Abstract
Purpose
Pemetrexed, an inhibitor of thymidylate synthase (TS) and additional folate-dependent enzymes, is clinically active in patients suffering from “non-squamous” non-small cell lung cancer (NSCLC). High expression of TS has been implied as biomarker predictive of resistance to pemetrexed. Against this background, we studied whether inhibition of mTOR could lower expression of TS and thus sensitize NSCLC cells to pemetrexed.
Methods and results
Using squamous cell carcinoma and adenocarcinoma NSCLC cell lines, we observed that constitutive TS expression levels failed to correlate with sensitivity to growth inhibition or apoptosis imposed by pemetrexed in vitro. Interestingly, pemetrexed strongly induced TS RNA and protein expression in all cell lines. The allosteric “rapalogue” mTOR inhibitor everolimus suppressed constitutive, but not pemetrexed-induced TS expression. Surprisingly, cotreatment with everolimus protected NSCLC cells against pemetrexed-induced apoptosis. This resulted in increased long-term clonogenic survival of NSCLC cells treated with pemetrexed plus everolimus as compared to pemetrexed alone. No such negative interaction was observed when everolimus was combined with recombinant TRAIL, a proliferation-independent proapoptotic agent.
Conclusions
Rapalogues may suppress the antitumor activity of pemetrexed by slowing cell cycle progression. This should be considered when combining pemetrexed and mTOR inhibitors in NSCLC treatment.
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References
Alvarez RH, Valero V, Hortobagyi GN (2010) Emerging targeted therapies for breast cancer. J Clin Oncol 28(20):3366–3379
An X, Tiwari AK, Sun Y, Ding PR, Ashby CR Jr, Chen ZS (2010) BCR-ABL tyrosine kinase inhibitors in the treatment of Philadelphia chromosome positive chronic myeloid leukemia: a review. Leuk Res 34(10):1255–1268
Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, Klevernic I, Arthur JS, Alessi DR, Cohen P (2007) The selectivity of protein kinase inhibitors: a further update. Biochem J 408(3):297–315
Baselga J, Semiglazov V, van Dam P, Manikhas A, Bellet M, Mayordomo J, Campone M, Kubista E, Greil R, Bianchi G, Steinseifer J, Molloy B, Tokaji E, Gardner H, Phillips P, Stumm M, Lane HA, Dixon JM, Jonat W, Rugo HS (2009) Phase II randomized study of neoadjuvant everolimus plus letrozole compared with placebo plus letrozole in patients with estrogen receptor-positive breast cancer. J Clin Oncol 27(16):2630–2637
Bepler G, Sommers KE, Cantor A, Li X, Sharma A, Williams C, Chiappori A, Haura E, Antonia S, Tanvetyanon T, Simon G, Obasaju C, Robinson LA (2008) Clinical efficacy and predictive molecular markers of neoadjuvant gemcitabine and pemetrexed in resectable non-small cell lung cancer. J Thorac Oncol 3(10):1112–1118
Ceppi P, Volante M, Saviozzi S, Rapa I, Novello S, Cambieri A, Lo Iacono M, Cappia S, Papotti M, Scagliotti GV (2006) Squamous cell carcinoma of the lung compared with other histotypes shows higher messenger RNA and protein levels for thymidylate synthase. Cancer 107(7):1589–1596
Chou TC (1991) The median-effect principle and the combination index for quantitation of synergism and antagonism. In: Chou TCaR DC (ed) Synergism and antagonism in chemotherapy. Academic Press, San Diego, pp 61–102
Chou TC, Talalay P (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22:27–55
Coppin C (2010) Everolimus: the first approved product for patients with advanced renal cell cancer after sunitinib and/or sorafenib. Biologics 4:91–101
Davies SP, Reddy H, Caivano M, Cohen P (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351(Pt 1):95–105
