- Email: [email protected]
The potential benefits of rapamycin on renal function, tolerance, fibrosis, and malignancy following transplantation
mini review
http://www.kidney-international.org & 2010 International Society of Nephrology
The potential benefits of rapamycin on renal function, tolerance, fibrosis, and malignancy following transplantation Edward K. Geissler1 and Hans J. Schlitt1 1
Department of Surgery, University Hospital Regensburg, University of Regensburg, Regensburg, Germany
Use of the mammalian target of rapamycin (mTOR) inhibitor rapamycin in organ transplantation has evolved through different phases over the past two decades. After its discovery in the mid 1970s, antifungal and cytotoxic effects were the first of its properties to be explored, but the most significant advancement was found in its use as an immunosuppressive agent to reduce transplant rejection. This was viewed as an important step forward for immunosuppression, as early studies suggested that rapamycin was less nephrotoxic than calcineurin inhibitors (CNIs). Later, detrimental effects of rapamycin on kidney function were found in some patients. Nonetheless, a fascination with the mTOR pathway and its central role in multiple cellular processes has ensued. Among the potential positive clinically relevant effects is rapamycin’s capacity to interfere with fibrotic processes that often accompany transplant rejection, and to influence the preferential development of immunological tolerance. A feature of increasing importance is that the mTOR pathway is central for vital aspects of tumor development, including angiogenesis and cell growth; rapamycin, therefore, has anticancer activities, which may prove critical in the fight against high cancer rates in transplant recipients. The final chapters defining the value of rapamycin have not been written yet, and indeed remain a work in progress. Only further research will reveal the full potential of rapamycin in organ transplantation. Kidney International (2010) 78, 1075–1079; doi:10.1038/ki.2010.324; published online 22 September 2010 KEYWORDS: fibrosis; immunosuppression; malignancy; rapamycin; tolerance
Correspondence: Edward K. Geissler, Department of Surgery, University Hospital Regensburg, University of Regensburg, Franz-Josef-Strauss-Allee 11, Regensburg 93053, Germany. E-mail: [email protected] Received 29 April 2010; revised 24 June 2010; accepted 13 July 2010; published online 22 September 2010 Kidney International (2010) 78, 1075–1079
EARLY VERSUS MORE RECENT IMPRESSIONS OF RAPAMYCIN USE IN TRANSPLANTATION
Rapamycin was first discovered in the early 1970s from a soil sample taken on Easter Island (Rapa Nui). Based on the molecule’s readily apparent antiproliferative effects, it was first tested as an antimicrobial agent and for its cytotoxicity against tumors when used at high concentrations.1 However, its actual medicinal potential was not revealed until used continuously at low concentrations as an immunosuppressant in organ transplantation. Several pharmacological rapamycin analogs (e.g., sirolimus and CCI-779 (Pfizer, New York, NY, USA), RAD001 (Novartis, Basel, CH, USA), AP23573 (Ariad Pharma, Cambridge, MA, USA)) have been developed, as a result, all of which will simply be hereafter referred to as ‘rapamycin’. Regarding its use as an immunosuppressant, it was discovered early that T cells are inhibited by rapamycin as interleukin-2 triggers T-cell proliferation via the mammalian target of rapamycin (mTOR) signaling pathway. Expectations became increasingly high that this new immunosuppressive drug would be effective at preventing rejection, although not causing nephrotoxicity associated with calcineurin inhibitors (CNIs) use. Early animal experiments supported this notion, and initial clinical studies also pointed towards reduced nephrotoxicity with rapamycin use.2 Studies that followed, however, where rapamycin was clinically tested to reduce or eliminate CNI use, particularly in the setting of delayed or chronic renal allograft dysfunction, showed that mTOR inhibition can, in some patients, cause proteinuria.3 The exact effects of mTOR inhibition on cells of the kidney are not completely understood, but evidence suggests that tubular cell regeneration is affected, as well as podocytes.4 Interestingly, podocytes produce vascular endothelial cell growth factor, which is an important factor for maintaining the glomerular filtration barrier; as rapamycin potently inhibits vascular endothelial cell growth factor gene transcription and signaling,5 proteinuria could be a consequence of its therapeutic use. With proper monitoring and patient selection criteria, proteinuria is avoidable or controllable in most cases with rapamycin use; in more malignant proteinuria situations, rapamycin use needs to be withdrawn. The question of rapamycin effectiveness against rejection also must be considered. In renal transplantation, rapamycin has proved quite effective against kidney rejection when used 1075
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de novo in combination with CNIs, but to avoid excessive side effects, both substances need to be used at reduced levels. Although an improvement in glomerular filtration rate is reproducible when CNIs are tapered away,6 the trade-off is that rejection rates may increase. An alternative approach gaining more favor recently is to convert CNI-based immunosuppression to an mTOR inhibitor-based regimen at strategic periods within the first year after transplantation. Early clinical data suggest that this option does not increase rejection rates, improves renal function,7 and provides some protection against chronic allograft nephropathy.8 To date, however, the issue of whether rapamycin use alone can adequately prevent rejection and improve long-term renal allograft function remains an open question, and is beyond the scope of debate within this concise review.
