1 Department of Oncology, Hospital Saint Louis, Paris 2 Department of Medical Oncology, Beaujon University Hospital, Clichy, France
* Correspondence to: Dr E. Raymond, Department of Medical Oncology, Beaujon University Hospital, 100 Boulevard du General Leclerc, 92100 Clichy cedex, France. Tel: +33-01-4087-5617; Fax: +33-01-4087-5487; Email: eric.raymond{at}bjn.ap-hop-paris.fr
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Abstract |
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Key words: cell signal inhibitors, phase I trial, rapamycin, signal transduction inhibitors, sirolimus
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Introduction |
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Rapamycin (sirolimus) is a macrolide antibiotic produced by Streptomyces hygroscopicus, which binds FKBP-12 (FK506 binding protein). Thereby, the rapamycinFKBP12 complex can inhibit mTOR preventing further phosphorylation of P70S6K, 4E-BP1 and, indirectly, other proteins involved in transcription and translation and cell cycle control. Rapamycin is currently used alone or in combination with cyclosporine as an immunosuppressive drug to prevent renal graft rejection.
Rapamycin analogues currently selected for clinical development are CCI-779 (intravenous formulation currently in phase III from Wyeth Ayest), RAD001 (oral formulation currently in phase I-II from Novartis Pharma) and AP23573 (intravenous formulation currently in phase I from Ariad Pharma). In clinical settings, using intermittent administration of CCI-779, RAD001 and AP23576, no evidence of immunosuppressive effects has been observed. Dose-limiting toxicities consist of skin reactions, mucositis and minimal myelosuppression. Evidence of antitumour activity has been reported in several patients with renal clear cell carcinoma and breast cancer. Interestingly, rapamycin and its analogues antagonise tumour growth induced by loss of the PI3K antagonist, PTEN. Selection of patients based on the detection of activated P70S6K/AKT and/or loss of PTEN expression might help to predict the sensitivity of tumour cells to rapamycin analogues. Pharmacodynamic monitoring of the biological activity of rapamycin in clinical trials using molecular endpoints such as the phosphorylation of AKT, P70S6K and/or 4E-BP1 might also help to determine biological relevant dose(s) and plasma concentration(s) in individuals treated with rapamycin analogues. In addition, rapamycin and its analogues may sensitise cancer cells to apoptosis induction by cisplatin and gemcitabine.
In this review, we will describe the molecular pathways involved in rapamycin activity and we will present recent preclinical and clinical data on rapamycin and its analogues. We will then discuss the current and future placement of those molecules into current therapeutic strategies against cancer.
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PI3K signaling pathway and mTOR |
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The PTEN product has a protein tyrosine phosphatase domain and extensive homology to tensin (related protein with focal adhesions), suggesting that PTEN suppresses tumour cell growth by antagonising protein tyrosine kinases, and regulates tumour cell invasion and metastasis through interactions at focal adhesions. Davies et al. [16] and others, have demonstrated that PTEN plays an important role in anchorage-dependant cell survival. Additionally, loss of PTEN protects cells from apoptosis triggered by matrix detachment (anoikis), and the re-expression of PTEN in PTEN-mutated cells causes apoptosis in cells in suspension.
PTEN is involved in the regulation of the PI3K pathway [3]. There is evidence that PTEN dephosphorylates phosphatydilinositol 3,4,5-triphosphate while mutated PTEN cannot dephosphorylate phosphoinositides at the D3 position (D3-PPI). PTEN ± mice spontaneously develop neoplasia, associated with loss of the normal PTEN allele and an increased activation of AKT, mTOR and P70S6K. In vitro and in vivo, the growth of PTEN-deleted human cancer cells and PTEN/ mouse cells can be preferentially inhibited by pharmacologic mTOR inhibition [17
]. This growth inhibition then involves both a decrease in proliferation and an increase in apoptosis.
