Review

10.1517/14728222.12.2.209 © 2008 Informa UK Ltd ISSN 1472-8222 209

Oncologic, Endocrine & Metabolic

Mammalian target of rapamycin as a therapeutic target in oncology Robert T Abraham † & Christina H Eng Oncology Discovery Research, Wyeth, 401 N. Middletown Road, Pearl River, NY 10965, USA

Background : The mammalian target of rapamycin (mTOR) has emerged as a validated therapeutic target in cancer and mTOR inhibitors alter tumor cell responses to mitogenic signals and microenvironmental stress. Objectives : The aims of this review are to describe the mTOR signaling path-way and the rationale for the use of rapamycin analogs and other mTOR inhibitors for oncology indications. Methods : This review presents informa-tion from recent publications, as well as some more conjectural viewpoints stemming from the early clinical experience with mTOR inhibitors in cancer patients. Results/conclusions : A thorough understanding of the antitumor mechanisms of the existing mTOR inhibitors will drive the development of effective combination therapies to overcome tumor resistance to these agents. Furthermore, the development of second-generation inhibitors of this critical protein target may yield deeper and broader therapeutic activities in human cancers.

Keywords: Akt , autophagy , cancer , mammalian target of rapamycin , mammalian target of rapamycin complex 1 , mammalian target of rapamycin complex 2 , phosphoinositol 3-kinase , metabolism , rapamycin

Expert Opin. Ther. Targets (2008) 12(2):209-222

1. Introduction

The target of rapamycin (TOR) signaling pathway has emerged as a convergent area of interest for cell biologists, pharmaceutical scientists and clinicians engaged in the treatment of diseases ranging from neurodegenerative disorders to cancer. This review focuses on TOR as a therapeutic target in cancer, a long-standing concept that has now marked a major, long-awaited milestone, with the recent approval of the mammalian target of rapamycin (mTOR) inhibitor, temsirolimus (Wyeth; also known as CCI-779), by the FDA for the treatment of patients with renal cancer. In addition to temsirolimus, two distinct mTOR inhibitors, everolimus (RAD001; Novartis) and deferolimus (AP23573; Ariad/Merck), are under active clinical development for the treatment of hematopoietic, mesenchymal and epithelial neoplasms, in a variety of single agent and combination therapy protocols.

The clinical validation of mTOR as an oncology drug target consummates a journey of > 30 years that began with the identification of a bacterial product, rapamycin, as a potent antifungal agent. Investigations of the mechanism of action of this drug led to the characterization of the TOR proteins in eukaryotes, ranging from single-celled fungi to humans. So far, rapamycin remains a powerful chemical probe for studies of TOR function in myriad cell types and organisms. Moreover, rapamycin (Sirolimus; Wyeth) is an effective clinical agent in its own right, with applications in organ transplantation, psoriasis, and arterial stenosis [1-3] . This review focuses on the underlying rationale for the development of mTOR

1. Introduction

2. Studies of rapamycin uncover a

novel family of signaling kinases

3. Mammalian target of rapamycin

signaling complexes

4. Regulation and functions

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5. Regulation and functions

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6. Mammalian target of rapamycin

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inhibitors as anticancer agents, the clinical results obtained so far with these compounds and the future of this thera-peutic strategy in oncology. However, the clinical application of mTOR inhibitors in oncology does not represent the end of this journey, as ongoing clinical studies of cancer patients treated with mTOR inhibitors will probably raise provocative new questions regarding the role of TOR signaling in tumorigenesis, as well as the mechanism of action of the inhibitors themselves.

2. Studies of rapamycin uncover a novel family of signaling kinases

Although rapamycin was not developed as an antifungal agent, the potent growth–inhibitory activity of the drug against the single-celled fungus, Saccharomyces cerevisiae (budding yeast), provoked a seminal series of experiments that laid the foundation for studies in human cells and tissues [4-6] . Investigations of ‘rapamycin resistance genes’ in yeast uncovered two highly related genes encoding novel signaling proteins that appeared to be direct targets of rapamycin. Although these findings were greeted with considerable fanfare, their full biologic impact was not appreciated until it became apparent that the mechanism of action of rapamycin was conserved and that TOR orthologs were expressed in all eukaryotic cell types. Furthermore, the yeast TORs were the founding members of a small family of signaling proteins, termed phosphoinositol 3-kinase related kinases (PIKKs), which play important roles in cell growth control and stress responses [7,8] .

Several years after the yeast TORs were defined genetically, three laboratories isolated mammalian TOR (mTOR) orthologs using rapamycin-based affinity chromatography to

capture the polypeptide from cell or tissue extracts [9-11] . The mTOR cDNA encodes a ∼ 290 kDa polypeptide bearing a carboxyl-terminal, phosphoinositol 3-kinase (PI3K)-related kinase domain, a common feature of all PIKK family members ( Figure 1 ). A unique and pivotal structural feature of mTOR is the FKBP12•rapamycin-binding (FRB) domain, which represents the binding site for the inhibitory FKBP12•rapamycin complex [12] . Structural studies suggest that the initial binding of rapamycin to FKBP12 locks rapamycin into an optimal configuration for insertion into a hydrophobic binding cleft in the FRB domain [12,13] . Interestingly, the FRB domain resides upstream of the actual phosphotransferase domain, which suggests that FKBP12•rapamycin serves as an allosteric inhibitor of mTOR kinase activity. Although the exact mechanism remains unclear, the specificity of this inhibitory complex is extraordinary, in that it recognizes mTOR only when it is associated with a particular set of partner proteins.

3. Mammalian target of rapamycin signaling complexes

At least two functionally distinct, mTOR-containing complexes are expressed in mammalian cells [14,15] . Interestingly, yeast cells, unlike mammalian cells, express two TOR proteins, TOR1p and TOR2p, only one of which (TOR2p) is essential for cell viability. These observations indicated that the two TOR proteins carried out different signaling functions in yeast and subsequent experiments confirmed TOR1p and TOR2p resided in two different multi-protein complexes in this organism. TOR complex 1 (TORC1) contained the yeast TOR1p protein, whereas the second ortholog, TOR2p, partnered with a different set of

Figure 1 . Domain structure of mammalian target of rapamycin, Raptor and Rictor. The mTOR kinase, like other members of the phosphoinositol 3-kinase related kinase family, is a large ( ∼ 290 kDa) polypeptide bearing a carboxyl-terminal catalytic domain with signifi cant homology to that of PI3K. The amino-terminal region of mTOR comprises a series of Huntingtin, EF3, A subunit of PP2A and TOR1 repeats, which probably mediate intra- and/or inter-molecular protein interactions. The catalytic domain functions as a protein serine–threonine kinase, and is fl anked by two PIKK-specifi c supporting elements, the FRAP, ATM and TRAP (FAT) and FATC domains. The FRB domain resides immediately upstream of the kinase domain. The mTORC1-specifi c subunit, Raptor, is a 150 kDa protein containing Huntingtin, EF3, A subunit of PP2A and TOR1 repeats, C-terminal WD40 repeats, and a unique raptor N-terminal conserved domain. Multiple domains of Raptor are apparently required for the interaction with mTOR during mTORC1 assembly. The mTORC2-specifi c subunit, Rictor, is a ∼ 200 kDa protein whose amino acid sequence provides no insights into the structural architecture of this protein. FAT: FRAP, ATM and TRAP; FATC: FRAP, ATM and TRAP C-terminal; FRB: FKBP12•rapamycin-binding; HEAT: Huntingtin, EF3, A subunit of PP2A and TOR1; mTOR: Mammalian target of rapamycin; RNC: Raptor N-terminal conserved.

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proteins to form TORC2 [16] . TORC1 is necessary for optimal cell growth but not for viability and is sensitive to rapamycin, whereas TORC2 is essential for cell viability and refractory to rapamycin. Although mammalian cells express only one TOR protein, the strategy of segregating this protein into two complexes, or mTORCs, is conserved in these cells [17-19] .

