Rapamycin: An anti-cancer immunosuppressant?

Critical Reviews in Oncology/Hematology 56 (2005) 47–60

Rapamycin: An anti-cancer immunosuppressant? Brian K. Law ∗ Department of Pharmacology and Therapeutics and the Shands Cancer Center, University of Florida, P.O. Box 100267, R5-136, ARB, 1600 SW Archer Road, Gainesville, FL 32610, USA Accepted 24 September 2004

Contents 1. 2.

3. 4.

5.

6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mTOR mediates control of the cell cycle by nutrient levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. TOR and mTOR regulate cell proliferation in response to nutrient availability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. The mTOR signaling network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. mTOR regulation of the mammalian cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rapamycin as an immunosuppressant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rapamycin and rapamycin analogs as anti-cancer agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. The mTOR signaling pathway is activated in cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Rapamycin anti-tumor actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Rapamycin inhibition of angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future directions: harnessing the anti-cancer potential of mTOR inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. The mTOR pathway as a target of multiple existing anti-cancer agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Potential synergy between mTOR inhibitors and other anti-cancer agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

48 48 48 48 49 50 51 51 52 53 53 53 53 54 54 54 60

Abstract Rapamycin and its derivatives are promising therapeutic agents with both immunosuppressant and anti-tumor properties. These rapamycin actions are mediated through the specific inhibition of the mTOR protein kinase. mTOR serves as part of an evolutionarily conserved signaling pathway that controls the cell cycle in response to changing nutrient levels. The mTOR signaling network contains a number of tumor suppressor genes including PTEN, LKB1, TSC1, and TSC2, and a number of proto-oncogenes including PI3K, Akt, and eIF4E, and mTOR signaling is constitutively activated in many tumor types. These observations point to mTOR as an ideal target for anti-cancer agents and suggest that rapamycin is such an agent. In fact, early preclinical and clinical studies indicate that rapamycin derivatives have efficacy as anti-tumor agents both alone, and when combined with other modes of therapy. Rapamycin appears to inhibit tumor growth by halting tumor cell proliferation, inducing tumor cell apoptosis, and suppressing tumor angiogenesis. Rapamycin immunosuppressant actions result from the inhibition of T and B cell proliferation through the same mechanisms that rapamycin blocks cancer cell proliferation. Therefore, one might think that rapamycin-induced immunosuppression would be detrimental to the use of rapamycin as an anti-cancer agent. To the contrary, rapamycin decreases the frequency of tumor formation that occurs in organ transplant experiments when combined with the widely used immunosuppressant cyclosporine compared with the tumor incidence observed when cyclosporine is used alone. The available evidence indicates that with respect to tumor growth, rapamycin anti-cancer activities are dominant over rapamycin immunosuppressant effects. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Rapamycin; RAD001; CCI-779; mTOR; Cancer; Cell cycle; Cyclin-dependent kinase; Immunosuppressant ∗

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B.K. Law / Critical Reviews in Oncology/Hematology 56 (2005) 47–60

1. Introduction

2.2. The mTOR signaling network

Rapamycin is perhaps best known as a potent immunosuppressive agent. It is unclear from this vantage point what utility rapamycin might have as an anti-cancer agent. This perspective however stems from the historical development of rapamycin use rather than the intrinsic nature of rapamycin action. The aim of this review is to rationalize how rapamycin is able to act both as an immunosuppressive agent and an anticancer agent. Rapamycin was first identified as an anti-fungal agent produced by the bacterium Streptomyces hygroscopicus [1,2] and was subsequently demonstrated to be a potent immunosuppressive agent [3–6]. The target of rapamycin (TOR) was identified in a screen of yeast mutants able to proliferate in the presence of rapamycin [7]. A mammalian protein homologous to TOR (mTOR) was isolated later based on its ability to bind the FK506 binding protein FKBP12 [8–10]. TOR bound to FKBP12 in the presence of rapamycin, but not in the presence of the related immunosuppressant bacterial macrolide FK506. mTOR is a 289 kDa protein that is evolutionarily related to lipid kinases, but exhibits protein serine/threonine kinase activity (reviewed in [11]).

