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MAPK pathways in treating a tuberous sclerosis complex cell model
JOURNAL OF
GENETICS AND GENOMICS J. Genet. Genomics 36 (2009) 355361
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Efficacy of combined inhibition of mTOR and ERK/MAPK pathways in treating a tuberous sclerosis complex cell model Ruifang Mi a, b, Jianhui Ma a, Dechang Zhang c, Limin Li b, Hongbing Zhang a, * a
Department of Physiology, Institute of Basic Medical Sciences and School of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China b Department of Pathology, Institute of Basic Medical Sciences and School of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China c Department of Pharmacology, Institute of Basic Medical Sciences and School of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China Received for publication 1 April 2009; revised 19 April 2009; accepted 20 April 2009
Abstract Tuberous sclerosis complex (TSC) is an autosomal dominant tumor syndrome which afflicts multiple organs and for which there is no cure, such that TSC patients may develop severe mental retardation and succumb to renal or respiratory failure. TSC derives from inactivating mutations of either the TSC1 or TSC2 tumor suppressor gene, and the resulting inactivation of the TSC1/TSC2 protein complex causes hyperactivation of the mammalian target of rapamycin (mTOR), leading to uncontrolled cell growth and proliferation. Recent clinical trials of targeted suppression of mTOR have yielded only modest success in TSC patients. It was proposed that abrogation of a newly identified mTOR-mediated negative feedback regulation on extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling pathway and on the well-documented RTK-PI3K-AKT signaling cascade could limit the efficacy of mTOR inhibitors in the treatment of TSC patients. Therefore, we speculate that dual inhibition of mTOR and ERK/MAPK pathways may overcome the disadvantage of single agent therapies and boost the efficacy of mTOR targeted therapies for TSC patients. Investigation of this hypothesis in a TSC cell model revealed that mTOR suppression with an mTOR inhibitor, rapamycin (sirolimus), led to up-regulation of ERK/MAPK signaling in mouse Tsc2 knockout cells and that this augmented signaling was attenuated by concurrent administration of a MEK1/2 inhibitor, PD98059. When compared with monotherapy, combinatorial application of rapamycin and PD98059 had greater inhibitory effects on Tsc2 deficient cell proliferation, suggesting that combined suppression of mTOR and ERK/MAPK signaling pathways may have advantages over single mTOR inhibition in the treatment of TSC patients. Keywords: TSC; mTOR; ERK/MAPK; rapamycin; PD98059
Introduction Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome affecting approximately 1 in 6,000 births, characterized by the development of distinctive * Corresponding author. Tel: +86-10-6529 6495; Fax: +86-10-6529 6491. E-mail address: [email protected] DOI: 10.1016/S1673-8527(08)60124-1
tumors and malformations in multiple organs (Crino et al., 2006; Curatolo et al., 2008), with a TSC patient’s prognosis depending on the severity of symptoms. While brain lesion is the major cause of morbidity, renal angiomyolipoma (AML) is the leading cause of death. Although rare, TSC related lung lymphangioleiomyomatosis (LAM) is often detrimental for females in their child-bearing years. At the present time, there is no cure for this disease beyond the treatment of its symptoms (Crino, 2008).
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Inactivating mutations in one of the two tumor suppressor genes, TSC1 and TSC2, cause TSC; TSC2 mutations are 5–6 times more common than TSC1 mutations and have a more severe phenotype (Dabora et al., 2001). TSC1 and TSC2 form a protein complex which promotes GTPase activity of the mammalian target of rapamycin (mTOR) activator Rheb and therefore suppresses mTOR activity (Castro et al., 2003; Garami et al., 2003; Inoki et al., 2003; Tee et al., 2003). Under physiological conditions, mTOR is a serine/threonine kinase which integrates various signals from growth factors, nutrients, energy, and stress to regulate protein synthesis, cell growth, and proliferation. With aberrant mTOR activation frequently observed in various tumors (Manning and Cantley, 2007) and the loss of either TSC1 or TSC2 leading to hyperactivation of mTOR, mTOR inhibition was anticipated to be effective in the treatment for TSC patients (Kwiatkowski et al., 2002; Potter et al., 2002; El-Hashemite et al., 2003; Zhang et al., 2003; Chan et al., 2004). In recent years, the targeted suppression of mTOR functions has been employed for treating a broad spectrum of cancer patients in numerous clinical trials, but only a subset of cancers, such as advanced renal cell carcinoma, respond well to mTOR inhibitors (Brachmann et al., 2009). Only half of phosphatase and tensin homolog deleted on chromosome ten (PTEN) mutant glioblastoma patients were responsive to the mTOR inhibitor rapamycin, a macrolide antibiotic (also called sirolimus) produced by Streptomyces hagroscopicus, in a phase one clinical trial (Cloughesy et al., 2008). Although targeted mTOR suppression may hold promise in treating TSC in preclinical studies and clinical trials (Lee et al., 2005; Bissler et al., 2008; Ehninger et al., 2008; Paul and Thiele, 2008), in recent TSC clinical trials, lesions tended to relapse after rapamycin withdrawal (Bissler et al., 2008; Paul and Thiele, 2008). Thus the utility of the suppression of mTOR signaling is limited to ameliorative treatments for TSC or sporadic LAM (Davies et al., 2008). One of the possible mechanisms underlying the cytostatic activity of rapamycin is its relief of mTOR mediated negative feed-back regulation on receptor tyrosine kinases (RTK), such as platelet-derived growth factor receptor (PDGFR), or downstream effectors, such as insulin receptor substrate 1 (IRS-1), and then on the phosphatidylinositol-3-kinase/protein kinases B (PI3K/AKT) cascade (Zhang et al., 2003, 2007; Harrington et al., 2004; Shah et al., 2004). Rapamycin mediated disruption of the feedback suppression of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK)
pathway from mTOR may also render cells resistant to mTOR inhibition (Carracedo et al., 2008). Because both the AKT/mTOR and ERK/MAPK signaling pathways are prosurvival signals, the combined inhibition of mTOR and either PI3K or MAPK may hold greater promise than a single agent in the treatment of TSC. In this study, we investigated the efficacy of combinatorial inhibition of mTOR and ERK/MAPK in Tsc2/ mouse embryonic fibroblast cells (MEF), a widely used TSC cell model (Zhang et al., 2003) and found that dual suppression of mTOR and ERK/MAPK is effective in inhibiting TSC in vitro.
Material and methods Cell culture The Tsc2/ MEF cell line was derived from a Tsc2/ mouse embryo in Dr. Kwiatkowski’s Laboratory at Brigham & Women’s Hospital, Boston, MA, USA (Zhang et al., 2003) and cultured in DMEM with 10% FCS in a humidified atmosphere of 5% CO2 at 37°C.
Antibodies and other reagents Antibodies against ERK and ȕ-actin were obtained from Santa Cruz Biotechnology, USA, and antibodies against phospho-ERK, S6 ribosomal protein, and phospho-S6 from Cell Signaling Technology, USA. Rapamycin (Cell Signaling Technology, USA) was dissolved in 100% ethanol for a 10 mmol/L stock solution and PD98059 (an inhibitor of ERK phosphorylation, EMD Biosciences, USA) dissolved in 100% dimethylsulfoxide (DMSO) for a 20 mmol/L stock solution. The MTT Cell Proliferation Assay kit was obtained from BioDev Technology, China.
Western blotting analysis Cells were harvested in lysis buffer (2% SDS, 0.1 mol/L DTT, 10% glycerol, and 60 mmol/L Tris, pH 6.8) and boiled at 98°C for 10 min. The protein lysates were separated by 4%12% Bis-Tris SDS-PAGE gel (Invitrogen, USA) and transferred onto PVDF membranes (Pierce Chemical, USA). Nonspeci¿c reactivity was blocked by 5% non-fat dry milk in TBST buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05% Tween 20, and pH 7.5) at room temperature for 1 h, followed by incubation with the primary antibodies for phospho-ERK (1:1,000), ERK
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(1:5,000), phospho-S6 (1:4,000), S6 (1:6,000), and ȕ-actin (1:8,000) at 4°C for 12 h, and subsequently incubated with a secondary HRP-conjugated second antibody (Santa Cruz Biotechnology, USA) at 1:5,000 dilution at room temperature for 2 h.
Cell proliferation assay To assess the IC50 and the combination effects of rapamycin and PD98059, Tsc2/ MEF cells were seeded in triplicate 96-well plates at 4,000 cells/well and incubated for 6 h and then the medium was removed and replaced with fresh medium containing either rapamycin, PD98059, or both in varying concentrations. At various time points, 20 ȝL of MTT dye (3-[4,5-dimehyl-2-thiazolyl]-2,5diphenyl-2H-tetrazolium bromide, 5 mg/mL in PBS) was added to each well, incubated at 37°C for 4 h, the culture medium removed, 150 ȝL DMSO added to each well, and shaken at room temperature for 10 min. The spectrometric absorbance at 570 nm was then determined with a microplate reader. The viability ratio was calculated as: (OD value of the sample/OD value of control) × 100% (Kuang et al., 2009).
Fig. 1. Inhibitor of MEK compromised rapamycin-induced MEK-ERK activation. Tsc2/ MEF cells treated with 1 nmol/L mTOR inhibitor rapamycin, 50 ȝmol/L MEK inhibitor PD98059, or both for 48 h. Whole cell extracts used for activities of mTOR (phospho-S6) and ERK/MAPK (phospho-ERK1/2) were analyzed by Western blotting.
it was proposed here that combined inhibition of mTOR and ERK/MAPK might be a promising regimen for the treatment of TSC.
