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mTOR and cancer: reason for dancing at the crossroads?
mTOR and cancer: reason for dancing at the crossroads? George V Thomas Recent successes using Gleevec for the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors have provided proof that strategies to target signal transduction pathways mutated in human cancers can work. However, the application of this strategy to other cancers has been slow. Central to alleviating this impedance is the molecular characterization of the tumors. There is an urgent need to translate basic scientific findings into relevant, clinically applicable molecular diagnostic assays. Addresses Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at the University of California Los Angeles, A7-149 Center for Health Sciences, 10833 Le Conte Avenue, Los Angeles, CA 90095-1732, USA Corresponding author: Thomas, George V ([email protected])
Current Opinion in Genetics & Development 2006, 16:78–84 This review comes from a themed issue on Oncogenes and cell proliferation Edited by Allan Balmain and Denise Montell Available online 15th December 2005 0959-437X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2005.12.003
Introduction How do we extend to our patients the promise of molecular targeted therapies and the major strides made in the post-genome era? This review summarizes recent advances that have helped us to better understand the function and regulation of mTOR (mammalian target of rapamycin). Using preclinical and emerging clinical data from kidney cancers, I develop a paradigm for the translational application of mTOR inhibitors that can be applied to other tumors, and I give my perspective on how the goal of personalized molecularly targeted therapies can be achieved.
mTOR: at the crossroads of multiple signaling pathways Like all good puzzles, the mTOR dynamic has been one of discoveries, twists, turns, challenges and hope. To understand the central role of mTOR, we start at the cell membrane, where growth factor-mediated activation of their cognate receptor tyrosine kinases (RTKs) recruits PI3K (phosphatidylinositol 3-kinase), a lipid kinase, to Current Opinion in Genetics & Development 2006, 16:78–84
the cell membrane (Figure 1). PI3K converts PIP2 to PIP3 (phosphatidylinositol-3, 4, 5-triphosphate), which is actively opposed by its phosphatase, PTEN (phosphatase and tensin homologue deleted on chromosome ten). PIP3 recruits PDK1 (phosphoinositide-dependent kinase-1) and AKT to the membrane; PDK1 activates AKT at the Thr308 position [1]. Once activated, AKT phosphorylates a myriad of proteins, with the net result of increased cellular survival, proliferation, growth and metabolism. Specifically, phosphorylation of mTOR by AKT occurs through inactivation of the tuberous sclerosis complex (TSC). The TSC complex, a heterodimer consisting of unphosphorylated TSC2 (tuberin) and TSC1 (hamartin), acts as a GTPase-activating protein (GAP), inhibiting the small G-protein Rheb (Ras homolog enriched in brain). By phosphorylating TSC2, AKT disrupts the TSC complex, enabling Rheb to bind to ATP and convert itself from the inactive GDP state to the active GTP state. GTP-bound Rheb, in turn, activates mTOR [2,3]. In a parallel pathway, 50 AMP-activated protein kinase (AMPK) phosphorylates and activates TSC2, thereby inhibiting mTOR activation in response to changes in the intracellular ATP/AMP ratio [4]. This links the energy-sensing pathway (i.e. through amino acids and ATP) to mTOR. STK11 (serine threonine kinase 11; also called LKB1), a tumor suppressor inactivated in Peutz-Jeghers syndrome, is an upstream, AMPK-activating kinase and serves as an important inhibitor of the mTOR pathway in response to energy starvation [5,6]. Loss of LKB1 protein results in hyperactivation of mTOR signaling — similar to the effect of loss of PTEN on the growth factor-mediated pathway. In addition, amino acids regulate mTOR activity directly, through the TSC complex, although the mechanisms have not been fully deciphered [7]. mTOR, strategically positioned at the intersection of multiple growth factors (e.g. insulin, IGF [insulin-like growth factor] and PDGF [platelet-derived growth factor]) and nutrient (e.g. amino acids) signaling networks, functions as a gatekeeper, regulating cell growth, metabolism and proliferation. Rapamycin, the archetypical mTOR-inhibitor, specifically complexes with FKBP12 (FK506-binding protein 12), and it is this complex that interacts with and inhibits mTOR [8]. Although mTOR has several functions, it appears that its regulation of translation is the best studied in relation to oncogenesis. In particular, protein synthesis is regulated www.sciencedirect.com
mTOR and cancer: reason for dancing at the crossroads? Thomas 79
Figure 1
mTOR: regulation and function. This schematic outlines how mTOR integrates multiple signaling inputs into cohesive cell growth, proliferation, metabolism and survival end-points. Growth factor RTKs recruit PI3K, with subsequent activation of AKT. AKT phosphorylates and inactivates the TSC1–2 complex, resulting in constitutive activation of mTOR and S6K, and inactivation of 4E-BP1, ultimately driving translation. Inhibition of mTOR by rapamycin results in downregulation of these signals.