It has long been known that FKBP12 is an intracellular receptor for rapamycin [29], that the FKBP12–rapamycin complex binds to TOR/mTOR [7–10], and that the formation of this ternary complex inhibits the intrinsic kinase activity of mTOR toward exogenous substrates [30–33]. It is also well known that p70s6k and 4EBP1 are two critical downstream targets of mTOR signaling and that rapamycin is a dominant inhibitor of p70s6k and 4EBP1 function [34–37]. However, the mechanisms by which mTOR is regulated, and in turn regulates downstream signaling, has largely been a “black box” until quite recently. Raptor was the first protein shown to bind directly to mTOR that is required to mediate mTOR regulation of p70s6k and 4EBP1 activities [38,39]. Strikingly, Raptor forms nutrient-sensitive complexes with mTOR and another protein, GßL, that is required for the nutrient-sensitive interaction between Raptor and mTOR [40]. In addition to participating in a nutrient-sensing complex, Raptor acts as a scaffolding protein that facilitates mTOR-dependent phosphorylation of p70s6k and 4EBP1 by binding the evolutionarily conserved TOR signaling motifs (TOS) present in both p70s6k and 4EBP1 [41,42]. Together, these studies provide the first mechanistic glimpses into the basis for the nutrient sensitivity of the mTOR signaling network and how mTOR is coupled to the downstream effectors p70s6k and 4EBP1. The mTOR signaling network as it is currently understood (Fig. 1) involves a large number of different signaling intermediates. The upstream regulators of mTOR fall into at least two independent signaling cascades. The first involves the lipid kinase phosphatidylinositol 3-kinase (PI3K), the phosphoinositide-dependent protein kinase 1 (PDK1), the proto-oncogenic protein kinase Akt, the tumor suppressive kinase LKB1, and the AMP-dependent protein kinase (AMPK), the tumor suppressive tuberous sclerosis complex proteins 1 and 2 (TSC1/TSC2), and the Ras related Rheb GTPase. The second cascade involves the small GTPase Cdc42 and phospholipase D1 (PLD1).

2. mTOR mediates control of the cell cycle by nutrient levels 2.1. TOR and mTOR regulate cell proliferation in response to nutrient availability Cell proliferation must be regulated such that cell division occurs only when adequate levels of all necessary nutrients are available. However, despite detailed knowledge of how cells respond to growth factors, relatively little is known concerning how cells respond to changes in nutrient levels. In budding yeast, the TOR proteins mediate responses to levels of nutrients such as nitrogen [12,13]. In mammals, mTOR integrates signals and mediates biological responses to growth factors and mitogens [14,15], amino acid levels [16–20], phosphatidic acid levels [21], ATP levels [22], inorganic polyphosphate levels [23], and AMP levels [24,25]. The observation that TOR homologs are responsive to nutrients in both yeast and metazoans suggests that this function of TOR is conserved throughout eukaryotic evolution, and that the TOR proteins play a fundamental role in coupling nutrient availability with cell cycle regulation. In this sense, mTOR can be considered to act as a key component of a nutrient-responsive cell cycle regulatory signaling pathway. Interestingly, all other members of the PIKK family, including ATM and ATR [26–28], act in some capacity as genome/transcriptome surveillance proteins with mTOR being the only direct participant in mitogenic signaling pathways. Since the focus of this review is on rapamycin as an immunosuppressant and anti-cancer agent, subsequent sections will focus on mTOR and its biological functions in cells of the immune system and cancer cells.

Fig. 1. The mTOR cell signaling network.

B.K. Law / Critical Reviews in Oncology/Hematology 56 (2005) 47–60

PI3K is activated by a number of receptor tyrosine kinases and catalyzes the conversion of phosphatidylinositol bisphosphate (PIP2 ) to PIP3 by phosphorylating the 3 position of the inositol ring. Consistent with the notion that PI3K plays a critical role in regulating cell proliferation, the enzyme that catalyzes the dephosphorylation of PIP3 at the 3 position, the phosphatase and Tensin homologous protein (PTEN), is encoded by a well known tumor suppressor gene whose expression is lost in a number of human cancers. PIP3 residing in the plasma membrane docks PDK1 and Akt to the membrane through their respective pleckstrin homology (PH) domains. While colocalized at the membrane, PDK1 phosphorylates and activates Akt. The activity of TSC1/TSC2 is in turn regulated via inhibition by Aktmediated phosphorylation [43,44]. The activity of TSC2 is dominantly inhibited by AMPK-mediated phosphorylation independent of the level of Akt activity [25]. AMPK is activated coordinately by LKB1-dependent phosphorylation and by increased levels of AMP during times of metabolic stress [45–48]. The TSC1 and TSC2 proteins are also referred to as Hamartin and Tuberin, respectively. Several recent reports have elucidated the role that TSC1/TSC2 play in mTOR regulation. Rheb GTPase activity is regulated by the GTPase activating protein (GAP) function of TSC2 as part of the TSC1/TSC2 complex [49–51]. Active GTP-bound Rheb stimulates mTOR kinase activity through mechanisms that are not completely characterized [49,50,52]. In a parallel pathway, GTP-bound Cdc42 binds and activates PLD1 [53]. PLD1 in turn catalyzes the production of phosphatidic acid. Phosphatidic acid stimulates mTOR kinase activity and the phosphorylation of its downstream effectors [21,54–56]. The most well studied downstream effectors of mTOR are eiF4E binding protein 1 (4EBP1) [57,58], which was independently discovered and termed phosphorylated heat and acid stable protein-I (PHAS-I) [59–61], and the protein kinase, p70s6k . 4EBP1 binds and inhibits the activity of the translation factor eiF4E. eiF4E recognizes the 5 -7-Me-GTP cap of mRNAs and is thus required for cap-dependent translation (reviewed in [11]). Growth factor stimulation or stimulation of cells with high amino acid levels induces the multi-site phosphorylation of 4EBP1, resulting in the release of eiF4E and the activation of protein synthesis. Importantly, several 4EBP1 phosphorylation sites are directly phosphorylated by mTOR, and phosphorylation of these sites is inhibited by rapamycin [32,58,62]. p70s6k was one of the earliest characterized protein kinases [63–67] and its activity is strongly stimulated by growth factors including insulin and PDGF [68], and nutrients such as amino acids [16–19]). p70s6k activation in response to all of these stimuli is blocked by rapamycin, implicating p70s6k as a downstream effector of mTOR. It is still a matter of debate as to whether p70s6k is regulated by mTOR directly, indirectly, or both. p70s6k regulation is complex and involves the phosphorylation of a number of different sites by several kinases