Results MEK inhibitor impaired rapamycin-induced MEK-ERK activation In rapamycin treatment of TSC cells, disruption of the mTOR mediated suppression on the ERK-PI3K-AKT signaling cascade has been identified here and in other laboratories (Zhang et al., 2003, 2007; Harrington et al., 2004; Shah et al., 2004) and it was also thought here that the reactivation of ERK/MAPK might have compromised the effectiveness of rapamycin in the TSC treatment. Thus, to prevent rapamycin mediated hyperactivation of ERK/ MAPK, PD98059, an inhibitor of MEK1/2 kinase upstream of ERK, was used to produce inhibition of ERK /MAPK signaling. After treatment with rapamycin alone or in combination with PD98059, Tsc2/ cells were harvested for Western blotting analysis, and it was observed that mTOR inhibition by rapamycin produced up-regulation of ERK1/2 phosphorylation, suggesting that mTOR indeed suppressed the ERK/MAPK pathway in Tsc2/ MEF cells (Fig. 1). Furthermore, it appeared that rapamycin-reactivated ERK1/2 was blunted with addition of PD98059. Thus,
Rapamycin and PD98059 had additive effects on Tsc2/ cell proliferation The potential benefit of dual inhibition of mTOR and MAPK in Tsc2 deficient cells was explored by initial quantification of the individual effects of rapamycin and PD98059 on Tsc2/ MEF cell proliferation using a dose course evaluation. Both drugs individually inhibited Tsc2/ cell proliferation with an IC50 of 0.172 nmol/L and 79.13 ȝmol/L for rapamycin and PD98059, respectively (Fig. 2). Interestingly, rapamycin at 10 nmol/L and greater inhibited Tsc2/ cell proliferation to a constant degree and the concurrent reduction of cell viability with rapamycin was never below 40% (Fig. 2A; Fig. 3A). In contrast, PD98059 exerted an inhibitory effect on Tsc2/ cells in a dose dependent manner (Fig. 2B; Fig. 3C). These data were consistent with the cytostatic action of rapamycin and the apoptotic effect of PD98059 observed in previous studies (Carracedo et al., 2008; Kinkade et al., 2008). Examination of the interactive action of these two drugs on cell proliferation revealed that the shapes (slopes) of the related cell viability curves of one drug were not altered or shifted by
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Fig. 2. Determination of IC50 for rapamycin and PD98059 in TSC cells. Tsc2/ MEF cells were treated with various concentrations of rapamycin (A) or PD98059 (B) for 48 h. IC50 of rapamycin, 0.172 nmol/L; IC50 of PD98059, 79.13 ȝmol/L.
Fig. 3. Rapamycin and PD98059 showed additive effects in cell culture. Tsc2/ MEF cells were treated with various concentrations of rapamycin (A and B) or PD98059 (C and D) in the presence of constant concentrations of PD98059 (A and B) or rapamycin (C and D) for 48 h. IC50 curves of rapamycin or PD98059 almost overlapped at fractional concentrations of other agent (A and C). IC50 of rapamycin and PD98059 was nearly constant at higher concentrations of other agent (B and D). IC50 of rapamycin, 0.172, 0.204, and 0.156 nmol/L rapamycin with addition of 0, 10, and 100 ȝmol/L PD98059, respectively (B). IC50 of PD98059, 79.1, 86.7, and 78.7 ȝmol/L PD98059 with addition of 0, 1, and 20 nmol/L rapamycin, respectively (D).
the other drug (after subtraction of the effect from that drug alone; Fig. 3, A and C). The IC50 of rapamycin and PD98059 had no significant differences in most combinations at various concentrations (Fig. 3, B and D), indicating that these two drugs exhibited additive rather than synergistic effects on Tsc2/ cell proliferation.
Suppression of MAPK signaling strengthened the inhibition of TSC cell proliferation by rapamycin The present observation that rapamycin and PD98059 individually inhibited the proliferation of Tsc2/ cells and that there was no antagonism from combinations of these
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drugs suggested that inhibition of MAPK and mTOR simultaneously could be more effective than targeting either a single pathway. Thus, the efficacy of combinatorial applications of rapamycin and PD98059 was evaluated in terms of Tsc2/ cell proliferation. Application of both drugs for 3 days yielded a nearly complete blockage of Tsc2/ cell proliferation (90.2% reduction) in an additive fashion (Fig. 4), which indicated that the pharmacological blockade of the ERK/MAPK pathway increased rapamycinmediated Tsc2/ MEF cell growth inhibition in vitro.