EK Geissler and HJ Schlitt: Pros of rapamycin use in organ transplantation
Growth factors: e.g., IL-2, VEGF
P13K PTEN PDK1
Nutrients
AKT IKKβ
mTOR
TSC1/2
mTORC2
PC-1 Rheb
MOLECULAR INTRIGUE
Perhaps the most fascinating aspect related to rapamycin is the central significance of the mTOR pathway, which it influences. Indeed, the past decade of research on the mTOR pathway has revealed its pivotal role in controlling responses to a wide variety of growth factors, cytokines (e.g., interleukin-2), and nutrients. Recent studies indicate that even life span is affected (positively) by mimicking dietary restriction.9 Through the mTOR axis, basic cellular processes such as cell growth and proliferation are regulated, controlling the behavior and development of normal as well as abnormal tissues. In some instances it becomes even more complex because common processes such as angiogenesis are dependent on mTOR signaling, affecting both normal tissue repair (e.g., wound healing) and pathological tumor expansion. It is not surprising, therefore, that mTOR is highly regulated and integrated with other intracellular signaling pathways. The complexities of this molecular pathway have produced a high degree of molecular intrigue, motivating researchers to investigate the more exact effects of mTOR inhibition by rapamycin and other mTOR inhibitors. Here, we can only highlight the most critical elements of the mTOR signaling pathway (Figure 1). mTOR is part of two signaling complexes, mTOR complex 1 (mTORC1) and mTORC2. Interestingly, AKT provides signals to mTORC1, but mTORC2 actually regulates AKT through Ser473 phosphorylation. This review focuses on mTORC1, as it is the primary target of rapamycin. Phosphoinositide-dependent kinase-1 phosphorylates AKT at a second site, Thr308, further potentiating AKT activity. A wide variety of growth factors and cytokines can trigger this pathway. Key upstream signaling molecules include phosphoinositide 3-kinase, which converts phosphatidylinositiol-4,5-phosphate to phosphatidylinositiol3,4,5-phosphate and recruits phosphoinositide-dependent kinase-1 to AKT. Another important molecule is PTEN (phosphatase and tensin homolog deleted on chromosome 10), which is a tumor suppressor that reverses the phosphatidylinositiol-4,5-phosphate-to-phosphatidylinositiol-3,4,5-phosphate reaction, thus reducing AKT activity. Indeed, AKT has a critical role by mediating mTORC1 activation via inhibition of 1076
Other signaling pathways (e.g., MAPK)
mTOR
Rapamycin
mTORC1
S6K1
4E-BP1
eIF-4E
Transcription
Translation
Cell growth and metabolism
Figure 1 | mTORC1 pathway basics. Abbreviations: eIF-4E, eukaryotic initiation factor 4E; IKKb, inhibitory kB kinase-b; IL-2, interleukin-2; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; mTORC, mTOR complex; PC-1, polycystin-1; PDK1, phosphoinositide-dependent kinase-1; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homolog deleted on chromosome 10; Rheb, Ras homolog enriched in brain; S6K1, S6 kinase 1; TSC, tuberous sclerosis complex; VEGF, vascular endothelial cell growth factor; 4E-BP1, eIF-4E-binding protein-1.
the tumor suppressors, hamartin tuberous sclerosis complex (TSC)1) and tuberin (TSC2). The TSC1/2 complex inactivates Rheb (Ras homolog enriched in brain), thereby inhibiting mTORC1 activation. The primary downstream effectors of mTORC1 are S6 kinase 1 and eukaryotic initiation factor 4E-binding protein-1, which are important regulators of mRNA translation. Interestingly, to apparently protect against overactivation of mTORC1, active S6 kinase 1 negatively feeds back to inhibit AKT, and thus activation of mTORC1. The mTOR pathway is tangentially influenced in many ways. For instance, inhibitory kB kinase-b, a kinase downstream of tumor necrosis factor-a, integrates with mTORC1 by inhibiting TSC1/2, molecularly linking inflammation and cell proliferation.10 The mitogen-activated protein kinase cascade is also linked to mTOR, as inhibition of one pathway affects the activation of the other. Additionally, mTOR, because of its effects on cell growth, is intimately involved in energy metabolism. For instance, mTORC1 activity is regulated by sensing cellular adenosine triphosphate levels (via AMP-activated protein kinase and TSC1/2) and by sensing amino acids.11 Even lipid metabolism is influenced by Kidney International (2010) 78, 1075–1079
EK Geissler and HJ Schlitt: Pros of rapamycin use in organ transplantation
mTORC1, positively affecting peroxisome proliferator-activated receptor-g, which has an important role in adipogenesis and lipid accumulation.12 Therefore, mTOR’s central intracellular signaling position in essential cell processes make it an attractive therapeutic target for conditions where it is desirable to reduce cell growth and proliferation (e.g., T cells—immune suppression, neoplastic cells—oncology, endothelial and smooth muscle cells—vasculopathy). DISCOVERY OF ANTITUMOR EFFECTS
Besides the well-known immunosuppressive properties of rapamycin, there is a rapidly expanding literature base on its anticancer effects. Although early toxicology studies showed some modest effects on tumors, the concentrations of rapamycin used in these experiments were high and carried significant toxicity.1 Enthusiasm for rapamycin use as a pure oncologic agent faded until early in the present decade. A better understanding of the mTOR signaling pathway brought new hypotheses as to how rapamycin could be used more effectively against cancer. One breakthrough came in 2002 when Guba et al.5 discovered in mice that rapamycin potently inhibits tumor angiogenesis, and thus tumor growth, at relatively low, sustainable, concentrations that are compatible with long-term immunosuppression in transplant recipients. Evidence suggested that the antiangiogenic effect was related to inhibition of vascular endothelial cell growth factor production and endothelial cell responses to vascular endothelial cell growth factor, which are both dependent on the mTOR pathway. This key finding was later confirmed by others, and tumors highly dependent on angiogenesis have since shown positive responses to rapamycin treatment.13,14 A natural question in the context of organ transplantation is whether rapamycin could cause a tumor to regress in immunosuppressed patients. Indeed, it has been shown that rapamycin can prevent allograft rejection in mice through its immunosuppressive effects, while at the same time inhibiting tumor growth.15 At least in the situation of Kaposi’s sarcoma in renal transplant recipients, this idea has shown clinical results.13 These examples are an extraordinary demonstration of the multiple effects of rapamycin, and they stress the central importance of this pathway for essential cellular processes. The anticancer effects of rapamycin are not limited to angiogenesis. Tumor cells are often directly dependent on the mTOR pathway because of their need to use its proliferationpromoting mechanisms and because gene mutations within the pathway can constitutively activate mTOR. There are many examples that illustrate this latter point. For example, PTEN and TSC1/2 can be oncogenic through mutations that result in a release of their inhibitory effects on mTORC1; e.g., tuberous sclerosis is a specific hyperproliferative condition that results from mutations in the tuberous sclerosis suppressor complex. Overexpression of the receptor tyrosine kinase human epidermal growth factor receptor 2 in malignancies such as breast and gastric cancer is another example of promoting pathological signaling through the AKT/mTORC1 axis. Polycystic kidney disease is a condition Kidney International (2010) 78, 1075–1079