The subunit composition of mTORC1 is schematized in Figure 2 , with the defining subunit of this complex being Raptor. The association of mTOR with Raptor in mTORC1 apparently places mTOR in a conformation that allows FKBP12–rapamycin to access the FRB domain. The mTORC2 contains two unique subunits, Rictor and mammalian stress-activated protein kinase interaction protein 1 (mSin1), in place of the mTORC1-specific Raptor subunit, and is not directly susceptible to inhibition by rapamycin [16,17] . Recent studies suggest that differential mRNA splicing gives rise to three different mSin1 isoforms

that in turn define three mTORC2 complexes [20] . Whether the differential expression of mSin1 splice variants further subdivides mTORC2 into a series of functionally distinct subcomplexes remains unclear. Additional proteomic experiments will probably reveal additional mTORC1 and mTORC2 subunits, and possibly additional mTORCs in mammalian cells.

The mTORC1 seems to carry out all of the rapamycin-sensitive signaling functions of mTOR ( Figure 3 ), although different mTORC1-dependent events exhibit variable sensitivities to rapamycin [21] . Like mTOR itself, the key mTORC1 subunit, Raptor, is essential for embryonic development in the mouse [22] . A critical function of Raptor is to act as a docking site for the known substrates of mTORC1, which include eIF4E-binding protein-1 (4E-BP1), p70 S6 kinase (S6K1) and proline-rich Akt substrate of 40 kDa (PRAS-40) [18,23,24] . Interestingly, recruitment of these substrates to Raptor is mediated through a pentapeptide

Figure 2 . The protein kinase B–mammalian target of rapamycin signaling pathway. Activation of the mTOR pathway is initiated through ligation of growth factor receptors, such as the IGF receptor, which leads to the phosphorylation of the multi-functional signaling scaffold, insulin receptor substrate 1 (IRS1), which, in turn drives the activation of PI3K and Akt. Akt phosphorylates the TSC2 component of the TSC heterodimer, which inhibits the Rheb–GAP activity of TSC, thereby promoting the accumulation of Rheb–GTP and the consequent activation of mTORC1. Substrates of mTORC1, including S6K1, 4E-BP and PRAS-40, are recruited to the complex via the TOS motif on Raptor. In addition, activation of mTORC1 and S6K inhibits the tyrosine phosphorylation and signaling functions of insulin receptor substrate 1 through a negative feedback mechanism, resulting in the attenuation of PI3K–Akt signaling. The signaling functions of mTORC2 may also be stimulated by PI3K, but this pathway does not involve TSC or Rheb. Akt is both an upstream activator of mTORC1 and a downstream substrate of mTORC2. 4E-BP: eIF-4E-binding protein; Akt: Protein kinase B; IGF-R: Insulin-like growth factor receptor; IRS1: Insulin receptor substrate 1; mLST8: Mammalian counterpart of yeast Lst8; mTORC: Mammalian target of rapamycin complex; PDK: Phosphoinositol-dependent protein kinase; PI3K: Phosphatidylinositol 3-kinase; PIP2: Phosphatidylinositol (4,5)-bisphosphate; PIP3: Phosphatidylinositol (3,4,5)-trisphosphate; PRAS-40: Proline-rich Akt substrate of 40 kDa; PTEN: Phosphatase and tensin homolog deleted on chromosome ten; REDD1: Regulated in development and DNA damage responses; S6K: p70 S6 kinase; SIN1: Stress-activated protein kinase interaction protein 1; TSC: Tuberous sclerosis.

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sequence termed the TOR-signaling motif [25,26] . Recent evidence indicates that the TOR-signaling motif-containing mTORC1 substrates compete with one another for binding to Raptor and sub sequent phosphorylation by mTOR [26] . This finding could have important implications for mTORC1 function, as the relative concentrations of these proteins are quite variable in different cell populations. In the case of PRAS-40, the conclusion that this mTORC1-binding protein functions as a repressor of mTORC1 signal-ing [24,27] has been challenged, due to potential artifacts associated with the overexpression of PRAS-40 in the con-text of endogenous levels of Raptor and the competing proteins, eIF-4E-binding protein 1 and S6K1 [26] . Additional work is clearly needed to understand the functions of PRAS-40 during mTORC1 signaling.

The signaling functions of mTORC1 are relatively well defined, due in large part to the availability of the specific inhibitor, rapamycin. Unfortunately, no such selective probe is available for mTORC2. The presence of the Rictor–mSin1 subunits defines mTORC2 but the functions of these subunits in mTORC2 signaling remain unclear. Rictor knockout mice die at a later stage of embryonic development than raptor -/- embryos, underscoring the idea that Raptor and Rictor direct different mTOR-dependent signaling activities [20,22,28,29] . In tissue culture, Rictor- or mSin1-deficient fibroblasts are viable and proliferation competent but display increased sensitivity to stress [28,29] . As stated above, the signaling functions of

mTORC2 are resistant to rapamycin ( Figure 3 ), although the actual mechanism underlying this drug resistance is unresolved. The most plausible explanation is that asso-ciation of Rictor–mSin1 with mTOR either sterically hinders access of FKBP12•rapamycin to the FRB domain or that the conformation of the FKBP12•rapamycin-binding pocket is allosterically altered when mTOR associates with the other mTORC2 subunits. The mTORC2 also targets a distinct set of substrates relative to mTORC1, namely two protein serine-threonine kinases, protein kinase B (Akt) and PKC α [22,30] . Finally, mTORC2 also plays a direct or indirect role in the regulation of the actin cytoskeleton in mitogen-stimulated cells [17,19] .

4. Regulation and functions of mammalian target of rapamycin complex 1

Four general types of stimuli modulate mTOR signaling ( Figure 4 ). Growth factors stimulate mTOR, largely through the activation of Class I PI3Ks. These PI3Ks generate the bioactive metabolite, phosphatidylinositol-3,4,5-trisphosphate, which activates an array of cytoplasmic signaling proteins that govern cell growth, proliferation, migration and survival responses. The other three known mTOR modulators, hypoxia, amino acids and intracellular ATP concentrations, are all related to cellular anabolic metabolism, which is required for normal cell growth and repair. Thus, the upstream stimuli that converge on mTOR provide critical

Figure 3 . Two complexes mediate mammalian target of rapamycin signaling. In mammalian cells, mTOR is segregated into two functionally distinct complexes: mTORC1 and mTORC2. Activation of mTORC1 induces eIF-4E-dependent translation, ribosome biogenesis and upregulation of amino acid transporters, and suppresses autophagic activity. mTORC2 activation leads to the phosphorylation of the regulatory hydrophobic motifs on PKC α and Akt, and also triggers reorganization of the actin cytoskeleton. The rapalogs in clinical development at present bind to and inhibit mTORC1 only, whereas direct inhibitors of the mTOR kinase domain are predicted to interfere with signaling from both mTORCs. Akt: Protein kinase B; eIF-4E: Eukaryotic initiation factor 4E; mTORC: Mammalian target of rapamysin complex; PKC: Protein kinase C.

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information related to growth factor and amino acid availability, as well as the internal bioenergetic status of the cell. When nutrient and ATP supplies are low, growth factors fail to activate mTORC1 and cells do not progress from G1 to S phase of the cell cycle. The mitotic cell cycle is a metabolically demanding process and the suppression of mTORC1 activity by anabolic and bioenergetic signals protects cells from the potentially catastropic consequences of attempting a cell division cycle when metabolism is unable to support these events.