Discussion Because inactive mutations of either TSC1 or TSC2 tumor suppressor gene cause hyperactivation of mTOR, which is in turn responsible for the uncontrolled cell growth and proliferation in TSC patients, TSC patients were predicted to be extremely sensitive to mTOR inhibition. However, in recent clinical trials the efficacy of mTOR inhibitors in TSC patients was modest (Bissler et al., 2008; Paul and Thiele, 2008). Identification of the factors that compromised the therapeutic potential of mTOR inhibitors is thus critical for the improvement of targeted TSC therapies. Consistent with a recent report (Carracedo et al., 2008), the observations here showed that the negative feedback regulation of ERK/MAPK pathway exerted by mTOR was abolished with rapamycin treatment of Tsc2/ MEF cells (Fig. 1). The resulting augmented activation of MAPK signaling was attenuated by concomitant administration of a MEK1/2 inhibitor, PD98059. In addition, combinatorial application of rapamycin and PD98059 had greater inhibitory effect on Tsc2 deficient cell proliferation than these agents used singly (Fig. 4). Tumorigenesis is a multiple-step process involving numerous genetic alterations and it is not surprising that these complex, activated, prosurvival machineries may confer resistance to any single agent used in a targeted therapy for cancer. Thus, cotargeting of survival signaling pathways is an important advancement in the treatment of cancer (Stommel et al., 2007; Grant, 2008). Under physiological conditions, mTOR is governed by a panel of proto-oncogenes, such as epidermal growth factor receptor (EGFR), Ras protein, PI3K, and AKT, and tumor suppressors, such as PTEN and TSC1/2 complex, so it is frequently activated in variety of tumors (Manning and Cantley, 2007). In recent years, much time and effort has been committed to the discovery and clinical validation of specific inhibitors of PI3K and mTOR for patients with various kinds of tumors
Fig. 4. Combined blockade of mTOR and ERK/MAPK enhanced the inhibitory effect of single inhibitors on Tsc2/ cell proliferation. Tsc2/ MEF cells were treated with 1nmol/L rapamycin, 100 ȝmol/L PD98059, or both up to 3 days. Cell proliferation was assayed by MTT method (* means P < 0.01 compared with control cells, # means P < 0.01 compared with rapamycin-treated cells, and & means P < 0.01 compared with PD98059-treated cells.
(Brachmann et al., 2009). Unfortunately, the majority of treated cancer patients failed to benefit from these novel therapies. Some genetically stratified cancer patients with PTEN or TSC1/TSC2 mutations responded only moderately to mTOR inhibition (Lee et al., 2005; Bissler et al., 2008; Ehninger et al., 2008; Paul and Thiele, 2008). Elucidation of these confounding factors, which attenuate the response to rapamycin, has become the focus of cancer targeted therapy research. The targeted suppression of mTOR by rapamycin and its analogs, inevitably leading to the disruption of the newly identified mTOR-mediated negative feedback regulation on MAPK signaling pathway, as well as on the well-documented RTK-PI3K-AKT signaling cascade, supports the study of combined targeting of mTOR and MEK as a potential anticancer strategy (Zhang et al., 2003; Carracedo et al., 2008). In agreement with these findings, several preclinical studies of PTEN deficient prostate cancer and cancer cell lines have shown that the combined inhibition of mTOR and MAPK pathways is a promising regimen in the treatment of cancers (Carracedo et al., 2008; Kinkade et al., 2008). We propose here that the undesired activation of RTK-PI3K-AKT and ERK/MAPK by mTOR suppression could yield cells refractory to mTOR inhibitors and thus limit their efficacy in the treatment of TSC patients. On the other hand, the dual inhibition of mTOR and MAPK pathways would overcome the disadvantage of single agent therapy and boost the efficacy of mTOR targeted
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therapy in TSC patients (Brachmann et al., 2009). It was observed here that, indeed, double inhibition of mTOR and MAPK signaling with respective inhibitors achieved significant suppression of TSC cell proliferation, albeit in an additive, not synergistic, way. The limitation of this therapeutic strategy is that the reactivated ERK-PI3K-AKT function produced by rapamycin might still confer TSC cells with some resistance to the combined therapy of mTOR and MAPK. Thus, we suggest that dual pan-PI3K/ mTOR inhibitors, such as NVP-BEZ235 combined with MEK inhibitors, should be explored to achieve a triple intervention of mTOR, PI3K, and MEK MAPK functions in TSC cells (Engelman et al., 2008; Serra et al., 2008; Brachmann et al., 2009). Our findings demonstrate that dual inhibition of mTOR and MAPK signaling pathways effectively blocked Tsc2/ cell proliferation in vitro and provided a proof-of-concept demonstration that combination therapy targeting both mTOR and MAPK signaling pathways may have the potential to become a novel strategy in the treatment of TSC patients. Preclinical studies are therefore warranted to define the benefits and risks of the application of such combinational therapy in TSC animal models.
Acknowledgements This work was supported in part by the National Natural Science Foundation of China (No. 30788004). We thank David J. Kwiatkowski for providing Tsc2/ MEF cells, Xinxin Chen and Jingmei Jiang for help with statistical analysis.