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including PDK1 [69,70], Cdc2 [71], and likely mTOR itself [41,72]. Interestingly, a large number of protein kinases, including p70s6k , require PDK1-dependent phosphorylation for activation. Thus, p70s6k is regulated directly by PDK1-dependent phosphorylation at Thr229 [69] and indirectly through PDK1dependent activation of the Akt–TSC1/TSC2–Rheb–mTOR axis. Several p70s6k phosphorylation sites are rapamycin sensitive and therefore regulated via mTOR, however, it has been argued that the Thr389 phosphorylation site is the primary rapamycin-sensitive phosphorylation site [73]. Once activated, p70s6k phosphorylates several proteins that regulate translation and RNA splicing including the ribosomal S6 protein and the CBP80 cap-binding protein [74]. Since 4EBP1 and p70s6k participate in controlling protein synthesis, and amino acid levels control protein synthesis by regulating the mTOR signaling pathway, the well accepted model has emerged that rapamycin acts primarily by inhibiting mTOR protein kinase activity, resulting in the inhibition of protein synthesis. While this model is accurate, it is likely an oversimplification. Multiple PKC isoforms appear to serve as effectors of mTOR and to participate in the regulation of other mTOR downstream effectors, including p70s6k and 4EBP1. An early report indicated that phorbol ester-sensitive PKC isoforms play a role in p70s6k activation by EGF [75]. More recently, the novel PKC isoforms ␦ and ␧ were shown to have regulatory phosphorylation sites whose phosphorylation is inhibited by rapamycin treatment, including Ser662 of PKC␦ [76]. Intriguingly, PKC␦ interacts physically with mTOR and this interaction is required for mTOR-dependent regulation of 4EBP1 phosphorylation [77]. The atypical PKC isoforms PKC␭ and PKC␨ exist in complexes with p70s6k and PDK1, and kinase dead mutants of PKC␭ or PKC␨ antagonize p70s6k activation [78]. PKC␨ also phosphorylates PKC␦ on the rapamycin sensitive site Ser662 [79], suggesting that novel and atypical PKC isoforms collaborate in their participation in the mTOR signaling network. These observations suggest that PKC isoforms play an important, but largely unexplored role in signaling downstream of mTOR. Other recent reports have indicated that the transcriptional activity of STATs 1 [80] and 3 [81,82] are also regulated by mTOR-dependent phosphorylation, and that rapamycin regulates Pol I-dependent transcription [83]. Thus, there is likely still much to learn about the mechanisms of rapamycin action and the mTOR signaling pathway. 2.3. mTOR regulation of the mammalian cell cycle The entry of eukaryotic cells into the cell cycle is controlled primarily through the activation of the cyclindependent kinases (Cdks) Cdk4/Cdk6 and Cdk2 by their respective cyclin partners Cyclins D1, D2, D3, and E and A (Fig. 2). Increased expression of the D-type cyclins is a rapid response to growth factor stimulation and is mediated by several different signaling pathways including the Ras/Map

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B.K. Law / Critical Reviews in Oncology/Hematology 56 (2005) 47–60

Fig. 2. Rapamycin down-regulates proteins that drive cell proliferation.

