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Histone Deacetylase Inhibitor Romidepsin Enhances Anti-Tumor Effect of Erlotinib in Non-small Cell Lung Cancer (NSCLC) Cell Lines
ORIGINAL ARTICLE
Histone Deacetylase Inhibitor Romidepsin Enhances Anti-Tumor Effect of Erlotinib in Non-small Cell Lung Cancer (NSCLC) Cell Lines Wei Zhang, PhD,* Michael Peyton, PhD,* Yang Xie, PhD,†‡ Junichi Soh, MD,* John D. Minna, MD,*†§储 Adi F. Gazdar, MD,*†¶ and Eugene P. Frenkel, MD†§
Introduction: Most epidermal growth factor receptor (EGFR) mutant non-small cell lung cancers (NSCLCs) are sensitive to EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib or gefitinib, but many EGFR wild type NSCLCs are resistant to TKIs. In this study, we examined the effects of the histone deacetylase inhibitor, romidepsin, in combination with erlotinib, in NSCLC cell lines and xenografts. Methods: For in vitro studies, nine NSCLC cell lines with varying mutation status and histology were treated with erlotinib and romidepsin alone or in combination. 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays were performed to determine the concentration that inhibits 50% (IC50) value of each drug or the combination. For in vivo studies, NCIH1299 xenografts were inoculated subcutaneously into athymic nude mice. Romidepsin and/or erlotinib were injected intraperitoneally after tumors developed and tumor sizes were measured. Results: We found that romidepsin increased the sensitivity of erlotinib synergistically in all nine NSCLC cell lines including EGFR and KRAS wild type cell lines, KRAS mutant cell lines, and TKI resistant EGFR mutant cell lines. This effect was partially due to enhanced apoptosis. Furthermore, cotreatment of erlotinib and romidepsin inhibited NCIH1299 xenograft growth in athymic nude mice. Conclusions: These observations support a role for the combination of a histone deacetylase inhibitor and a TKI in the treatment of NSCLCs. Key Words: Erlotinib, Romidepsin, Non-small cell lung cancer, Epidermal growth factor receptor, KRAS. (J Thorac Oncol. 2009;4: 161–166)
*Hamon Center for Therapeutic Oncology Research, †Harold C. Simmons Comprehensive Cancer Center, ‡Department of Clinical Sciences, §Division of Hematology/Oncology, Department of Internal Medicine, Departments of 储Pharmacology, and ¶Pathology, University of Texas Southwestern Medical Center, Dallas, Texas. Disclosure: The authors declare no conflicts of interest. Address for correspondence: Eugene P. Frenkel, MD, Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75235. E-mail: [email protected] Copyright © 2009 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/09/0402-0161
T
he epidermal growth factor receptor (EGFR) belongs to the EGFR family of tyrosine kinase receptors.1 Upon activation, EGFR can promote cell proliferation and survival through Ras/MEK/MAPK and/or PI3K/AKT signaling pathways.2 In recent years, non-small cell lung cancers (NSCLCs) containing EGFR mutations have been found to be sensitive to EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib (Tarceva) or gefitinib (Iressa), and these drugs have been successfully used in the therapy for such patients.3,4 Unfortunately, some patients with cancers containing EGFR mutations develop resistance to TKIs after a period of time. Although the mechanisms of resistance are not completely understood, the existence of the secondary T790M mutation activity appears to be the major mechanism.5 Furthermore, most of the NSCLCs containing wild type EGFR receptor are resistant to therapy with EGFR TKIs.6 In addition, the presence of KRAS mutations is associated with primary resistance to TKIs.7 Thus, the role of erlotinib and gefitinib for the treatment of NSCLC is limited. Acetylation of histones plays an important role in chromatin organization and gene expression.8 Acetylation is determined by both histone acetyltransferases and histone deacetylases (HDACs).9 HDACs remove the acetyl groups from the lysine residues of the histone tail leading to compaction of chromatin and resultant repression of gene transcription.9 Many nonhistone proteins can be HDAC substrates such as p53, cMyc, Stat3, and Hsp90.10 Such potential targets have provided a new strategy for the role of HDAC inhibitors in cancer therapy. HDAC inhibitors can induce growth arrest, differentiation, and apoptosis in tumor cells.11 Several HDAC inhibitors have been used in clinical trials.12 HDAC inhibitors are also excellent candidates for combination therapies. The HDAC inhibitor MS-275 was shown to increase the sensitivity of gefitinib in NSCLC cell lines.13 Romidepsin (previously termed FK228), also known as depsipeptide, is a natural cyclic peptide HDAC inhibitor. It bears the molecular structure of depsipeptides, namely sequences of alternating amino- and hydroxy-carboxylic acid residues.14 It has shown important inhibition of in vitro growth of several tumor cell types including lung and prostate cancers, lymphomas and leukemias.14 –17 Romidepsin has recently been studied in a clinical trial for treatment of lung cancer.18 In this article, we investigated the effects of combining
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TABLE 1. The IC50 Values of Romidepsin and Erlotinib in NSCLC Cell Lines Mutation Statusa