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GENETICS AND GENOMICS J. Genet. Genomics 36 (2009) 355361
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Efficacy of combined inhibition of mTOR and ERK/MAPK pathways in treating a tuberous sclerosis complex cell model Ruifang Mi a, b, Jianhui Ma a, Dechang Zhang c, Limin Li b, Hongbing Zhang a, * a
Department of Physiology, Institute of Basic Medical Sciences and School of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China b Department of Pathology, Institute of Basic Medical Sciences and School of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China c Department of Pharmacology, Institute of Basic Medical Sciences and School of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China Received for publication 1 April 2009; revised 19 April 2009; accepted 20 April 2009
Abstract Tuberous sclerosis complex (TSC) is an autosomal dominant tumor syndrome which afflicts multiple organs and for which there is no cure, such that TSC patients may develop severe mental retardation and succumb to renal or respiratory failure. TSC derives from inactivating mutations of either the TSC1 or TSC2 tumor suppressor gene, and the resulting inactivation of the TSC1/TSC2 protein complex causes hyperactivation of the mammalian target of rapamycin (mTOR), leading to uncontrolled cell growth and proliferation. Recent clinical trials of targeted suppression of mTOR have yielded only modest success in TSC patients. It was proposed that abrogation of a newly identified mTOR-mediated negative feedback regulation on extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling pathway and on the well-documented RTK-PI3K-AKT signaling cascade could limit the efficacy of mTOR inhibitors in the treatment of TSC patients. Therefore, we speculate that dual inhibition of mTOR and ERK/MAPK pathways may overcome the disadvantage of single agent therapies and boost the efficacy of mTOR targeted therapies for TSC patients. Investigation of this hypothesis in a TSC cell model revealed that mTOR suppression with an mTOR inhibitor, rapamycin (sirolimus), led to up-regulation of ERK/MAPK signaling in mouse Tsc2 knockout cells and that this augmented signaling was attenuated by concurrent administration of a MEK1/2 inhibitor, PD98059. When compared with monotherapy, combinatorial application of rapamycin and PD98059 had greater inhibitory effects on Tsc2 deficient cell proliferation, suggesting that combined suppression of mTOR and ERK/MAPK signaling pathways may have advantages over single mTOR inhibition in the treatment of TSC patients. Keywords: TSC; mTOR; ERK/MAPK; rapamycin; PD98059
Introduction Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome affecting approximately 1 in 6,000 births, characterized by the development of distinctive * Corresponding author. Tel: +86-10-6529 6495; Fax: +86-10-6529 6491. E-mail address: [email protected] DOI: 10.1016/S1673-8527(08)60124-1
tumors and malformations in multiple organs (Crino et al., 2006; Curatolo et al., 2008), with a TSC patient’s prognosis depending on the severity of symptoms. While brain lesion is the major cause of morbidity, renal angiomyolipoma (AML) is the leading cause of death. Although rare, TSC related lung lymphangioleiomyomatosis (LAM) is often detrimental for females in their child-bearing years. At the present time, there is no cure for this disease beyond the treatment of its symptoms (Crino, 2008).
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Inactivating mutations in one of the two tumor suppressor genes, TSC1 and TSC2, cause TSC; TSC2 mutations are 5–6 times more common than TSC1 mutations and have a more severe phenotype (Dabora et al., 2001). TSC1 and TSC2 form a protein complex which promotes GTPase activity of the mammalian target of rapamycin (mTOR) activator Rheb and therefore suppresses mTOR activity (Castro et al., 2003; Garami et al., 2003; Inoki et al., 2003; Tee et al., 2003). Under physiological conditions, mTOR is a serine/threonine kinase which integrates various signals from growth factors, nutrients, energy, and stress to regulate protein synthesis, cell growth, and proliferation. With aberrant mTOR activation frequently observed in various tumors (Manning and Cantley, 2007) and the loss of either TSC1 or TSC2 leading to hyperactivation of mTOR, mTOR inhibition was anticipated to be effective in the treatment for TSC patients (Kwiatkowski et al., 2002; Potter et al., 2002; El-Hashemite et al., 2003; Zhang et al., 2003; Chan et al., 2004). In recent years, the targeted suppression of mTOR functions has been employed for treating a broad spectrum of cancer patients in numerous clinical trials, but only a subset of cancers, such as advanced renal cell carcinoma, respond well to mTOR inhibitors (Brachmann et al., 2009). Only half of