The afferent signaling inputs into mTORC1 converge on the tuberous sclerosis (TSC) complex, which contains two subunits, TSC1 and TSC2 ( Figure 4 ). Germline loss of function mutations in the TSC1 or TSC2 genes give rise to tuberous sclerosis; a tissue overgrowth syndrome that leads to multiple organ dysfunction [31] . Cells from these patients exhibit constitutive mTORC1 signaling, suggesting that the TSC serves as a negative regulator of mTORC1 in wild type cells. In actuality, TSC does not act directly on mTORC1 but rather on the Ras-related GTPase, Rheb [31-33] . TSC functions as a GTPase-activating protein (GAP) for Rheb, stimulating the conversion of active, GTP-bound

Rheb to the inactive GDP-bound state. The GAP activity of TSC is stimulated by environmental cues associated with limiting growth conditions, such as nutrient deprivation or hypoxia, and results in the accumulation of GDP-bound Rheb. Conversely, pro-proliferative conditions, exemplified by ample supplies of growth factors and anabolic precursors, inhibit TSC activity and promote the formation of activated, GTP-bound Rheb. Recent evidence indicates that active Rheb–GTP binds directly to mTORC1, thereby stimulating mTOR kinase activity [24] . Thus, the Rheb – GAP activity of TSC indirectly controls mTORC1 kinase activity through modulation of the level of Rheb–GTP levels in response to changes in growth factor receptor signaling or metabolic conditions.

The upstream signals that regulate mTORC1 converge on TSC through several distinct pathways. Growth factor receptors are coupled to TSC through the PI3K–Akt pathway. Akt phosphorylates the TSC2 subunit, thereby disrupting the interaction with TSC1, abolishing Rheb–GAP activity and activating mTORC1 [34,35] . Amino acids also activate mTORC1 through a poorly understood pathway that may involve a Class III PI3K (distinct from the Class I

Figure 4 . Stressful microenvironments suppress mTORC signaling. The activity of mTORC1 is regulated by stress-inducing conditions associated with the tumor microenvironment. Suboptimal conditions for cellular anabolic metabolism (e.g., amino acid starvation, glucose defi ciency or hypoxia) or cell-cycle progression (e.g., growth factor deprivation) inactivate mTORC1 through a several pathways converging on the TSC complex, which, through its Rheb–GAP activity, inactivates Rheb, thereby suppressing mTORC1. Subsequently, energy consumption is reduced through a reduction in protein synthesis, cells delay G1–S progression and cell viability is maintained through autophagic recycling of cellular macromolecules. Akt: Protein kinase B; AMPK: AMP-activated protein kinase; C3-PI3K: Class III phosphoinositide 3-kinase; MAP4K3: Mitogen-activated protein kinase kinase kinase kinase 3; mTORC1: Mammalian target of rapamycin complex 1; PTEN: Phosphatase and tensin homolog deleted on chromosome ten; REDD1: ; TSC: Tuberous sclerosis.

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PI3Ks activated by growth factors) and mitogen-activated protein kinase kinase kinase kinase 3, a member of the Ste20 kinase family [36-39] . How these two signaling kinases communicate with one another, and with mTORC1, remains somewhat controversial, with evidence for and against the involvement of TSC. Limiting supplies of oxygen and intracellular ATP create a bioenergetic state that is generally unfavorable for cell growth. These parameters deliver signals that lead to an increase in the Rheb–GAP activity of TSC and a corresponding reduction in mTORC1 activity. Hypoxic conditions trigger the transcriptional activation of the hypoxia-induced factor-1 target gene, REDD1 (also called RTP801 ) [40] , which inhibits mTORC1 in a TSC-dependent fashion. A recent report suggests that TSC integrates signals that reflect the availability of growth factors and intracellular ATP to support cell growth and proliferation. This study demon-strated that full activation of TSC (and consequent inhibition of Rheb–mTORC1) is achieved through sequential phosphorylation of TSC2 by AMP-dependent protein kinase (AMPK) and glycogen synthase kinase 3 (GSK3) [41] . AMPK is activated by an increase in the intracellular AMP:ATP ratio, which reflects a decline in the stores of metabolic energy, whereas GSK3 activity rises as growth factor receptor signaling wanes. Hence, AMPK- and GSK3-dependent phosphorylation of TSC2 is maximal under conditions that are not conducive to cell growth and inappropriate for Rheb–mTORC1 signaling.

The efferent signaling functions of mTORC1 focus on the eIF-4E-binding proteins 1 – 3 and ribosomal S6Ks 1 and 2. 4EBPs act as repressors of cap-dependent translation by binding to eIF-4E, and phosphorylation of 4EBP by mTORC1 disrupts this interaction, allowing eIF-4E to orchestrate the assembly of a functional translation initiation complex at the 5 ′ -cap structure of mRNAs [42] . An important consideration is that, while the vast majority of mammalian mRNAs contain a 5 ′ -cap site, transcripts containing lengthy and/or highly structured 5 ′ -untranslated regions (UTRs) are more dependent on eIF-4E for efficient translation. Interestingly, mRNAs encoding proteins involved in the regulation of cell growth, survival, and proliferation areoverrepresented in this highly eIF-4E-dependent subset. Two relevant examples are the G1 progression factors, cyclin D1 and c-Myc. The positive impact of eIF-4E on cell proliferation and survival is documented by the finding that forced overexpression of eIF-4E induces cell transformation in culture and that eIF-4E levels are often elevated in human tumors [43,44] .

The mTORC1 controls S6K1 activity through phosphorylation of this protein kinase within a ‘hydrophobic motif ’ (HM) sequence located downstream of the S6K1 catalytic domain [45-47] . Phosphorylation of S6K1 by mTORC1 is a critical intermediate step in a series of phosphorylation events that ultimately lead to full activation of the S6K1 catalytic domain.

Although the canonical S6K1 substrate is ribosomal protein S6, additional S6K1 targets, including eukaryotic initiation factor 4B and eukaryotic elongation factor-2 kinase, also contribute to the translation- and cell growth-promoting activities of S6K1 [23,48] . A striking observation is that inhibition of mTORC1 by rapalogs triggers almost immediate dephosphorylation of the HM and consequent inactivation of S6K1, which suggests that mTORC1 inhibition disrupts a delicate balance between the upstream kinases that stimulate S6K1 activity and protein phosphatases that reverse this effect [49] . The intricate connections among mTORC1, S6K1, and the translational machinery are highlighted by a report that the preinitiation complex itself provides a platform for the interaction between mTORC1 and its S6K1 substrate [50] . From the translational medicine perspective, rapalog-induced S6K1 dephosphorylation is an attractive readout for mTOR1 inhibition but should not be considered an efficacy biomarker in rapalog-treated patients. Human cells (normal and transformed) exhibit widely varying sensitivities to the antiproliferative effects of the rapalogs, in spite of the fact that profound inhibition of S6K1 is observed in virtually all cells exposed to these drugs [51] .

5. Regulation and functions of mammalian target of rapamycin complex 2

The upstream regulatory pathways that govern mTORC2 activity are considerably less well understood than those that control mTORC1. Unlike mTORC1, mTORC2 activity is not controlled by the metabolic state of the host cell. In addition, whereas Raptor is the only pivotal subunit (other than mTOR itself ) needed for mTORC1 activity, all of the components of mTORC2 – Rictor, mammalian counterpart of yeast Lst8, mSin1, and mTOR – are needed for mTORC2 activity [15,20,21,27] . Existing evidence suggests that mTORC2 is embedded within the PI3K pathway, functioning as a downstream effector that contributes to the phosphorylation and activation of Akt. Accumulation of the PI3K metabolite, PtdIns-3, 4,5-P 3 , in plasma membranes triggers the recruitment of cytoplasmic Akt. The membrane-localized protein kinase then undergoes phosphorylation in its catalytic loop (Thr308 in human Akt1) by phosphoinositide-dependent kinase 1 and in a carboxyl-terminal HM at Ser473 [52] . Both modifications are needed for full activation of the enzyme and consider-able excitement was generated with the discovery that mTORC2 was a major Akt (Ser473) kinase in mammalian cells [30] . These observations underscore the complexity of the PI3K signaling network. In one type of complex (mTORC2), mTOR serves as an upstream regulator of Akt activation; in another guise (mTORC1), mTOR is itself regulated by Akt. At least one additional protein kinase A/protein kinase G/protein kinase C (AGC) kinase family member, PKC α , is also phosphorylated in its HM by mTORC2 [17] .