by the phosphorylation of two mTOR substrates: ribosomal S6 kinase (S6K) and the eukaryotic initiation factor 4E binding protein-1 (4EBP-1) [9,10]. S6K phosphorylates S6 ribosomal protein, resulting in the translation of mRNAs containing specific oligopyrimidine tracts, referred to as terminal oligopyrimidine tracts (TOPs), within their 50 untranslated region (UTR); these mRNAs, in turn, encode proteins essential for ribosome biogenesis, including eIF-4G (eukaryotic initiation factor-4G), eEF-1A (eukaryotic elongation factor-1A) and eEF-2A, thus enhancing the cell’s overall translational machinery. Although recent data have added another level of complexity to the definition of the mechanism of S6K-mediated TOP-dependent translation, the data consistently show that rapamycin inhibits both S6 activawww.sciencedirect.com
tion and 50 TOP mRNA translation, confirming a role for mTOR in this process [11–13]. The second translational regulator targeted by mTOR is 4EBP-1, which acts as a translational repressor, binding and inhibiting the eukaryotic translation initiation factor4E (eIF-4E). eIF-4E binds the m7GTP-containing cap structure of the mRNA located in the 50 UTR, and, subsequently, this complex interacts with eIF-4G — the resulting complex is called eIF-4F — thereby driving cap-dependent translation. mTOR-mediated signaling causes 4EBP-1 to become highly phosphorylated and to dissociate from eIF-4E. eIF-4E then drives the translation of 50 cap mRNAs, including several oncogenic proteins such as FGF (fibroblast growth factor), c-Myc, VEGF (vascular endothelial growth factor) and cyclin Current Opinion in Genetics & Development 2006, 16:78–84
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D1 [9]. Recent data point to eIF-4E having an additional role, in suppressing apoptosis [14,15]. Additionally, mTOR regulates polypeptide chain elongation, indirectly, through phosphorylation of eEF-2. It does this by phosphorylating eEF-2 kinase — one input is through S6K — making it less active and, subsequently, relieving the inhibition on its substrate, eEF-2. This might account for the global inhibition of translation of specific mRNA transcripts by rapamycin (e.g. short-lived proteins, such as p21 and HIF-a (hypoxia-inducible factor-a) subunits [16,17]. Recent discoveries of a mTOR kinase domain-interacting protein, GßL, as well as two scaffold proteins, Raptor and Rictor, are shedding some light on how mTOR achieves its downstream effect s. The GßL–Raptor– mTOR or the GßL–Rictor–mTOR multimeric complexes are able to bind to S6K and 4E-BP1, through mTOR signaling motifs (TOSs) present on both these effectors [18,19]. Rather than being redundant, these complexes are strikingly distinct, in that the GßL–Raptor–mTOR complex is rapamycin-sensitive, whereas the GßL–Rictor–mTOR complex is rapamycin-resistant [20]. Furthermore, it has been shown recently that the Rictor–mTOR complex is actually the elusive PDK2, which phosphorylates AKT at the Ser473 position, resulting in its full activation [21]. The clinical hallmark of the TSC, Peutz-Jeghers syndrome and PTEN loss-of-function mutations is the development of benign hamartomatous syndromes. By contrast, activation of the PI3K–AKT pathway results in malignant tumors. Recent data, using Tsc1/ or Tsc1/ mouse embryonic fibroblasts have shown that insulin- or IGF-1-induced hyperactivation of mTOR in these isogenic systems produces a negative feedback loop, resulting in the inhibition of PI3K–AKT signaling [22,23]. This is mediated by S6K, which phosphorylates IRS1 and IRS2 (insulin receptor substrates 1 and 2), inhibiting their function and promoting their degradation [24]. Insulin- or IGF-1-stimulated phosphorylation of AKT can be rescued by rapamycin treatment. The depletion of the IRS protein by hyperactivated mTOR might explain, at least in part, the benign nature of tumors associated with the TSC syndromes. There might be the requirement for additional mutation(s), either in an epistatic or in a parallel pathway, to ensure malignant transformation. These new findings raise more questions than answers regarding the suitability of mTOR inhibitors as anticancer agents: would rapamycin treatment promote tumor formation by downregulating the S6K negative feedback on IRS proteins? Which of the mTOR stoichiometric complexes are more stable or more abundant in biological systems? Given that the GßL–Rictor–mTOR complex is Current Opinion in Genetics & Development 2006, 16:78–84
rapamycin resistant, should the focus be more on inhibiting targets upstream of mTOR, such as PI3K or AKT? In addition, mTOR does not fit the classic targetable kinase paradigm, in that there is no evidence that mTOR is either mutated or amplified in cancers. Despite these reservations, I would find it hard to ignore a target that lies downstream of signaling pathways that read like cancer’s most wanted: Ras, PI3K, AKT (oncogenes); PTEN, TSC, LKB1, NF (tumor suppressor genes); S6K, eIF-4E, Cyclin D1, HIF-a, VEGF and c-MYC (effectors). The preclinical and emerging clinical data show that mTOR inhibitors have promising results, with minimal toxicity (more below).
Insights from preclinical studies Rapamycin is a natural macrolide product originally studied because of its immunosuppressant activity. Its ability to suppress T-cell activation led to it being approved by the FDA for the treatment of allograft rejection in transplant patients. Rapamycin also induces G1 growth arrest — and, in some cases, apoptosis — in certain tumor cell lines, leading to its current evaluation as a possible anti-cancer agent. Presently, there are at least three additional mTOR inhibitors that have been widely characterized in preclinical models, and that are in clinical development as anti-cancer agents: CCI-779, RAD001 and AP23573 [25]. All have comparable activity against mTOR in biochemical and biological assays, and differences between the three agents lie mainly in bioavailability and formulation protocols. To date, all of the currently available data on CCI-779 and rapamycin from preclinical trials support the hypothesis that the anti-tumor activity is explained by inhibition of mTOR. Assuming that mTOR is the relevant therapeutic target, what might be the molecular basis of the clinical responses seen? At least in models in which there is constitutive activation of the PI3K–AKT pathway (e.g. through loss of PTEN), it appears that these tumors are mTOR-dependent [26,27]. Contextually, these effects are probably mediated through the ability of mTOR to enhance translation of key factors that control cell proliferation, anti-apoptotic effects, anti-angiogenic effects or a combination of these. These preclinical studies have also highlighted some aspects of mTOR inhibitors that should hopefully guide the design of rational clinical trials that use these agents individually or in combination. First, the predominant biological endpoint was one of cytostasis rather than cytotoxicity, and this is consistent with the known ability of rapamycin to induce G1 arrest [10]. Going into clinical trials, this should shift the focus from readouts based on maximum tolerated dose and tumor shrinkage to one of disease stabilization and time to progression. Second, these studies have identified potential biomarkers for pathway activation as well as for target inhibition (e.g. phosphorylated-S6 ribosomal protein) [26–28]. www.sciencedirect.com
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To date, the natural history of targeted therapies is early response followed by emergence of resistance [29]. This should spur the search for rational combinations that have additive and/or synergistic effects for transfer to the clinic. Although targeted therapies might be the antithesis of conventional chemotherapies, emerging data are showing how rapamycin can function as a chemotherapeutic sensitizer in this context [17].