phosphatase and tensin homolog deleted on chromosome ten (PTEN) mutant glioblastoma patients were responsive to the mTOR inhibitor rapamycin, a macrolide antibiotic (also called sirolimus) produced by Streptomyces hagroscopicus, in a phase one clinical trial (Cloughesy et al., 2008). Although targeted mTOR suppression may hold promise in treating TSC in preclinical studies and clinical trials (Lee et al., 2005; Bissler et al., 2008; Ehninger et al., 2008; Paul and Thiele, 2008), in recent TSC clinical trials, lesions tended to relapse after rapamycin withdrawal (Bissler et al., 2008; Paul and Thiele, 2008). Thus the utility of the suppression of mTOR signaling is limited to ameliorative treatments for TSC or sporadic LAM (Davies et al., 2008). One of the possible mechanisms underlying the cytostatic activity of rapamycin is its relief of mTOR mediated negative feed-back regulation on receptor tyrosine kinases (RTK), such as platelet-derived growth factor receptor (PDGFR), or downstream effectors, such as insulin receptor substrate 1 (IRS-1), and then on the phosphatidylinositol-3-kinase/protein kinases B (PI3K/AKT) cascade (Zhang et al., 2003, 2007; Harrington et al., 2004; Shah et al., 2004). Rapamycin mediated disruption of the feedback suppression of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK)
pathway from mTOR may also render cells resistant to mTOR inhibition (Carracedo et al., 2008). Because both the AKT/mTOR and ERK/MAPK signaling pathways are prosurvival signals, the combined inhibition of mTOR and either PI3K or MAPK may hold greater promise than a single agent in the treatment of TSC. In this study, we investigated the efficacy of combinatorial inhibition of mTOR and ERK/MAPK in Tsc2/ mouse embryonic fibroblast cells (MEF), a widely used TSC cell model (Zhang et al., 2003) and found that dual suppression of mTOR and ERK/MAPK is effective in inhibiting TSC in vitro.
Material and methods Cell culture The Tsc2/ MEF cell line was derived from a Tsc2/ mouse embryo in Dr. Kwiatkowski’s Laboratory at Brigham & Women’s Hospital, Boston, MA, USA (Zhang et al., 2003) and cultured in DMEM with 10% FCS in a humidified atmosphere of 5% CO2 at 37°C.
Antibodies and other reagents Antibodies against ERK and ȕ-actin were obtained from Santa Cruz Biotechnology, USA, and antibodies against phospho-ERK, S6 ribosomal protein, and phospho-S6 from Cell Signaling Technology, USA. Rapamycin (Cell Signaling Technology, USA) was dissolved in 100% ethanol for a 10 mmol/L stock solution and PD98059 (an inhibitor of ERK phosphorylation, EMD Biosciences, USA) dissolved in 100% dimethylsulfoxide (DMSO) for a 20 mmol/L stock solution. The MTT Cell Proliferation Assay kit was obtained from BioDev Technology, China.
Western blotting analysis Cells were harvested in lysis buffer (2% SDS, 0.1 mol/L DTT, 10% glycerol, and 60 mmol/L Tris, pH 6.8) and boiled at 98°C for 10 min. The protein lysates were separated by 4%12% Bis-Tris SDS-PAGE gel (Invitrogen, USA) and transferred onto PVDF membranes (Pierce Chemical, USA). Nonspeci¿c reactivity was blocked by 5% non-fat dry milk in TBST buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05% Tween 20, and pH 7.5) at room temperature for 1 h, followed by incubation with the primary antibodies for phospho-ERK (1:1,000), ERK
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(1:5,000), phospho-S6 (1:4,000), S6 (1:6,000), and ȕ-actin (1:8,000) at 4°C for 12 h, and subsequently incubated with a secondary HRP-conjugated second antibody (Santa Cruz Biotechnology, USA) at 1:5,000 dilution at room temperature for 2 h.
Cell proliferation assay To assess the IC50 and the combination effects of rapamycin and PD98059, Tsc2/ MEF cells were seeded in triplicate 96-well plates at 4,000 cells/well and incubated for 6 h and then the medium was removed and replaced with fresh medium containing either rapamycin, PD98059, or both in varying concentrations. At various time points, 20 ȝL of MTT dye (3-[4,5-dimehyl-2-thiazolyl]-2,5diphenyl-2H-tetrazolium bromide, 5 mg/mL in PBS) was added to each well, incubated at 37°C for 4 h, the culture medium removed, 150 ȝL DMSO added to each well, and shaken at room temperature for 10 min. The spectrometric absorbance at 570 nm was then determined with a microplate reader. The viability ratio was calculated as: (OD value of the sample/OD value of control) × 100% (Kuang et al., 2009).
Fig. 1. Inhibitor of MEK compromised rapamycin-induced MEK-ERK activation. Tsc2/ MEF cells treated with 1 nmol/L mTOR inhibitor rapamycin, 50 ȝmol/L MEK inhibitor PD98059, or both for 48 h. Whole cell extracts used for activities of mTOR (phospho-S6) and ERK/MAPK (phospho-ERK1/2) were analyzed by Western blotting.
it was proposed here that combined inhibition of mTOR and ERK/MAPK might be a promising regimen for the treatment of TSC.