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where mutations in polycystin-1 have very recently been shown to cause a lack of tethering of TSC2 to the cell membrane, decreasing the ability of TSC2 to repress mTORC1.16 These and other mTORC1 pathway-promoting gene mutations make mTOR an attractive target for antitumor therapy. Recent studies also suggest that rapamycin has even broader ranging effects that influence the growth and development of tumors. For instance, rapamycin treatment markedly reduces spontaneous de novo intestinal tumor formation in APCmin/ þ mice, with a near complete correction in the expression of otherwise upregulated oncogenic Na þ and K þ ion channels;17 the mechanism for the normalization of oncogenic ion channels is unknown. Rapamycin can also strongly affect the pattern of ultraviolet-signature p53 mutations, which are thought to be important in skin cancer development.18 Therefore, there is even evidence that mTOR influences processes that affect DNA damage repair. With more research and a better understanding of the mTOR pathway, the spectrum of rapamycin effects that may impact tumor growth will likely continue to expand. Finally, we cannot leave this discussion without addressing the potential impact that rapamycin has on immunity against tumor cells and viruses. An intuitive reaction to this point is that rapamycin inhibits the immune system in general and therefore must diminish immunity against invasive neoplasms. Although this is a reasonable argument, the conclusion appears premature and full of complexities. In favor of this hypothesis are two main factors, the well-known general suppressive effect that rapamycin has on T-cell proliferation, and its recently discovered promotion of T-regulatory cell development.19 It can be reasonably speculated that T-regulatory cells could act locally within a tumor, or systemically towards a virus, to protect against invading immune cells; much the same idea as the protection against rejection that is expected with T-regulatory cells in a patient with a tolerant organ allograft. Although it is concerning that rapamycin treatment could promote immunological tolerance to a tumor entity or virus, there is recent data that takes a completely opposite perspective. Rao et al.20 have recently reported that mTOR inhibition with rapamycin inhibits Tbet and promotes eomesodermin expression, which is associated with an enhanced memory phenotype in CD8 þ cells; rapamycin-treated CD8 þ cells showed impressive antitumor activity in vivo. Data also suggest that rapamycin-exposed CD8 þ cells could work favorably against BK virus,21 cytomegalovirus,22 or other viruses23 associated with infectious and neoplastic diseases in transplant recipients (e.g., Epstein–Barr virus in posttransplant lymphoproliferative disease; herpesvirus in Kaposi’s sarcoma). Indeed, a recent organ transplantation guideline publication suggests that there may be a lower incidence of cytomegalovirus infections when using mTOR inhibitors.24 It has to be added, however, that conflicting data and much controversy surrounds this topic, so further studies will be critical in determining the balance of effects rapamycin has on the development of malignancies and viral infections. 1077
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TOLERANCE-INDUCTION POTENTIAL
There is much debate about which immunosuppressive agents and regimens may possess the most favorable properties for tolerance induction in organ transplantation. Early experimental data indicate that CNI use, through blocking T-cell activation, may hinder the development of immunological tolerance.25 In contrast, allowing T-cell activation, but not proliferation, by using rapamycin could potentially promote tolerance. Moreover, recent publications indicate that as mTORC1 is essential for the development of conventional effector T cells, blocking mTORC1 with rapamycin likely favours T-regulatory cell expansion.26 Indeed, expanding T-regulatory cells either through drug therapy or ex vivo cell therapy is being considered in novel transplant tolerance-induction strategies. Rapamycin may also indirectly promote T-cell anergy and regulation by inhibiting the maturation of dendritic cells.27 However, the immune response depending on mTOR is as complex as that when considering its influence on tumor formation. For instance, it has been shown that molecules involved in the innate immune response (e.g., toll-like receptor 4) can actually inhibit the production of inflammatory cytokines through mTOR, thus predicting that mTOR inhibition could be proinflammatory.28 Therefore, the timing of therapeutic rapamycin application may be critical in determining its potential in inducing immunological tolerance. These considerations notwithstanding, mTOR has a central, albeit complex, role in directing the immune response. CONTROL OF FIBROTIC PROCESSES
Preventing development of interstitial renal fibrosis remains an unsolved problem following organ transplantation. Although we have learned how to control early and late acute rejection reactions against allografts, chronic pathological processes such as fibrosis have not proven to be controllable. When considering renal fibrosis, transforming growth factor-b is a well-known primary mediator through induction of fibroblast proliferation and myofibroblast transition. Although smad-2 and -3 are the most commonly known substrates of the transforming growth factor-b receptor, recent evidence also suggests that transforming growth factor-b signals via other pathways, including the AKT/mTORC1 axis.29 Indeed, blocking mTORC1 with rapamycin has been recently shown to reduce renal interstitial fibrosis in an obstructive nephropathy rodent model by diminishing the number of interstitial fibroblasts and myofibroblasts.30 The question remains, however, whether rapamycin can reduce fibrosis in transplant recipients. Data have been analyzed from different trials where patients on CNIs were later converted to rapamycin, but the results are conflicting. Pontrelli et al.31 have reported that rapamycin can substantially reduce interstitial fibrosis in renal transplant recipients after a chronic allograft nephropathy diagnosis, whereas Servais et al.32 did not find a significant reduction in fibrosis after 1 year when patients were converted from CNIs to rapamycin 12 weeks after renal transplantation. A number of 1078