Clinical development Wyeth Pharmaceuticals has conducted several phase I and II clinical trials of intravenous CCI-779 (Temsirolimus1), to evaluate its safety and anti-tumor activity in a range of tumor types. Two phase I dose escalation studies were conducted in a range of solid tumors, with CCI-779 administered as a 30-minute intravenous (IV) infusion weekly (7.5 to 220 mg/ml/week) or as a 30-minute IV infusion daily for five days (0.75 to 24 mg/ml/day) every two weeks. These studies opted for intermittent IVdosing to minimize the potential risk of immunosuppression. Objective responses and disease stabilization were observed in several patients with advanced stage kidney, breast cancer and non-small cell lung cancers; minor responses were observed in subsets of lymphomas, sarcomas, and endometrial and skin carcinomas. The drug was well tolerated, with the main adverse effects being mucocutaneous (i.e. rash, mucositis and stomatitis). Significantly, the maximum tolerated dose was not reached — signifying a high therapeutic index — and no immunosuppressive effects were seen [25]. On the basis of the promising data seen in kidney cancers (renal cell carcinomas), a phase II trial was conducted to evaluate the safety and efficacy of three dose-levels of CCI-779 in 110 previously treated patients with advanced renal cell carcinomas. Patients were randomized to receive a weekly dose of 25, 75 or 250mg of CCI-779 by intravenous infusion. The objective response rate was only 7% (one complete response and seven partial responses), but there was a remarkably high rate of minor responses and disease stabilization (i.e. complete response, partial response, minor response or stable disease 24 weeks was noted in approximately 50% of patients; median time to progression was close to six months; and median survival was 15 months). These findings were even more striking when intermediate and high-risk patients were examined — the survival rates in this subset of patients were 1.6- to 1.7-fold greater than those of interferon-a-treated patients [30]. These findings are consistent with the cytostatic activity of this compound in most models and have led to a phase III randomized trial using time to progression as the primary endpoint. Notably, equal responses were observed across all doses. All doses tested in these trials gave peak plasma concentrations well above that required for inhibition of mTOR, but the intermittent nature of the dosing enabled troughs to fall below mTOR inhibition levels. These www.sciencedirect.com
clinical data are consistent with the notion that mTOR is the relevant drug target in renal cell carcinomas. The striking results seen with renal cell carcinomas require further consideration, especially given that they have led to phase III clinical trials being carried out, and yet there is no genuine understanding of the underlying molecular mechanisms of response. The current, randomized phase III study compares CCI-779 with interferona or a combination of both. Although this clinical development strategy makes sense on the basis of available clinical data, there is the real possibility that the trial could be negative because there is no current molecular insight into the group of patients who have mTORdependent tumors. The parallels to the recent EGFR (epidermal growth factor receptor)-inhibitor trials in unselected lung cancer patients are striking. EGFR inhibitors (e.g. Iressa and Tarveca) have low but consistent single-agent objective response rates in refractory lung cancer but fail to add to chemotherapy in large randomized trials. Two recent studies showing that Iressa responders have EGFR mutations in their lung tumors clarifies the confusion surrounding these clinical datasets and makes it clear that molecular phenotyping of lung cancer patients will be essential for the selection of appropriate therapy [31,32]. Are there clues that might suggest which kidney tumors would be more sensitive to mTOR inhibitors? Surprisingly, some of the answers can be found in a mouse model of pre-invasive prostate cancer. The Sellers laboratory engineered the selective overexpression of activated AKT in the murine prostate; these mice develop a highly penetrant murine prostatic intraepithelial neoplasia (mPIN) phenotype. Treatment with RAD001 completely reversed the mPIN phenotype in two weeks, confirming that these tumors were mTOR-dependent [33]. The mechanism of response was through the induction of apoptosis. When they examined the transcriptional profile of the vehicle-treated mice, they found that HIF-1a target genes, especially those involved in regulating angiogenesis and glycolysis were upregulated and that these were downregulated following RAD001 treatment. These findings point to an additional function for mTOR — either directly or indirectly — in the regulation transcription. Recent data clearly place HIF downstream of mTOR in the PI3K–AKT pathway [34,35]. For example, constitutively active alleles of PI3K or AKT are sufficient to induce the production of VEGF mRNA and even angiogenesis [36–38]. These effects are blocked by rapamycin. Similarly, the receptor tyrosine kinase Her-2/neu acts through the PI3K–AKT–mTOR pathway to upregulate expression of HIF protein in breast cancer cells, and this upregulation is blocked by the PI3K inhibitor LY294002 and by rapamycin [39]. In these experiments, HIF is most Current Opinion in Genetics & Development 2006, 16:78–84
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probably upregulated by increased translation of HIF mRNA, which contains 50 -UTR sequences sensitive to the mTOR effector S6K. Therefore, the rapamycin analogue CCI-779 can have anti-angiogenic activity by downregulation of HIF in tumor cells, particularly in the setting of PI3K–AKT pathway dysregulation. In addition, recent data have shown that rapamycin is able to decrease HIF-1 levels in TSC2-null cells, indicating that TSC2 regulates HIF by inhibiting mTOR [40]. We already know that treatment with the anti-VEGF monoclonal antibody results in significant delays in time to progression in patients with metastatic renal cell carcinomas [41]. Similarly, in another tumor in which VEGF is upregulated, treatment with rapamycin completely inhibited the progression of Kaposi’s sarcoma [42]. This implies a combinatorial role in renal cell carcinomas between VEGF or VEGFR inhibitors and mTOR inhibitors. The upregulation of HIF-regulated glycolytic pathway transcripts (e.g. Glut-1) can be exploited both as a biomarker for patient selection and as a target for inhibition using FDG–PET ([18F] 2-fluro-2-deoxy-D-glucose–positron emission tomography). The increased glucose uptake imaged with FDG is dependent largely on the rate of glycolysis. FDG and trapping occurs because of upregulation of glucose transporters (e.g. Glut-1) and phosphorylation by hexokinases [43]. Given that subsets of renal cell carcinomas (e.g. clear-cell tumors) have increased levels of HIF, one would predict that they would exhibit higher uptake of FDG than that exhibited by non-clear cell tumors. Since the primary effect of mTOR inhibitors is G1 arrest, functional molecular imaging would be predicted to be superior, compared with conventional anatomic imaging, in elucidating biologically effective doses of mTOR inhibitors. Finally, developing non-invasive molecular assays enables repeatable, quantitative assessment of drug effect, thus sparing patients from additional surgical procedures to obtain tissue. Emerging data from phase II clinical trials using CCI-779 in brain and breast cancers show subsets of patients responding [44–46]. Reassuringly, when used as a single agent, it has shown considerable anti-tumor activity in patients with relapsed and/or refractory mantle-cell lymphoma, a hematological malignancy characterized by overexpression of cyclin D1 [47].