Dematteo RP, Heinrich MC, El-Rifai WM, Demetri G (2002) Clinical management of gastrointestinal stromal tumors: before and after STI-571. Hum Pathol 33(5):466–477
Dobashi Y, Watanabe Y, Miwa C, Suzuki S, Koyama S (2011) Mammalian target of rapamycin: a central node of complex signaling cascades. Int J Clin Exp Pathol 4(5):476–495
Druker BJ (2002) Inhibition of the Bcr-Abl tyrosine kinase as a therapeutic strategy for CML. Oncogene 21(56):8541–8546
Engelman JA, Janne PA, Mermel C, Pearlberg J, Mukohara T, Fleet C, Cichowski K, Johnson BE, Cantley LC (2005) ErbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines. Proc Natl Acad Sci USA 102(10):3788–3793
Fan QL, Zou WY, Song LH, Wei W (2005) Synergistic antitumor activity of TRAIL combined with chemotherapeutic agents in A549 cell lines in vitro and in vivo. Cancer Chemother Pharmacol 55(2):189–196
Frese S, Brunner T, Gugger M, Uduehi A, Schmid RA (2002) Enhancement of Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis in non-small cell lung cancer cell lines by chemotherapeutic agents without correlation to the expression level of cellular protease caspase-8 inhibitory protein. J Thorac Cardiovasc Surg 123(1):168–174
Giles FJ, Albitar M (2005) Mammalian target of rapamycin as a therapeutic target in leukemia. Curr Mol Med 5(7):653–661
Guix M, Faber AC, Wang SE, Olivares MG, Song Y, Qu S, Rinehart C, Seidel B, Yee D, Arteaga CL, Engelman JA (2008) Acquired resistance to EGFR tyrosine kinase inhibitors in cancer cells is mediated by loss of IGF-binding proteins. J Clin Investig 118(7):2609–2619
Hahnel PS, Thaler S, Antunes E, Huber C, Theobald M, Schuler M (2008) Targeting AKT signaling sensitizes cancer to cellular immunotherapy. Cancer Res 68(10):3899–3906
Hanauske AR, Eismann U, Oberschmidt O, Pospisil H, Hoffmann S, Hanauske-Abel H, Ma D, Chen V, Paoletti P, Niyikiza C (2007) In vitro chemosensitivity of freshly explanted tumor cells to pemetrexed is correlated with target gene expression. Investig New Drugs 25(5):417–423
Jiang BH, Liu LZ (2008) Role of mTOR in anticancer drug resistance: perspectives for improved drug treatment. Drug Resist Updat 11(3):63–76
Johnston SR (2009) Enhancing the efficacy of hormonal agents with selected targeted agents. Clin Breast Cancer 9(Suppl 1):S28–S36
Laplante M, Sabatini DM (2009) mTOR signaling at a glance. J Cell Sci 122(Pt 20):3589–3594
LoPiccolo J, Blumenthal GM, Bernstein WB, Dennis PA (2008) Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations. Drug Resist Updat 11(1–2):32–50
Neal JW, Sequist LV (2010) First-line use of EGFR tyrosine kinase inhibitors in patients with NSCLC containing EGFR mutations. Clin Adv Hematol Oncol 8(2):119–126
Ozasa H, Oguri T, Uemura T, Miyazaki M, Maeno K, Sato S, Ueda R (2010) Significance of thymidylate synthase for resistance to pemetrexed in lung cancer. Cancer Sci 101(1):161–166
Reck M, von Pawel J, Zatloukal P, Ramlau R, Gorbounova V, Hirsh V, Leighl N, Mezger J, Archer V, Moore N, Manegold C (2009) Phase III trial of cisplatin plus gemcitabine with either placebo or bevacizumab as first-line therapy for nonsquamous non-small-cell lung cancer: AVAil. J Clin Oncol 27(8):1227–1234
Righi L, Papotti MG, Ceppi P, Bille A, Bacillo E, Molinaro L, Ruffini E, Scagliotti GV, Selvaggi G (2010) Thymidylate synthase but not excision repair cross-complementation group 1 tumor expression predicts outcome in patients with malignant pleural mesothelioma treated with pemetrexed-based chemotherapy. J Clin Oncol 28(9):1534–1539
Ross JS, Slodkowska EA, Symmans WF, Pusztai L, Ravdin PM, Hortobagyi GN (2009) The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine. Oncologist 14(4):320–368