However, although PTEN inactivation might be required, it might not be sufficient to explain the sensitivity to rapamycin since there is also evidence to show that cancer cells with PTEN inactivation might remain resistant to rapamycin. Conversely, a dose dependent tumour growth delay is observed in mice bearing PTEN proficient cancer cells. In that case, reports have suggested that the effects of rapamycin might be related to the inhibitory effects against endothelial cells blocking tumour angiogenesis.
Several studies have suggested that genomic integrity, transcript and protein levels, phosphorylation and activity of all the multiples components of the PI3K pathway, should be evaluated to determine whether they predict prognosis or response to therapy in several cancers. The members of the PIKK family are key components of signals that coordinate the activity of the cell cycle and their functional characterisation gives important insights into cell growth and cell cycle checkpoint function. Further, development of molecular therapeutics targeting the PI3K pathway is clearly warranted in different types of cancer. Wortmannin inhibits the multiple effects of PI3Ks and yield anti-inflammatory, immunosuppressive, cytotoxic and radio-sensitising properties with potential as an anti-neoplastic drug [18, 19
]. The multiple molecular targets inhibited by this agent (PI3, PI4 and PIKK) raise caution about its clinical use. Besides, wortmannin presents chemical instability and hepatotoxicity, limiting its development. Thus, we need to evaluate more specific molecules in this pathway as individual targets.
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Focus on mTOR (Figure 3) |
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In humans, mTOR primarily appears to be a nutrient-sensing protein: mTOR is constitutively activated in the presence of growth factor and nutrients and acts as a master switch of cellular catabolism and anabolism [12, 24
]. mTOR is also regulated by hypoxia and by AMP levels. mTOR is inhibited through deacetylated tRNA species accumulating as a result of amino acid shortage (but the exact pathway remains to be elucidated) and via C-ABL protein tyrosine kinase that phosphorylates mTOR and inhibits its action. mTOR is also activated by TSC2 mutations or loss of LKB1.
As discussed above, upregulation of mTOR can be related to loss of the tumour suppressor gene PTEN and activation of AKT.
Translational control by mTOR
mTOR modulates translation of specific mRNAs via the regulation of the phosphorylation state of several different translation proteins, mainly 4E-BP1, P70S6K and eEF2.
4E-BP1. Protein synthesis is regulated in many instances at the initiation phase, when a ribosome is recruited to the 5' end of an mRNA. Eukaryotic ribosomes do not have the ability to locate and bind to the 5' end of mRNA and need translation initiation factors to guide them. The cap structure at the 5' end of an mRNA is recognised by the eukaryotic translation initiation factor 4E(eIF4E). eIF4E, in association with eIF4G, directs the translation machinery to the 5' end of the mRNA. The 4E-binding proteins (4E-BP) are essential in the regulation of the interaction between eIF4E and eIF4G. The mTOR signalling pathway modulates 4E-BP1 phosphorylation and mediates its dissociation from eIF-4E [25, 26
]. This dissociation is a crucial step toward activating translation of mRNAs with specific regulatory elements in the 5'-untranslated terminal region (5'UTR), especially c-myc, cyclin D1 and ornithine decarboxylase. In contrast, when growth factor or nutrients are lacking, or in the presence of mTOR inhibitors, 4E-BP1 becomes hypophosphorylated, which increases its binding with EIF-4E and prevents initiation of translation.
P70S6K. mTOR also phosphorylates and activates P70S6K to favour the recruitment of the 40S ribosomal subunit into actively translating polysomes and enhance the translation of mRNAs with 5' terminal oligopyrimidine tracts. These transcripts can encode up to 20% of the mRNAs [27].
eEF2. Finally, mTOR also acts at the level of the elongation phase. The eukaryotic elongation factor 2 (eEF2) promotes translocation of the mRNA and mTOR regulates the activity of eEF2 kinase, apparently via regulation of a phosphatase activity (PP2A) [28].
