Rapamycin and tumor growth: mechanisms behind its anticancer activity

Transplantation Reviews 19 (2005) 20 – 31 www.elsevier.com/locate/trre

Rapamycin and tumor growth: mechanisms behind its anticancer activityB Gudrun E. Koehl, Hans J. Schlitt, Edward K. GeisslerT Department of Surgery, University of Regensburg, 93053 Regensburg, Germany

Abstract In the past few years, a heightened awareness has developed toward the problem of cancer occurrence and treatment in organ transplant recipients under immunosuppression. Treatment and prevention of cancer in transplant patients with an intentionally suppressed immune system are generally not considered an ideal situation because intact immunity is important for recognizing and destroying potentially neoplastic cells. Nonetheless, recent studies indicate that fighting tumors is not impossible under conditions of immune suppression, and in fact, immunosuppressive agents have been discovered that possess potent anticancer effects. In particular, one class of immunosuppressants, referred to as mammalian target of rapamycin (mTOR) inhibitors (mTORi), has experimentally shown an ability to suppress the immune system to protect allografts from rejection while simultaneously inhibiting tumor growth. To gain a better understanding of this dual effect, we will review the key intracellular signaling pathways controlled by mTOR. We will discuss how mTORi affects the growth and survival of a variety of nonimmune cells, with a special emphasis on cancer. A better understanding of mTOR-related pathways and the repertoire of normal and neoplastic cells affected by mTORi will likely improve our ability to treat transplant rejection, cancer, and other pathological conditions. D 2005 Elsevier Inc. All rights reserved.

1. Introduction Cancer in patients after organ transplantation is a wellknown life-threatening complication [1]. Malignancies develop in those immunosuppressed patients at a younger age and tend to be more aggressive. The incidence of cancer increases dramatically with time after transplantation [2- 4]. While the risk for cancer is generally increased, certain types of cancer including lymphomas, nonmelanoma skin tumors, and buncommonQ tumors, such as Kaposi sarcoma, are much more prevalent [3,5]. Depending on the type of organ transplantation, the risk for posttransplant non-Hodgkin lymphomas is 12- to 240-fold increased (for kidney and heart/lung transplant patients, respectively [6-8]). The most common malignancy is skin cancer [9]. Not surprisingly the skin cancer rate is exceptionally high in Australia and New Zealand [10]. Other types of cancer also tend to vary geographically [3-5]. For instance, in western countries Kaposi sarcoma has a high incidence in transplant patients,

B

This work has been supported by the Roche Organ Transplantation Research Foundation. T Corresponding author. Tel.: +49 941 6964; fax: +49 941 944 6886. E-mail address: [email protected] (E.K. Geissler). 0955-470X/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.trre.2005.01.001

whereas it is basically undetected in Japan. Japan in turn has a high incidence of Epstein-Barr virus (EBV) and T-cell lymphotrophic virus type I (HTLV-1) – related tumors in kidney transplant patients. Regardless of the prevalence and cause for cancer in transplant recipients, treatment of tumors that do occur is complicated by the need to prevent graft rejection with immunosuppressive drugs. Immunosuppression is thought to be the primary culprit in transplant-related cancer [11]. In particular, azathioprine and cyclosporine-based immunosuppression have been clinically linked to the development of malignancies [3]. Experimentally, cyclosporine has been shown to promote tumor growth by different research groups [12-14], including ours [15,16]. Meanwhile, the development of other agents with different modes of action, such as mammalian target of rapamycin inhibitors (mTORi) has opened the possibility that not all immunosuppressive drugs promote tumor growth and, in fact, may even be used to treat cancer. Indeed, rapamycin has shown potent antitumor effects in several experimental tumor models (Table 1). Moreover, case reports and the first results of clinical studies (Table 2) indicate that rapamycin may be effective against tumors in transplant and nontransplant patients. These studies are critical because they open the way to adjust immunosuppressive regimens in a way to fight

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Table 1 Tumor-related nonimmune cells and experimental tumors responsive to the effect of mTOR inhibitors Cell type Nonneoplastic cells Endothelial cells

Cell line

Smooth muscle cells

Bovine aortic [17], HUVEC [15,18] HuAoSMC [19-21]

Fibroblasts Neurons

3T3 [17,19] embryonic neurons [22]

Tumor cells B-cell lymphoma

AB5, JB7, MF4 (PTLD-derived cell lines) [23], BKS-2, L1.2, NFS.1.1, WEHI-279 [24]

B-cell lymphoma B-lymphoblastoid cells

Bladder cell carcinoma Brain cancer Brain cancer (glioma) Breast cancer

Colon carcinoma

Colon carcinoma Colon carcinoma Hepatoma/hepatocellular carcinoma Mast cell –derived tumor Medulloblastoma Melanoma

