekar, N. et al. Downregulation of uPA inhibits Biochem. 88, 470–476 (2003). 132. Humar, R. et al. Hypoxia enhances vascular cell migration and PI3k/Akt signaling in glioblastoma cells. 108. Li, V. S. et al. Mutations of PIK3CA in gastric proliferation and angiogenesis in vitro via rapamycin Oncogene 22, 392–400 (2003). adenocarcinoma. BMC Cancer 5, 29 (2005). (mTOR)-dependent signaling. FASEB J. 16, 771–780 83. Frisk, T. et al. Silencing of the PTEN tumor-suppressor 109. Oki, E. et al. Akt phosphorylation associates with LOH (2002). gene in anaplastic thyroid cancer. Genes of PTEN and leads to chemoresistance for gastric A report on the role of hypoxia and consequences Chromosomes Cancer 35, 74–80 (2002). cancer. Int. J. Cancer 117, 376–380 (2005). in activation of cell signalling in endothelial cells, 84. Virolle, T. et al. The Egr-1 transcription factor directly 110. Zhang, L., Yu, Q., He, J. & Zha, X. Study of the PTEN proliferation and tumour angiogenesis. activates PTEN during irradiation-induced signalling. gene expression and FAK phosphorylation in human 133. Castedo, T. et al. Sequential involvement of Cdk1, Nature Cell Biol. 3, 1124–1128 (2001). hepatocarcinoma tissues and cell lines. Mol. Cell mTOR and p53 in apoptosis induced by the HIV-1 85. Tell, G. et al. Control of phosphatase and tensin Biochem. 262, 25–33 (2004). envelope. EMBO J. 21, 4070–4080 (2002). homolog (PTEN) gene expression in normal and 111. Ma, D. Z. et al. Down-regulation of PTEN expression 134. Decaudin, D. et al. Bcl-2 and Bcl-XL antagonize the neoplastic thyroid cells. Endocrinology 145, due to loss of promoter activity in human mitochondrial dysfunction preceding nuclear 4660–4666 (2004). hepatocellular carcinoma cell lines. World J. apoptosis induced by chemotherapeutic agents. 86. Lee, H. Y. et al. Evidence that phosphatidylinositol Gastroenterol. 11, 4472–4477 (2005). Cancer Res. 57, 62–67 (1997). 3-Kinase-and mitogen-activated protein kinase kinase- 112. Vezina, C., Kudelski, A. & Sehgal, S. N. Rapamycin (AY- 135. Zangemeister-Wittke, U. et al. A novel bispecific 4/c-Jun NH2-terminal kinase-dependent pathways 22,989), a new antifungal antibiotic. I. Taxonomy of antisense oligonucleotide inhibiting both BCL-2 and cooperate to maintain lung cancer cell survival. J. Biol. the producing streptomycete and isolation of the active BCL-xL expression efficiently induces apoptosis in Chem. 278, 23630–23638 (2003). principle. J. Antibiot. (Tokyo) 28, 721–726 (1975). tumor cells. Clin. Cancer Res. 6, 2547–2555 (2000). 87. Marsit, C. J. et al. PTEN expression in non-small-cell 113. Yatscoff, R. W., LeGatt, D. F. & Kneteman, N. M. 136. Shinjyo, T. et al. Downregulation of bim, a lung cancer: evaluating its relation to tumor Therapeutic monitoring of rapamycin: a new proapoptotic relative of Bcl-2, is a pivotal step in characteristics, allelic loss, and epigenetic alteration. immunosuppressive drug. Ther. Drug Monit. 15, cytokine-initiated survival signaling in murine Hum. Pathol. 36, 768–776 (2005). 478–482 (1993). hematopoietic progenitors. Mol. Cell Biol. 21, 88. Ferraro, B., Bepler, G., Sharma, S., Cantor, A. & 114. Davies, C. B. et al. Effect of a short course of 854–864 (2001). Haura, E. B. EGR1 predicts PTEN and survival in rapamycin, cyclosporin A, and donor-specific 137. Shinoura, N. et al. Expression level of bcl-2 patients with non-small-cell lung cancer. J. Clin. Oncol. transfusion on rat cardiac allograft survival. determines anti- or proapoptotic function. Cancer Res. 23, 1921–1926 (2005). Transplantation 55, 1107–1112 (1993). 59, 4119–4128 (1999).

