erlotinib or sorafenib alone were identical to concentrations detected after drug combination (data not shown).
During the last decade novel anticancer treatments have emerged from advances in understanding of tumor biology, and identification of a number of key molecular targets in cancer signaling [24]. However, the multilevel cross-stimulation among the targets of these new agents along several pathways of signal transduction involved in neoplastic events allows other pathways to act as salvage mechanisms for cancer cells [34]. To overcome this problem one strategy might be to fine-tune the right mixture of drugs that target specific molecules. Therefore, drug combination treatment strategies should be explored in preclinical settings for the development of more effective combination treatment regimens.
Figure 4. Analysis of emerging targets of erlotinib-sorafenib combination from the Pamchip array. (A) Overlap analysis of kinases significantly inhibited by erlotinib-sorafenib in A549 and H1975 cells. (B) Signal transduction scheme of several key kinases in NSCLC, including kinases annotated as being able to phosphorylate the peptides emerging from the previous Pamchip array analyses. (C) Representative blots of at least two independent western blotting analyses performed as described in the Material and Methods, in A549 cells.
By targeting the EGFR- and RAF- dependent cancer cell proliferation and the VEGFR-2-dependent tumor angiogenesis pathways, the combination of erlotinib with sorafenib offers the potential advantage of blocking key pathways in different cell types, namely cell proliferation in the tumor and angiogenesis in endothelial cells. Several studies demonstrated that NSCLC is characterized by dysregulation of molecular mechanisms involved in cell proliferation and angiogenesis [35]. NSCLC has one of the highest EGFR levels among solid tumors, K-Ras mutations are detected in more than 30% of NSCLC samples [36], and most studies showed that an increased VEGF expression is a poor prognostic factor in lung cancer patients [37].
Therefore, combination of small molecule inhibitors that target these factors, such as the erlotinib-sorafenib combination, may be very useful for NSCLC treatment.
In a recent study a strong synergism of erlotinib and sorafenib was found in colorectal and lung cancer cells, associated by a marked inhibition of MEK signal and in vitro migration in the H1299 NSCLC cells [11].
Similar synergistic effects were observed in in vitro studies on cetuximab-sorafenib combination, as well as
in H1299 xenografts [11], while another study provided evidences that sorafenib can inhibit the growth of the gefitinib/erlotinib/vandetanib-resistant variant of Calu-3 NSCLC cells, which showed a significant increase in the levels of activated Akt, MAPK and surviving [16]. However, our findings are novel because they show that the synergistic interaction seems to be mediated by several other mechanisms, which enhanced both the sensitivity to erlotinib and to sorafenib, and should be further evaluated as potential predictive biomarkers for the future clinical development of this or similar combinations.
The main downstream mediators of the EGFR signal transduction pathway were affected by the sorafenib-erlotinib combination, which significantly reduced both ERK and Akt phosphorylation. These data fit with the synergistic interaction suggesting that combinations of specific signal transduction inhibitors targeting different steps of EGFR-PI3K pathways may be a successful strategy. There are several genetic and laboratory studies suggesting that the MAPK and PI3K-Akt pathways are vital to the growth and survival of cancer cells, and inhibitors targeting these pathways are entering the clinic at a rapid pace [38-39].
Moreover, since phospho-Akt downregulation correlated with the enhancement of drug-induced apoptosis and antitumor activity in lung cancer cells [28], the reduction of activated-Akt may explain the increased apoptosis found in the erlotinib-sorafenib combination.
The erlotinib-sorafenib combination was also able to reduce the mRNA and protein expression of E2F-1.
These results may be related to the nuclear effects of EGFR-TKIs, which influence the activity of some cell cycle proteins and transcription factors, including E2F-1 [40], This effect might be exerted directly or via down-regulation of cyclin D1 [41-42]. However, the Ras/MAPK/ERK dependent pathway is also implicated in the modulation of the expression of the cyclin D1 gene, and cyclin D1 down-regulation results in E2F-1 inhibition [42]. E2F-1 belongs to the p53-Rb pathway and acts as a growth-promoting factor associated with adverse prognosis in NSCLC [43]. Since adenovirus-mediated E2F-1 gene transfer in NSCLC cells induces cell growth arrest and apoptosis [44], we might hypothesize that E2F-1 inhibition caused by the erlotinib-sorafenib combination plays an important role in the synergistic inhibition of cell growth and apoptosis observed in our NSCLC cells. Moreover, the Rb-E2F-1 pathway contributes to the expression of VEGF receptors [45], suggesting that a reduction in the amount of E2F-1 mediated by the erlotinib-sorafenib combination might also affect the expression of genes involved in other aspects of tumor growth and progression, such as angiogenesis.