BJAB [27], PTLD-like EBV infected cell lines (panel) [25] T2 [28] DAOY [29]

Rhabdomyosarcoma T-cell lymphoma

AB5 [23], PTLD-derived Human tumors [24], PTLD-like EBV- infected cell lines [25]

EBV infection

breast cancer cell line panels [32-35]

SF295 [30], U-251 [31] MDA-MB-468 MDA-MB-435 [32]

Human Human

CT26 [15]

CT26 [15,16,18]

Murine

CT38 [36,37] GC3 [30]

Murine Human

H4IIEC [38], HepG2 [39], SK-Hep1, HepB3, PCL/PRF/5 [40], H4IIE [41] V2D1, V3D6, V3D8 (autocrine tumor cell lines) [42]

HL60 [27] DU-145, PC-3 PPC-1, TSU, LNCap [30,53,54] 786-O [56] RH18, RH30 [30,57-59] HTLV-I–transformed cells [19], YAC-1 [60], EL-4 [61]

Immature and mature lymphomas, EBV infection

Murine

Murine

B16 [15]

Additional information

Regulation of translation

T24 [28]

Muscle BC3H1 [44] Neuroblastoma KP-N-RT [45] Non–small cell lung cancer H69, H345, H510 [46], KLN-205, A-549 [47,48] Ovarian cancer IGROV1, OVCAR3, A2780, SKOV3, OVCAR4, OVCAR5 [49,50] Pancreatic cancer L3.6pl [51]

Renal cell cancer

Tumor origin

bFGF and VEGF induction, wound-healing, angiogenesis bFGF and PDGF induction, angiogenesis, wound healing

genetically induced lymphoma models [26]

Multiple myeloma

Promyelotic cells Prostate cancer

Tumor

Sensitive and resistant cell lines/tumors, depending on oncogene and Akt-pathway status Angiogenesis, combination with cyclosporine and 5-fluorouracil, combination with transplantation

Autophagy

v-H-ras DAOY, TE-671 [29] B16 [15,16,37]

Human Murine

8226, OPM-2, U266 [43]

Human

Combination with cyclosporine, combination with transplantation Reduction of tumor cell size induction of differentiation

KLN-205 [46], A-549 [47] OVCAR5 [30]

L3.6pl [51], CA20948 [52] DU-145, PC-3 [30], PIN [55] spontaneous murine tumor [28], 786-O [56] RH18 [30]

HTLV-I, T-cell lymphotrophic virus type I; PDGF, platelet-derived growth factor.

Murine, human No apoptosis, reduction in tumor size and metastasis Human Some resistant cell lines, regulation of apoptosis pathways, no significant effect on tumor growth Human, rat Thrombosis in the tumor, combination with anti-VEGF antibody, 5-fluorouracil Human

Induction of Akt, HIF1a neoplasms induced by Akt overexpression Murine, human Combination with cyclosporine, reduced number of tumors in metastasis model Human Dependency on IGF-1/IGF receptor Cell lines with acquired resistance

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Table 2 Clinical studies, case reports, and analysis of patient material regarding mTORi Type

Tumors

Drug

Additional information

Patient samples Case report

Head and neck cancer [62] Kaposi sarcoma [63]

No drug Sirolimus

Phase I

Hepatocellular carcinoma, metastatic renal cell carcinoma, metastatic sarcoma [64] Renal cell carcinoma, breast adenocarcinoma [65] Advanced solid tumors (including gastrointestinal tumors) [66] Fibrosarcoma, non–small cell lung carcinoma [67] Advanced gastrointestinal tumors [68] Refractory renal cell carcinoma [69,70] Renal transplant patients [71]

AP23573

Akt pathway, no rapamycin Conversion to rapamycin leads to tumor regression Dose escalation, preliminary meeting reports

Phase I Phase I Phase I Phase I/II Phase II Results from 5 multicenter studies (phases II and III) Retrospective single-center analyses

PTLD, hepatoblastoma (pediatric liver transplant patients) [72]