REVIEWS

138. Aguirre, D. et al. Bcl-2 and CCND1/CDK4 expression 155. Atkins, M. B. et al. Randomized phase II study of 172. Sankhala, K. K. et al. Early response evaluation of levels predict the cellular effects of mTOR inhibitors in multiple dose levels of CCI-779, a novel mammalian therapy with AP23573 (an mTOR inhibitor) in human ovarian carcinoma. Apoptosis 9, 797–805 target of rapamycin kinase inhibitor, in patients with sarcoma using [18F]2-fluoro-2-deoxy-D-glucose (FDG) (2004). advanced refractory renal cell carcinoma. J. Clin. positron emission tomography (PET) scan. Proc. Am. This paper underlines the role of BCL2 expression Oncol. 22, 909–198 (2004). Soc. Clin. Oncol. 24, 823s, A9028 (2005). as a molecular factor of resistance to mTOR 156. Hudes, G. et al. A phase 3, randomised, 3-arm study 173. Cho, D. C. et al. Low expression of surrogates for inhibitors in human cancer cells. of temsirolimus (TEMSR) or interferon-α (IFN) or the mTOR pathway activation predicts resistance to CCI- 139. Boya, P. et al. Inhibition of macroautophagy combination of TEMSR + IFR in the treatment of first- 779 in patients with advanced renal cell carcinoma triggers apoptosis. Mol. Cell Biol. 25, 1025–1040 line, poor-risk patients with advanced renal cell (RCC). Proc. 17th Symp. Mol. Targets Cancer Thera. (2005). carcinoma. J. Clin. Oncol. 24, Abs. LBA4, 18S (2006). Philadelphia, USA, November, 233 C137 (2005). 140. Kroemer, G. & Jaattela, M. Lysosomes and 157. Amato, R. J., Misellati, A., Khan, M. & Chiang, S. 174. Diehl, K. M. et al. In-vitro studies of rapamycin autophagy in cell death control. Nature Rev. Cancer. A phase II trial of RAD001 in patients wih metastatic treatment of osteosarcoma cell lines. Proc. 17th 11, 886–897 (2005). renal cell carcinoma. J. Clin. Oncol. 24, A4530, 18S Symp. Mol. Targets Cancer Thera., Philadelphia, USA, 141. Hamada, K. et al. The PTEN/PI3K pathway governs (2006). November, 210 AC53 (2005). normal vascular development and tumor 158. Chan, S. et al. Phase II study of tensirolimus (CCI- 175. Shah, O. J., Wang, Z. & Hunter, T. Inappropriate angiogenesis. Gene Dev. 19, 2054–2065 (2005). 779), a novel inhibitor of mTOR, in heavily pretreated activation of the TSC/Rheb/mTOR/S6K cassette induces 142. Guba, M. et al. Rapamycin inhibits primary and patients with locally advanced or metastatic breast IRS1/2 depletion, insulin resistance, and cell survival metastatic tumor growth by antiangiogenesis: cancer. J. Clin. Oncol. 23, 5314–5322 (2005). deficiencies. Curr. Biol. 14, 1650–1656 (2004). involvement of vascular endothelial growth factor. 159. Witzig, T. E. et al. Phase II trial of single-agent 176. Sarbassov, D. D., Guertin, D. A., Ali, S. M. & Sabatini, Nature Med. 8, 128–135 (2002). tensirolimus (CCI-779) for relapsed mantle cell D. M. Phosphorylation and regulation of Akt/PKB by Describes the modulation of cross-talk between lymphoma. J. Clin. Oncol. 23, 5347–5356 (2005). the rictor-mTOR complex. Science 307, 1098–1101 PI3K/AKT and VEGF signalling pathways by A report of the remarkable antitumour activity of (2005). rapamycin. CCI-779 (temsirolimus) in mantle cell lymphoma, a 177. Cheng, J. Q. et al. The Akt/PKB pathway: molecular 143. Humar, R., Kiefer, F. N., Berns, H., Resink, T. J. & disease driven by cyclin D1 overexpression that can target for cancer drug discovery. Oncogene 24, Battegay, E. J. Hypoxia enhances vascular cell be controlled using mTOR inhibitors. 7482–7492 (2005). proliferation and angiogenesis in vitro via rapamycin 160. Ansell, S. M. et al. Anti-tumor activity of mTOR 178. Guertin, D. A. & Sabatini, D. M. An expanding role for (mTOR)-dependent signaling. FASEB J. 16, 771–780 inhibitor temsirolimus for relapse mantle cell mTOR in cancer. Trends Mol. Med. 11, 353–361 (2002). lymphoma: a phase II trial in the North Central Cancer (2005). 