kinase cascade and the PI3K/PDK1/Akt/GSK3 cascade (reviewed in [84]). Growth factor-induced D-type cyclins form catalytically active kinase complexes with Cdk4 and Cdk6 and initiate phosphorylation and inactivation of the tumor suppressor Rb. Partial phosphorylation of Rb allows some E2F-dependent transcription that results in the expression of Cyclins E and A. Cyclin E forms complexes with Cdk2 causing more complete phosphorylation and inactivation of Rb, leading to irreversible entry into the S-phase of the cell cycle. Given the potent anti-proliferative effects of rapamycin, it is not surprising that the mTOR signaling pathway regulates the levels of several proteins directly involved in controlling cell division. In a number of studies, rapamycin has been shown to inhibit the expression of Cyclins D1 [85–88] and D3 [89,90] and to suppress c-Myc translation [91]. In hepatocytes, rapamycin-induced cell cycle arrest is mediated by Cyclin D1 down-regulation [92]. Likewise, Cyclin A and PCNA expression are suppressed by rapamycin [88,93–97], although the mechanisms involved in this suppression are unclear. Recently, Cyclin E expression was shown to be under the control of p70s6k and therefore under the control of mTOR signaling [98]. It is not known to what extent changes in the levels of these individual proteins contribute to rapamycininduced cell cycle arrest, and which changes are causal to cell cycle arrest or a result of cell cycle arrest. The observation that rapamycin inhibits E2F-dependent transcription [78,99,100] and that several rapamycin-suppressed genes are E2F-dependent, including Cyclins E, A, and PCNA, suggests that rapamycin suppression of the levels of these proteins may be at least partially a result of cell cycle arrest. The cyclin-dependent kinase inhibitor proteins, p21 and p27, are also downstream effectors of rapamycin action and may mediate rapamycin cell cycle effects. Rapamycin inhibition of T cell mitogenesis correlates with decreased p21 expression and increased p27 expression, p27 association with Cdk2, and Cdk2 inhibition [101]. Subsequent studies have demonstrated that rapamycin inhibits increases in p21

levels in response to phorbol esters [102], vascular endothelial cell growth factor (VEGF) [103], and serum [104]. The interpretation of these results is complicated by the fact that p21 and p27 act as cyclin-dependent kinase “inhibitors” by blocking kinase activity [105–111], but also facilitate the assembly and nuclear localization during the maturation of cyclin-dependent kinase complexes and thus can also act as cyclin-dependent kinase “activators” [112–115]. The degree to which p21 or p27 serve as an “activator” or “inhibitor” may depend on the levels of p21 and p27 relative to the levels of the relevant cyclin-dependent kinases. This might explain why phorbol ester-induced increases in p21 is associated with growth arrest in human venous endothelial cells [102], while rapamycin-induced decreases in p21 levels in BP-A31 fibroblasts is associated with growth arrest [104]. Thus, with respect to p21, rapamycin induces p21 downregulation that may have differing effects on proliferation depending on the cellular context. As is the case with p21, the role of p27 in rapamycin action is complex. T cells from p27 null mice have impaired growth inhibitory responses to rapamycin, and rapamycin blocks serum-induced decreases in p27 levels [116]. Consistent with rapamycin down-regulating p27, Tsc2 null fibroblasts, that exhibit constitutively active mTOR signaling (Fig. 1), have very low levels of p27 expression and exhibit p27 mislocalization to the cytoplasm [117]. p27 may also be regulated by rapamycin through indirect mechanisms. It has been proposed that Cyclin D–Cdk complexes act as a “sink” to bind p27 and prevent it from binding and inhibiting Cdk2 [118–120]. Since rapamycin down-regulates Cyclins D1 and D3 [87–90,121,122], it has been suggested that rapamycin also inhibits cell proliferation by removing the p27 sink and allowing p27 to bind and inactivate Cdk2 [87]. Thus, p21 and p27 play important roles in rapamycin cell cycle effects but likely do so in a context-dependent manner. Rapamycin has been assumed to induce cell cycle arrest through the down-regulation of Cyclins D1, E, and A, inactivation of their corresponding cyclin-dependent kinase partners, and by decreasing Rb phosphorylation. Interestingly, rapamycin inhibition of cell proliferation does not always correlate with dramatic decreases in Rb phosphorylation. This lack of decreased Rb phosphorylation might be explained by Cdk-independent functions of the cyclins. Cyclin E, for example, has been proposed to stimulate proliferation through a Cdk-independent mechanism [123], and Cyclin D1 interacts with and regulates a number of different transcription factors including DMP1 and TAF(II)250 [124–126]. Therefore, rapamycin likely inhibits cell proliferation through both Cdk-dependent and Cdk-independent mechanisms.