cancer while simultaneously maintaining a safe level of immunosuppression to protect allografts —understandably, this concept can be viewed with skepticism. Nonetheless, a better knowledge of the biology of tumors highly prevalent in the transplant population will likely lead to the development of rationally balanced tumor-transplant drug treatment options. Therefore, a special focus should be placed on understanding how immunosuppressive drugs affect vital signaling pathways that promote tumor transformation, proliferation, angiogenesis, and metastasis. Recently, we have been interested in the potential for mTORi to play the dual role of immunosuppressive and antitumor agent in transplant recipients. In this review, we discuss the mode of action of mTORi with a main focus on their effects on intracellular signaling pathways. These effects will be discussed in view of mutational and biochemical changes that are typical for transformed cells leading to tumor genesis and growth. A special emphasis will be placed on tumor types with a high prevalence in transplant patients. 2. Rapamycin: discovery and characterization Today, the class of mTORi consists of rapamycin (Rapamune/sirolimus, Wyeth) and its derivatives (CCI779, Wyeth, RAD001/everolimus, Novartis) [73] and the analogue ap23573 (ARIAD Pharmaceuticals). Rapamycin was originally found in a discovery program for antimicrobial agents from natural resources [19]. A strain of Streptomyces hygroscopicus with antifungal properties was isolated from a soil sample collected at Easter Island (Rapa Nui). The active substance, named rapamycin, is a lipophilic macrocyclic lactone with strong antimicrobial and, as later found,

CCI779 CCI779 RAD001 RAD001 CCI779 Sirolimus

Sirolimus

Safety, tolerability, and pharmacokinetics Combination with leucovorin and 5-fluorouracil, discontinued because of toxicity Dose escalation, toxicity, pharmacokinetics, pharmacodynamics Combination with imatinib in imatinib refractory tumors Efficacy, safety, and pharmacokinetics of multiple doses Rate of malignancy after transplantation, 2-y results, sirolimus in addition to cyclosporine Only sirolimus-treated patients analyzed

immunosuppressive activity. Since first characterized in 1975, rapamycin’s structure, biosynthesis, and binding partner(s) have been reported [19]. Rapamycin binds to intracellular FK binding protein (FKBP) 12, forming a drug/ immunophilin complex that modulates the activity of intracellular targets. In a similar fashion, cyclosporine binds to the immunophilin cyclophilin A, causing inactivation of calcineurin and consequently blocking nuclear factor of activation of T cells (NFAT) translocation into the nucleus. This action basically defines the class of calcineurin inhibitors within the immunosuppressants [19,74]. Interestingly, FK506 (tacrolimus) also binds to FKBP12. Whereas the tacrolimus/FKBP12 complex forms a ternary complex with calcineurin, thereby exhibiting the same immunosuppressive mode of action as cyclosporine [74], rapamycin has a different target and consequently a different mode of action. The rapamycin/FKBP12 complex binds to mTOR (mammalian target of rapamycin) [19,75,76]. This 289-kDa protein (a member of the PI-kinase family) is a pivotal regulator of cell growth and proliferation for many cell types. Although mTOR inhibition interferes with several intracellular mechanisms [77], one of its best known effects is G1 cell cycle arrest [78]. Moreover, very diverse cell types ranging from cells of the immune system [19,79], to smooth muscle cells [36,80], to endothelial cells [15,80], and to tumor cells (Table 1) respond poorly to cytokines and growth factors in the presence of rapamycin. The effects on different cell types not only have led to a variety of potential therapeutic uses but also indicate a potential for undesired effects. Indeed, rapamycin’s diverse effects have made for a curious trail of therapeutic applications. Although originally discovered for its antifungal action [19], enthusiasm for rapamycin’s therapeutic usefulness against infections was

G.E. Koehl et al. / Transplantation Reviews 19 (2005) 20 –31

trolled by mTOR. One key pathway controlled by mTOR, which is particularly important for cancer cells, is the phosphatidylinositol-3 (PI3) kinase/Akt signaling pathway [19,76,77,81,85-87] (Fig. 1). Basically, PI3 kinase is activated by growth factor/cytokine-triggered receptors, leading to phosphorylation of Akt and subsequent downstream effectors including 40S ribosomal p70S6 kinase and 4E-BP1 (eukaryotic translation initiation factor 4E binding protein 1). In more detail, growth factors and cytokines [19], including interleukin 2, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF), lead into the mTOR pathway by activating PI3 kinase via receptor tyrosine kinases and G-protein–coupled receptors [88]. Activation of PI3 kinase leads to production of the second messenger phosphatidylinositol (3,4,5)-P3 (PIP3). PIP3 activates Akt, leading to its translocation to the cytoplasmic membrane. PTEN (phosphatase and tensin homolog deleted from chromosome 10) counteracts Akt activation by eliminating PIP3 and can thereby bswitch offQ the PI3 kinase/Akt pathway [89]. Loss of PTEN function occurs in many cancers and is consequently correlated with increased Akt activity, making cancer cells resistant to apoptosis [90], but potentially highly sensitive to mTORi [26,91]. Akt in turn phosphorylates tuberoses sclerosis complex II (TSCII) leading to inactivation of the TSCI/TSCII complex, which negatively regulates mTOR. TSCI and TSCII are associated with dominant genetic or spontaneous mutations causing a disorder called tuberous sclerosis [81]. Loss-of-function mutations in TSCI or II cause enlarged cells and organs in Drosophila, whereas overexpression of both proteins dramatically slows cell growth. Deficiency in TSCII leads to constitutive phosphorylation of p70S6 kinase, but cells remain sensitive to rapamycin. Therefore, both Akt and TSCI/II