Anti-apoptotic and pro-apoptotic effects
There is evidence that the downstream target of mTOR, P70S6K, binds to mitochondrial membranes and phosphorylates the pro-apoptotic molecule BAD [29]. The binding of P70S6K to BAD inactivates BAD and increases cell survival. In contrast, mTOR might translocate from the cytoplasm to the nucleus shortly after the formation of syncitium between cells expressing the HIV envelope and CD4 cells. Once in the nucleus, it causes phosphorylation of P53, transcriptional activation and induction of pro-apoptotic proteins such as BAX, and activation of the intrinsic cell death pathway [30
].
Cell cycle regulation
mTOR inhibition results in an increase in the turnover of cyclin D1, at both mRNA and protein levels [31], and a decrease in the elimination of the cyclin dependant kinase inhibitor P27. Additionally, mTOR downregulates cyclin-A-dependent kinase activity in exponentially growing cells. The pharmacological inhibition of mTOR decreases G1 transit in the cell cycle [32
].
Metabolic modulation
Cells have the ability to adapt to the dynamic pool of nutrients in their immediate environment. Mammalian cells respond continually to changes in available blood glucose and amino acids. mTOR plays an important role in the modulation of metabolic pathways, including those related to insulin [12, 33
]. The initiation of translation appears to be the limiting phase in protein synthesis. The central role played by mTOR in protein translation leads to the control of skeletal muscle protein synthesis. Because mTOR inhibition causes cellular responses indicating the physiological state of starvation, these proteins are thought to be mediators of nutrient-sensing pathways. mTOR can detect nutrients such as carbon and nitrogen, signaling cells to grow and proliferate, a fact particularly important in tumour cells that proliferate aggressively. In yeast, TOR functions are well established genetically, with somewhat less compelling data in mammalian cells. In yeast, TOR signaling modulates the transcription of genes that are involved in amino acid biosynthesis, regulates the activity of amino acid permeases and represses autophagy. In the absence of the TOR signal, ribosomal biosynthesis is inhibited and autophagy is activated.
Neuronal function and role in brain development
Recent evidence also suggests that mTOR may be involved in neuronal protein synthesis. mTOR could play a role in embryonic brain development and in the learning and memory process [34]. mTOR could inhibit eEF2 phosphorylation in active synapses to locally unrepress translation, whereas some studies have reported an increase of eEF2 phosphorylation in response to various neurotransmitters.
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Rapamycin and analogues |
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In the meantime, rapamycin was developed as an immunosupressive agent and those studies have enabled us to understand the mechanism of action of this agent. Rapamycin, via its methoxy group, crosslinks the immunophilin FK506 binding protein (FKBP12). The rapamycinFKBP12 complex specifically interacts with mTOR to inhibit mTOR signalling to downstream targets [38]. Rapamycin inhibits T-cell proliferation induced by antigen, mitogenic lectins, alloantigen and crosslinking of T-cell surface markers with monoclonal antibodies. Rapamycin can inhibit proliferative responses induced by cytokines, including IL-1, IL-2, IL-3, IL-4 and IL-6, IGF, PDGF and CSFs.
The preclinical development of rapamycin as an immunosuppressor has been extensively reviewed [39, 40
]. It has demonstrated a high degree of synergy with cyclosporin [41
] both in vitro and in vivo, lowering the dose of cyclosporin necessary for immunosuppression, enhancing the rejection prevention in renal transplantation and minimising cyclosporin-induced toxicity [42
, 43
]. There are observations that high doses of rapamycin block the proliferative responses to cytokines by vascular and smooth muscle cells after mechanical injury, such as balloon angioplasty or allo-rejection [44
, 45
]. In a non-human primate model, supra-therapeutic concentration of rapamycin stabilised and possibly reversed the intimal vascular lesion caused by the progression of immune injury in aortic allograft [46
]. Rapamycin treatment concomitant with monoclonal antibody blockade of the co-stimulatory signal by anti-CD154 in mice induces tolerance, and the combination of rapamycin with anti-B7 in non-human primates seems to facilitate tolerance induction [47
]. IC50 values of rapamycin as an immunosuppressor are in the range of 0.1300 nM.