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6. Mammalian target of rapamycin as a therapeutic target in cancer

Given the widespread involvement of the PI3K–Akt–mTOR signaling network in cancer development and progression, the entry of the first generation of mTOR inhibitors into the oncology clinics was eagerly anticipated. However, do we really understand how these drugs suppress tumor growth and why the inhibition of this broadly conserved pathway gives such variable and often disappointingly modest anti-tumor responses in human patients? As is the case for most of the anticancer drugs, much of the research on rapalogs has focused on cancer cell-autonomous effects of these drugs. However, although fewer in absolute number, other studies have generated clear evidence that these drugs alter host responses, such as angiogenesis, which are crucial for progressive tumor growth. In this section, the authors highlight a subset of the alterations imposed by rapalog therapy that, in the authors’ opinion, make important contributions to therapeutic responsiveness or resistance.

6.1 Mutational activation of the phosphoinositol 3-kinase pathway as a cell-autonomous indicator of mammalian target of rapamycin inhibitor sensitivity Loss of the phosphatase and tensin homolog deleted on chromosome ten (PTEN) tumor suppressor is extremely common in human cancers, supporting the concept that deregulated signaling through the PI3K pathway is highly selected for during tumorigenesis [53] . PTEN-deficient cancer cells exhibit constitutive activation of Akt and mTORC1 signaling, together with an aggressive phenotype and resis-tance to most forms of cancer therapy [54] . The notion that loss of PTEN during tumorigenesis creates a strong dependence on mTOR signaling, potentially addressable with rapalogs, has been proposed and debated in numerous publications [55-59] . The equivocal results obtained in the clinic underscore the idea that cause and effect relationships in sporadic human diseases, oncology in particular, are rarely straightforward. For example, the results of Phase II clinical trials in melanoma and glioblastoma, two cancers that commonly exhibit loss of PTEN function, yielded disappointingly low response rates to Temsirolimus [60-62] . A number of theoretical explanations can be offered to explain this outcome. A ‘systems-level’ possibility is that loss of PTEN creates an addiction only in certain genetic contexts. Many tumor types, including melanoma and glioblastoma, tend to lose PTEN function at a relatively late stage of development. Although these tumor types obviously derive a selective benefit from deregulated PI3K signaling, their genomes and transcriptomes probably evolved under selective pressures imposed by ‘founder’ lesions that occurred much earlier in the tumorigenic process. The authors hypothesize that pathologic PI3K pathway activation at more advanced stages of malignancy would increase the

probability that mTORC1-independent pathways would be called into play during an earlier phase of carcinogenesis. Such cells might be more prone to resist mTORC1-targeted therapy than tumor cells in which pathologic PI3K activation stems from a founder lesion (e.g., PTEN loss or an activating mutation in Class I PI3K), leading to lineage dependence on mTORC1 function. The concept of founder lesion dependence is supported by studies in genetically engineered, tumor-prone mice, in which reversal of the original oncogenic lesion causes regression of advanced tumors [63] . Similar findings were reported in mice bearing germline (i.e., founder) lesions in Pten . The hyperplastic and malig-nant tissues seen in these mice are remarkably responsive to rapalog therapy [56,57,59] . In the clinic, endometrial carcinoma may be one tumor type that shows a strong lineage dependence on loss of PTEN function, as loss of PTEN expression is seen in a significant proportion of the hyperplastic foci that eventually give rise to full-blown cancers [64] . Interestingly, a recent Phase II study with Temsirolimus indicated a remarkable 26% objective response rate for patients with endometrial carcinoma [65] . Obviously, this hypothesis requires deeper investigation, but, if correct, molecular profiling of cancer models in which PI3K activation occurs at early versus late stages of transformation could yield more predictive signatures of tumors that are more likely to respond to rapalog therapy.

Although lineage dependence determines the intrinsic responsiveness of tumor cells to rapalog therapy, cellular exposure to these drugs triggers homeostatic responses that may also affect the antitumor response. In certain settings, treatment with a rapalog provokes a rebound increase in PI3K and Akt activities [66,67] , which has been attributed to the operation of a negative feedback loop involving the mTORC1 substrate, S6K1 (see Figure 2 ). Disruption of this negative feedback mechanism by mTORC1 inhibitors explains the unexpected increase in PI3K–Akt activity seen in IGF-dependent tumor cells. However, a recent publication indicates that interruption of the negative feedback loop during rapamycin treatment does not necessarily translate into reduced efficacy in a preclinical model system [68] . Clinical experience will eventually reveal whether the rebound activation of PI3K–Akt actually limits the response to rapalog therapy in cancer patients. A second homeostatic response observed in ∼ 20% of tumor cell lines [14] might yield the opposite phenotype: an increased therapeutic response to rapalog treatment. As discussed, the rapalogs selectively target mTORC1 and show no direct interactions with mTORC2. Given the role of mTORC2 in Akt activation, it has been speculated that inhibition of both mTORC complexes could boost antitumor activity more than that observed with mTORC1 inhibition alone. An unanticipated finding was that certain transformed cell lines suffer a loss of mTORC2, as well as mTORC1 function in response to chronic rapamycin exposure [14,69] . A plausible model to explain these findings is that persistent inhibition of

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mTORC1 triggers a homeostatic response that pushes newly translated mTOR polypeptides into additional mTORC1 complexes, at the cost of mTORC2 complex assembly. Whatever the mechanism, it will be important to determine whether long-term rapalog treatment induces the loss of mTORC2 in human tumor tissues and, if so, whether drug exposures that trigger this response lead to better therapeutic outcomes for cancer patients.

6.2 Metabolic effects of mammalian target of rapamycin complex 1 inhibitors on cancer cells Accumulating evidence argues that disruption of cancer cell metabolism plays an important role in the antitumor mechanism of the mTORC1 inhibitors. In mammalian cells, growth factors regulate both the metabolic alterations and the cell-cycle events needed to support mitotic cell division. In normal cells, particularly those geared for explosive growth responses (e.g., T lymphocytes), mitogenic stimulation triggers a rapid switch from mitochondria-dependent oxidative phosphorylation to mitochondria-independent glycolysis [70] . However, cancer cells exhibit a constitutive overdependence on glycolytic metabolism, which allows for more rapid production of ATP but delivers a much lower energy yield per molecule of glucose consumed than oxidative phosphorylation [71] . It turns out that the PI3K–Akt–mTORC1 pathway controls numerous facets of energy production (increased glycolysis) and consumption (increased protein synthesis) during the cellular response to growth factor receptor stimulation. Akt plays a particularly important role in directing the shift toward increased glucose uptake and glycolytic metabolism [72] . Through the activation of mTORC1, Akt also orchestrates the increases in amino acid uptake and translational activity that are required to support tumor cell growth and division [21,73,74] . Clearly, chronic hyperactivation of PI3K signaling in evolving malignant clones offers some critical selective advantages, including the ability to out-compete their neighbors for nutrients and to generate bioenergy (ATP) under suboptimal microenvironmental conditions.