Biomarker-driven clinical trials How do we identify the patients who are most likely to benefit from mTOR inhibitors? We already know which cancers should respond. Prior work has shown that PI3K–AKT pathway-driven transformation is mTOR-dependent, because mTOR is a crucial component of a downstream kinase cascade. This result is consistent with the broader notion of ‘pathway Current Opinion in Genetics & Development 2006, 16:78–84
addiction’ to explain why tumor cells might be at a disadvantage, when faced with a pathway-specific inhibitor, compared with normal cells [48]. Based on this, I predict that a clinical trial using mTOR inhibitors in glioma, prostate or endometrial cancer patients with documented loss of PTEN would achieve significant disease stabilization and meaningfully delay the time to progression. However, we are not very good at implementing what we know. This is because we still rely on traditional definitions of cancers made on the basis of anatomical and histological parameters. Yes, these certainly have prognostic value in relation to disease recurrence and progression, but they add very little in reliably identifying individuals who will benefit from targeted therapies. There needs to be a re-evaluation of our perception of cancer. Solid tumors show great inter- and intra-tumoral heterogeneity, each with a distinct genetic abnormality and subsequent response to therapy. This then presents us with unique opportunities to make a therapeutic shift from the traditional view of treating cancers on the basis of their tissue or organ of origin — a move from ‘tissuespecific’ towards a more ‘target-specific’ strategy. Next, we need to identify the patients most likely to respond to these targeted drugs (i.e. matching the inhibitor to the patient). This requires the development of robust, clinically applicable assays. The tools for these assays are already at hand. Examples include global gene-expression profiles; proteomic approaches that are able to identify post-translational modifications such as phosphorylation; mutation analysis; methylation patterns, single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) arrays that are able to identify gain or loss in copy number; and molecular imaging. After therapy, the individual molecular signature can be monitored and, in cases of relapse, repeated — akin to the on-going kinome resequencing projects — and will become the basis for a revised personalized treatment strategy. In the first iteration of this, we have already identified a molecular signature of PTEN loss and/or AKT pathway activation in gliomas and prostate cancer [49,50]. Ideally, we would have a suite of biomarkers, some of which would be secreted proteins that could be measured in the serum (e.g. peIF-4G, p4E-BP1, HIF-1, GLUT-1 and VEGF), in addition to having molecular imaging probes (e.g. FDG– PET, FLT–PET {[18F] 3-deoxy-3-fluorothymidine– positron emission tomography}), to document inhibition of mTOR in tumor cells after administration of the mTOR inhibitor. Additionally, as with RAD001 studies in prostate cancer models, systematic expression-profiling experiments of tumor tissue before and after therapy to generate signatures of mTOR inhibition in tumor tissue will need to be performed and validated [33]. The alternative is the status quo, with investigators rolling www.sciencedirect.com
mTOR and cancer: reason for dancing at the crossroads? Thomas 83
out the mandatory CT (computed tomography) scans of anecdotal patient responses, which at best gives us a glimpse of what can be achieved.
Conclusions mTOR inhibitors have shown promising clinical efficacy in subsets of cancers, with low toxicity profiles. The challenge before us is to develop robust, reproducible biomarkers that can be used in the clinic to identify patients most likely to benefit, and to document modulation of the drug target. Our goal? Transform the way cancers are diagnosed, treated and cured.
Update We have recently shown that loss of the von HippelLindau (VHL) tumor suppressor gene sensitizes kidney cancer cells to the mTOR inhibitor CCI-779, both in vitro and in mouse models. Growth arrest caused by CCI-779 correlates with translational inhibition ofHIF mRNA and is rescued by expression of a VHL-resistant HIF cDNA lacking the 50 untranslated region [51].
Acknowledgements I thank Charles Sawyers for the science; Ingo Mellinghoff for helpful discussions; Moya Costello for graphics support. My work is supported by grants from the NCI–UCLA Prostate SPORE, US Department of Defense, University of California Cancer Research Coordinating Committee, the Stein-Oppenheimer Family Endowment and the Wendy Will Case Foundation. I am also indebted to Jonathan Braun for the academic and research support.