diminished because of its immunosuppressive effect. Sehgal [79] insightfully pursued the drug’s immunosuppressive potential to actually use rapamycin against the immune system, and it has since been fully developed in transplantation medicine for this purpose. Another property recognized early in rapamycin’s development was its anticancer activity [37]. Less than desirable potency at cytotoxic doses and its known immunosuppressive activity undoubtedly reduced early enthusiasm for its use in oncology. However, in recent years, the antitumor activities of mTORi have come back into focus with the development of rapamycin analogues CCI779 and RAD001, which are now being tested as pure oncological agents in phases I and II trials (Table 2). Phases I and II trials with ap23573 are also under way. In addition, the transplant community has become increasingly aware of the high cancer incidence in transplant recipients and is seeking a means of immunosuppression that could decrease this problem —mTORi could provide new hope for this purpose. 3. Mammalian target of rapamycin action and the phosphatidylinositol-3 kinase/Akt pathway Mammalian target of rapamycin is a central factor for integrating adaptive responses to external and internal signals [81] (eg, growth factors, hypoxia [80,82], and amino acid/ nutrient levels [83]) with regulation of transcription [77] and translation [84]. These responses in many cases alter critical processes such as cell cycle progression and cell proliferation. Evolutionary conservation of mTOR, as seen by wellcharacterized homologues in yeast (Saccharomyces cerevisiae) and Drosophila, underscores its important role in the regulation of cell functions in response to nutrient status. In mammals, nutrient status has been integrated with highly developed regulatory mechanisms, including those conGF

23

IL-2, VEGF, PDGF, IGF, EGF, FGF

ErbB2 receptor

P

PIP2

PI3-kinase

PTEN Akt

br a

integrins

em

P

lm

PIP3

ce l

ras

ne

nutrients TSCI TSCII P

upstream

Rheb

rapamycin FKBP12 mTOR raptor Fig. 1. PI3-kinase Akt pathway upstream of mTOR.

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brain) [81]. Small G-proteins, are only active in the GTP (guanosine triphosphate) – bound state, generally using a specific GTPase-activating protein as a bhelper Q to convert it to the inactive GDF (guanosine diphosphate) – bound state. The GTPase-activating protein for Rheb is TSCII, thus linking Akt and mTOR. Finally, to phosphorylate and therefore activate the downstream effectors of this pathway (p70S6 kinase and 4E-BP1), mTOR must be in a complex with raptor (regulatory associated protein of mTOR) [77]. Raptor serves as a docking site for p70S6 kinase and 4E-BP1 and brings the target proteins in position for phosphorylation by mTOR. Rapamycin causes disruption of the raptor/ mTOR complex, leading to mTOR inactivation (Fig. 1).

rapamycin FKBP12 mTOR raptor

P

P

downstream

4E-BP1

p70S6K

eIF4E

eIF4B

cyclins translation

CDKs p53

P

pRb

ribosomal biosynthesis

apoptosis

4. What can be expected of mTORi use?

cell proliferation

tumor immune system Fig. 2. Downstream targets of mTOR I: cell cycle and apoptosis.

are upstream of mTOR and rapamycin inhibition. More recently, it has been shown that the interaction of the TSCI/II complex with mTOR is indirectly mediated by the small G-protein, Rheb (Ras homologue enriched in

Inhibition of mTOR leads to a 15% to 20% decrease in overall protein synthesis in many cell types [91], with its main effect being, as mentioned above, cell cycle arrest in G1. In yeast, mTOR signaling modulates transcription of genes involved in nutrient use, respiration, protein degradation, and amino acid biosynthesis [76,77]. Other effects include apoptosis (depending on retinoblastoma protein [pRB] and p53 status) [91], inhibition of T-cell activation [92], inhibition of cell migration [93] and invasion [93], changes in the (actin-)cytoskeleton [78], membrane trafficking [78], autophagy [94], and decreased expression of specific proteins such as VEGF [95]. The two major downstream effectors of mTOR are p70S6 kinase and eukaryotic translation initiation factor 4E (eIF4E). Both of these proteins and their targets are

rapamycin FKBP12 mTOR raptor

P

c-Myc

STAT3 P

downstream

transcription

p70S6K

HIF1α

HIF1α

glucose use genes

VEGF

P

translation

VHL

ribosomal biosynthesis cell proliferation

degradation

signalling to other cells

tumor angiogenesis

immune system

Fig. 3. Downstream targets of mTOR II: physiology and adaptive responses.