A relevant point of rapamycin as an immunosuppressor is the absence of the vasomotor renal side effects exhibited by CsA and tacrolimus. Treatment with rapamycin preserves glomerular filtration and renal blood flow in normal, salt-depleted and spontaneously hypertensive rats [48]. The renal tissue seems to be protected during the rapamycin treatment by an inhibition of the intrarenal angiotensin II cascade. However, rapamycin does produce a dose-dependent tubular toxicity in rats, which is related to the delayed recovery of tubular epithelial function after injury [49
].
Over the last 8 years, rapamycin has undergone clinical trials as an immunosuppressive agent, progressing from phase I safety, tolerability and pharmacokinetic investigation to phase II dose-finding studies and limited sized evaluations of drug combination regimens. The completion of phase III trials led to approval of rapamycin by the Food and Drug Administration (FDA) of the USA in 1999 to prevent acute rejection in combination with cyclosporin and steroids. One year later, the drug was approved by the European Agency as an alternative to calcineurin antagonists for long-term maintenance therapy to avoid graft rejection. Interestingly, rapamycin, unlike cyclosporin, does not seem to increase the risk of malignancy but rather to decrease the risk of post-transplant lymphoproliferative disorders.
Apart from its immunosupressive capacity, rapamycin was also recently shown to be capable of preventing coronary artery re-stenosis [50, 51
]. Growth, migration and differentiation of vascular smooth-muscle cells are two major features of neointimal proliferation after vascular injury. The proposed mechanism of inhibition of proliferation of vascular smooth-muscle cells by sirolimus includes binding of the immunophilin FKBP12, blockage of P70S6K, impairment of retinoblastoma protein phosphorylation, and prevention of p27 downregulation. Additionally, rapamycin has been shown to be effective in inhibiting PDGF-induced migration of human vascular smooth cells in vitro, without affecting the ability of these cells to bind collagen and without disrupting their cytoskeletal components [52
, 53
]. To avoid the systemic effects of rapamycin, it has been used locally in an impregnated stent to prevent coronary restenosis [51
].
Pharmacokinetic and metabolic information
These data were initially obtained from studies that evaluated rapamycin as an immunosuppressor [54, 55
]. The systemic bio-availability of rapamycin is approximately 15%, it has a maximal concentration at about 1 h and is widely distributed in tissues compared with plasma. The ratio blood cells/plasma ranges between 36 in renal transplant cases to 79 in healthy volunteers. In vitro experiments using human liver microsomes suggest that cytochrome Cyp450 3A4 is the major biotransformation system, generating the inactive metabolites, hydroxy, dihydroxy, hydroxy-demethyl, didemethyl, 7-0 demethyl and 41-0 demethyl. More than 90% of the drug is recovered in the faeces. Urine represents only 2% of the drug elimination. The average elimination half life is variable, ranging from 10 h in children to 110 h in patients with hepatic impairment.
Rapamycin exposure is increased by diltiazem and ketoconazole and decreased by rifamycin and anticonvulsants [56]. Regarding the interaction between rapamycin and cyclosporin (CsA), rapamycin concentrations are increased by concomitant administration of Neoral, the microemulsion formulation of CsA, and rapamycin increases CsA exposure approximately 2-fold, presumably because of competition for metabolism by Cyp450 3A4 and, possibly, drug extrusion by P-glycoprotein [57
].
Initial clinical studies show that a dose-dependent reversible reduction in mean platelet number and, to a far lesser extent, leukocyte count, was accompanied by increased serum cholesterol and triglyceride values. There were no changes in blood pressure, kidney or liver function test results.
Corroborating preclinical studies, rapamycin does not affect glomerular filtration, but hypokalemia and hypophosphatemia has been reported as evidence of renal tubular abnormalities [58, 59
].