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30. Atkins MB, Hidalgo M, Stadler WM, Logan TF, Dutcher JP, Hudes GR, Park Y, Liou SH, Marshall B, Boni JP et al.: Randomized phase II study of multiple dose levels of CCI-779, a novel mammalian target of rapamycin kinase inhibitor, in patients with advanced refractory renal cell carcinoma. J Clin Oncol 2004, 22:909-918. 31. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG et al.: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004, 350:2129-2139. See annotation [32]. 32. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ et al.: EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004, 304:1497-1500. This study and that by Lynch et al. [31] uncover the mechanisms of sensitivity to EGFR inhibitors in subsets of lung cancer. 33. Majumder PK, Febbo PG, Bikoff R, Berger R, Xue Q, McMahon LM, Manola J, Brugarolas J, McDonnell TJ, Golub TR et al.: mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1dependent pathways. Nat Med 2004, 10:594-601. Using preclinical information that either PTEN loss or AKT overexpression sensitizes xenograft tumors to mTOR inhibitors, these authors showed that treatment with RAD001was able to reverse the mPIN phenotype in their AKT transgenic mice. They go on to identify a biological readout of RAD001 activity (i.e. the HIF-regulated family of target genes). 34. Hudson CC, Liu M, Chiang GG, Otterness DM, Loomis DC, Kaper F, Giaccia AJ, Abraham RT: Regulation of hypoxiainducible factor 1a expression and function by the mammalian target of rapamycin. Mol Cell Biol 2002, 22:7004-7014. 35. Semenza GL: Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003, 3:721-732. 36. Jiang BH, Zheng JZ, Aoki M, Vogt PK: Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of
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44. Chang SM, Kuhn J, Wen P, Greenberg H, Schiff D, Conrad C, Fink K, Robins HI, Cloughesy T, De Angelis L et al.: Phase I/ pharmacokinetic study of CCI-779 in patients with recurrent malignant glioma on enzyme-inducing antiepileptic drugs. Invest New Drugs 2004, 22:427-435. 45. Chang SM, Wen P, Cloughesy T, Greenberg H, Schiff D, Conrad C, Fink K, Robins HI, De Angelis L, Raizer J et al.: Phase II study of CCI-779 in patients with recurrent glioblastoma multiforme. Invest New Drugs 2005, 23:357-361. 46. Galanis E, Buckner JC, Maurer MJ, Kreisberg JI, Ballman K, Boni J, Peralba JM, Jenkins RB, Dakhil SR, Morton RF et al.: Phase II trial of temsirolimus (CCI-779) in recurrent glioblastoma multiforme: a North Central Cancer Treatment Group Study. J Clin Oncol 2005, 23:5294-5304. 47. Witzig TE, Geyer SM, Ghobrial I, Inwards DJ, Fonseca R, Kurtin P, Ansell SM, Luyun R, Flynn PJ, Morton RF et al.: Phase II trial of single-agent temsirolimus (CCI-779) for relapsed mantle cell lymphoma. J Clin Oncol 2005, 23:5347-5356. 48. Weinstein IB: Cancer. Addiction to oncogenes — the Achilles heal of cancer. Science 2002, 297:63-64. 49. Choe G, Horvath S, Cloughesy TF, Crosby K, Seligson D, Palotie A, Inge L, Smith BL, Sawyers CL, Mischel PS: Analysis of the phosphatidylinositol 30 -kinase signaling pathway in glioblastoma patients in vivo. Cancer Res 2003, 63:2742-2746. 50. Thomas GV, Horvath S, Smith BL, Crosby K, Lebel LA, Schrage M, Said J, De Kernion J, Reiter RE, Sawyers CL: Antibody-Based Profiling of the Phosphoinositide 3-Kinase Pathway in Clinical Prostate Cancer. Clin Cancer Res 2004, 10:8351-8356. This study and that by Choe et al. [49] describe a molecular signature for the PI3K–AKT pathway activation that can be probed in clinical samples and, thus, serve as a biomarker for patient selection and target inhibition. 51. Thomas GV, Tran C, Mellinghoff IK, Welsbie DS, Chan E, Fueger B, Czernin J, Sawyer CL: Hypoxia-inducible factor determines sensitivity to mTOR inhibitors in kidney cancer. Nat Med 2006, in press.
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Current Opinion in Genetics & Development 2006, 16:78–84 This review comes from a themed issue on Oncogenes and cell proliferation Edited by Allan Balmain and Denise Montell Available online 15th December 2005 0959-437X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2005.12.003
Introduction How do we extend to our patients the promise of molecular targeted therapies and the major strides made in the post-genome era? This review summarizes recent advances that have helped us to better understand the function and regulation of mTOR (mammalian target of rapamycin). Using preclinical and emerging clinical data from kidney cancers, I develop a paradigm for the translational application of mTOR inhibitors that can be applied to other tumors, and I give my perspective on how the goal of personalized molecularly targeted therapies can be achieved.
mTOR: at the crossroads of multiple signaling pathways Like all good puzzles, the mTOR dynamic has been one of discoveries, twists, turns, challenges and hope. To understand the central role of mTOR, we start at the cell membrane, where growth factor-mediated activation of their cognate receptor tyrosine kinases (RTKs) recruits PI3K (phosphatidylinositol 3-kinase), a lipid kinase, to Current Opinion in Genetics & Development 2006, 16:78–84
the cell membrane (Figure 1). PI3K converts PIP2 to PIP3 (phosphatidylinositol-3, 4, 5-triphosphate), which is actively opposed by its phosphatase, PTEN (phosphatase and tensin homologue deleted on chromosome ten). PIP3 recruits PDK1 (phosphoinositide-dependent kinase-1) and AKT to the membrane; PDK1 activates AKT at the Thr308 position [1]. Once activated, AKT phosphorylates a myriad of proteins, with the net result of increased cellular survival, proliferation, growth and metabolism. Specifically, phosphorylation of mTOR by AKT occurs through inactivation of the tuberous sclerosis complex (TSC). The TSC complex, a heterodimer consisting of unphosphorylated TSC2 (tuberin) and TSC1 (hamartin), acts as a GTPase-activating protein (GAP), inhibiting the small G-protein Rheb (Ras homolog enriched in brain). By phosphorylating TSC2, AKT disrupts the TSC complex, enabling Rheb to bind to ATP and convert itself from the inactive GDP state to the active GTP state. GTP-bound Rheb, in turn, activates mTOR [2,3]. In a parallel pathway, 50 AMP-activated protein kinase (AMPK) phosphorylates and activates TSC2, thereby inhibiting mTOR activation in response to changes in the intracellular ATP/AMP ratio [4]. This links the energy-sensing pathway (i.e. through amino acids and ATP) to mTOR. STK11 (serine threonine kinase 11; also called LKB1), a tumor suppressor inactivated in Peutz-Jeghers syndrome, is an upstream, AMPK-activating kinase and serves as an important inhibitor of the mTOR pathway in response to energy starvation [5,6]. Loss of LKB1 protein results in hyperactivation of mTOR signaling — similar to the effect of loss of PTEN on the growth factor-mediated pathway. In addition, amino acids regulate mTOR activity directly, through the TSC complex, although the mechanisms have not been fully deciphered [7]. mTOR, strategically positioned at the intersection of multiple growth factors (e.g. insulin, IGF [insulin-like growth factor] and PDGF [platelet-derived growth factor]) and nutrient (e.g. amino acids) signaling networks, functions as a gatekeeper, regulating cell growth, metabolism and proliferation. Rapamycin, the archetypical mTOR-inhibitor, specifically complexes with FKBP12 (FK506-binding protein 12), and it is this complex that interacts with and inhibits mTOR [8]. Although mTOR has several functions, it appears that its regulation of translation is the best studied in relation to oncogenesis. In particular, protein synthesis is regulated www.sciencedirect.com
mTOR and cancer: reason for dancing at the crossroads? Thomas 79
Figure 1
mTOR: regulation and function. This schematic outlines how mTOR integrates multiple signaling inputs into cohesive cell growth, proliferation, metabolism and survival end-points. Growth factor RTKs recruit PI3K, with subsequent activation of AKT. AKT phosphorylates and inactivates the TSC1–2 complex, resulting in constitutive activation of mTOR and S6K, and inactivation of 4E-BP1, ultimately driving translation. Inhibition of mTOR by rapamycin results in downregulation of these signals.