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needed for assembly of translation machinery and for the recruitment of ribosomes to mRNA (Figs. 2 and 3). The effect of mTOR on eIF4E is indirectly mediated via 4E-BP1 [77,84,91], which is a suppressor of eIF4E. Phosphorylation of 4E-BP1 by mTOR leads to release of suppression of eIF4E and thereby to efficient initiation of CAP-dependent translation of mRNA. Rapamycin treatment keeps 4E-BP1 in its suppressor state and blocks not only efficient translation of key proteins for the translation machinery, but also for the synthesis of proteins necessary for cell cycle progression (eg, cyclin D1 [90]) and survival (eg, c-Myc [96]). Thus, rapamycin use leads to reduced cell growth and cell cycle arrest in G1. Interestingly, phosphorylation of 4E-BP1 by protein kinase Cd can be blocked by rapamycin, although it is Akt-independent [97]. Indeed, signaling through MAP-kinase pathways [98] (eg, the Erk and p38 families) also leads to eIF4E activation. Therefore, other important signal transduction pathways have the potential to interact with mTOR signaling or possibly provide a bypass around the effects of rapamycin [77,91]. Activated p70S6 kinase increases translation of the so-called 5VTOP (terminal oligopyrimidine) tract mRNAs [84], which encode ribosomal proteins, elongation factors, and other proteins of the translational machinery. Notably, several proteins involved in recruiting ribosomes including eIF4B, eIF4G, and indirectly by its repressor, eIF4E, are sensitive to rapamycin [77]. Therefore, mTOR has a profound effect on cell growth and cell cycle progression by regulating ribosomal protein synthesis and recruitment. In terms of sufficient use of resources and energy, it is only logical that mTOR can regulate the upstream processes of translation and transcription. In effect, mTOR can stimulate the transcription of rRNA genes, ribosomal proteins, tRNA genes, and some transcription factors [77]. With specific regard to the cell cycle (Fig. 2), mTOR is needed for efficient translation of cyclin D1 [90]. Cyclin D1 in complex with cell cycle –dependent kinase (CDK) 4 is needed for phosphorylation of pRB [91]. pRB is involved in the regulation of Pol I/II/III –dependent transcription and provides a link to the apoptosis pathways. In addition, rapamycin stabilizes p27, which inhibits the activity of the cyclin/CDK complexes [91]. Moreover, it has been shown that rapamycin inhibits phosphorylation of STAT3 (signal transducer and activator of transcription 3) via mTOR [99]. The transcription factor STAT3 mediates stabilization of cyclin D1 and up-regulates c-Myc. Other cyclins and CDKs are involved in mTOR-regulated events, and once again, mTOR/rapamycin acts as an important regulator in several different ways. For example, by inhibiting CDK1 rapamycin induces G1 arrest in T lymphocytes [91], thereby preventing T-cell proliferation, which contributes to an immunosuppressive effect exploited in transplant patients. From a different perspective, inhibition of the cell cycle in tumor and endothelial cells provides 1 potential explanation for rapamycin’s antitumor and antiangiogenic properties. Interestingly, the cell cycle also plays a role in rapamycin’s

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ability to promote cell apoptosis. Of special interest with regard to tumors is the fact that the p53 status of a cell can determine its fate with regard to rapamycin-induced apoptosis. For instance, cells with wild-type p53 arrest in G1 and remain viable, whereas p53 mutant rhabdomyosarcoma cells undergo apoptosis [100]. This underscores the importance of the G1/S –phase checkpoint control by p53, which is lost in many tumor cells and may be a mechanism for rapamycin to more or less selectively target tumor cells by sending them into death by unsuccessful cell division. Therefore, although rapamycin is generally considered an antiproliferative agent, there are circumstances where it also promotes cell death. Integration of these intracellular pathways with the environment surrounding individual cells is controlled in a large part by growth factors. Growth factors not only activate the PI3 kinase/Akt pathway; expression of these molecules is controlled by the same signaling pathway. For example, inhibition of mTOR leads to less production and release of VEGF [53], thus having the potential to systemically reduce growth factor–dependent responses. VEGF is regulated at the translational and transcriptional level by rapamycin via 4EB-P1 [101] and hypoxia-induced factor 1a (HIF1a) [53], respectively. The transcription factor HIF1a is activated and translocated to the nucleus during hypoxic conditions. Mammalian target of rapamycin induces a protein stabilization function that leads to accumulation of activated HIF1a [102]. Consequently, rapamycin can potentially reduce accumulation of HIF1a in the nucleus independent of the cell reaction to hypoxia (Fig. 3). This could be important for tumor growth, where hypoxia leads to increased VEGF production, which in turn promotes tumor angiogenesis. In addition, in cancers where HIF1a is constitutively boverexpressed,Q rapamycin can have an antitumor effect independent of hypoxia by reducing HIF1a-induced transcription of growth factors, glycolytic enzymes, and glucose transporters [103]. For instance, this type of HIF1a overactivation occurs in cells where mutations in the von HippelLindau (VHL) gene prevent efficient HIF1a degradation. Interestingly, patients with renal cell carcinoma (often with VHL mutations) have recently been shown to respond to the rapamycin analogue CCI-779 [69]. In summary, rapamycin inhibits pivotal downstream regulators of cell growth, division, and survival and therefore can be expected to affect many normal, as well as neoplastic, cell types. The response of each cell type to rapamycin treatment likely depends substantially on the physiological and genetic state of the cell. Most affected will be not only cells depending on growth factor– or cytokine-induced cell growth and division, such as T cells and tumor cells, but also cells needed for proliferative processes such as angiogenesis and wound healing. Different genetic mutations may make tumor cells susceptible to direct rapamycin effects, particularly apoptosis. Screening for such tumors and tumors highly dependent on angiogenesis will make best use of rapamycin’s antitumor efficacy.