Circulating VEGF levels have attracted considerable interest as a potential pharmacodynamic marker of VEGFR-2 inhibition, and recent studies reported that sorafenib significantly increased plasma levels of VEGF [23, 46]. Although our in vitro models cannot represent the complexity of the interaction of tumor cells with the tumor neovasculature, sorafenib alone and in combination with erlotinib did not significantly affect VEGF mRNA expression nor VEGF secretion into the medium in our NSCLC cells, reflecting the slight non-significant increase in plasma VEGF during 21 days of treatment in patients treated with erlotinib-sorafenib combination [47].
In addition to the effects on EGFR cytoplasmatic and nuclear signaling pathways, the present study also shows that erlotinib interfered with the activity of other key determinants of sorafenib, such RKIP, which was significantly correlated with sorafenib sensitivity in our panel of NSCLC cells. The increased expression of RKIP after erlotinib treatment could be explained by the reduced nuclear factor κB (NF-κB) activation caused by EGFR inhibition [48]. Activation of NF-κB requires the phosphorylation and degradation of IKB, which
allows NF-κB to translocate into the nucleus. Yeung and colleagues demonstrated that RKIP dephosphorylated IKB, leading to inhibition of NF-κB activation [49]. Therefore, the reduced NF-κB activation and enhancement of IKB phosphorylation may cause a compensatory increase of RKIP expression, facilitating sorafenib activity.
After the analysis of several candidate biomarkers, we also evaluated whether a commercially available 144-peptide array might be useful to detect other tyrosine kinases that can be inhibited by the erlotinib-sorafenib combination and might represent new potential interesting druggable targets. The main limitations of this study included 1) the limited subset of the peptides spotted on the array, 2) the limited specificity, since for the majority of kinases it is difficult to have a single peptide substrate that is specific to a single kinase, and 3) the fact that cell lysates cannot reproduce subcellular compartmentalization and protein docking or scaffolding [50]. However, we were able to identify a list of tyrosine kinase peptide substrates which were significantly inhibited by erlotinib and sorafenib, respectively, including several common targets.
In agreement with the synergistic interaction, these peptides were also inhibited by the drug combination, which additionally inhibited other 11 common peptides in the two studied cell lines. A validation of some of these results was obtained by subsequent western blot analysis of two of the kinases that can phosphorylate these peptides and constitute appealing targets as central nodes of the signaling pathways involved in NSCLC proliferation, SRC and CDK2 [51-52]. Finally, we were able to find modulation of kinase activity of a few kinases in tumor tissues, supporting further ex vivo studies. In particular, detection of key deregulated signaling pathways may pave the way for novel therapeutics design and appropriate therapy selection in individual patients. However, since DNA and microRNA samples are easier to be collected and stored than frozen tumor-biopsy samples, the data from these protein-array studies should be integrated with the results of novel pharmaco/epigenetic analyses [53-54].
CONCLUSIONS
The present study characterizes several molecular mechanisms and determinants involved in the synergistic interaction of erlotinib and sorafenib against NSCLC cells, focusing on cells harbouring K-Ras and EGFR T790M mutations. Sorafenib reduced ERK and Akt phosphorylation, which was additionally reduced by drug combination, and favoured apoptosis induction. Erlotinib and sorafenib significantly reduced E2F-1 expression, while erlotinib and the combination increased RKIP expression, favouring sorafenib activity. The modulation of all these determinants influences the cytotoxicity of this combination and, although the extrapolation of in vitro data to the clinical setting should be considered with caution, these results may have implications for the rational development of chemotherapeutic regimes including erlotinib and sorafenib or other emerging multikinase inhibitor of angiogenic, stromal and oncogenic kinases, such as regorafenib [55].