EK Geissler and HJ Schlitt: Pros of rapamycin use in organ transplantation
factors can explain the results of the latter study, as the negative effects of CNIs can occur very early (before randomization) and the readout was for a short period, possibly not allowing for detection of a difference within the observed time frame. Nonetheless, more trials testing this hypothesis will clearly need to be performed to determine whether fibrosis can be influenced by rapamycin following renal transplantation. We should add to this discussion that hepatic fibrosis associated with liver disease may also be targetable with rapamycin treatment, as a recent experimental study in rats has demonstrated.33 Interestingly, multiple profibrinogenic pathways are operable within liver tissue, suggesting that the mechanism of rapamycin inhibition of fibrosis in the liver is different than that which takes place in the kidney. PITFALLS OF RAPAMYCIN USE IN TRANSPLANTATION
Although this review concentrates on the positive aspects of rapamycin use in transplantation, it is important to include some discussion of the pitfalls encountered with the use of this substance or its derivatives. The most common problems are associated with side effects that can decrease the quality of life for some transplant recipients. In particular, oral ulcers, skin lesions (e.g., acne) and various forms of edema can present distressing problems for patients. In these cases, dose reductions can be helpful, along with specific counteractive treatments, but the problems may continue to persist and eventually result in rapamycin discontinuation. Furthermore, other serious side effects include wound-healing problems after surgery, delayed graft function, anemia, pneumonitis, and the problem described earlier with proteinuria in some patients. These types of side effects have sometimes resulted in elevated dropout rates in clinical studies. Although a legitimate argument can be made that physicians prematurely switch patients off mTOR inhibitors before making enough efforts to manage the side effects, it is also fair to make the argument that treatment with mTOR inhibitors requires substantial medical management. Therefore, assessment of the risk/benefit profile of rapamycin use in individual renal transplant recipients has become a central, and decisive, issue. CLINICAL PERSPECTIVES
When trying to calculate the future of rapamycin use in organ transplantation, there are many factors that have to go into the equation. Factors of particular importance that have to be considered are the development of malignancy and chronic fibrosis, both of which are critical problems facing transplantation medicine. Based on our present knowledge of the mTOR pathway, including experimental and clinical data that are presently available, an argument can be made for rapamycin treatment as a means to diminish these serious and otherwise untreatable post-transplant complications. Moreover, of the immunosuppressants currently available for transplantation, rapamycin has shown the highest potential to support the development of immunological tolerance in experimental models, especially those that espouse tolerance through the induction of anergy and Kidney International (2010) 78, 1075–1079
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EK Geissler and HJ Schlitt: Pros of rapamycin use in organ transplantation
T-regulatory cells. This is not an insignificant consideration, given that the induction of at least some level of stable immunological tolerance remains the ‘holy grail’ of transplantation medicine. If future clinical trials can definitively show that any of these potential positive attributes are veritable, mTOR inhibitors could gain a more broader base of acceptance in organ transplantation. The value of rapamycin in the situation where a malignancy occurs, or has a high probability of occurrence, in transplant recipients is especially promising. In this respect, one important ongoing multicentre clinical study is testing whether rapamycin treatment can reduce hepatocellular carcinoma-free survival in liver transplant recipients with a pretransplant hepatocellular carcinoma.34 Demonstration of a clear long-term benefit from this and other ongoing or planned clinical studies will give motivation for transplant physicians to better manage the side effects associated with rapamycin use, rather than prematurely switching to other immunosuppressants that possess their own undesirable side effect profiles. In the meantime, intrigue regarding the central role mTOR has in multiple cellular processes will continue to shed new light onto the potential therapeutic usefulness of mTOR inhibitors in organ transplantation. What is known so far is that rapamycin is a drug with potent immunosuppressive, antiproliferative, and antitumor effects, indicating that this substance influences a wide range of cell types and cellular processes. The level of this complexity is shown by rapamycin’s ability to treat multiple conditions simultaneously, as demonstrated by its combined immunosuppressive and anticancer action in transplant patients with a malignancy. The question that still needs to be answered is whether the balance of positive effects outweighs the side effects that often accompany treatment. Only further experimental and clinical studies can answer this question. DISCLOSURE
7.
8. 9. 10.
11. 12.
13. 14. 15.
16. 17.
18.
19. 20.
21.
22. 23. 24.
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26.
All the authors declared no competing interests. 27.
ACKNOWLEDGMENTS
Source of support: RISET: A European Union FP6 program aimed at resetting the immune system for the establishment of tolerance.
28.
REFERENCES 1. Eng CP, Sehgal SN, Ve´zina C. Activity of rapamycin (AY-22,989) against transplanted tumors. J Antibiot 1984; 37: 1231–1237. 2. Morales JM, Wramner L, Kreis H et al. Sirolimus does not exhibit nephrotoxicity compared to cyclosporine in renal transplant recipients. Am J Transplant 2002; 2: 436–442. 3. Ruiz JC, Campistol JM, Sanchez-Fructuoso A et al. Increase of proteinuria after conversion from calcineurin inhibitor to sirolimus-based treatment in kidney transplant patients with chronic allograft dysfunction. Nephrol Dial Transplant 2006; 21: 3252–3257. 4. Torras J, Herrero-Fresneda I, Gulias O et al. Rapamycin has dual opposing effects on proteinuric experimental nephropathies: is it a matter of podocyte damage? Nephrol Dial Transplant 2009; 24: 3632–3640. 5. 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: 128–135. 6. Webster AC, Lee VW, Chapman JR et al. Target of rapamycin inhibitors (sirolimus and everolimus) for primary immunosuppression of kidney transplant recipients: a systematic review and meta-analysis of randomized trials. Transplantation 2006; 81: 1234–1248.