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5. Which nonimmune cells respond to rapamycin? mTOR’s central role in controlling cell proliferation and cell cycle progression in living organisms from yeast and Drosophila to mammals suggests that rapamycin affects several cell types. Besides effects on T cells, rapamycin influences at least 4 other types of cells: (1) tumor cells, (2) vascular cells, (3) virally infected cells, and (4) developmentally or regeneratively active cells. Effects on tumor cells were reported shortly after rapamycin’s discovery [37]. Subsequent screening of the National Cancer Institute human tumor cell line panel [73,85] has shown significant growth inhibition in breast cancer, prostate cancer, renal cell cancer, pancreatic cancer, melanoma, leukemia, and glioblastoma. At present, different antiproliferative effects and changes in signal transduction, as well as changes in translation and transcription, have been elucidated in many cancer cell types (Table 1). Studies with murine and human tumors in mice have shown an antitumor effect of rapamycin as a single agent or in combination with other chemotherapeutics (Tables 1 and 2). In general, good targets for rapamycin treatment would theoretically be tumors with mutations in proteins with a close connection to the PI3 kinase/Akt pathway. These include gain-offunction receptor mutations, such as ErbB2 overexpression in breast cancer. PTEN mutations occurs in up to 50% of human solid tumors [104] and have been extensively studied in melanoma [89], breast cancer [105], glioblastoma [89], and prostate carcinoma [106]. Both ErbB2 and PTEN mutations lead to constitutive activation of PI3 kinase and Akt, therefore making those tumors potential targets for treatment by mTORi [90,107,108]. Other, somewhat unexpected, activators of PI3 kinase include the oncogenic small G-protein ras [90] and integrins [109] (Fig. 1). Because ras generally transmits signals to the MAP kinase pathway, and the integrins provide a link to cell movements, two other aspects of cell function and tumor biology are linked to mTOR. As discussed earlier, mutations in the tumor suppressor p53 may promote rapamycin-induced apoptosis of different tumor cells, thereby potentially enhancing its cytotoxic properties (Fig. 2). Another oncogene that is often associated with renal cell cancer [78] and could be a key factor for mTORi sensitivity is VHL. Despite being blocatedQ downstream of mTOR, HIF1a activation by VHL mutations [107] may be counterbalanced by rapamycin (Fig. 3). Of particular importance to transplant recipients, skin cancer has a high correlation to sun exposure and ultraviolet-induced damage. The combination of general immunosuppression and promotion of aberrant repair mechanisms by drugs such as cyclosporine [110] is thought to promote transformation of cells. Interestingly, studies indicate that ultravioletinduced damage may activate mTOR-dependent p70S6 kinase, providing a potential target for mTORi [111]. Even cancers highly refractory to treatment, including pancreatic cancer, may be responsive to rapamycin therapy. In vitro studies and xenogenic (human to mouse) models have