Additionally, rapamycin augments reactions to CsA: hypertension, acne and hirsutism. It has been associated with minor adverse effects such as diarrhea, tachycardia and arthralgia, as well as with non-infectious pneumonitis [60].
Rapamycin as an anticancer drug
Rapamycin was shown to inhibit the growth of several murine and human cancer cell lines in a concentration-dependent manner, both in tissue culture and xenograft models: B16 melanoma, P388 leukemia, MiaPaCa-2 and Panc-1 human pancreatic carcinomas and others [6163
]. In the 60 tumour cell lines screened at the National Cancer Institute in the USA (COMPARE program), the average GI50 obtained for rapamycin over all cell lines was 8.2 nM when the highest concentration tested is 1000 nM and 1800 nM when the highest concentration tested is 106 nM. In practical terms, they found general sensitivity to the drug at doses under 2000 ng/ml, more evident in leukemia, ovarian, breast, central nervous system and small cell lung cancer cell lines.
Rapamycin induces P53-independent apoptosis in childhood rhabdomyosarcoma [64] and enhances the apoptosis induced in vitro by cisplatin in murine T-cell and human HL-60 promyelocytic leukemias and human ovarian SKOV3 carcinoma [65
]. On the contrary, it inhibits taxol-induced apoptosis in human B-cell lines, probably through preventing BCL-2 inactivation and can inhibit hybridoma cell death in bioreactors, thereby increasing the production of monoclonal antibody [66
].
In addition, rapamycin inhibits the oncogenic transformation of human cells induced by either PI3K or AKT and has shown metastatic tumour growth inhibition and anti-angiogenic effect in in vivo mouse models [67]. Considering this effect, there is evidence that the PI3KP70S6K intracellular signaling pathway is required for HIF1 and VEGF expression and also for VEGF stimulation of endothelial cells. The anti-angiogenic effect of rapamycin seems to be related to the inhibition of these effects [68
].
Based on these pre-clinical results, studies with rapamycin as an anticancer drug were begun and rapamycin analogues were developed with more favourable pharmaceutical properties.
Rapamycin derivatives (Table 1)
CCI-779, a more water-soluble ester derivative of sirolimus, was identified by investigators at Wyeth Ayerst as a noncytotoxic agent that delayed tumour proliferation. CCI-779 was designed to increase the solubility of rapamycin making this compound readily available for intravenous formulation. At several non-toxic doses, CCI-779 demonstrated antitumour activity alone or in combination with cytotoxic agents in a variety of human cancer models such as gliomas, rhabdomyosarcoma, primitive neuroectodermal tumour such as medulloblastoma, head and neck, prostate, pancreatic and breast cancer cells [6973
]. Treatment of mice with CCI-779 inhibits P70S6K activity and reduces neoplastic proliferation. As with sirolimus, PTEN-deficient human tumours are more sensitive to CCI-779-mediated growth inhibition than PTEN-expressing cells. Specifically, studies in vitro in a panel of eight human breast cancer cell lines showed that six of eight cancer lines studied were inhibited by CCI-779 with IC50 in the low nanomolar range. Two lines, however, were found to be resistant with IC50>1 µM. The sensitive cell lines were estrogen receptor positive, or overexpressed HER-2/Neu, or had lost the tumour suppressor gene product PTEN [74
]. Interestingly, preclinical studies indicate that intermittent administration of CCI-779 reduces its immunosuppressive properties while retaining its antitumour activity.
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Another phase II study is currently ongoing in patients with advanced breast cancer [82]. The majority of patients in this trial received more than two lines of prior chemotherapy containing anthracyclines and taxanes. The study was designed to explore two doses of weekly intravenous administration of CCI-779 (75 and 250 mg/m2). Toxicity was significantly higher in patients receiving higher doses (requiring dose reduction in 45.1% of those patients). At the higher dose, psychic disorders were reported, including lethargy and depression. Activity was similar in the two treatment groups with an overall response rate of 10%. Immunohistochemistry was performed in samples from patients treated with CCI-779 in this trial. In this study, patients who responded to CCI-779 did not express PTEN but overexpressed HER-2/neu in tumour cells.