The selective benefits derived from the PI3K–Akt-dependent switch to aerobic glycolysis in malignant cells may contribute to positive or negative therapeutic outcomes with mTORC1 inhibitors in cancer patients. Pathologic activation of PI3K creates a persistent anabolic drive that may be disconnected from the supply of anabolic precursors. When nutrient avail-ability is insufficient to meet the needs of the cell growth and division cycle, inappropriate Akt activation may lead to ‘metabolic catastrophe’ and cell death [71] . Exposure of meta-bolically stressed tumor cells to mTORC1 inhibitors, which trigger a starvation-like response in their own right, may push stressed cancer cells into death by excessive self-digestion, known as autophagy (see below). Conversely, inhibition of mTORC1 could, theoretically, aid cell survival by suppressing inappropriate protein synthesis and, in turn, sparing cellular energy stores under conditions of extreme energetic stress. Once again, additional preclinical and clinical studies are

needed to determine as to whether the pseudo-starvation response contributes to or limits the antitumor activities of the rapalogs in patients with phenotypically distinct cancers.

6.3 Autophagy: friend or foe to cancer therapy? Cellular exposure to certain rapalogs triggers a starvation-related stress response, termed autophagy, which may play a pivotal role in tumor responsiveness to rapalog therapy. Under nutrient-replete conditions, autophagy helps to maintain normal cell function and lifespan by removing protein aggregates and dysfunctional mitochondria [75] . When cells experience starvation conditions, autophagy increases to promote cell survival by recycling endogenous biomolecules into metabolic precursors needed to maintain energy production and critical cellular functions. Although starvation-induced autophagy is clearly a protective response to stress, excessive autophagy can lead to cell death and recent studies suggest an intimate relationship between autophagy and the apoptotic machinery [76] . Consistent with its activation under growth factor- and nutrient-replete conditions, the PI3K–Akt–mTORC1 pathway suppresses autophagy [75,77,78] . Observations that mTORC1 inhibitors stimulate autophagy in certain cell types have raised some unanticipated clinical opportunities for these inhibitors, particularly in the treatment of neurodegenerative diseases, such as Huntington, Parkinson’s and Alzheimer’s disease [75] . In preclinical models, certain rapalogs increase the autophagic clearance of protein aggregates that mediate neuronal dysfunction and cell death in these diseases [75] .

At this stage, autophagy appears to be a double-edged sword with respect to tumor responsiveness to cancer therapy. Solid tumor cells are commonly exposed to hypoxic- and nutrient-limited conditions, in which autophagy could serve as a protective mechanism [79,80] . Furthermore, increased levels of autophagy might allow tumor cells to rid themselves of defective mitochondria and misfolded protein aggregates that could limit tumor growth. In this setting, rapalog therapy might have the undesirable effect of promoting the survival of stressed cancer cells, just as it protects neurons from toxic protein aggregates [81] . On the other hand, a therapy-induced boost in autophagic activity in cells that are already heavily engaged in self-digestion might lead to a lethal loss of cell mass and eventual lethality. Death by autophagy may be a rational strategy for killing of metabolically stressed cancer cells that are resistant apoptotic cell death [71,82] . Clearly, there is much more to learn about the relationship between mTORC1 and autophagy, and the contribution of increased autophagic activity to the outcome of therapy with both PI3K and mTOR inhibitors.

6.4 Effects of mammalian target of rapamycin complex 1 inhibitors on the tumor microenvironment Malignant clones evolve in close apposition to resident epithelial cells, fibroblasts, cellular components of the

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immune system, as well as a matrix of connective tissue and other stromal elements [83,84] . During tumor evolution, a continuous stream of regulatory signals flows between the developing tumor mass and the local microenvironment. It is now recognized that progressive tumor growth is accompanied by alterations in the local host tissue that collectively resemble the chronic response to tissue injury [85] . A well-documented example is inflammatory breast cancer, a disease characterized by intensive immune cell infiltration, an aggressive phenotype and a poor prognosis [86] . The tumor microenvironment is now considered fertile ground for cancer drug discovery with agents targeted against tumor-associated angiogenesis, exemplified by bevacizumab (Genentech-Roche), leading the way. To fully understand the antitumor mechanisms of the rapalogs, one needs to consider how and in which tumor types these drugs alter the tumor microenvironment and the contributions of host effects to rapalog treatment successes or failures in cancer patients. As potent immunosuppressants and anti-inflammatory agents, rapalogs may disengage corrupted interactions between malignant cells and cells of the immune and inflammatory systems that support progressive tumor growth. The idea that the anti-inflammatory effects of the rapalogs may be of significant therapeutic benefit against tumor tissues bearing a strong inflammatory signature merits further testing in preclinical models.

The effects of mTORC1 inhibitors on tumor-induced angiogenesis have received considerably more attention from researchers and clinicians. An elegant tumor imaging study provided striking visual evidence that rapamycin treatment disrupted the vascularization of tumor implants in immuno-competent mice [87] . The authors attributed the antiangiogenic effects of the rapalogs to the inhibition of signaling from VEGF receptors (VEGFRs). VEGFR ligands are produced by many tumors, and the resulting activation of VEGRs on endothelial cells and lymphatic precursor cells delivers survival- and growth-promoting signals that support tumor vascularization. Accumulating evidence indicates that mTORC1 inhibitors interfere with tumor-induced angiogenesis at multiple levels [88-90] .

A key transcription factor that drives hypoxia-induced VEGF gene expression in cancer cells and other cell types is HIF-1, a heterodimeric complex consisting of an oxygen-regulated subunit (HIF-1 α or HIF-2 α ) and a constitutively expressed subunit (HIF-1 β ) [91] . Levels of HIF-1 α /2 α sub-units are tightly linked to the ambient oxygen tension. Under normoxic conditions, HIF-1 α /2 α proteins are expressed at low levels, due to their continuous prolyl hydroxylation, which marks these proteins for recognition by the von Hippel–Lindau protein–ubiquitin E3 ligase com-plex and subsequent degradation via the ubiquitin–protea-some pathway. Hypoxic conditions, such as those found in poorly vascularized tumors, block this destabilization mecha-nism, leading to the accumulation of transcriptionally active heterodimers in hypoxic cell nuclei. HIF-1 regulates the

expression of > 100 genes that generally promote cellular adaptation to tissue hypoxia [92] . In addition to directing a metabolic shift from oxidative phosphorylation to glycolysis, HIF-1 also stimulates expression of several pro-angiogenic factors, including the VEGFs. Rapalog therapy interferes with HIF-1-dependent VEGF production during hypoxia by decreasing the accumulation of HIF-1 α /2 α , due, in part, to the inhibition HIF-1 α /2 α mRNA translation [93-95] . Chronic inhibition of mTORC1 may also interfere with HIF-1 α stabilization in certain cancer cell types [95] . Compelling preclinical evidence indicates that inhibition of HIF-1 function is centrally involved in the antiangiogenic effect of rapamycin. Indeed, the suppressive effects of rapamycin may, at least partially, explain the significant thera peutic activity of temsirolimus in renal cell carcinoma [96,97] because the most prevalent subtype of renal cell carcinoma (the clear cell subtype) is characterized by loss of function mutations in the von Hippel–Lindau protein, which leads to abnormal HIF-1 accumulation and activation [98] .

7. Expert opinion

The identification of the eukaryotic TOR proteins in the mid-1990s launched a decade of discovery research that defined key mechanisms, whereby cells tune anabolic metabolism and proliferation to the availability of growth factors and nutrients. Investigations that began with the mTORC1 inhibitor, rapamycin, and advanced with increasingly sophisticated genetic, biochemical and proteomic strategies, have highlighted the PI3K–Akt–mTOR pathway as a central player in coordinating cell metabolism with cell growth and cell cycle progression. Pathologic activation of the PI3K signaling network is a hallmark attribute of malignant cells and is centrally involved in progressive tumor growth, metastasis and resistance to many anticancer therapies. The present crop of mTORC1 inhibitors represent promising first entries in what will undoubtedly become a battery of chemotherapeutic drugs targeting various facets of the pathologic PI3K signaling network in cancer cells.