demonstrated effective rapamycin monotherapy against pancreatic cancer that synergizes with standard chemotherapy (gemcitabine [51], 5-fluorouracil [52]) — curiously, rapamycin also works well in combination with anti-VEGF antibody [112]. In addition, we have shown that one mechanism of the effect of rapamycin on pancreatic cancer relates to the promotion of thrombosis specifically in tumor vessels, thus reducing the tumor blood supply and inhibiting tumor growth; no effects on normal blood vessels were observed in these experiments [51]. Whether this activity has any relationship to rare cases of thrombosis in rapamycin-coated vascular stents or to hepatic artery thrombosis after liver transplantation will require additional research. Recently, we have correlated mTORi anticancer effects with inhibition of angiogenesis in animal models [15]. In fact, vascular-associated tissue contains a third group of cells targeted by mTORi. The mechanisms of this antiangiogenic effect are not completely understood, but as discussed above, HIF1a-dependent signaling can be inhibited by rapamycin, thereby hindering angiogenic signaling at its origin. In addition, vascular endothelial and smooth muscle cells are affected by mTOR inhibition. Rapamycin not only inhibits the proliferation and migration of these vascular cells in response to VEGF, but it can also promote endothelial cell death under proliferative conditions [51]. These antiangiogenic effects have been correlated with markedly reduced tumor growth in different mouse models [15,18,28,51]. The effects of rapamycin on bnormalQ vascular cells embedded within a neoplasm suggest that although cancer cells may not necessarily be directly affected by rapamycin, indirect effects on angiogenesis could help to control tumor growth [15,51,112]. In a different respect, angiogenesis is essential for tissue regeneration and for processes such as wound healing [113,114]. Therefore, caution should be taken when considering mTORi use in patients with wounds or transplant recipients demanding tissue regeneration (eg, period immediately after living-related liver transplantation). With regard to virally infected cells, it is of special interest that transplant patients infected with EBV tend to develop posttransplant lymphoproliferative disorders (PTLDs). Interestingly, it has been shown that mTOR inhibition blocks proliferation of human EBV-transformed B cells in immunodeficient mice and induces their apoptosis in culture [23]. A key effect of rapamycin on EBV-infected B cell proliferation described in this study is through an inhibitory action on interleukin 10 secretion. Therefore, mTORi may prove to be valuable in treating PTLD. Viral infections may also be important for other types of cancer in transplant patients, and the effects of immunosuppression could be critical. For instance, hepatitis C virus (HCV) is thought to be important for development and recurrence of hepatocellular carcinoma (after liver transplantation). Interestingly, it has recently been reported that the mTOR pathway controls the phosphorylation/activation of a critical HCV-replication protein (nonstructural protein 5A, NS5A) [115]. Moreover, rapamycin was shown to inhibit NS5A activity, potentially impacting

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HCV replication, and therefore cancer recurrence in hepatocellular carcinoma patients receiving a liver transplant. A fourth group of cells depending on mTOR is developmental [94] and regenerative cells. For example, a mutation in mTOR can cause severe brain defects in mice and is homozygous embryonic lethal; the same phenotype is induced by rapamycin injections during pregnancy. Although the mechanism by which rapamycin causes such severe developmental problems is not clear, it has been hypothesized that the drug induces autophagy through mTOR dysregulation. Indeed, nutrient-scavenging autophagy plays an important role in developmental processes requiring controlled cell death (eg, insect morphogenesis and Xenopus spinal cord development). Nonetheless, mTOR has also been implicated in bnormalQ neuronal function because neurotransmitters, such as serotonin and dopamine, and stimulation of the l-opioid receptors (mediating morphine effects) operate via the PI3 kinase/p70S6 kinase/4E-BP1 pathway. There are also a number of examples of how mTORi affects cell regeneration. For instance, bone marrow suppression is a well-known side effect of mTORi use in transplant recipients [116]. Cell regeneration could be critical as well for liver regeneration in patients who receive either a living-related or split liver transplant. In fact, in an experimental rat model where a partial hepatectomy is performed, it has been reported that liver regeneration is impaired by rapamycin treatment [117]. In summary, mTORi has diverse effects on normal cells that are important for tissue-organ development and regeneration. These potential effects need to be taken into consideration when using mTORi during periods where these processes are critical. 6. Will mTORi synergize with standard chemotherapy against cancer? A practical aspect of mTORi as anticancer agents is that they will likely be best used in combination with additional chemotherapy. Examples of this strategy are emerging with time. For instance, we have recently reported experimentally that rapamycin works best against pancreatic cancer when combined with gemcitabine chemotherapy [51]. This work has led to the initiation of a clinical pilot study at our institution using this combination treatment strategy for pancreatic cancer. For colon cancer, we have shown that rapamycin synergizes well with 5-fluorouracil chemotherapy in mice [18]. In this case, we could show mechanistically that rapamycin blocks a proangiogenic escape pathway that may allow tumors to compensate for 5-fluorouracil treatment. Other studies have also shown that mTORi treatment can be effective to break resistance that tumor cells may develop against other chemotherapeutics. One example is that chronic myelogenous leukemia cells tend to become resistant to the effects of the tyrosine kinase inhibitor imatinib (STI571, Gleevec, Novartis). However, resistant chronic myelogenous leukemia cells are sensitive to the effects of imatinib when combined with rapamycin because the cells use mTOR-