ACKNOWLEDGEMENTS
This work was partially supported by grants from the Netherlands Organization for Scientific Research (NWO-VENI grant, Elisa Giovannetti), and AIRC-Marie Curie International Fellowship (Elisa Giovannetti). We would like to thank Rob Ruijtenbeek (Pamgene International B.V, Inc.), who provided initial technical support to conduct this project.
REFERENCES
1. Azzoli CG, Temin S, Aliff T, et al. 2011 Focused Update of 2009 American Society of Clinical Oncology Clinical Practice Guideline Update on Chemotherapy for Stage IV Non-Small-Cell Lung Cancer. J Clin Oncol 2011; 29: 3825-31.
2. Dorans K. Outpacing cancer. Nat Med. 2009;15:718-22.
3. Sequist LV, Martins RG, Spigel D, et al. First-line gefitinib in patients with advanced non-small-cell lung cancer harboring somatic EGFR mutations. J Clin Oncol. 2008;26:2442-9.
4. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Rosell R, Carcereny E, Gervais R, et al.
Lancet Oncol 2012;13:239-46.
5. Scoccianti C, Vesin A, Martel G, et al. Prognostic value of TP53, KRAS and EGFR mutations in Non-Small Cell Lung Cancer:
EUELC cohort. Eur Respir J. 2012 Jan 20. [Epub ahead of print]
6. Roberts PJ, Stinchcombe TE, Der CJ, Socinski MA. Personalized medicine in non-small-cell lung cancer: is KRAS a useful marker in selecting patients for epidermal growth factor receptor-targeted therapy? J Clin Oncol. 2010;28:4769-77.
7. Adnane L, Trail PA, Taylor I, Wilhelm SM. Sorafenib (BAY 43-9006, Nexavar), a dual-action inhibitor that targets RAF/MEK/ERK pathway in tumor cells and tyrosine kinases VEGFR/PDGFR in tumor vasculature. Methods Enzymol. 2006;407:597-612.
8. Escudier B, Eisen T, Stadler WM, et al. Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med. 2007;356:125-34.
9. Llovet JM, Ricci S, Mazzaferro V, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 2008;359:378-90.
10. Wilhelm SM, Carter C, Tang L, et al. BAY 43-9006 exhibits broad-spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 2004;64:7099-109.
11. Martinelli E, Troiani T, Morgillo F, et al. Synergistic antitumor activity of sorafenib in combination with epidermal growth factor receptor inhibitors in colorectal and lung cancer cells. Clin Cancer Res. 2010;16:4990-5001.
12. Viloria-Petit A, Crombet T, Jothy S, et al. Acquired resistance to the antitumor effect of epidermal growth factor receptor-blocking antibodies in vivo: a role for altered tumor angiogenesis. Cancer Res 2001;61:5090-101.
13. Ciardiello F, Bianco R, Caputo R, et al. Antitumor activity of ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in human cancer cells with acquired resistance to antiepidermal growth factor receptor therapy. Clin Cancer Res 2004;10:784-93.
14. Tortora G, Ciardiello F, Gasparini G. Combined targeting of EGFR-dependent and VEGF-dependent pathways: rationale, preclinical studies and clinical applications. Nat Clin Pract Oncol 2008;5:521–30.
15. Naumov GN, Nilsson MB, Cascone T, et al. Combined vascular endothelial growth factor receptor and epidermal growth factor receptor (EGFR) blockade inhibits tumor growth in xenograft models of EGFR inhibitor resistance. Clin Cancer Res 2009;15:3484-94.
16. Morgillo F, Martinelli E, Troiani T, Orditura M, De Vita F, Ciardiello F. Antitumor activity of sorafenib in human cancer cell lines with acquired resistance to EGFR and VEGFR tyrosine kinase inhibitors. PLoS One. 2011;6:e28841.
17. Lind JS, Dingemans AM, Groen HJ, et al. A multicenter phase II study of erlotinib and sorafenib in chemotherapy-naive patients with advanced non-small cell lung cancer. Clin Cancer Res. 2010;16:3078-87.
18. Gridelli C, Morgillo F, Favaretto A, et al. Sorafenib in combination with erlotinib or with gemcitabine in elderly patients with advanced non-small-cell lung cancer: a randomized phase II study. Ann Oncol. 2011;22:1528-34.