The clinical development of rapamycin-related compounds as antitumor agents offers unprecedented opportunities to explore the mechanisms through which these drugs suppress tumor growth. As discussed, the therapeutic effects of these compounds probably stem from direct actions on the tumor cells, as well as drug-induced alterations in the tumor microenvironment. With regard to drug responsiveness, constitutive activation of the PI3K pathway (e.g., through loss of PTEN) remains a promising harbinger of tumor sensitivity to rapalog treatment. However, the experience so far, indicates that this ‘efficacy biomarker’ is only partially predictive and additional research is needed to unravel why tumors of seemingly similar PI3K phenotypes respond so variably to rapalog therapy. We have proposed that the timing of PI3K pathway activation, relative to the evolution

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of the tumor, is a critical determinant of mTORC1 inhibitor responsiveness in human patients. This hypothesis should be subjected to rigorous testing in preclinical models and human tumor tissues with the potential payoff that rapalog therapy can be more accurately directed to those patients who are likely to benefit from treatment with these drugs.

The rapamycin derivatives presently in clinical development will probably prove most effective when administered in combination with other anticancer agents and several rational combination strategies will be explored in the coming years. Treatment with rapalogs enhances the pro-apoptotic activities of the cytotoxic agents, cisplatin and paclitaxel [99,100] . A particularly intriguing, non-cytotoxic combination therapy involves rapalog administration together with the Bayer drug, sorafenib, a multikinase inhibitor that targets Raf kinases and tumor vasculature [101,102] . An equally intriguing strategy involves the co-administration of mTORC1 inhibitors with drugs that target other components of the PI3K pathway. Inhibitors of the Class IA PI3Ks are moving through the development pipeline in several companies and combination of these agents with a rapalog could deliver an effective one-two punch to the PI3K pathway in tumor cells resistant to therapy with mTORC1 inhibitors alone. Finally, preliminary observations that mTORC1 inhibitors inhibit pericyte association with the tumor microvasculature merit more intensive investigation [88] . If these findings are confirmed, then combinations involving the rapalogs and endothelial cell-directed therapies, such as bevacizumab, become appealing strategies to force tumor starvation through multi-factorial disruption of tumor angiogenesis.

The next logical step in the development of mTOR-targeted therapies is the discovery of compounds that

directly interact with and inhibit the mTOR kinase domain. These agents would have a theoretical advantage over the rapalogs, in that they would disable both mTORC1 and mTORC2 signaling in tumor tissues (see Figure 3 ). We expect that the added inhibition of mTORC2 will broaden and deepen the antitumor effects of these agents, relative to the mTORC1 inhibitors. However, an accompanying risk is that a broadened suppression of mTOR function will lead to increased toxicity to normal host tissues, which might obviate any efficacy advantage gained against the tumor tissue. We should temper our enthusiasm regarding these second-generation mTOR inhibitors with the knowledge that the present crop of rapalogs sets a high bar for therapeutic strategies. The specificity of the rapalogs for their protein target is unlikely to be equaled by more traditional, small-molecule inhibitors of the mTOR kinase domain. Moreover, the clinically active rapalogs (when bound to FKBP12) are, for all intents and purposes, irreversible inhibitors of mTORC1 [103] . It will be difficult to achieve a similar duration of action against mTOR with the reversible, ATP-competitive compounds now in preclinical development. Will the added suppressive effects on mTORC2 confer a substantial clinical advantage to the second-generation mTOR kinase inhibitors? Time will tell but if past history is any indication, the drugs themselves will be pivotal tools in the ongoing journey towards a complete understanding of the mTOR signaling pathway in human health and disease.

Declaration of interest

The authors are employees of Wyeth Pharmaceuticals, a company that has developed an mTOR inhibitor (Torisel) for oncology indications.

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Bibliography Papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.

1. Sehgal SN. Sirolimus: its discovery, biological properties, and mechanism of action. Transplant Proc 2003 ; 35 : S7 -S14

• Excellent historical overview of the development and actions of rapamycin (Sirolimus).

2. Parry TJ, Brosius R, Thyagarajan R, et al. Drug-eluting stents: sirolimus and paclitaxel differentially affect cultured cells and injured arteries. Eur J Pharmacol 2005 ; 524 : 19 -29

3. Young DA, Nickerson-Nutter CL. mTOR – beyond transplantation. Curr Opin Pharmacol 2005 ; 5 : 418 -23

4. Hall MN. The TOR signalling pathway and growth control in yeast. Biochem Soc Trans 1996 ; 24 : 234 -9

5. Schmelzle T, Hall MN. TOR, a central controller of cell growth. Cell 2000 ; 103 : 253 -62

6. Jacinto E, Hall MN. Tor signalling in bugs, brain and brawn. Nat Rev Mol Cell Biol 2003 ; 4 : 117 -26

• A well-written comparative review of TOR signaling in yeast and mammalian cells.

7. Tibbetts RS, Abraham RT. PI3K-related kinases – roles in cell-cycle regulation and DNA damage responses. Signaling Networks and Cell Cycle Control: The Molecular Basis of Cancer and Other Diseases . Bethesda, MD: Humana Press; 2000 . p. 267-301

8. Abraham RT. PI 3-kinase related kinases: ‘big’ players in stress-induced signaling pathways. DNA Repair (Amst) 2004 ; 3 : 883 -7

9. Sabers CJ, Martin MM, Brunn GJ, et al. Isolation of a protein target of the FKBP12 – rapamycin complex in mammalian cells. J Biol Chem 1995 ; 270 : 815 -22

10. Brown EJ, Albers MW, Shin TB, et al. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 1994 ; 369 : 756 -8

11. Sabatini DM, Erdjument-Bromage H, Lui M, et al. RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell 1994 ;78: 35 -43

12. Choi J, Chen J, Schreiber SL, Clardy J. Structure of the FKBP12–rapamycin complex interacting with the binding domain of human FRAP. Science 1996 ; 273 : 239 -42

13. Leone M, Crowell KJ, Chen J, et al. The FRB domain of mTOR: NMR solution structure and inhibitor design. Biochemistry 2006 ; 45 : 10294 -302

14. Sabatini DM. mTOR and cancer: insights into a complex relationship. Nat Rev Cancer 2006 ; 6 : 729 -34

15. Guertin DA, Sabatini DM. Defi ning the role of mTOR in cancer. Cancer Cell 2007 ; 12 : 9 -22

• An updated overview of the mTOR signaling pathway in cancer cells and the effects of mTOR inhibitors on tumor biology and cancer cell signaling.

16. Loewith R, Jacinto E, Wullschleger S, et al. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol Cell 2002 ; 10 : 457 -68

17. Sarbassov DD, Ali SM, Kim DH, et al. Rictor, a novel binding partner of mTOR, defi nes a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 2004 ; 14 : 1296 -302

• References [16] and [17] are historically important papers that reveal a second mTOR signaling complex.

18. Kim DH, Sabatini DM. Raptor and mTOR: subunits of a nutrient-sensitive complex. Curr Top Microbiol Immunol 2004 ; 279 : 259 -70

19. Jacinto E, Loewith R, Schmidt A, et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol 2004 ; 6 : 1122 -8

20. Frias MA, Thoreen CC, Jaffe JD, et al. mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms defi ne three distinct mTORC2s. Curr Biol 2006 ; 16 : 1865 -70

21. Edinger AL, Linardic CM, Chiang GG, et al. Differential effects of rapamycin on mammalian target of rapamycin signaling functions in mammalian cells. Cancer Res 2003 ; 63 : 8451 -60

22. Guertin DA, Stevens DM, Thoreen CC, et al. Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1. Dev Cell 2006 ; 11 : 859 -71

23. Fingar DC, Blenis J. Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene 2004 ; 23 : 3151 -71

24. Sancak Y, Thoreen CC, Peterson TR, et al. PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol Cell 2007 ; 25 : 903 -15

25. Schalm SS, Blenis J. Identifi cation of a conserved motif required for mTOR signaling. Curr Biol 2002 ; 12 : 632 -9

26. Oshiro N, Takahashi R, Yoshino KI, et al. The proline-Rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mTOR complex 1. J Biol Chem 2007 ; 282 : 20329 -39

27. Haar EV, Lee SI, Bandhakavi S, et al. Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat Cell Biol 2007 ; 9 : 316 -23

• Together with reference [24] and [26] , this report identifi es PRAS-40 as a novel mTORC1-binding protein that appears to be dually regulated by Akt and mTORC1.