by the phosphorylation of two mTOR substrates: ribosomal S6 kinase (S6K) and the eukaryotic initiation factor 4E binding protein-1 (4EBP-1) [9,10]. S6K phosphorylates S6 ribosomal protein, resulting in the translation of mRNAs containing specific oligopyrimidine tracts, referred to as terminal oligopyrimidine tracts (TOPs), within their 50 untranslated region (UTR); these mRNAs, in turn, encode proteins essential for ribosome biogenesis, including eIF-4G (eukaryotic initiation factor-4G), eEF-1A (eukaryotic elongation factor-1A) and eEF-2A, thus enhancing the cell’s overall translational machinery. Although recent data have added another level of complexity to the definition of the mechanism of S6K-mediated TOP-dependent translation, the data consistently show that rapamycin inhibits both S6 activawww.sciencedirect.com
tion and 50 TOP mRNA translation, confirming a role for mTOR in this process [11–13]. The second translational regulator targeted by mTOR is 4EBP-1, which acts as a translational repressor, binding and inhibiting the eukaryotic translation initiation factor4E (eIF-4E). eIF-4E binds the m7GTP-containing cap structure of the mRNA located in the 50 UTR, and, subsequently, this complex interacts with eIF-4G — the resulting complex is called eIF-4F — thereby driving cap-dependent translation. mTOR-mediated signaling causes 4EBP-1 to become highly phosphorylated and to dissociate from eIF-4E. eIF-4E then drives the translation of 50 cap mRNAs, including several oncogenic proteins such as FGF (fibroblast growth factor), c-Myc, VEGF (vascular endothelial growth factor) and cyclin Current Opinion in Genetics & Development 2006, 16:78–84
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D1 [9]. Recent data point to eIF-4E having an additional role, in suppressing apoptosis [14,15]. Additionally, mTOR regulates polypeptide chain elongation, indirectly, through phosphorylation of eEF-2. It does this by phosphorylating eEF-2 kinase — one input is through S6K — making it less active and, subsequently, relieving the inhibition on its substrate, eEF-2. This might account for the global inhibition of translation of specific mRNA transcripts by rapamycin (e.g. short-lived proteins, such as p21 and HIF-a (hypoxia-inducible factor-a) subunits [16,17]. Recent discoveries of a mTOR kinase domain-interacting protein, GßL, as well as two scaffold proteins, Raptor and Rictor, are shedding some light on how mTOR achieves its downstream effect s. The GßL–Raptor– mTOR or the GßL–Rictor–mTOR multimeric complexes are able to bind to S6K and 4E-BP1, through mTOR signaling motifs (TOSs) present on both these effectors [18,19]. Rather than being redundant, these complexes are strikingly distinct, in that the GßL–Raptor–mTOR complex is rapamycin-sensitive, whereas the GßL–Rictor–mTOR complex is rapamycin-resistant [20]. Furthermore, it has been shown recently that the Rictor–mTOR complex is actually the elusive PDK2, which phosphorylates AKT at the Ser473 position, resulting in its full activation [21]. The clinical hallmark of the TSC, Peutz-Jeghers syndrome and PTEN loss-of-function mutations is the development of benign hamartomatous syndromes. By contrast, activation of the PI3K–AKT pathway results in malignant tumors. Recent data, using Tsc1/ or Tsc1/ mouse embryonic fibroblasts have shown that insulin- or IGF-1-induced hyperactivation of mTOR in these isogenic systems produces a negative feedback loop, resulting in the inhibition of PI3K–AKT signaling [22,23]. This is mediated by S6K, which phosphorylates IRS1 and IRS2 (insulin receptor substrates 1 and 2), inhibiting their function and promoting their degradation [24]. Insulin- or IGF-1-stimulated phosphorylation of AKT can be rescued by rapamycin treatment. The depletion of the IRS protein by hyperactivated mTOR might explain, at least in part, the benign nature of tumors associated with the TSC syndromes. There might be the requirement for additional mutation(s), either in an epistatic or in a parallel pathway, to ensure malignant transformation. These new findings raise more questions than answers regarding the suitability of mTOR inhibitors as anticancer agents: would rapamycin treatment promote tumor formation by downregulating the S6K negative feedback on IRS proteins? Which of the mTOR stoichiometric complexes are more stable or more abundant in biological systems? Given that the GßL–Rictor–mTOR complex is Current Opinion in Genetics & Development 2006, 16:78–84
rapamycin resistant, should the focus be more on inhibiting targets upstream of mTOR, such as PI3K or AKT? In addition, mTOR does not fit the classic targetable kinase paradigm, in that there is no evidence that mTOR is either mutated or amplified in cancers. Despite these reservations, I would find it hard to ignore a target that lies downstream of signaling pathways that read like cancer’s most wanted: Ras, PI3K, AKT (oncogenes); PTEN, TSC, LKB1, NF (tumor suppressor genes); S6K, eIF-4E, Cyclin D1, HIF-a, VEGF and c-MYC (effectors). The preclinical and emerging clinical data show that mTOR inhibitors have promising results, with minimal toxicity (more below).