Sandler A, Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A, Lilenbaum R, Johnson DH (2006) Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 355(24):2542–2550
Sarbassov DD, Ali SM, Sabatini DM (2005) Growing roles for the mTOR pathway. Curr Opin Cell Biol 17(6):596–603
Scagliotti GV, Parikh P, von Pawel J, Biesma B, Vansteenkiste J, Manegold C, Serwatowski P, Gatzemeier U, Digumarti R, Zukin M, Lee JS, Mellemgaard A, Park K, Patil S, Rolski J, Goksel T, de Marinis F, Simms L, Sugarman KP, Gandara D (2008) Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer. J Clin Oncol 26(21):3543–3551
Schiller JH, Harrington D, Belani CP, Langer C, Sandler A, Krook J, Zhu J, Johnson DH (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 346(2):92–98
Schuler W, Sedrani R, Cottens S, Haberlin B, Schulz M, Schuurman HJ, Zenke G, Zerwes HG, Schreier MH (1997) SDZ RAD, a new rapamycin derivative: pharmacological properties in vitro and in vivo. Transplantation 64(1):36–42
Shih C, Chen VJ, Gossett LS, Gates SB, MacKellar WC, Habeck LL, Shackelford KA, Mendelsohn LG, Soose DJ, Patel VF, Andis SL, Bewley JR, Rayl EA, Moroson BA, Beardsley GP, Kohler W, Ratnam M, Schultz RM (1997) LY231514, a pyrrolo[2, 3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes. Cancer Res 57(6):1116–1123
Sigmond J, Backus HH, Wouters D, Temmink OH, Jansen G, Peters GJ (2003) Induction of resistance to the multitargeted antifolate Pemetrexed (ALIMTA) in WiDr human colon cancer cells is associated with thymidylate synthase overexpression. Biochem Pharmacol 66(3):431–438
Sinnberg T, Lasithiotakis K, Niessner H, Schittek B, Flaherty KT, Kulms D, Maczey E, Campos M, Gogel J, Garbe C, Meier F (2009) Inhibition of PI3K-AKT-mTOR signaling sensitizes melanoma cells to cisplatin and temozolomide. J Investig Dermatol 129(6):1500–1515
Vignot S, Faivre S, Aguirre D, Raymond E (2005) mTOR-targeted therapy of cancer with rapamycin derivatives. Ann Oncol 16(4):525–537
Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabolism. Cell 124(3):471–484
Acknowledgments
We thank all members of the Laboratory of Molecular Oncology, Department of Medical Oncology, West German Cancer Center, for their help and stimulating discussions. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SCHU1541/5-1) and Deutsche Krebshilfe (No. 107993).
Conflict of interest
Stephan Herbertz is an employee of Novartis Pharma; Martin Schuler has served as consultant to and has received research support from Novartis.
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432_2011_1123_MOESM1_ESM.pdf
Supplemental Figure 1. Thymidylate synthase expression in lung cancer cells following treatment with everolimus. NSCLC cells were cultured in the absence or presence (+) of everolimus (10 nM) for 24 h. Cell lysates were analyzed by immunoblotting for the expression of thymidylate synthase (TS), S6 ribosomal protein phosphorylation (pS6) and actin as a loading control (PDF 344 kb)
432_2011_1123_MOESM2_ESM.pdf
Supplemental Figure 2. Everolimus treatment alters the cell cycle distribution of lung cancer cells. Cell cycle analyses of FaDu, NCl-H23 and A549 cells following 48 h of treatment with everolimus (10 nM) (PDF 113 kb)
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Markova, B., Hähnel, P.S., Kasper, S. et al. Pharmacologic inhibition of mTOR antagonizes the cytotoxic activity of pemetrexed in non-small cell lung cancer. J Cancer Res Clin Oncol 138, 545–554 (2012). https://doi.org/10.1007/s00432-011-1123-9
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DOI: https://doi.org/10.1007/s00432-011-1123-9