3. Rapamycin as an immunosuppressant Shortly after it was first described as an anti-fungal antibiotic [1,2] rapamycin was demonstrated to have

B.K. Law / Critical Reviews in Oncology/Hematology 56 (2005) 47–60

immunosuppressant properties as indicated by its ability to inhibit experimental allergic encephalomyelitis, adjuvant arthritis, and the humoral (IgE) immune response [127]. Several years later, rapamycin was shown to inhibit tumor growth in xenograft models [128], an observation seemingly inconsistent with rapamycin acting as an immunosuppressant. The observation that rapamycin shares structural similarities with a known immunosuppressant, FK506, probably led to the early exploration of the immunosuppressive actions of rapamycin. Studies demonstrating that rapamycin inhibits renal rejection in pig allograft models [4] led to cell biological assays showing that rapamycin suppresses T and B cell activation [129,130] and proliferation [131]. A recent report showing that rapamycin induces dendritic cell apoptosis by interfering with GM–CSF signaling suggests that rapamycin not only inhibits T cell activation directly, but may also decrease the population of dendritic cells that present antigen to T cells during activation [132]. Upon investigating the mechanisms of action of FK506 and rapamycin as immunosuppressive agents, it was observed that despite the structural similarity between FK506 and rapamycin, the two drugs antagonize each other’s binding to cells and therefore inhibit each other’s biological function [133]. These results indicate that FK506 and rapamycin bind to the same intracellular receptor(s), but elicit their actions through distinct mechanisms. The intracellular binding protein for FK506 and rapamycin in T cells was termed FK506 binding protein (FKBP). The rapamycin–FKBP12 complex inhibits the activity of the mTOR protein kinase (see Section 2.2), while the FK506–FKBP12 complex inhibits the activity of the protein phosphatase calcineurin [134,135]. mTOR inhibition by rapamycin results in the blockade of interleukin-2 (IL-2)-dependent activation of p70s6k and T cell mitogenesis [136,137], while calcineurin inhibits T cell activation by blocking IL-2 production [138]. Thus, nature has designed two related bacterial macrolides that act at distinct steps to abrogate T cell activation. The effectiveness of rapamycin as an immunosuppressant likely rests on the fact that IL-2-dependent T cell proliferation is selectively dependent on mTOR signaling to drive T cell mitogenesis [136]. Moreover, as discussed in Section 5.2 below, several different oncogenic lesions may render cancer cells dependent on the mTOR signaling for proliferation, cell survival, and angiogenesis. Further, as reviewed in Section 2.1, the mechanistic basis for rapamycin action is conserved from Saccharomyces cerevisiae to man. Thus, rapamycin

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cannot be viewed simply as an immunosuppressant but must be viewed as an inhibitor of an evolutionarily conserved pathway that couples nutrient sensing and growth factor signaling with cell proliferation. Rapamycin blockade of the mTOR signaling cascade can be viewed as mimicking nutrient deprivation, resulting in cell cycle arrest [139]. Cancer is fundamentally a disease of deregulated cell division suggesting that a cytostatic agent such as rapamycin might have anti-cancer efficacy. A reasonable concern regarding the use of rapamycin as an anti-cancer therapy is that suppression of the immune response might accentuate rather than halt tumor growth. The available empirical evidence argues to the contrary. As described below (Section 4.2), in clinical and preclinical studies rapamycin inhibits the growth of a broad array of tumor types with no reports of rapamycin accentuating tumor growth. In studies using a renal adenocarcinoma model rapamycin inhibits the growth of tumors in both the presence and absence of cyclosporine [140]. This result suggests that combining rapamycin with cyclosporine for immunosuppressant therapy may prevent or suppress the growth of tumors that arise during immunosuppression using cyclosporine alone. Consistent with the anti-proliferative actions of rapamycin on both immune cells and cancer cells, rapamycin and rapamycin derivatives are used as anti-rejection immunosuppressive agents [141–145] and are also in clinical trials as anti-cancer agents (Section 4.2). Although the short-term studies performed to date suggest that the anti-cancer actions of rapamycin derivatives are dominant over the immunosuppressant actions of rapamycin with respect to tumor growth, it is still a concern that the immunosuppressant effects of rapamycin may prove deleterious over the long-term.

4. Rapamycin and rapamycin analogs as anti-cancer agents 4.1. The mTOR signaling pathway is activated in cancers A hallmark of human cancer is deregulation of the cell cycle [146]. Consistent with the notion that the mTOR pathway is a key regulator of cell proliferation, several upstream activators and downstream effectors of mTOR are deregulated in cancers (Table 1). The two most well studied mTOR effectors are p70s6k and 4EBP1. p70s6k is overexpressed in

Table 1 Constitutive activation of the mTOR signaling network in cancers mTOR pathway signaling intermediates

Biochemical function

Biological function

Alteration in cancers

p70s6k PTEN eiF4E TSC1/TSC2 LKB1

Protein kinase PIP3 phosphatase Translation factor Rheb GAP Protein kinase

Stimulates translation Tumor suppressor Stimulates translation; oncogene Tumor suppressor Tumor suppressor