Insights from preclinical studies Rapamycin is a natural macrolide product originally studied because of its immunosuppressant activity. Its ability to suppress T-cell activation led to it being approved by the FDA for the treatment of allograft rejection in transplant patients. Rapamycin also induces G1 growth arrest — and, in some cases, apoptosis — in certain tumor cell lines, leading to its current evaluation as a possible anti-cancer agent. Presently, there are at least three additional mTOR inhibitors that have been widely characterized in preclinical models, and that are in clinical development as anti-cancer agents: CCI-779, RAD001 and AP23573 [25]. All have comparable activity against mTOR in biochemical and biological assays, and differences between the three agents lie mainly in bioavailability and formulation protocols. To date, all of the currently available data on CCI-779 and rapamycin from preclinical trials support the hypothesis that the anti-tumor activity is explained by inhibition of mTOR. Assuming that mTOR is the relevant therapeutic target, what might be the molecular basis of the clinical responses seen? At least in models in which there is constitutive activation of the PI3K–AKT pathway (e.g. through loss of PTEN), it appears that these tumors are mTOR-dependent [26,27]. Contextually, these effects are probably mediated through the ability of mTOR to enhance translation of key factors that control cell proliferation, anti-apoptotic effects, anti-angiogenic effects or a combination of these. These preclinical studies have also highlighted some aspects of mTOR inhibitors that should hopefully guide the design of rational clinical trials that use these agents individually or in combination. First, the predominant biological endpoint was one of cytostasis rather than cytotoxicity, and this is consistent with the known ability of rapamycin to induce G1 arrest [10]. Going into clinical trials, this should shift the focus from readouts based on maximum tolerated dose and tumor shrinkage to one of disease stabilization and time to progression. Second, these studies have identified potential biomarkers for pathway activation as well as for target inhibition (e.g. phosphorylated-S6 ribosomal protein) [26–28]. www.sciencedirect.com
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To date, the natural history of targeted therapies is early response followed by emergence of resistance [29]. This should spur the search for rational combinations that have additive and/or synergistic effects for transfer to the clinic. Although targeted therapies might be the antithesis of conventional chemotherapies, emerging data are showing how rapamycin can function as a chemotherapeutic sensitizer in this context [17].
Clinical development Wyeth Pharmaceuticals has conducted several phase I and II clinical trials of intravenous CCI-779 (Temsirolimus1), to evaluate its safety and anti-tumor activity in a range of tumor types. Two phase I dose escalation studies were conducted in a range of solid tumors, with CCI-779 administered as a 30-minute intravenous (IV) infusion weekly (7.5 to 220 mg/ml/week) or as a 30-minute IV infusion daily for five days (0.75 to 24 mg/ml/day) every two weeks. These studies opted for intermittent IVdosing to minimize the potential risk of immunosuppression. Objective responses and disease stabilization were observed in several patients with advanced stage kidney, breast cancer and non-small cell lung cancers; minor responses were observed in subsets of lymphomas, sarcomas, and endometrial and skin carcinomas. The drug was well tolerated, with the main adverse effects being mucocutaneous (i.e. rash, mucositis and stomatitis). Significantly, the maximum tolerated dose was not reached — signifying a high therapeutic index — and no immunosuppressive effects were seen [25]. On the basis of the promising data seen in kidney cancers (renal cell carcinomas), a phase II trial was conducted to evaluate the safety and efficacy of three dose-levels of CCI-779 in 110 previously treated patients with advanced renal cell carcinomas. Patients were randomized to receive a weekly dose of 25, 75 or 250mg of CCI-779 by intravenous infusion. The objective response rate was only 7% (one complete response and seven partial responses), but there was a remarkably high rate of minor responses and disease stabilization (i.e. complete response, partial response, minor response or stable disease 24 weeks was noted in approximately 50% of patients; median time to progression was close to six months; and median survival was 15 months). These findings were even more striking when intermediate and high-risk patients were examined — the survival rates in this subset of patients were 1.6- to 1.7-fold greater than those of interferon-a-treated patients [30]. These findings are consistent with the cytostatic activity of this compound in most models and have led to a phase III randomized trial using time to progression as the primary endpoint. Notably, equal responses were observed across all doses. All doses tested in these trials gave peak plasma concentrations well above that required for inhibition of mTOR, but the intermittent nature of the dosing enabled troughs to fall below mTOR inhibition levels. These www.sciencedirect.com
clinical data are consistent with the notion that mTOR is the relevant drug target in renal cell carcinomas. The striking results seen with renal cell carcinomas require further consideration, especially given that they have led to phase III clinical trials being carried out, and yet there is no genuine understanding of the underlying molecular mechanisms of response. The current, randomized phase III study compares CCI-779 with interferona or a combination of both. Although this clinical development strategy makes sense on the basis of available clinical data, there is the real possibility that the trial could be negative because there is no current molecular insight into the group of patients who have mTORdependent tumors. The parallels to the recent EGFR (epidermal growth factor receptor)-inhibitor trials in unselected lung cancer patients are striking. EGFR inhibitors (e.g. Iressa and Tarveca) have low but consistent single-agent objective response rates in refractory lung cancer but fail to add to chemotherapy in large randomized trials. Two recent studies showing that Iressa responders have EGFR mutations in their lung tumors clarifies the confusion surrounding these clinical datasets and makes it clear that molecular phenotyping of lung cancer patients will be essential for the selection of appropriate therapy [31,32]. Are there clues that might suggest which kidney tumors would be more sensitive to mTOR inhibitors? Surprisingly, some of the answers can be found in a mouse model of pre-invasive prostate cancer. The Sellers laboratory engineered the selective overexpression of activated AKT in the murine prostate; these mice develop a highly penetrant murine prostatic intraepithelial neoplasia (mPIN) phenotype. Treatment with RAD001 completely reversed the mPIN phenotype in two weeks, confirming that these tumors were mTOR-dependent [33]. The mechanism of response was through the induction of apoptosis. When they examined the transcriptional profile of the vehicle-treated mice, they found that HIF-1a target genes, especially those involved in regulating angiogenesis and glycolysis were upregulated and that these were downregulated following RAD001 treatment. These findings point to an additional function for mTOR — either directly or indirectly — in the regulation transcription. Recent data clearly place HIF downstream of mTOR in the PI3K–AKT pathway [34,35]. For example, constitutively active alleles of PI3K or AKT are sufficient to induce the production of VEGF mRNA and even angiogenesis [36–38]. These effects are blocked by rapamycin. Similarly, the receptor tyrosine kinase Her-2/neu acts through the PI3K–AKT–mTOR pathway to upregulate expression of HIF protein in breast cancer cells, and this upregulation is blocked by the PI3K inhibitor LY294002 and by rapamycin [39]. In these experiments, HIF is most Current Opinion in Genetics & Development 2006, 16:78–84