Results MEK inhibitor impaired rapamycin-induced MEK-ERK activation In rapamycin treatment of TSC cells, disruption of the mTOR mediated suppression on the ERK-PI3K-AKT signaling cascade has been identified here and in other laboratories (Zhang et al., 2003, 2007; Harrington et al., 2004; Shah et al., 2004) and it was also thought here that the reactivation of ERK/MAPK might have compromised the effectiveness of rapamycin in the TSC treatment. Thus, to prevent rapamycin mediated hyperactivation of ERK/ MAPK, PD98059, an inhibitor of MEK1/2 kinase upstream of ERK, was used to produce inhibition of ERK /MAPK signaling. After treatment with rapamycin alone or in combination with PD98059, Tsc2/ cells were harvested for Western blotting analysis, and it was observed that mTOR inhibition by rapamycin produced up-regulation of ERK1/2 phosphorylation, suggesting that mTOR indeed suppressed the ERK/MAPK pathway in Tsc2/ MEF cells (Fig. 1). Furthermore, it appeared that rapamycin-reactivated ERK1/2 was blunted with addition of PD98059. Thus,
Rapamycin and PD98059 had additive effects on Tsc2/ cell proliferation The potential benefit of dual inhibition of mTOR and MAPK in Tsc2 deficient cells was explored by initial quantification of the individual effects of rapamycin and PD98059 on Tsc2/ MEF cell proliferation using a dose course evaluation. Both drugs individually inhibited Tsc2/ cell proliferation with an IC50 of 0.172 nmol/L and 79.13 ȝmol/L for rapamycin and PD98059, respectively (Fig. 2). Interestingly, rapamycin at 10 nmol/L and greater inhibited Tsc2/ cell proliferation to a constant degree and the concurrent reduction of cell viability with rapamycin was never below 40% (Fig. 2A; Fig. 3A). In contrast, PD98059 exerted an inhibitory effect on Tsc2/ cells in a dose dependent manner (Fig. 2B; Fig. 3C). These data were consistent with the cytostatic action of rapamycin and the apoptotic effect of PD98059 observed in previous studies (Carracedo et al., 2008; Kinkade et al., 2008). Examination of the interactive action of these two drugs on cell proliferation revealed that the shapes (slopes) of the related cell viability curves of one drug were not altered or shifted by
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Fig. 2. Determination of IC50 for rapamycin and PD98059 in TSC cells. Tsc2/ MEF cells were treated with various concentrations of rapamycin (A) or PD98059 (B) for 48 h. IC50 of rapamycin, 0.172 nmol/L; IC50 of PD98059, 79.13 ȝmol/L.
Fig. 3. Rapamycin and PD98059 showed additive effects in cell culture. Tsc2/ MEF cells were treated with various concentrations of rapamycin (A and B) or PD98059 (C and D) in the presence of constant concentrations of PD98059 (A and B) or rapamycin (C and D) for 48 h. IC50 curves of rapamycin or PD98059 almost overlapped at fractional concentrations of other agent (A and C). IC50 of rapamycin and PD98059 was nearly constant at higher concentrations of other agent (B and D). IC50 of rapamycin, 0.172, 0.204, and 0.156 nmol/L rapamycin with addition of 0, 10, and 100 ȝmol/L PD98059, respectively (B). IC50 of PD98059, 79.1, 86.7, and 78.7 ȝmol/L PD98059 with addition of 0, 1, and 20 nmol/L rapamycin, respectively (D).
the other drug (after subtraction of the effect from that drug alone; Fig. 3, A and C). The IC50 of rapamycin and PD98059 had no significant differences in most combinations at various concentrations (Fig. 3, B and D), indicating that these two drugs exhibited additive rather than synergistic effects on Tsc2/ cell proliferation.
Suppression of MAPK signaling strengthened the inhibition of TSC cell proliferation by rapamycin The present observation that rapamycin and PD98059 individually inhibited the proliferation of Tsc2/ cells and that there was no antagonism from combinations of these
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drugs suggested that inhibition of MAPK and mTOR simultaneously could be more effective than targeting either a single pathway. Thus, the efficacy of combinatorial applications of rapamycin and PD98059 was evaluated in terms of Tsc2/ cell proliferation. Application of both drugs for 3 days yielded a nearly complete blockage of Tsc2/ cell proliferation (90.2% reduction) in an additive fashion (Fig. 4), which indicated that the pharmacological blockade of the ERK/MAPK pathway increased rapamycinmediated Tsc2/ MEF cell growth inhibition in vitro.