Overexpression Loss of expression or mutational inactivation Overexpression Loss of expression or mutational inactivation Loss of expression or mutational inactivation

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breast cancers and is constitutively activated in several types of cancers [121,147,148]. Significantly, p70s6k overexpression correlates with poor prognosis [149,150]. As described above, 4EBP1 binds and inhibits the translation factor eiF4E and in so doing blocks the translation of messages containing a 5 -7-Me-GTP cap. Since 4EBP1 is a titratable inhibitor of eiF4E, it has been proposed [151] that the ratio of eiF4E to 4EBP1 present in cancers should be determined rather than examining the absolute amount of either protein. mTOR-dependent phosphorylation of 4EBP1 releases eiF4E allowing the translation of a number of proteins, including proteins that stimulate proliferation such as Cyclin D1 [152] and c-Myc [153]. Thus, one way by which a cancer might bypass 4EBP1-mediated negative regulation is through the overexpression of eiF4E such that eiF4E is in molar excess of 4EBP1. Consistent with this idea, eiF4E is overexpressed in a number of tumor types including cancers of the colon [154], breast [155,156], and head and neck [157]. Cell culture experiments demonstrate that eiF4E overexpression is sufficient to transform cells [158], and that co-expression of 4EBP1 reverses eiF4E-induced transformation [159]. Additionally, eIF4E is overexpressed in a wide array of cancer cell lines [160]. Thus, the two most well studied mTOR downstream effectors, p70s6k and 4EBP1, are deregulated in cancers and likely contribute to the process of tumorigenesis. At least three tumor suppressors that act as negative upstream regulators of mTOR, LKB1, PTEN, and TSC1/TSC2, are deregulated in cancers and may contribute to tumorigenesis through inappropriate mTOR activation (Fig. 1). TSC1/TSC2 were discovered as genes whose inactivation gives rise to nonmalignant tumors called hamartomas [161–163]. Thus, by definition TSC1 and TSC2 are tumor suppressor genes. PTEN is a potent tumor suppressor gene that is inactivated in tumors of multiple organs including cancers of the prostate, brain (glioblastoma), thyroid, ovaries, lung, kidney, endometrium, and bladder [164–174]. Furthermore, genetic inactivation of PTEN in mice results in the formation of multiple types of cancers [175–181]. Similarly, the tumor suppressor LKB1 is inactivated in the Peutz–Jeghers hereditary cancer syndrome [182–184]. Additionally, inactivation of the LKB1 gene by somatic mutation [185–190], and epigenetic inactivation [191] have been observed. LKB1 inactivation is thought to contribute to the formation of melanoma [186,187], pancreatic and biliary cancer [185], ovarian cancer [188], lung cancer [189,190], and breast cancer [192]. LKB1 likely plays a causal role in driving tumorigenesis because heterozygous inactivation of LKB1 in mice results in hepatocellular carcinoma, and the tumors formed exhibit loss of the remaining LKB1 allele [193]. Together, these observations indicate that the mTOR signaling pathway frequently becomes inappropriately activated during tumor progression and contributes to tumorigenesis by deregulating cancer cell proliferation. Thus, targeting signaling through the mTOR pathway with rapamycin derivatives is a promising approach for inhibiting tumor growth.