Results of CCI-779 in combination with 5-fluorouracil, provided evidence that CCI-779 increased the mucous toxicity of cytotoxic agents [83]. Other phase I combination studies are pending to determine the feasibility of combining CCI-779 with gemcitabine.
RAD001, 40-O-(2-hydroxyethyl)-rapamycin, or Everolimus, is another analogue of rapamycin that can be administrated orally. The immunosuppressive activity of RAD001 in vitro is about 3-fold lower than rapamycin, but its activity in vivo seems comparable to rapamycin due to some more favourable pharmacokinetic properties. RAD001 inhibits the growth factor-stimulated in vitro proliferation of vascular smooth muscle cells and prevents allograft rejection in the rat [84]. A synergistic activity between RAD001 and cyclosporin has been reported [85
]. It has demonstrated activity and several tissue distribution advantages, including a decrease in cyclosporin toxicity, when they are co-administered. Its anti-neoplastic activity has been evaluated in different human cancer cell lines in vitro and in xenograft models in vivo with IC50 ranging from 5 to 1800 nM [86
]. P70S6K inhibition and anti-neoplastic effect have been shown in these models, an optimal effect being achieved with 2.5 mg/kg/day in melanoma, lung, pancreas and colon carcinoma. Similarly, RAD001 demonstrates a concentration-dependent anti-tumour activity in a syngenic rat pancreas carcinoma model with an intermittent dosing schedule. RAD001 has also shown antiangiogenic activity [87
] and inhibits the human vascular endothelial cells (HUVECs) proliferation. RAD001 also has an inhibitory effect on in vitro growth in six different PTLD (post-transplant lymphoproliferative disorders) associated with EpsteinBarr virus and lymphoblastoid B cell lines [88
, 89
]. RAD001 led to an increase in the number of cells in the G0/G1 phase of the cell cycle. At a concentration of 10 nM, there was a 1070% increase indicating a delay in cell cycle progression at the early G0/G1 phase. An in vivo xenograft model confirmed this observation.
RAD001 is currently undergoing evaluation in phase I studies as an anti-neoplastic agent and is already in phase III trials as an immunosuppressor acting in synergy with cyclosporin in kidney transplantation. Free interval from acute rejection was significantly related to minimal concentration (Cmin) with an incidence of 68% at 1.03.4 ng/ml, 8185% at 3.57.7 ng/ml and 91% at 7.815 ng/ml.
The toxicity reported for RAD001 includes hypercholesterolemia, hypertriglyceridemia, mild leukocytopenia and thrombocytopenia [90]. In a phase I trial performed in patients with advanced cancer, RAD001 displayed a good safety profile with mild to moderate skin and mucous toxicity up to 30 mg weekly. Pharmacokinetic and pharmacodynamic analysis and modeling performed during phase I trials revealed that inhibition of P70S6K might help to determine the biologically active doses of RAD001. Preliminary results on activity showed an objective response in a patient with a non small cell lung carcinoma. Current phase Ib trials are underway combining RAD001 with cytotoxic drugs.