144. Arsham, A. M., Plas, D. R., Thompson, C. B. & Simon, Treatment Group. J. Clin. Oncol. 24 18S, A7532 179. Takeuchi, H. et al. Synergistic augmentation of M. C. Phosphatidylinositol 3-kinase/Akt signaling is (2006). rapamycin-induced autophagy in malignant glioma neither required for hypoxic stabilization of HIF-1α nor 161. Yao, J. C. et al. Phase II study of RAD001 (everolimus) cells by phosphatidylinositol 3-kinase/protein kinase B sufficient for HIF-1-dependent target gene transcription. and depot octreotide (sandostatine LAR) in patients inhibitors. Cancer Res. 65, 3336–3346 (2005). J. Biol. Chem. 277, 15162–15170 (2002). with advanced low grade neuroendocrine carcinoma. 180. Sun, S. Y. et al. Activation of Akt and eIF4E survival 145. Trisciuoglio, D., Iervolino, A., Zupi, G. & Del Bufalo, D. J. Clin. Oncol. 24, 18S, A4042 (2006). pathways by rapamycin-mediated mammalian target of Involvment of PI3K and MAPK signaling in bcl-2- 162. Duran, I. et al. A phase II trial of temsirolimus in rapamycin inhibition. Cancer Res. 65, 7052–7058 induced vascular endothelial growth factor expression metastatic neuroendocrine carcinoma. Proc. Am. Soc. (2005). in melanoma cells. Mol. Biol. Cell 16, 4153–4162 Clin. Oncol. 24, 824s, A146 (2005). 181. Oki, E. et al. Akt phosphorylation associates with LOH (2005). 163. von Oosterom, A. et al. A phase I/II trial of the oral of PTEN and leads to chemoresistance for gastric 146. Costa, L. F., Balcells, M., Edelman, E. R., Nadler, L. M. mTOR-inhibitor everolimus (E) and imatinib mesylate cancer. Int. J. Cancer 117, 376–380 (2005). & Cardoso, A. A. Pro-angiogenic stimultation of bone (IM) in patients (pts) with gastrointestinal stromal 182. Aoki, K. et al. Cisplatin activates survival signals in marrow endothelium engages mTOR, and is inhibited tumor (GIST) refractory to IM: study update. Proc. Am. UM-SCC-23 squamous cell carcinoma and these signal by simultaneous blockade of mTOR and NF-κB. Blood Soc. Clin. Oncol. 24, 824s, A9033 (2005). pathways are amplified in cisplatin-resistant squamous 107, 285–292 (2006). 164. Chawla, S. P. et al. A phase II trial of AP23573, a cell carcinoma. Onc. Rep. 11, 375–379 (2004). 147. Thomas, G. V. et al. Hypoxia-inducible factor novel mTOR inhibitor, in patients (pts) with advanced 183. Beuvink, I. et al. The mTOR inhibitor RAD001 determines sensitivity to inhibitors of mTOR in soft tissue or bone sarcoma. Proc. 17th Symp. Mol. sensitizes tumor cells to DNA-damaged induced kidney cancer. Nature Med. 12, 122–127 (2006). Targets Cancer Thera., Philadelphia, USA, November, apoptosis through inhibition of p21 translation. Cell 148. Raymond, E. et al. Safety and pharmacokinetics of 268 AC272 (2005). 120, 747–759 (2005). escalated doses of weekly intravenous infusion of 165. Boulay, A. et al. Antitumor efficacy of intermittent 184. Dong, J. et al. Role of glycogen synthase kinase 3β in CCI-779, a novel mTOR inhibitor, in patients with treatment schedules with the rapamycin derivative rapamycin-mediated cell cycle regulation and cancer. J. Clin. Oncol. 16, 2336–2347 (2004). RAD001 correlates with prolonged inactivation of chemosensitivity. Cancer Res. 65, 1961–1972 (2005). This paper reports the first-in-man experience ribosomal protein S6 kinase 1 in peripheral blood 185. Nagata, Y. et al. PTEN activation contributes to tumor (phase I trial) using CCI-779 (temsirolimus, a mononuclear cells. Cancer Res. 64, 252–261 (2004). inhibition by trastuzumab, and loss of PTEN predicts potent mTOR inhibitor) in patients with advanced This research paper reports that S6K1 trastuzumab resistance in patients. Cancer Cell 6, cancers, describing the first evidence of phosphorylation can be used as a molecular 117–127 (2004). antitumour activity in patients with renal cell marker for mTOR inhibitors in peripheral blood An exploration of the functional role of PTEN for carcinoma. mononuclear cells, paving the way for the use of sensitivity to trastuzumab, a HER2 inhibitor, in 149. Hidalgo, M. et al. CCI-779, a rapamycin analog and S6K1 in translational clinical research. breast cancer cells. multifaceted inhibitor of signal transduction: a phase I 166. Duran, I. et al. Pharmacodynamic evaluations of paired 186. Kokubo, Y. et al. Reduction of PTEN protein and lss of study. Proc. Am. Soc. Clin. Oncol. 