4.2. Rapamycin anti-tumor actions Only recently has the anti-tumor potential of rapamycin become widely appreciated. Early reports [128,194] demonstrated rapamycin anti-tumor efficacy in glioma and colorectal carcinoma models. More recently, Wyeth-Ayerst Research and Novartis have developed rapamycin derivatives, CCI-779 and RAD001, respectively, as potential cancer therapeutics. CCI-779 and RAD001 are structurally similar to rapamycin and act through the same mechanisms as rapamycin. Anticancer efficacy of CCI-779, a drug formulated for intravenous use, has been demonstrated in preclinical models of rhabdomyosarcoma [195], medulloblastoma [196], and breast cancer [197]. A flurry of reports indicate that constitutive activation of the PTEN–PI3K–Akt axis renders several tumor types exquisitely sensitive to inhibition by CCI-779 [198–201]. The basis for the sensitizing effect of PTEN inactivation to rapamycin action was suggested in a recent report demonstrating that in cells with high levels of Akt activity rapamycin or CCI-779 potently represses the translation of Cyclin D1 and c-Myc in a very specific manner [202]. This result may also explain the rapamycin sensitivity of cells transformed by oncoproteins and growth factors including v-Src [203], Gli [204], PI3K and Akt [205], and EGF [206] that drive transformation and cell division in part by constitutive activation of the PI3K–Akt–mTOR axis. RAD001 is expected to have similar anti-cancer effects as rapamycin and CCI-779, and in fact potently inhibits the growth of pancreatic cancer in the CA20948 tumor model [207]. However, given the structural differences between RAD001, CCI-779, and rapamycin, along with the fact that RAD001 is formulated for oral administration, the pharmacokinetics of these three agents are likely to differ. The bioactivity of both RAD001 and CCI-779 is prolonged. A single dose of 10 mg/kg CCI-779 inhibits p70s6k activity in peripheral blood mononuclear cells (PBMCs) by greater than 80% 72 h after treatment [208]. CCI-779 effects on PBMC p70s6k activity paralleled effects observed in tumor tissue suggesting that p70s6k activity in PBMCs may be a useful surrogate marker for CCI-779 anti-tumor efficacy. Likewise, a dose of 5 mg/kg RAD001 inhibits p70s6k activity for at least 72 h, and levels of PBMC p70s6k activity serve as a useful surrogate marker for assessing RAD001 treatment schedules [207]. The results of two CCI-779 clinical trials were recently published. The first study [209] was a phase I clinical trial and involved weekly treatments of patients with refractory renal cell carcinoma with 25, 75, or 250 mg of CCI-779 weekly. Seven percent of patients exhibited an objective response and 26% of patients exhibited minor responses. It was observed that CCI-779 dosage did not influence efficacy or toxic side effects that included maculopapular rash, mucositis, asthenia, and nausea. The second study [210] was a randomized phase II clinical trial and involved weekly doses of CCI-779 ranging from 7.5 to 220 mg/m2 . This study reported toxicities similar to those observed in the first study and noted reversible thrombocytopenia as the main dose-limiting toxi-

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city. A partial response was observed in a patient with renal clear-cell carcinoma and a patient with breast adenocarcinoma. In summary, the availability of rapamycin derivatives and knowledge concerning their pharmacokinetics and useful surrogate endpoints for biological activity should rapidly accelerate the implementation of mTOR pathway inhibitors as anti-cancer therapeutics. 4.3. Rapamycin inhibition of angiogenesis In addition to the direct effects of rapamycin on tumor cells that are expected to vary from tumor to tumor, rapamycin also inhibits tumor angiogenesis [211]. Given the limited size that tumors can attain in the absence of their own vascular system [212], rapamycin-mediated inhibition of tumor angiogenesis would be expected to inhibit the growth of all solid tumors. Rapamycin inhibits tumor angiogenesis through at least two mechanisms. Rapamycin directly inhibits the proliferation of vascular endothelial cells driven by serum [103] and vascular endothelial growth factor (VEGF) [213]. Rapamycin also suppresses the induction of hypoxia-inducible factor 1␣ (HIF1) by growth factors and oncogenes [214–216], and by hypoxia [217]. HIF1 is a transcriptional regulator of VEGF expression. Thus, rapamycin inhibits vascular endothelial cell proliferation by both decreasing the expression of VEGF, and by inhibiting the mitogenesis of vascular endothelial cells in response to VEGF. Rapamycin also inhibits vascular smooth muscle cell proliferation and migration, likely through the same mechanisms that it inhibits the proliferation of cancer cells and immune cells (summarized in [218]). Coating cardiac stents with rapamycin could potentially revolutionize the use of cardiac stents by overriding their major limitation, restenosis [219,220].

5. Future directions: harnessing the anti-cancer potential of mTOR inhibitors 5.1. The mTOR pathway as a target of multiple existing anti-cancer agents As discussed above the PI3 kinase pathway and mTOR signaling are inextricably intertwined. To complicate matters, presumed PI3K inhibitors such as wortmannin and LY294004 also inhibit mTOR [31]. Thus, the antiproliferative, anti-cancer effects of PI3K inhibitors may be partially attributable to mTOR inactivation. We have shown that the aspirin metabolite salicylate inhibits p70s6k activation and induces many of the same biochemical cell cycle responses as rapamycin [88]. This suggests that some of the chemo-protective effects of salicylates may be due, in part, to suppression of p70s6k activity. We also demonstrated that the ability of the farnesyltransferase inhibitor (FTI) L-744,832 to inhibit p70s6k activity correlates best with its ability to induce cell cycle arrest [221,222].