28. Jacinto E, Facchinetti V, Liu D, et al. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specifi city. Cell 2006 ; 127 : 125 -37

29. Shiota C, Woo JT, Lindner J, et al. Multiallelic disruption of the rictor gene in mice reveals that mTOR complex 2 is essential for fetal growth and viability. Dev Cell 2006 ; 11 : 583 -9

30. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor – mTOR complex. Science 2005 ; 307 : 1098 -101

• Seminal report describing a critical function for mTORC2 in Akt activation.

31. Kwiatkowski DJ. Tuberous sclerosis: from tubers to mTOR. Ann Human Genet 2003 ; 67 : 87 -96

32. Li Y, Corradetti MN, Inoki K, Guan KL. TSC2: fi lling the GAP in the mTOR signaling pathway. Trends Biochem Sci 2004 ; 29 : 32 -8

33. Tee AR, Manning BD, Roux PP, et al. Tuberous sclerosis complex gene products, tuberin and hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb. Curr Biol 2003 ; 13 : 1259 -68

34. Manning BD, Tee AR, Logsdon MN, et al. Identifi cation of the tuberous sclerosis complex-2 tumor suppressor gene product

Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y L

akeh

ead

Uni

vers

ity o

n 10

/27/

14Fo

r pe

rson

al u

se o

nly.

Mammalian target of rapamycin as a therapeutic target in oncology

220 Expert Opin. Ther. Targets (2008) 12(2)

tuberin as a target of the phosphoinositide 3-kinase/Akt pathway. Mol Cell 2002 ; 10 : 151 -62

35. Hay N. The Akt-mTOR tango and its relevance to cancer. Cancer Cell 2005 ; 8 : 179 -83

36. Findlay GM, Yan L, Procter J, et al. A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling. Biochem J 2007 ; 403 : 13 -20

• An intriguing report describing a Ste20 family member as an amino acid-stimulated protein kinase and an upstream activator of mTORC1.

37. Cook SJ, Morley SJ. Nutrient-responsive mTOR signalling grows on sterile ground. Biochem J 2007 ; 403 : e1 -e3

38. Byfi eld MP, Murray JT, Backer JM. hVps34 is a nutrient-regulated lipid kinase required for activation of p70 S6-kinase. J Biol Chem 2005 ; 280 : 33076 -82

39. Nobukuni T, Joaquin M, Roccio M, et al. Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proc Natl Acad Sci USA 2005 ; 102 : 14238 -43

40. Abraham RT. TOR signaling: an odyssey from cellular stress to the cell growth machinery. Curr Biol 2005 ; 15 : R139 -R141

41. Inoki K, Ouyang H, Zhu T, et al. TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell 2006 ; 126 : 955 -68

42. Gingras AC, Raught B, Sonenberg N. Regulation of translation initiation by FRAP/mTOR. Genes Dev 2001 ; 15 : 807 -26

43. Bjornsti MA, Houghton PJ. Lost in translation: dysregulation of cap-dependent translation and cancer. Cancer Cell 2004 ; 5 : 519 -23

• An overview of the functional connections between mTORC1 and the translational machinery.

44. Wendel HG, De Stanchina E, Fridman JS, et al. Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy. Nature 2004 ; 428 : 332 -7

45. Pullen N, Thomas G. The modular phosphorylation and activation of p70s6k. FEBS Lett 1997 ; 410 : 78 -82

46. Burnett PE, Barrow RK, Cohen NA, et al. RAFT1 phosphorylation of the translational regulators p70 S6 kinase

and 4E-BP1. Proc Natl Acad Sci USA 1998 ; 95 : 1432 -7

47. Isotani S, Hara K, Tokunaga C, et al. Immunopurifi ed mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro. J Biol Chem 1999 ; 274 : 34493 -8

48. Ruvinsky I, Meyuhas O. Ribosomal protein S6 phosphorylation: from protein synthesis to cell size. Trends Biochem Sci 2006 ; 31 : 342 -8

49. Kuo CJ, Chung J, Fiorentino DF, et al. Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase. Nature 1992 ; 358 : 70 -3

50. Holz MK, Ballif BA, Gygi SP, Blenis J. mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events. Cell 2005 ; 123 : 569 -80

51. Hosoi H, Dilling MB, Liu LN, et al. Studies on the mechanism of resistance to rapamycin in human cancer cells. Mol Pharmacol 1998 ; 54 : 815 -24

52. Kandel ES, Hay N. The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB. Exp Cell Res 1999 ; 253 : 210 -29

53. Simpson L, Parsons R. PTEN: life as a tumor suppressor. Exp Cell Res 2001 ; 264 : 29 -41

54. Saal LH, Johansson P, Holm K, et al. Poor prognosis in carcinoma is associated with a gene expression signature of aberrant PTEN tumor suppressor pathway activity. Proc Natl Acad Sci USA 2007 ; 104 : 7564 -9

55. Mills GB, Lu Y, Kohn EC. Linking molecular therapeutics to molecular diagnostics: inhibition of the FRAP/RAFT/TOR component of the PI3K pathway preferentially blocks PTEN mutant cells in vitro and in vivo. Proc Natl Acad Sci USA 2001 ; 98 : 10031 -3

56. Wendel HG, Malina A, Zhao Z, et al. Determinants of sensitivity and resistance to rapamycin-chemotherapy drug combinations in vivo. Cancer Res 2006 ; 66 : 7639 -46

57. Podsypanina K, Lee RT, Politis C, et al. An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten(+/-) mice. Proc Natl Acad Sci USA 2001 ; 98 : 10320 -5

58. Neshat MS, Mellinghoff IK, Tran C, et al. Enhanced sensitivity of PTEN-defi cient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci USA 2001 ; 98 : 10314 -19

• Together with reference [57] , this paper provides evidence that early-stage loss of PTEN confers tumor cell sensitivity to rapamycin and its derivatives.

59. Hernando E, Charytonowicz E, Dudas ME, et al. The AKT-mTOR pathway plays a critical role in the development of leiomyosarcomas. Nat Med 2007 ; 13 : 748 -53

60. Chang SM, Wen P, Cloughesy T, et al. Phase II study of CCI-779 in patients with recurrent glioblastoma multiforme. Invest New Drugs 2005 ; 23 : 357 -61

61. Galanis E, Buckner JC, Maurer MJ, et al. Phase II trial of temsirolimus (CCI-779) in recurrent glioblastoma multiforme: a North Central Cancer Treatment Group Study. J Clin Oncol 2005 ; 23 : 5294 -304

62. Margolin K, Longmate J, Baratta T, et al. CCI-779 in metastatic melanoma: a Phase II trial of the California Cancer Consortium. Cancer 2005 ; 104 : 1045 -8

63. Chin L, Tam A, Pomerantz J, et al. Essential role for oncogenic Ras in tumour maintenance. Nature 1999 ; 400 : 468 -72

64. Baak JP, Van Diermen B, Steinbakk A, et al. Lack of PTEN expression in endometrial intraepithelial neoplasia is correlated with cancer progression. Hum Pathol 2005 ; 36 : 555 -61

65. Oza AM, Elit EL, Biagi J, et al. Molecular correlates associated with a Phase II study of temsirolimus (CCI-779) in patients with metastatic or recurrent endometrial cancer – NCIC IND 160. J Clin Oncol 2006 ; 24 : 3003

66. Sun SY, Rosenberg LM, Wang X, et al. Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res 2005 ; 65 : 7052 -8

67. O’Reilly KE, Rojo F, She QB, et al. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 2006 ; 66 : 1500 -8

68. Skeen JE, Bhaskar PT, Chen CC, et al. Akt defi ciency impairs normal cell proliferation and suppresses oncogenesis in a p53-independent and mTORC1-dependent manner. Cancer Cell 2006 ; 10 : 269 -80

69. Sarbassov DD, Ali SM, Sengupta S, et al. Prolonged rapamycin treatment inhibits

Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y L

akeh

ead

Uni

vers

ity o

n 10

/27/

14Fo

r pe

rson

al u

se o

nly.