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Kreis H, Oberbauer R, Campistol JM et al. Long-term benefits with sirolimus-based therapy after early cyclosporine withdrawal. J Am Soc Nephrol 2004; 15: 809–817. Rostaing L, Kamar N. mTOR inhibitor/proliferation signal inhibitors: entering or leaving the field? J Nephrol 2010; 23: 133–142. Harrison DE, Strong R, Sharp ZD et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 2009; 460: 392–395. Lee DF, Kuo HP, Chen CT et al. IKKb suppression of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway. Cell 2007; 130: 440–455. Nicklin P, Bergman P, Zhang B et al. Bidirectional transport of amino acids regulates mTOR and autophagy. Cell 2009; 136: 521–534. Kim JE, Chen J. Regulation of peroxisome proliferator-activated receptorg activity by mammalian target of rapamycin and amino acids in adipogenesis. Diabetes 2004; 53: 2748–2756. Stallone G, Schena A, Infante B et al. Sirolimus for Kaposi’s sarcoma in renal-transplant recipients. N Engl J Med 2005; 352: 1317–1323. Hudes G, Carducci M, Tomczak P et al. Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med 2007; 356: 2271–2281. Koehl G, Andrassy J, Guba M et al. Rapamycin protects allografts from rejection while simultaneously attacking tumors in immunosuppressed mice. Transplantation 2004; 77: 1319–1326. Dere R, Wilson PD, Sandford RN et al. Carboxy terminal tail of polycystin-1 regulates localization of TSC2 to repress mTOR. PLoS One 2010; 5: e9239. Koehl GE, Spitzner M, Ousingsawat J et al. Rapamycin inhibits oncogenic intestinal ion channels and neoplasia in APCMin/+ mice. Oncogene 2010; 29: 1553–1560. de Gruijl FR, Koehl GE, Voskamp P et al. Early and late effects of the immunosuppressants rapamycin and mycophenolate mofetil on UV carcinogenesis. Int J Cancer 2009; 127: 796–804. Battaglia M, Stabilini A, Roncarolo MG. Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood 2005; 105: 4743–4748. Rao RR, Li Q, Odunsi K et al. The mTOR kinase determines effector versus memory CD8+ T cell fate by regulating the expression of transcription factors T-bet and Eomesodermin. Immunity 2010; 32: 67–78. Egli A, Ko¨hli S, Dickenmann M et al. Inhibition of polyomavirus BK-specific T-cell responses by immunosuppressive drugs. Transplantation 2009; 88: 1161–1168. Mueller NJ. New immunosuppressive strategies and the risk of infection. Transpl Infect Dis 2008; 10: 379–384. Araki K, Youngblood B, Ahmed R. The role of mTOR in memory CD8+ T-cell differentiation. Immunol Rev 2010; 235: 234–243. Kotton CN, Kumar D, Caliendo AM et al. International consensus guidelines on the management of cytomegalovirus in solid organ transplantation. Transplantation 2010; 89: 779–795. Li Y, Zheng XX, Li XC et al. Combined costimulation blockade plus rapamycin but not cyclosporine produces permanent engraftment. Transplantation 1998; 66: 1387–1388. Haxhinasto S, Mathis D, Benoist C. The AKT-mTOR axis regulates de novo differentiation of CD4+Foxp3+ cells. J Exp Med 2008; 205: 565–574. Turnquist HR, Raimondi G, Zahorchak AF et al. Rapamycin-conditioned dendritic cells are poor stimulators of allogeneic CD4+ T cells, but enrich for antigen-specific Foxp3+ T regulatory cells and promote organ transplant tolerance. J Immunol 2007; 178: 7018–7031. Weichhart T, Costantino G, Poglitsch M et al. The TSC-mTOR signaling pathway regulates the innate inflammatory response. Immunity 2008; 29: 565–577. Rahimi RA, Andrianifahanana M, Wilkes MC et al. Distinct roles for mammalian target of rapamycin complexes in the fibroblast response to transforming growth factor-b. Cancer Res 2009; 69: 84–93. Wang S, Wilkes MC, Leof EB et al. Noncanonical TGF-b pathways, mTORC1 and Ab1, in renal interstitial fibrogenesis. Am J Physiol Renal Physiol 2010; 298: F142–F149. Pontrelli P, Rossini M, Infante B et al. Rapamycin inhibits PAI-1 expression and reduces interstitial fibrosis and glomerulosclerosis in chronic allograft nephropathy. Transplantation 2008; 85: 125–134. Servais A, Meas-Yedid V, Toupance O et al. Interstitial fibrosis quantification in renal transplant recipients randomized to continue cyclosporine or convert to sirolimus. Am J Transplant 2009; 9: 2552–2560. Bridle KR, Popa C, Morgan ML et al. Rapamycin inhibits hepatic fibrosis in rats by attenuating multiple profibrogenic pathways. Liver Transpl 2009; 15: 1315–1324. Schnitzbauer AA, Zuelke C, Graeb C et al. A prospective randomised, open-labeled, trial comparing sirolimus-containing versus mTOR-inhibitorfree immunosuppression in patients undergoing liver transplantation for hepatocellular carcinoma. BMC Cancer 2010; 10: 190.
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http://www.kidney-international.org & 2010 International Society of Nephrology
The potential benefits of rapamycin on renal function, tolerance, fibrosis, and malignancy following transplantation Edward K. Geissler1 and Hans J. Schlitt1 1
Department of Surgery, University Hospital Regensburg, University of Regensburg, Regensburg, Germany
Use of the mammalian target of rapamycin (mTOR) inhibitor rapamycin in organ transplantation has evolved through different phases over the past two decades. After its discovery in the mid 1970s, antifungal and cytotoxic effects were the first of its properties to be explored, but the most significant advancement was found in its use as an immunosuppressive agent to reduce transplant rejection. This was viewed as an important step forward for immunosuppression, as early studies suggested that rapamycin was less nephrotoxic than calcineurin inhibitors (CNIs). Later, detrimental effects of rapamycin on kidney function were found in some patients. Nonetheless, a fascination with the mTOR pathway and its central role in multiple cellular processes has ensued. Among the potential positive clinically relevant effects is rapamycin’s capacity to interfere with fibrotic processes that often accompany transplant rejection, and to influence the preferential development of immunological tolerance. A feature of increasing importance is that the mTOR pathway is central for vital aspects of tumor development, including angiogenesis and cell growth; rapamycin, therefore, has anticancer activities, which may prove critical in the fight against high cancer rates in transplant recipients. The final chapters defining the value of rapamycin have not been written yet, and indeed remain a work in progress. Only further research will reveal the full potential of rapamycin in organ transplantation. Kidney International (2010) 78, 1075–1079; doi:10.1038/ki.2010.324; published online 22 September 2010 KEYWORDS: fibrosis; immunosuppression; malignancy; rapamycin; tolerance