19. Cho B, Kim S, Heo DS, et al. A multicenter phase II study of sorafenib in combination with erlotinib in patients with advanced non-small cell lung cancer (NSCLC) J Clin Oncol 2010;28:7s
20. Spigel DR, Burris HA 3rd, Greco FA, et al. Randomized, double-blind, placebo-controlled, phase II trial of sorafenib and erlotinib or erlotinib alone in previously treated advanced non-small-cell lung cancer. J Clin Oncol. 2011;29:2582-9.
21. Smit EF, Dingemans AM, Thunnissen FB, Hochstenbach MM, van Suylen RJ, Postmus PE. Sorafenib in patients with advanced non-small cell lung cancer that harbor K-ras mutations: a brief report. J Thorac Oncol. 2010;5:719-20.
22. Lind JS, Meijerink MR, Dingemans AM, et al. Dynamic contrast-enhanced CT in patients treated with sorafenib and erlotinib for non-small cell lung cancer: a new method of monitoring treatment? Eur Radiol. 2010;20:2890-8.
23. Kelly RJ, Rajan A, Force J, et al. Evaluation of KRAS mutations, angiogenic biomarkers, and DCE-MRI in patients with advanced non-small-cell lung cancer receiving sorafenib. Clin Cancer Res. 2011 Mar 1;17(5):1190-9.
24. Bates SE, Amiri-Kordestani L, Giaccone G. Drug development: portals of discovery. Clin Cancer Res. 2012;18:23-32.
25. Giovannetti E, Zucali PA, et al. Association of polymorphisms in AKT1 and EGFR with clinical outcome and toxicity in non-small cell lung cancer patients treated with gefitinib. Mol Cancer Ther. 2010;9:581-93.
26. Tibaldi C, Giovannetti E, Tiseo M, et al. Correlation of cytidine deaminase polymorphisms and activity with clinical outcome in gemcitabine-/platinum-treated advanced non-small-cell lung cancer patients. Ann Oncol. 2012;23:670-7.
27. Granchi C, Roy S, Giacomelli C, et al. Discovery of N-hydroxyindole-based inhibitors of human lactate dehydrogenase isoform A (LDH-A) as starvation agents against cancer cells. J Med Chem. 2011;54:1599-612.
28. Giovannetti E, Lemos C, Tekle C, et al. Molecular mechanisms underlying the synergistic interaction of erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor, with the multitargeted antifolate pemetrexed in non-small-cell lung cancer cells. Mol Pharmacol. 2008;73:1290-300
29. Al-Mulla F, Bitar MS, Feng J, Park S, Yeung KC. A new model for raf kinase inhibitory protein induced chemotherapeutic resistance. PLoS One. 2012;7:e29532.
30. Giovannetti E, Mey V, Loni L, et al. Cytotoxic activity of gemcitabine and correlation with expression profile of drug-related genes in human lymphoid cells. Pharmacol Res. 2007;55:343-9.
31. Bianco C, Giovannetti E, Ciardiello F, et al. Synergistic antitumor activity of ZD6474, an inhibitor of vascular endothelial growth factor receptor and epidermal growth factor receptor signaling, with gemcitabine and ionizing radiation against pancreatic cancer.
Clin Cancer Res. 2006;12:7099-107.
32. Honeywell R, Yarzadah K, Giovannetti E, et al. Simple and selective method for the determination of various tyrosine kinase inhibitors used in the clinical setting by liquid chromatography tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2010;878:1059-68.
33. Labots M, Gotink KJ, Dekker H, et al. Measurement of kinase activity in cancer cell lines and tumor tissue using a tyrosine kinase peptide substrate array. Proceedings Annual Meeting American Association Cancer Research 2012
34. Garraway LA, Jänne PA. Circumventing Cancer Drug Resistance in the Era of Personalized Medicine. Cancer Discov 2012 [Epub ahead of print]
35. Giovannetti E, Honeywell R, Hanauske AR, et al. Pharmacological aspects of the enzastaurin-pemetrexed combination in non-small cell lung cancer (NSCLC). Curr Drug Targets. 2010;11:12-28.