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In retrospect this is not surprising since the small GTPase Rheb that regulates mTOR activity (Fig. 1) is a farnesylated protein, FTIs block Rheb function [223], and a farnesylationdefective Rheb mutant has a diminished ability to activate p70s6k [50]. Additionally, mTOR is activated by growth factors, so anti-cancer agents such as receptor tyrosine kinase inhibitors and receptor-specific neutralizing antibodies also likely act in part by blocking activation of mTOR. Likewise, anticancer agents that induce or mimic amino acid depletion such as asparaginase [224], and l-histidinol or l-leucinol [19] likely work in large part through inactivation of the mTOR pathway. Based on these examples, it is clear that in addition to rapamycin derivatives, several other classes of anti-cancer agents act in part through the inhibition of mTOR signaling. 5.2. Potential synergy between mTOR inhibitors and other anti-cancer agents Cancer is a complex disease resulting from the genetic and epigenetic alteration of multiple oncogenes and tumor suppressor genes. Therefore, inhibiting the growth and progression of tumors will likely require a multipronged approach that targets several cancer-promoting genetic alterations through a combination of mechanism-based signaling inhibitors and classical chemotherapeutic agents and radiotherapy. Along these lines, rapamycin is being tested in combination with conventional modes of cancer therapy. As discussed above, multiple receptor tyrosine kinases (RTKs) activate the mTOR signaling pathway through activation of the PI3 kinase pathway, suggesting that tumors harboring activated RTKs may depend on signaling through the mTOR pathway to drive cell proliferation. Consistent with this idea, rapamycin enhances the ability of the Bcr–Abl kinase inhibitor Imatinib to arrest the proliferation of Ba/F3 cells overexpressing Bcr–Abl, but has no effect on the parental cells [225]. Importantly, rapamycin in combination with Imatinib inhibits the growth of cells expressing Imatinib-resistant mutants of Bcr–Abl [226]. MEK inhibitors cooperate with rapamycin and RTK inhibitors (RTKIs) to halt the growth of tumor cells [226]. This might be expected based on a previous report demonstrating that the MEK inhibitor U0126 is more potent than the MEK inhibitor PD98059 in blocking K-Ras-mediated transformation, and this correlates with the ability of U0126 to block both p70s6k and Erk activation [227]. While PD98059 blocks only Erk activation, it cooperates with rapamycin to reverse K-Ras-mediated transformation. Rapamycin also cooperates with classical anti-cancer therapeutic approaches to inhibit tumor growth and progression. For example, rapamycin cooperates with radiation therapy to inhibit the growth of U87 xenografts [228]. Rapamycin also cooperates with DNA damaging therapeutic agents including cisplatin [196,229] and doxorubicin [200]. Significantly, rapamycin cooperates with doxorubicin to inhibit tumor

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growth even in cell lines rendered doxorubicin-resistant by PTEN inactivation [200]. As points of cross-talk between the mTOR signaling pathway and other pathways are discovered, and it is determined how these points of cross-talk influence tumor progression, it is likely that additional rationally chosen drug combinations involving rapamycin will be developed. Some signaling pathways/signaling elements where cross-talk with the mTOR signaling pathway has already been identified include: the TGFß signaling pathway [100,230], the p38 Map kinase pathway [221,231], STATs 1 and 3 [80–82], and various PKC isoforms [76,79].

[2]

[3] [4] [5] [6]

[7]

6. Conclusions Rapamycin targets an evolutionarily conserved nutrientresponsive cell cycle regulatory pathway. Thus, the potent cytostatic nature of rapamycin stems from its ability to trigger a nutrient deprivation-like response. The growth inhibitory properties of rapamycin prompted researchers to explore the potential anti-cancer properties of rapamycin. It is clear from the discussion above that rapamycin is both a potent immunosuppressant and a promising anti-cancer agent. The anti-tumor efficacy of rapamycin likely stems from the fact that even though it suppresses the immune system, rapamycin inhibits tumor cell proliferation and angiogenesis and promotes tumor cell apoptosis. Stated another way, the effect of rapamycin on tumor growth and progression outweighs the simultaneous suppression of the immune system. Judging by the reports published to date demonstrating rapamycin inhibition of tumor growth, rapamycin derivatives are poised to become important weapons in the war against cancer both alone and in combination with other treatment modalities.

[8]

[9]

[10]

[11] [12]

[13]

[14] [15]

[16]

Reviewers Dr. Iduna Fichtner, Max-Delbr¨uck-Centrum for Molecular Medicine, Experimental Pharmacology, Robert-R¨ossle-Str. 10, D-13125 Berlin-Buch, Germany. Prof. Jaap Verweij, Department of Oncology, Erasmus University Medical Center, Postbus 5201 (Groene Hilledijk 301), NL-3008AE Rotterdam, The Netherlands. Joon-Ho Sheen, Ph.D., Sabatini Lab, Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142-1479, USA. Robert T. Abraham, Ph.D., Professor and Director, Cancer Research Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.

[17]

[18]

[19]

[20]

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Biography Brian Law obtained his Ph.D. in biochemistry in 1996 from Purdue University, West Lafayette, IN, USA. He is currently an assistant professor in the Department of Pharmacology and Therapeutics at the University of Florida, Gainesville, FL, USA.