Correspondence: Edward K. Geissler, Department of Surgery, University Hospital Regensburg, University of Regensburg, Franz-Josef-Strauss-Allee 11, Regensburg 93053, Germany. E-mail: [email protected] Received 29 April 2010; revised 24 June 2010; accepted 13 July 2010; published online 22 September 2010 Kidney International (2010) 78, 1075–1079
EARLY VERSUS MORE RECENT IMPRESSIONS OF RAPAMYCIN USE IN TRANSPLANTATION
Rapamycin was first discovered in the early 1970s from a soil sample taken on Easter Island (Rapa Nui). Based on the molecule’s readily apparent antiproliferative effects, it was first tested as an antimicrobial agent and for its cytotoxicity against tumors when used at high concentrations.1 However, its actual medicinal potential was not revealed until used continuously at low concentrations as an immunosuppressant in organ transplantation. Several pharmacological rapamycin analogs (e.g., sirolimus and CCI-779 (Pfizer, New York, NY, USA), RAD001 (Novartis, Basel, CH, USA), AP23573 (Ariad Pharma, Cambridge, MA, USA)) have been developed, as a result, all of which will simply be hereafter referred to as ‘rapamycin’. Regarding its use as an immunosuppressant, it was discovered early that T cells are inhibited by rapamycin as interleukin-2 triggers T-cell proliferation via the mammalian target of rapamycin (mTOR) signaling pathway. Expectations became increasingly high that this new immunosuppressive drug would be effective at preventing rejection, although not causing nephrotoxicity associated with calcineurin inhibitors (CNIs) use. Early animal experiments supported this notion, and initial clinical studies also pointed towards reduced nephrotoxicity with rapamycin use.2 Studies that followed, however, where rapamycin was clinically tested to reduce or eliminate CNI use, particularly in the setting of delayed or chronic renal allograft dysfunction, showed that mTOR inhibition can, in some patients, cause proteinuria.3 The exact effects of mTOR inhibition on cells of the kidney are not completely understood, but evidence suggests that tubular cell regeneration is affected, as well as podocytes.4 Interestingly, podocytes produce vascular endothelial cell growth factor, which is an important factor for maintaining the glomerular filtration barrier; as rapamycin potently inhibits vascular endothelial cell growth factor gene transcription and signaling,5 proteinuria could be a consequence of its therapeutic use. With proper monitoring and patient selection criteria, proteinuria is avoidable or controllable in most cases with rapamycin use; in more malignant proteinuria situations, rapamycin use needs to be withdrawn. The question of rapamycin effectiveness against rejection also must be considered. In renal transplantation, rapamycin has proved quite effective against kidney rejection when used 1075
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de novo in combination with CNIs, but to avoid excessive side effects, both substances need to be used at reduced levels. Although an improvement in glomerular filtration rate is reproducible when CNIs are tapered away,6 the trade-off is that rejection rates may increase. An alternative approach gaining more favor recently is to convert CNI-based immunosuppression to an mTOR inhibitor-based regimen at strategic periods within the first year after transplantation. Early clinical data suggest that this option does not increase rejection rates, improves renal function,7 and provides some protection against chronic allograft nephropathy.8 To date, however, the issue of whether rapamycin use alone can adequately prevent rejection and improve long-term renal allograft function remains an open question, and is beyond the scope of debate within this concise review.
EK Geissler and HJ Schlitt: Pros of rapamycin use in organ transplantation
Growth factors: e.g., IL-2, VEGF
P13K PTEN PDK1
Nutrients
AKT IKKβ
mTOR
TSC1/2
mTORC2
PC-1 Rheb
MOLECULAR INTRIGUE
Perhaps the most fascinating aspect related to rapamycin is the central significance of the mTOR pathway, which it influences. Indeed, the past decade of research on the mTOR pathway has revealed its pivotal role in controlling responses to a wide variety of growth factors, cytokines (e.g., interleukin-2), and nutrients. Recent studies indicate that even life span is affected (positively) by mimicking dietary restriction.9 Through the mTOR axis, basic cellular processes such as cell growth and proliferation are regulated, controlling the behavior and development of normal as well as abnormal tissues. In some instances it becomes even more complex because common processes such as angiogenesis are dependent on mTOR signaling, affecting both normal tissue repair (e.g., wound healing) and pathological tumor expansion. It is not surprising, therefore, that mTOR is highly regulated and integrated with other intracellular signaling pathways. The complexities of this molecular pathway have produced a high degree of molecular intrigue, motivating researchers to investigate the more exact effects of mTOR inhibition by rapamycin and other mTOR inhibitors. Here, we can only highlight the most critical elements of the mTOR signaling pathway (Figure 1). mTOR is part of two signaling complexes, mTOR complex 1 (mTORC1) and mTORC2. Interestingly, AKT provides signals to mTORC1, but mTORC2 actually regulates AKT through Ser473 phosphorylation. This review focuses on mTORC1, as it is the primary target of rapamycin. Phosphoinositide-dependent kinase-1 phosphorylates AKT at a second site, Thr308, further potentiating AKT activity. A wide variety of growth factors and cytokines can trigger this pathway. Key upstream signaling molecules include phosphoinositide 3-kinase, which converts phosphatidylinositiol-4,5-phosphate to phosphatidylinositiol3,4,5-phosphate and recruits phosphoinositide-dependent kinase-1 to AKT. Another important molecule is PTEN (phosphatase and tensin homolog deleted on chromosome 10), which is a tumor suppressor that reverses the phosphatidylinositiol-4,5-phosphate-to-phosphatidylinositiol-3,4,5-phosphate reaction, thus reducing AKT activity. Indeed, AKT has a critical role by mediating mTORC1 activation via inhibition of 1076
Other signaling pathways (e.g., MAPK)
mTOR
Rapamycin
mTORC1
S6K1
4E-BP1
eIF-4E
Transcription
Translation
Cell growth and metabolism
Figure 1 | mTORC1 pathway basics. Abbreviations: eIF-4E, eukaryotic initiation factor 4E; IKKb, inhibitory kB kinase-b; IL-2, interleukin-2; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; mTORC, mTOR complex; PC-1, polycystin-1; PDK1, phosphoinositide-dependent kinase-1; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homolog deleted on chromosome 10; Rheb, Ras homolog enriched in brain; S6K1, S6 kinase 1; TSC, tuberous sclerosis complex; VEGF, vascular endothelial cell growth factor; 4E-BP1, eIF-4E-binding protein-1.