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probably upregulated by increased translation of HIF mRNA, which contains 50 -UTR sequences sensitive to the mTOR effector S6K. Therefore, the rapamycin analogue CCI-779 can have anti-angiogenic activity by downregulation of HIF in tumor cells, particularly in the setting of PI3K–AKT pathway dysregulation. In addition, recent data have shown that rapamycin is able to decrease HIF-1 levels in TSC2-null cells, indicating that TSC2 regulates HIF by inhibiting mTOR [40]. We already know that treatment with the anti-VEGF monoclonal antibody results in significant delays in time to progression in patients with metastatic renal cell carcinomas [41]. Similarly, in another tumor in which VEGF is upregulated, treatment with rapamycin completely inhibited the progression of Kaposi’s sarcoma [42]. This implies a combinatorial role in renal cell carcinomas between VEGF or VEGFR inhibitors and mTOR inhibitors. The upregulation of HIF-regulated glycolytic pathway transcripts (e.g. Glut-1) can be exploited both as a biomarker for patient selection and as a target for inhibition using FDG–PET ([18F] 2-fluro-2-deoxy-D-glucose–positron emission tomography). The increased glucose uptake imaged with FDG is dependent largely on the rate of glycolysis. FDG and trapping occurs because of upregulation of glucose transporters (e.g. Glut-1) and phosphorylation by hexokinases [43]. Given that subsets of renal cell carcinomas (e.g. clear-cell tumors) have increased levels of HIF, one would predict that they would exhibit higher uptake of FDG than that exhibited by non-clear cell tumors. Since the primary effect of mTOR inhibitors is G1 arrest, functional molecular imaging would be predicted to be superior, compared with conventional anatomic imaging, in elucidating biologically effective doses of mTOR inhibitors. Finally, developing non-invasive molecular assays enables repeatable, quantitative assessment of drug effect, thus sparing patients from additional surgical procedures to obtain tissue. Emerging data from phase II clinical trials using CCI-779 in brain and breast cancers show subsets of patients responding [44–46]. Reassuringly, when used as a single agent, it has shown considerable anti-tumor activity in patients with relapsed and/or refractory mantle-cell lymphoma, a hematological malignancy characterized by overexpression of cyclin D1 [47].
Biomarker-driven clinical trials How do we identify the patients who are most likely to benefit from mTOR inhibitors? We already know which cancers should respond. Prior work has shown that PI3K–AKT pathway-driven transformation is mTOR-dependent, because mTOR is a crucial component of a downstream kinase cascade. This result is consistent with the broader notion of ‘pathway Current Opinion in Genetics & Development 2006, 16:78–84
addiction’ to explain why tumor cells might be at a disadvantage, when faced with a pathway-specific inhibitor, compared with normal cells [48]. Based on this, I predict that a clinical trial using mTOR inhibitors in glioma, prostate or endometrial cancer patients with documented loss of PTEN would achieve significant disease stabilization and meaningfully delay the time to progression. However, we are not very good at implementing what we know. This is because we still rely on traditional definitions of cancers made on the basis of anatomical and histological parameters. Yes, these certainly have prognostic value in relation to disease recurrence and progression, but they add very little in reliably identifying individuals who will benefit from targeted therapies. There needs to be a re-evaluation of our perception of cancer. Solid tumors show great inter- and intra-tumoral heterogeneity, each with a distinct genetic abnormality and subsequent response to therapy. This then presents us with unique opportunities to make a therapeutic shift from the traditional view of treating cancers on the basis of their tissue or organ of origin — a move from ‘tissuespecific’ towards a more ‘target-specific’ strategy. Next, we need to identify the patients most likely to respond to these targeted drugs (i.e. matching the inhibitor to the patient). This requires the development of robust, clinically applicable assays. The tools for these assays are already at hand. Examples include global gene-expression profiles; proteomic approaches that are able to identify post-translational modifications such as phosphorylation; mutation analysis; methylation patterns, single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) arrays that are able to identify gain or loss in copy number; and molecular imaging. After therapy, the individual molecular signature can be monitored and, in cases of relapse, repeated — akin to the on-going kinome resequencing projects — and will become the basis for a revised personalized treatment strategy. In the first iteration of this, we have already identified a molecular signature of PTEN loss and/or AKT pathway activation in gliomas and prostate cancer [49,50]. Ideally, we would have a suite of biomarkers, some of which would be secreted proteins that could be measured in the serum (e.g. peIF-4G, p4E-BP1, HIF-1, GLUT-1 and VEGF), in addition to having molecular imaging probes (e.g. FDG– PET, FLT–PET {[18F] 3-deoxy-3-fluorothymidine– positron emission tomography}), to document inhibition of mTOR in tumor cells after administration of the mTOR inhibitor. Additionally, as with RAD001 studies in prostate cancer models, systematic expression-profiling experiments of tumor tissue before and after therapy to generate signatures of mTOR inhibition in tumor tissue will need to be performed and validated [33]. The alternative is the status quo, with investigators rolling www.sciencedirect.com
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out the mandatory CT (computed tomography) scans of anecdotal patient responses, which at best gives us a glimpse of what can be achieved.
Conclusions mTOR inhibitors have shown promising clinical efficacy in subsets of cancers, with low toxicity profiles. The challenge before us is to develop robust, reproducible biomarkers that can be used in the clinic to identify patients most likely to benefit, and to document modulation of the drug target. Our goal? Transform the way cancers are diagnosed, treated and cured.
Update We have recently shown that loss of the von HippelLindau (VHL) tumor suppressor gene sensitizes kidney cancer cells to the mTOR inhibitor CCI-779, both in vitro and in mouse models. Growth arrest caused by CCI-779 correlates with translational inhibition ofHIF mRNA and is rescued by expression of a VHL-resistant HIF cDNA lacking the 50 untranslated region [51].
Acknowledgements I thank Charles Sawyers for the science; Ingo Mellinghoff for helpful discussions; Moya Costello for graphics support. My work is supported by grants from the NCI–UCLA Prostate SPORE, US Department of Defense, University of California Cancer Research Coordinating Committee, the Stein-Oppenheimer Family Endowment and the Wendy Will Case Foundation. I am also indebted to Jonathan Braun for the academic and research support.
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