36. Chowdhuri SR, Xi L, Pham TH, et al. EGFR and KRAS mutation analysis in cytologic samples of lung adenocarcinoma enabled by laser capture microdissection. Mod Pathol. 2012;25:548-55.
37. Farhat FS, Tfayli A, Fakhruddin N, et al. Expression, prognostic and predictive impact of VEGF and bFGF in non-small cell lung cancer. Crit Rev Oncol Hematol. 2012 Apr 9. [Epub ahead of print]
38. Schubbert S, Shannon K, Bollag G. Hyperactive Ras in developmental disorders and cancer. Nat Rev Cancer 2007;7:295-308.
39. Engelman JA. Targeting PI3K signalling in cancer: opportunities, challenges and limitations. Nat Rev Cancer 2009;9:550-62.
40. Lo HW, Hung MC. Nuclear EGFR signalling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival. Br J Cancer 2006;94:184-8.
41. Kobayashi S, Shimamura T, Monti S, et al. Transcriptional profiling identifies cyclin D1 as a critical downstream effector of mutant epidermal growth factor receptor signaling. Cancer Res 2006;66:11389-98.
42. Suenaga M, Yamaguchi A, Soda H, et al Antiproliferative effects of gefitinib are associated with suppression of E2F-1 expression and telomerase activity. Anticancer Res 2006;26:3387-91.
43. Gorgoulis VG, Zacharatos P, Mariatos G, et al. Transcription factor E2F-1 acts as a growth-promoting factor and is associated with adverse prognosis in non-small cell lung carcinomas. J Pathol. 2002;198:142-56.
44. Kuhn H, Liebers U, Gessner C, et al. Adenovirus-mediated E2F-1 gene transfer in non small-cell lung cancer induces cell growth arrest and apoptosis. Eur Respir J. 2002;20:703-9.
45. Pillai S, Kovacs M, Chellappan S. Regulation of vascular endothelial growth factor receptors by Rb and E2F1: role of acetylation.
Cancer Res 2010;70:4931-40.
46. Raut CP, Boucher Y, Duda DG, et al. Effects of sorafenib on intra-tumoral interstitial fluid pressure and circulating biomarkers in patients with refractory sarcomas (NCI protocol 6948). PLoS One 2012;7:e26331.
47. Vroling L, Lind JS, de Haas RR, et al. CD133+ circulating haematopoietic progenitor cells predict for response to sorafenib plus erlotinib in non-small cell lung cancer patients. Br J Cancer 2010;102:268-75.
48. Li Y, Sarkar FH. Inhibition of nuclear factor kappaB activation in PC3 cells by genistein is mediated via Akt signaling pathway. Clin Cancer Res 2002;8:2369-77.
49. Yeung KC, Rose DW, Dhillon AS, et al. Raf kinase inhibitor protein interacts with NFkappaB-inducing kinase and TAK1 and inhibits NF-kappaB activation. Mol Cell Biol 2001;21:7207-17.
50. Piersma SR, Labots M, Verheul HM, Jiménez CR. Strategies for kinome profiling in cancer and potential clinical applications:
chemical proteomics and array-based methods. Anal Bioanal Chem 2010;397:3163-71.
51. Giaccone G, Zucali PA. Src as a potential therapeutic target in non-small-cell lung cancer. Ann Oncol. 2008;19:1219-23.
52. Cai D, Latham VM Jr, Zhang X, Shapiro GI. Combined depletion of cell cycle and transcriptional cyclin-dependent kinase activities induces apoptosis in cancer cells. Cancer Res 2006;66:9270-80.
53. Danesi R, Altavilla G, Giovannetti E, Rosell R. Pharmacogenomics of gemcitabine in non-small-cell lung cancer and other solid tumors. Pharmacogenomics. 2009;10:69-80.
54. Giovannetti E, Erozenci A, Smit J, Danesi R, Peters GJ. Crit Rev Oncol Hematol. 2012;81:103-22.
55. Mross K, Frost A, Steinbild S, et al. A Phase I Dose-Escalation Study of Regorafenib (BAY 73-4506), an Inhibitor of Oncogenic, Angiogenic, and Stromal Kinases, in Patients with Advanced Solid Tumors. Clin Cancer Res. 2012;18:2658-67.