AP23573 is the latest developed rapamycin analog. It is a phosphorus-containing compound synthesised with the aid of computational modelling studies (modification of C-43 secondary alcohol moiety of the cyclohexyl group of rapamycin with substituted phosphonate and phosphinate groups with retention of high-affinity binding to FKBP and mTOR). AP23573 was found to be stable in organic solvents, aqueous solutions at a variety of pHs, and in plasma and whole blood, both in vitro and in vivo. These stability studies along with in vitro metabolism studies also indicated that AP23573 is not a rapamycin pro-drug [91]. AP23573 has shown potent inhibition of diverse human tumour cell lines in vitro and as xenografts implanted into nude mice, alone or in combination with cytotoxic or targeted agents [92
, 93
]. In an ongoing phase I trial [94
], AP23573 is administered intravenously daily for 5 days every 2 weeks. Dose-limiting toxicity is severe grade 3 oral mucositis occurring during the first cycle. Others side effects seem to be moderate, including minor to moderate episodes of mucositis, fatigue, nausea, rash, anemia, neutropenia, diarrhea, hyperlipidemias and thrombocytopenia. Preliminary antitumour activity is observed at all dose levels. Enrolment is ongoing at 18.75 mg/day levels and further studies are planned.
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Designing studies with mTOR inhibitors |
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The screening of tumour biopsy specimens by immunohistochemistry, fluorescence in situ hybridization, gene sequencing or DNA micro-array, looking for PTEN loss of function, AKT activation and mTOR phosphorylation may provide the rational basis for identification of those cancer patients most likely to benefit from therapy with mTOR inhibitors (Figure 6).
The PI3K/AKT/mTOR pathway is thought to be activated in 3050% of prostate cancer, 3060% of malignant gliomas, 3050% of endometrial carcinoma, >50% of melanoma, >30% of renal cell carcinoma and 10% of breast cancer. Apparently, the activity of mTOR inhibitors as antitumour agents is more remarkable in highly proliferative cancer cells of neuroectodermic origin such as neuroblastoma, meduloblastoma, and those with high expression of IGF such as alveolar rhabdomyosarcoma [64
]. In vitro, some human cancer cell lines (breast, gliomas, leukemia, prostate, ovary, kidney) are more sensitive than others to the effects of rapamycin and its derivatives [95
, 96
]. Likely, tumours that depend on activation of AKT/PI3K signaling pathway seem to have a better response, such as those with mutation in the PTEN tumour suppressor gene [17
, 97
]. Genes involved in cell cycle control have been recently evaluated in several human ovarian cancer cell lines in order to enable the prediction of sensitivity/resistance to rapamycin. In cells exposed to rapamycin, a dose-dependent downregulation of CCND1 (cyclin D1) and CDK4 gene expression, evaluated by RTPCR, and late G1 cell cycle arrest are observed [98
, 99
]. Those genes could be useful as surrogate markers to predict sensitivity to rapamycin.
Resistance mechanisms
The relationships between cellular sensitivity to rapamycin, drug accumulation, expression of mTOR, inhibition of growth factor activation of P70S6K and dephosphorylation of 4E-BP-1 have been explored [100, 101
]. Some cell lines are highly sensitive to growth inhibition by rapamycin, whereas others have intrinsic resistance. Both sensitive and resistant cells seem to have the same intracellular drug concentration and the same level of P70S6K inhibition. Genomic resources offer a thorough and relatively simple approach to find some reasons for intrinsic resistance to rapamycin derivatives. Authors have suggested that the ability of rapamycin to inhibit c-myc induction correlates with intrinsic sensitivity, whereas failure of rapamycin to inhibit induction or overexpression of c-myc correlates with resistance [100
]. It has been also suggested that rapamycin upregulates the cyclin-dependent kinase inhibitor P27 at both mRNA and protein levels in cells sensitive to rapamycin [102
, 103
]. More recently, we and others observed that expression of BCL-2 was associated with resistance to rapamycin and RAD001 (Figure 7) [99
, 104
]. Furthermore, BCL-2 antisense was shown to restore sensitivity to rapamycin in cell that expressed BCL-2 [99
]. Mechanisms associated with rapamycin-induced apoptosis in cancer cell lines are poorly understood and will require further laboratory studies to help in identifying patients unlikely to respond to rapamycin derivatives.
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Conclusion |
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Notes |
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Received for publication August 6, 2004. Revision received November 12, 2004. Accepted for publication November 15, 2004.
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References |
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