the tumor suppressors, hamartin tuberous sclerosis complex (TSC)1) and tuberin (TSC2). The TSC1/2 complex inactivates Rheb (Ras homolog enriched in brain), thereby inhibiting mTORC1 activation. The primary downstream effectors of mTORC1 are S6 kinase 1 and eukaryotic initiation factor 4E-binding protein-1, which are important regulators of mRNA translation. Interestingly, to apparently protect against overactivation of mTORC1, active S6 kinase 1 negatively feeds back to inhibit AKT, and thus activation of mTORC1. The mTOR pathway is tangentially influenced in many ways. For instance, inhibitory kB kinase-b, a kinase downstream of tumor necrosis factor-a, integrates with mTORC1 by inhibiting TSC1/2, molecularly linking inflammation and cell proliferation.10 The mitogen-activated protein kinase cascade is also linked to mTOR, as inhibition of one pathway affects the activation of the other. Additionally, mTOR, because of its effects on cell growth, is intimately involved in energy metabolism. For instance, mTORC1 activity is regulated by sensing cellular adenosine triphosphate levels (via AMP-activated protein kinase and TSC1/2) and by sensing amino acids.11 Even lipid metabolism is influenced by Kidney International (2010) 78, 1075–1079
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mTORC1, positively affecting peroxisome proliferator-activated receptor-g, which has an important role in adipogenesis and lipid accumulation.12 Therefore, mTOR’s central intracellular signaling position in essential cell processes make it an attractive therapeutic target for conditions where it is desirable to reduce cell growth and proliferation (e.g., T cells—immune suppression, neoplastic cells—oncology, endothelial and smooth muscle cells—vasculopathy). DISCOVERY OF ANTITUMOR EFFECTS
Besides the well-known immunosuppressive properties of rapamycin, there is a rapidly expanding literature base on its anticancer effects. Although early toxicology studies showed some modest effects on tumors, the concentrations of rapamycin used in these experiments were high and carried significant toxicity.1 Enthusiasm for rapamycin use as a pure oncologic agent faded until early in the present decade. A better understanding of the mTOR signaling pathway brought new hypotheses as to how rapamycin could be used more effectively against cancer. One breakthrough came in 2002 when Guba et al.5 discovered in mice that rapamycin potently inhibits tumor angiogenesis, and thus tumor growth, at relatively low, sustainable, concentrations that are compatible with long-term immunosuppression in transplant recipients. Evidence suggested that the antiangiogenic effect was related to inhibition of vascular endothelial cell growth factor production and endothelial cell responses to vascular endothelial cell growth factor, which are both dependent on the mTOR pathway. This key finding was later confirmed by others, and tumors highly dependent on angiogenesis have since shown positive responses to rapamycin treatment.13,14 A natural question in the context of organ transplantation is whether rapamycin could cause a tumor to regress in immunosuppressed patients. Indeed, it has been shown that rapamycin can prevent allograft rejection in mice through its immunosuppressive effects, while at the same time inhibiting tumor growth.15 At least in the situation of Kaposi’s sarcoma in renal transplant recipients, this idea has shown clinical results.13 These examples are an extraordinary demonstration of the multiple effects of rapamycin, and they stress the central importance of this pathway for essential cellular processes. The anticancer effects of rapamycin are not limited to angiogenesis. Tumor cells are often directly dependent on the mTOR pathway because of their need to use its proliferationpromoting mechanisms and because gene mutations within the pathway can constitutively activate mTOR. There are many examples that illustrate this latter point. For example, PTEN and TSC1/2 can be oncogenic through mutations that result in a release of their inhibitory effects on mTORC1; e.g., tuberous sclerosis is a specific hyperproliferative condition that results from mutations in the tuberous sclerosis suppressor complex. Overexpression of the receptor tyrosine kinase human epidermal growth factor receptor 2 in malignancies such as breast and gastric cancer is another example of promoting pathological signaling through the AKT/mTORC1 axis. Polycystic kidney disease is a condition Kidney International (2010) 78, 1075–1079
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where mutations in polycystin-1 have very recently been shown to cause a lack of tethering of TSC2 to the cell membrane, decreasing the ability of TSC2 to repress mTORC1.16 These and other mTORC1 pathway-promoting gene mutations make mTOR an attractive target for antitumor therapy. Recent studies also suggest that rapamycin has even broader ranging effects that influence the growth and development of tumors. For instance, rapamycin treatment markedly reduces spontaneous de novo intestinal tumor formation in APCmin/ þ mice, with a near complete correction in the expression of otherwise upregulated oncogenic Na þ and K þ ion channels;17 the mechanism for the normalization of oncogenic ion channels is unknown. Rapamycin can also strongly affect the pattern of ultraviolet-signature p53 mutations, which are thought to be important in skin cancer development.18 Therefore, there is even evidence that mTOR influences processes that affect DNA damage repair. With more research and a better understanding of the mTOR pathway, the spectrum of rapamycin effects that may impact tumor growth will likely continue to expand. Finally, we cannot leave this discussion without addressing the potential impact that rapamycin has on immunity against tumor cells and viruses. An intuitive reaction to this point is that rapamycin inhibits the immune system in general and therefore must diminish immunity against invasive neoplasms. Although this is a reasonable argument, the conclusion appears premature and full of complexities. In favor of this hypothesis are two main factors, the well-known general suppressive effect that rapamycin has on T-cell proliferation, and its recently discovered promotion of T-regulatory cell development.19 It can be reasonably speculated that T-regulatory cells could act locally within a tumor, or systemically towards a virus, to protect against invading immune cells; much the same idea as the protection against rejection that is expected with T-regulatory cells in a patient with a tolerant organ allograft. Although it is concerning that rapamycin treatment could promote immunological tolerance to a tumor entity or virus, there is recent data that takes a completely opposite perspective. Rao et al.20 have recently reported that mTOR inhibition with rapamycin inhibits Tbet and promotes eomesodermin expression, which is associated with an enhanced memory phenotype in CD8 þ cells; rapamycin-treated CD8 þ cells showed impressive antitumor activity in vivo. Data also suggest that rapamycin-exposed CD8 þ cells could work favorably against BK virus,21 cytomegalovirus,22 or other viruses23 associated with infectious and neoplastic diseases in transplant recipients (e.g., Epstein–Barr virus in posttransplant lymphoproliferative disease; herpesvirus in Kaposi’s sarcoma). Indeed, a recent organ transplantation guideline publication suggests that there may be a lower incidence of cytomegalovirus infections when using mTOR inhibitors.24 It has to be added, however, that conflicting data and much controversy surrounds this topic, so further studies will be critical in determining the balance of effects rapamycin has on the development of malignancies and viral infections. 1077
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TOLERANCE-INDUCTION POTENTIAL