Abraham & Eng

Expert Opin. Ther. Targets (2008) 12(2) 221

mTORC2 assembly and Akt/PKB. Mol Cell 2006 ; 22 : 159 -68

• First report to defi ne an indirect effect of mTORC1 inhibitors on mTORC2 functions.

70. Frauwirth KA, Thompson CB. Regulation of T lymphocyte metabolism. J Immunol 2004 ; 172 : 4661 -5

71. Jin S, DiPaola RS, Mathew R, White E. Metabolic catastrophe as a means to cancer cell death. J Cell Sci 2007 ; 120 : 379 -83

• Raises the possibility that cancer cell metabolism can be targeted with inhibitors of mTOR and other agents to trigger autophagic cell death.

72. Plas DR, Thompson CB. Akt-dependent transformation: there is more to growth than just surviving. Oncogene 2005 ; 24 : 7435 -42

73. Fuchs BC, Bode BP. Amino acid transporters ASCT2 and LAT1 in cancer: partners in crime? Semin Cancer Biol 2005 ; 15 : 254 -66

74. Edinger AL, Cinalli RM, Thompson CB. Rab7 prevents growth factor-independent survival by inhibiting cell-autonomous nutrient transporter expression. Dev Cell 2003 ; 5 : 571 -82

75. Rubinsztein DC, Gestwicki JE, Murphy LO, Klionsky DJ. Potential therapeutic applications of autophagy. Nat Rev Drug Discov 2007 ; 6 : 304 -12

76. Pattingre S, Tassa A, Qu X, et al. Bcl-2 antiapoptotic proteins inhibit beclin 1-dependent autophagy. Cell 2005 ; 122 : 927 -39

• Provides evidence for a direct interaction between the autophagic and apoptotic machinery in mammalian cells.

77. Iwamaru A, Kondo Y, Iwado E, et al. Silencing mammalian target of rapamycin signaling by small interfering RNA enhances rapamycin-induced autophagy in malignant glioma cells. Oncogene 2007 ; 26 : 1840 -51

78. Kim KW, Mutter RW, Cao C, et al. Autophagy for cancer therapy through inhibition of pro-apoptotic proteins and mammalian target of rapamycin signaling. J Biol Chem 2006 ; 281 : 36883 -90

• This paper suggests that natural or therapy-induced defects in apoptosis render tumor cells more sensitive to inhibitors of autophagy.

79. Karantza-Wadsworth V, Patel S, Kravchuk O, et al. Autophagy mitigates metabolic stress and genome damage

in mammary tumorigenesis. Genes Dev 2007 ; 21 : 1621 -35

• Demonstrates a protective role for autophagy during environment- or therapy-associated stress.

80. Levine B. Cell biology: autophagy and cancer. Nature 2007 ; 446 : 745 -7

81. Cuervo AM. Autophagy: in sickness and in health. Trends Cell Biol 2004 ; 14 : 70 -7

82. Jin S, White E. Role of autophagy in cancer: management of metabolic stress. Autophagy 2007 ; 3 : 28 -31

83. Bissell MJ, Radisky D. Putting tumours in context. Nat Rev Cancer 2001 ; 1 : 46 -54

84. Bierie B, Moses HL. Tumour microenvironment: TGFbeta: the molecular Jekyll and Hyde of cancer. Nat Rev Cancer 2006 ; 6 : 506 -20

85. Langowski JL, Zhang X, Wu L, et al. IL-23 promotes tumour incidence and growth. Nature 2006 ; 442 : 461 -5

86. Nguyen DM, Sam K, Tsimelzon A, et al. Molecular heterogeneity of infl ammatory breast cancer: a hyperproliferative phenotype. Clin Cancer Res 2006 ; 12 : 5047 -54

87. Guba M, von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 2002 ; 8 : 128 -35

• Striking visual documentation of the anti-vascular effects of rapamycin.

88. Liu M, Howes A, Lesperance J, et al. Antitumor activity of rapamycin in a transgenic mouse model of ErbB2-dependent human breast cancer. Cancer Res 2005 ; 65 : 5325 -36

• A mechanistic study of mTORC1 inhibitor activity in a murine model of breast cancer.

89. Phung TL, Ziv K, Dabydeen D, et al. Pathological angiogenesis is induced by sustained Akt signaling and inhibited by rapamycin. Cancer Cell 2006 ; 10 : 159 -70

90. Frost P, Shi Y, Hoang B, Lichtenstein A. AKT activity regulates the ability of mTOR inhibitors to prevent angiogenesis and VEGF expression in multiple myeloma cells. Oncogene 2007 ; 26 : 2255 -62

91. Semenza GL. Hypoxia and cancer. Cancer Metastasis Rev 2007 ; 26 : 223 -4

92. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003 ; 3 : 721 -32

93. Laughner E, Taghavi P, Chiles K, et al. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1 alpha (HIF-1 alpha) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol 2001 ; 21 : 3995 -4004

94. Thomas GV, Tran C, Mellinghoff IK, et al. Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer. Nat Med 2006 ; 12 : 122 -7

95. Hudson CC, Liu M, Chiang GG, et al. Regulation of hypoxia-induced factor-1 expression and function by the mammalian target of rapamycin. Mol Cell Biol 2002 ; 22 : 7004 -14

96. Atkins DJ, Gingert C, Justenhoven C, et al. Concomitant deregulation of HIF1alpha and cell cycle proteins in VHL-mutated renal cell carcinomas. Virchows Arch 2005 ; 447 : 634 -42

97. Hudes G, Carducci M, Tomczak P, et al. Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med 2007 ; 356 : 2271 -81

98. Haase VH. The VHL/HIF oxygen-sensing pathway and its relevance to kidney disease. Kidney Int 2006 ; 69 : 1302 -7

99. Beuvink I, Boulay A, Fumagalli S, et al. The mTOR inhibitor RAD001 sensitizes tumor cells to DNA-damaged induced apoptosis through inhibition of p21 translation. Cell 2005 ; 120 : 747 -59

100. Faried LS, Faried A, Kanuma T, et al. Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin increases chemosensitivity of CaSki cells to paclitaxel. Eur J Cancer 2006 ; 42 : 934 -47

101. Wilhelm S, Carter C, Lynch M, et al. Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat Rev Drug Discov 2006 ; 5 : 835 -44

102. Molhoek KR, Brautigan DL, Slingluff CL Jr. Synergistic inhibition of human melanoma proliferation by combination treatment with B-Raf inhibitor BAY43-9006 and mTOR inhibitor rapamycin. J Transl Med 2005 ; 3 : 39

103. Hosoi H, Dilling MB, Shikata T, et al. Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells. Cancer Res 1999 ; 59 : 886 -94

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Mammalian target of rapamycin as a therapeutic target in oncology

222 Expert Opin. Ther. Targets (2008) 12(2)

Affi liation Robert T Abraham † PhD & Christina H Eng PhD †Author for correspondence Oncology Discovery Research, Wyeth, 401 N. Middletown Road, Pearl River, NY 10965, USA Tel: +1 845 602 4595 ; Fax: +1 845 602 5557 ; E-mail: abrahar@wyeth.com

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