19, 187a, A726 tumor biopsies in advanced neuroendocrine carcinomas epidermal growth factor receptor gene mutation in (2000). (NECs) treated with the mTOR inhibitor tensirolimus. lung cancer with natural resistance to gefitinib 150. O’Donnell, A. et al. A phase I study of the oral mTOR Proc. 17th Symp. Mol. Targets Cancer Thera., (IRESSA). Br. J. Cancer 92, 1711–1719 (2005). inhibitor RAD001 as monotherapy to identify the Philadelphia, USA, November, 230 AC128 (2005). 187. Milton, D. T. et al. PhaseI/II trial of gefitinib and optimal biologically effective dose using toxicity, 167. Paralba, J. M. et al. Pharmacodynamic evaluation of RAD001 (everolimus) in patients (pts) with advanced pharmacokinetic (PK) and pharmacodynamic (PD) CCI-779, an inhibitor of mTOR, in cancer patients. non-small cell lung cancer (NSCLC). Proc. Am. Soc. endpoints in patients with solid tumors. Proc. Am. Clin. Cancer Res. 9, 2887–2892 (2003). Clin. Oncol. 24, 646s, A7104 (2005). Soc. Clin. Oncol. 22, 200, A803 (2003). 168. Tabernero, J. et al. A phase I study with tumor 188. deGraffenried, L. A. et al. Inhibition of mTOR activity 151. Mita, M. AP23573, an mTOR inhibitor, molecular pharmacodynamic (MPD) evaluation of restores tamoxifene response in breast cancer cells administered IV daily x 5 every other week in dose and schedule of the oral mTOR inhibitor with aberant Akt activity. Clin. Cancer Res. 10, patients with refractory or advanced malignancies — everolimus (RAD001) in patients (pts) with advanced 8059–8067 (2004). a phase I, pharmacokinetic (PK), and solid tumors. Proc. Am. Soc. Clin. Oncol. 24, 193s, Competing interests statement pharmacodynamic (PD) study. Proc. 16th Symp. A3007 (2005). The authors declare no competing financial interests. Mol. Targets Cancer Thera. Geneva, Switzerland, 169. Reardon, D. A. et al. A phase I trial of AP23573, October, 122 A409 (2004). a novel mTOR inhibitor, in patients (pts) with recurrent 152. Oza, A. M. et al. A phase II study or tensirolimus malignant glioma. Proc. 17th Symp. Mol. Targets DATABASES (CCI-779) in patients with metastatic and/or locally Cancer Thera., Philadelphia, USA, November, 105 The following terms in this article are linked online to:

recurrent endometrial cancer. Proc. 17th Symp. Mol. AC195 (2005). Entrez Gene: Targets Cancer Thera. Philadelphia, USA, November, 170. Rivera, V. et al. Pharmacodynamic evaluation of the http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene 197 AB269 (2005). mTOR inhibitor AP23573 in phase I clinical trials. 4EBP1 | BAD | BCL2 | EGR1 | c-MYC | eIF4E | eIF4F | HIF1α | 153. Galanis, E. et al. Phase II trial of tensirolimus (CCI- Proc. 16th Symp. Mol. Targets Cancer Thera. Geneva, IGF1 | IGF1R | mTOR | p21 | p27 | p53 | p85 | p110 | PDK1 | 779) in recurrent glioblastoma multiforme: a North Switzerland, October, 123 A411 (2004). PTEN | RICTOR | TSC 1 | TSC2 | Central Cancer Treatment Group Study. J. Clin. Oncol. 171. Rivera, V. M. et al. Pharmacodynamic study of skin OMIM: 23, 5294–5304 (2005). biopsy specimens in patients (pts) with refractory or http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM 154. Chang, S. M. et al. Phase II study of CCI-779 in advanced malignancies following administration of Cowden syndrome patients with recurrent glioblastoma multiforme. AP23573, an mTOR inhibitor. Proc. Am. Soc. Clin. Access to this links box is available online. Invest. New Drugs 23, 357–361 (2005). Oncol. 24, 200s, A3033 (2005).