Discussion Because inactive mutations of either TSC1 or TSC2 tumor suppressor gene cause hyperactivation of mTOR, which is in turn responsible for the uncontrolled cell growth and proliferation in TSC patients, TSC patients were predicted to be extremely sensitive to mTOR inhibition. However, in recent clinical trials the efficacy of mTOR inhibitors in TSC patients was modest (Bissler et al., 2008; Paul and Thiele, 2008). Identification of the factors that compromised the therapeutic potential of mTOR inhibitors is thus critical for the improvement of targeted TSC therapies. Consistent with a recent report (Carracedo et al., 2008), the observations here showed that the negative feedback regulation of ERK/MAPK pathway exerted by mTOR was abolished with rapamycin treatment of Tsc2/ MEF cells (Fig. 1). The resulting augmented activation of MAPK signaling was attenuated by concomitant administration of a MEK1/2 inhibitor, PD98059. In addition, combinatorial application of rapamycin and PD98059 had greater inhibitory effect on Tsc2 deficient cell proliferation than these agents used singly (Fig. 4). Tumorigenesis is a multiple-step process involving numerous genetic alterations and it is not surprising that these complex, activated, prosurvival machineries may confer resistance to any single agent used in a targeted therapy for cancer. Thus, cotargeting of survival signaling pathways is an important advancement in the treatment of cancer (Stommel et al., 2007; Grant, 2008). Under physiological conditions, mTOR is governed by a panel of proto-oncogenes, such as epidermal growth factor receptor (EGFR), Ras protein, PI3K, and AKT, and tumor suppressors, such as PTEN and TSC1/2 complex, so it is frequently activated in variety of tumors (Manning and Cantley, 2007). In recent years, much time and effort has been committed to the discovery and clinical validation of specific inhibitors of PI3K and mTOR for patients with various kinds of tumors
Fig. 4. Combined blockade of mTOR and ERK/MAPK enhanced the inhibitory effect of single inhibitors on Tsc2/ cell proliferation. Tsc2/ MEF cells were treated with 1nmol/L rapamycin, 100 ȝmol/L PD98059, or both up to 3 days. Cell proliferation was assayed by MTT method (* means P < 0.01 compared with control cells, # means P < 0.01 compared with rapamycin-treated cells, and & means P < 0.01 compared with PD98059-treated cells.
(Brachmann et al., 2009). Unfortunately, the majority of treated cancer patients failed to benefit from these novel therapies. Some genetically stratified cancer patients with PTEN or TSC1/TSC2 mutations responded only moderately to mTOR inhibition (Lee et al., 2005; Bissler et al., 2008; Ehninger et al., 2008; Paul and Thiele, 2008). Elucidation of these confounding factors, which attenuate the response to rapamycin, has become the focus of cancer targeted therapy research. The targeted suppression of mTOR by rapamycin and its analogs, inevitably leading to the disruption of the newly identified mTOR-mediated negative feedback regulation on MAPK signaling pathway, as well as on the well-documented RTK-PI3K-AKT signaling cascade, supports the study of combined targeting of mTOR and MEK as a potential anticancer strategy (Zhang et al., 2003; Carracedo et al., 2008). In agreement with these findings, several preclinical studies of PTEN deficient prostate cancer and cancer cell lines have shown that the combined inhibition of mTOR and MAPK pathways is a promising regimen in the treatment of cancers (Carracedo et al., 2008; Kinkade et al., 2008). We propose here that the undesired activation of RTK-PI3K-AKT and ERK/MAPK by mTOR suppression could yield cells refractory to mTOR inhibitors and thus limit their efficacy in the treatment of TSC patients. On the other hand, the dual inhibition of mTOR and MAPK pathways would overcome the disadvantage of single agent therapy and boost the efficacy of mTOR targeted
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therapy in TSC patients (Brachmann et al., 2009). It was observed here that, indeed, double inhibition of mTOR and MAPK signaling with respective inhibitors achieved significant suppression of TSC cell proliferation, albeit in an additive, not synergistic, way. The limitation of this therapeutic strategy is that the reactivated ERK-PI3K-AKT function produced by rapamycin might still confer TSC cells with some resistance to the combined therapy of mTOR and MAPK. Thus, we suggest that dual pan-PI3K/ mTOR inhibitors, such as NVP-BEZ235 combined with MEK inhibitors, should be explored to achieve a triple intervention of mTOR, PI3K, and MEK MAPK functions in TSC cells (Engelman et al., 2008; Serra et al., 2008; Brachmann et al., 2009). Our findings demonstrate that dual inhibition of mTOR and MAPK signaling pathways effectively blocked Tsc2/ cell proliferation in vitro and provided a proof-of-concept demonstration that combination therapy targeting both mTOR and MAPK signaling pathways may have the potential to become a novel strategy in the treatment of TSC patients. Preclinical studies are therefore warranted to define the benefits and risks of the application of such combinational therapy in TSC animal models.
Acknowledgements This work was supported in part by the National Natural Science Foundation of China (No. 30788004). We thank David J. Kwiatkowski for providing Tsc2/ MEF cells, Xinxin Chen and Jingmei Jiang for help with statistical analysis.
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