There is much debate about which immunosuppressive agents and regimens may possess the most favorable properties for tolerance induction in organ transplantation. Early experimental data indicate that CNI use, through blocking T-cell activation, may hinder the development of immunological tolerance.25 In contrast, allowing T-cell activation, but not proliferation, by using rapamycin could potentially promote tolerance. Moreover, recent publications indicate that as mTORC1 is essential for the development of conventional effector T cells, blocking mTORC1 with rapamycin likely favours T-regulatory cell expansion.26 Indeed, expanding T-regulatory cells either through drug therapy or ex vivo cell therapy is being considered in novel transplant tolerance-induction strategies. Rapamycin may also indirectly promote T-cell anergy and regulation by inhibiting the maturation of dendritic cells.27 However, the immune response depending on mTOR is as complex as that when considering its influence on tumor formation. For instance, it has been shown that molecules involved in the innate immune response (e.g., toll-like receptor 4) can actually inhibit the production of inflammatory cytokines through mTOR, thus predicting that mTOR inhibition could be proinflammatory.28 Therefore, the timing of therapeutic rapamycin application may be critical in determining its potential in inducing immunological tolerance. These considerations notwithstanding, mTOR has a central, albeit complex, role in directing the immune response. CONTROL OF FIBROTIC PROCESSES
Preventing development of interstitial renal fibrosis remains an unsolved problem following organ transplantation. Although we have learned how to control early and late acute rejection reactions against allografts, chronic pathological processes such as fibrosis have not proven to be controllable. When considering renal fibrosis, transforming growth factor-b is a well-known primary mediator through induction of fibroblast proliferation and myofibroblast transition. Although smad-2 and -3 are the most commonly known substrates of the transforming growth factor-b receptor, recent evidence also suggests that transforming growth factor-b signals via other pathways, including the AKT/mTORC1 axis.29 Indeed, blocking mTORC1 with rapamycin has been recently shown to reduce renal interstitial fibrosis in an obstructive nephropathy rodent model by diminishing the number of interstitial fibroblasts and myofibroblasts.30 The question remains, however, whether rapamycin can reduce fibrosis in transplant recipients. Data have been analyzed from different trials where patients on CNIs were later converted to rapamycin, but the results are conflicting. Pontrelli et al.31 have reported that rapamycin can substantially reduce interstitial fibrosis in renal transplant recipients after a chronic allograft nephropathy diagnosis, whereas Servais et al.32 did not find a significant reduction in fibrosis after 1 year when patients were converted from CNIs to rapamycin 12 weeks after renal transplantation. A number of 1078
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factors can explain the results of the latter study, as the negative effects of CNIs can occur very early (before randomization) and the readout was for a short period, possibly not allowing for detection of a difference within the observed time frame. Nonetheless, more trials testing this hypothesis will clearly need to be performed to determine whether fibrosis can be influenced by rapamycin following renal transplantation. We should add to this discussion that hepatic fibrosis associated with liver disease may also be targetable with rapamycin treatment, as a recent experimental study in rats has demonstrated.33 Interestingly, multiple profibrinogenic pathways are operable within liver tissue, suggesting that the mechanism of rapamycin inhibition of fibrosis in the liver is different than that which takes place in the kidney. PITFALLS OF RAPAMYCIN USE IN TRANSPLANTATION
Although this review concentrates on the positive aspects of rapamycin use in transplantation, it is important to include some discussion of the pitfalls encountered with the use of this substance or its derivatives. The most common problems are associated with side effects that can decrease the quality of life for some transplant recipients. In particular, oral ulcers, skin lesions (e.g., acne) and various forms of edema can present distressing problems for patients. In these cases, dose reductions can be helpful, along with specific counteractive treatments, but the problems may continue to persist and eventually result in rapamycin discontinuation. Furthermore, other serious side effects include wound-healing problems after surgery, delayed graft function, anemia, pneumonitis, and the problem described earlier with proteinuria in some patients. These types of side effects have sometimes resulted in elevated dropout rates in clinical studies. Although a legitimate argument can be made that physicians prematurely switch patients off mTOR inhibitors before making enough efforts to manage the side effects, it is also fair to make the argument that treatment with mTOR inhibitors requires substantial medical management. Therefore, assessment of the risk/benefit profile of rapamycin use in individual renal transplant recipients has become a central, and decisive, issue. CLINICAL PERSPECTIVES
When trying to calculate the future of rapamycin use in organ transplantation, there are many factors that have to go into the equation. Factors of particular importance that have to be considered are the development of malignancy and chronic fibrosis, both of which are critical problems facing transplantation medicine. Based on our present knowledge of the mTOR pathway, including experimental and clinical data that are presently available, an argument can be made for rapamycin treatment as a means to diminish these serious and otherwise untreatable post-transplant complications. Moreover, of the immunosuppressants currently available for transplantation, rapamycin has shown the highest potential to support the development of immunological tolerance in experimental models, especially those that espouse tolerance through the induction of anergy and Kidney International (2010) 78, 1075–1079
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T-regulatory cells. This is not an insignificant consideration, given that the induction of at least some level of stable immunological tolerance remains the ‘holy grail’ of transplantation medicine. If future clinical trials can definitively show that any of these potential positive attributes are veritable, mTOR inhibitors could gain a more broader base of acceptance in organ transplantation. The value of rapamycin in the situation where a malignancy occurs, or has a high probability of occurrence, in transplant recipients is especially promising. In this respect, one important ongoing multicentre clinical study is testing whether rapamycin treatment can reduce hepatocellular carcinoma-free survival in liver transplant recipients with a pretransplant hepatocellular carcinoma.34 Demonstration of a clear long-term benefit from this and other ongoing or planned clinical studies will give motivation for transplant physicians to better manage the side effects associated with rapamycin use, rather than prematurely switching to other immunosuppressants that possess their own undesirable side effect profiles. In the meantime, intrigue regarding the central role mTOR has in multiple cellular processes will continue to shed new light onto the potential therapeutic usefulness of mTOR inhibitors in organ transplantation. What is known so far is that rapamycin is a drug with potent immunosuppressive, antiproliferative, and antitumor effects, indicating that this substance influences a wide range of cell types and cellular processes. The level of this complexity is shown by rapamycin’s ability to treat multiple conditions simultaneously, as demonstrated by its combined immunosuppressive and anticancer action in transplant patients with a malignancy. The question that still needs to be answered is whether the balance of positive effects outweighs the side effects that often accompany treatment. Only further experimental and clinical studies can answer this question. DISCLOSURE
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All the authors declared no competing interests. 27.
ACKNOWLEDGMENTS
Source of support: RISET: A European Union FP6 program aimed at resetting the immune system for the establishment of tolerance.
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