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dependent pathways as an escape mechanism [118]. In ovarian cancer models, inhibition of the PI3 kinase/Akt pathway increases the sensitivity of tumors to paclitaxel [119] and cisplatin [49]. In yet another example, chemoresistance to tamoxifen because of overactivation of Akt can be reversed in breast cancer xenografts by mTORi (CCI779) [120]. Therefore, the activity of molecules in the PI3 kinase/ Akt/mTOR pathway may not only be involved in tumor chemoresistance, but their activation status may be indicative of tumor cell sensitivity to mTORi and potential synergism with standard chemotherapy. 7. Will mTORi be effective against cancer in transplant recipients? When considering the treatment of cancer in transplant recipients, a complicated situation emerges. The goal is to attack a tumor in the face of immunosuppression used to protect the allograft. Because of the known antitumor effects of the immune system, this seems an uphill battle. However, the emergence of immunosuppressants with both immunosuppressive and antitumor effects (at least experimentally) could provide one solution to the problem. To better understand the potential for mTORi to function simultaneously as an immunosuppressant and anticancer treatment, we have developed mouse models mimicking the scenario in humans [16]. Indeed, we have recently reported that a mouse allograft can be protected by rapamycin against immunologic rejection with simultaneous antitumor effects. Furthermore, we show that the antitumor effect remains fully intact when rapamycin is used in combination with cyclosporine, which otherwise promotes tumor growth in these models. Our results demonstrate in principle that mTORi can exhibit a dual function against allograft rejection and tumors in an organ transplant situation. At present, controlled prospective clinical data are not yet available to confirm these experimental results. However, some hints toward a potential effect have been recently published. For example, Campistol et al [63] have reported two renal transplant recipients who had developed Kaposi sarcoma under cyclosporine treatment and showed complete tumor regression when the patients were switched to a sirolimus/mTORi immunosuppressive regimen. Although it is not yet clear whether the tumor regression can be attributed to sirolimus in these cases (because this was not a controlled study), this report points to the fact that tumor regression can occur simultaneously with sirolimus immunosuppressive treatment. Interestingly, Kaposi sarcoma is a highly vascular tumor where mTORi might be expected to exhibit antiangiogenic effects. There are also clues in the literature that sirolimus might be useful in liver transplant patients with a pretransplant hepatocellular carcinoma. In a pilot study from Kneteman et al [121] using sirolimus-based immunosuppression, two groups of patients were examined; one group of patients was within the Milan criteria (n = 19), whereas a second group of patients presented with extended hepatocel-

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lular carcinoma (n = 21). Tumor recurrence rate in the Milan group at a median follow-up of 17 months was 5.3%, which compares favorably to published results of patient cohorts adhering to the Milan criteria under conventional immunosuppression. The tumor recurrence rate in the extended criteria group was 19%, which again compares favorably to data from patient cohorts with extended criteria under standard immunosuppression [122]. Although this study only reports on a small number of patients and is not controlled, it suggests a potential for sirolimus in a tumortransplant setting. Finally, regarding the prevention of de novo cancer in transplant recipients, only early data are available. Kauffman et al [123] from United Network for Organ Sharing have examined more than 33 000 renal transplant patients over a 2-year period and found that the relative risk for developing de novo cancer was decreased by 59% in patients on an mTORi-based (used together with a calcineurin inhibitor) versus calcineurin inhibitor–based immunosuppressive therapy. Whether this early trend will continue to diverge in favor of mTORi, narrow, or even reverse itself, as these patients enter the middle to late posttransplant period (when most cancers occur), is simply unknown. Skin cancer, in particular, will be interesting to observe over time because this most common type of transplant-related cancer tends to occur only after several years of immunosuppression. 8. Conclusions mTOR is emerging as a central molecule for integrating several important internal and external signals with essential cell functions such as biosynthesis of ribosomes, translation, and even transcription. Therefore, mTOR plays an important role for survival and differentiation at the single-cell level and for complex multicellular entities such as the immune system and tumors. Because operational mTOR pathways are needed by the immune system, mTORi has been incorporated successfully into immunosuppressive regimens to prevent organ allograft rejection. The effects of mTORi on endothelial cells and smooth muscle cells led also to the development of rapamycin-coated vascular stints for vascular stenosis. These devises have been used with great success clinically in preventing restenosis of blood vessels. A major focus of this review has been the potential for mTORi to be used against cancer. Clearly, mTORi not only blocks normal tumor cell proliferation, but they can also mitigate the effects oncogenic mutations have on intracellular pathways connected to cell proliferation and apoptosis. Furthermore, the normal process of angiogenesis, essential for tumor survival, is inhibited by drugs such as rapamycin, thus providing yet another avenue of anticancer activity. Because of this broad spectrum of effects, mTORi must be used with caution in clinical situations where processes such as tissue regeneration or wound healing are necessary. To minimize these side effects and to achieve its most favorable therapeutic effects in patients, we must continue to better

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