NCI-H1299 NCI-2882 HCC95 NCI-H23 NCI-H157 NCI-H460 NCI-H1975 NCI-H820 NCI-H1650
Histology
EGFR
KRAS
Romidepsinb IC50 ⴞ SD (ng/ml)
LC AD SQ AD SQ LC AD AD AD
wt wt wt wt wt wt mut mut mut
wt wt wt mut mut mut wt wt wt
4.6 ⫾ 0.2 1.6 ⫾ 0.04 2.5 ⫾ 0.05 2.9 ⫾ 0.2 1.6 ⫾ 0.02 2.1 ⫾ 0.07 1.3 ⫾ 0.04 2.4 ⫾ 0.1 4.9 ⫾ 0.3
Erlotinib IC50 ⴞ SD (M)b No Romidepsin
Romidepsin (1 ng/ml)
CIc
90.0 ⫾ 2.2 115.0 ⫾ 3.7 107.0 ⫾ 2.2 52.8 ⫾ 4.7 75.0 ⫾ 3.0 105.3 ⫾ 4.4 14.4 ⫾ 0.1 8.6 ⫾ 0.6 28.3 ⫾ 1.4
2.6 ⫾ 0.01 1.9 ⫾ 0.4 5.7 ⫾ 0.9 0.07 ⫾ 0.01 0.3 ⫾ 0.03 1.7 ⫾ 0.2 0.01 ⫾ 0.02 1.8 ⫾ 0.02 4.8 ⫾ 0.8
0.25 0.65 0.45 0.34 0.63 0.50 0.77 0.63 0.37
a
The mutation status of NSCLC cell lines were determined as described in Materials and Methods. The IC50 values of erlotinib and romidepsin were defined as the concentration needed for a 50% reduction in the absorbance calculated based on the cell viability curves. CI combination index. Values ⬍1 are interpreted as synergism. (see Materials and Methods). EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer; LC, large cell; AD, adenocarcinoma; SQ, squamous carcinoma; wt, wild type; mut, mutant; IC50, concentration that inhibits 50%. b c
romidepsin and erlotinib on the sensitivity of EGFR wild type and mutant NSCLC cell lines resistant to TKI therapy.
MATERIALS AND METHODS Cell Lines and Drugs Nine NSCLC cell lines (Table 1) were cultured in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 5% fetal bovine serum and incubated in humidified air and 5% CO2 at 37°C. The cell lines were established by us, DNA fingerprinted, and free of mycoplasma by molecular tests (iNtRON Biotechnology, Korea). Romidepsin was provided by Fujisawa Pharmaceutical Co. (Japan) (This drug is now the property of Gloucester Co., MA). Erlotinib (Tarceva) (OSI Pharmaceuticals, Inc., NY) was purchased from UT Southwestern pharmacy.
DNA Isolation and Polymerase Chain Reaction DNA was isolated from NSCLC cell lines and polymerase chain reaction was performed to determine the KRAS and EGFR mutation status of each cell line according to previously reported methods.19
MTS Assay Drug sensitivity was measured by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTS) assay (Promega, Madison, WI). Two ⫻ 103 cells were seeded into each well of a 96-well microtiter plate. After 24 hours, the cells were treated with varying concentrations (0.01–250 M) of erlotinib either in the absence or presence of 1 ng/ml romidepsin for 72 hours at 37°C. MTS solution was added to each well, and the plates were incubated for 1 hour at 37°C. Optical density was measured at 490 nm using a SpectraMax 190 spectrophotometer (Molecular Devices, Sunnywale, CA). Each experiment was carried out in eight replicate wells for each drug concentration and repeated twice. The concentration that inhibits 50% (IC50) value was defined as the concentration needed for a 50% reduction in the absorbance calculated based on the cell viability curves.
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Apoptosis Assay Apoptosis assay was preformed using cell death detection enzyme-linked immunosorbent assay (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s protocol.
Xenograft Animal Models Five ⫻ 106 NCI-H1299 cells were resuspended in 200 l of RPMI 1640 and inoculated into the subcutaneous tissue of the flanks of 25-week-old female Bagg Albino (BALB/c) athymic nude mice (Charles river Laboratories, Wilmington, MA). After tumors developed, romidepsin was injected intraperitoneally into the mice three times at 4 day intervals (1.2 mg/kg body weight). Erlotinib was injected daily 5 days a week (50 mg/kg body weight). The 1 ⫻ phosphate-buffered saline solution was used as a control solution. Tumor sizes were measured and calculated from the following formula: tumor size ⫽ L ⫻ W2/2, where L and W represent the length and the width of the tumor mass respectively.
Statistical Analysis The Loewe additivity model was used to characterize drug interaction between erlotinib and romidepsin. In this model, the combination index (CI) was defined as CI ⫽ d1/Dy,1 ⫹ d2/Dy,2, where d1 and d2 are the doses of drug 1 and drug 2 in mixture, which produces an effect y, Dy,1 and Dy,2 are the doses that produce the same effect y when using alone.20,21 If the CI is equal, less than, or greater than 1, the combination dose (d1, d2) is called as additive, synergistic or antagonistic, respectively. We also used isobologram to illustrate the drug interaction geometrically.20 The Student’s t test was used to determine the potential differences between the control group and the drug treatment groups. p value ⬍0.05 was considered statistically significant.
Copyright © 2009 by the International Association for the Study of Lung Cancer
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RESULTS Romidepsin Decreases the IC50 Values of Erlotinib in NSCLC Cell Lines Nine NSCLC cell lines of varying EGFR and KRAS mutation status were chosen to study, including three cell lines containing wild type EGFR and KRAS, three containing mutant KRAS and three containing mutant EGFR but resistant to TKIs. These NSCLC cell lines also had various histologies including adenocarcinoma, squamous carcinoma and large cell types (Table 1). We first examined the cytotoxicity of romidepsin in these NSCLC cell lines. The IC50 values of romidepsin ranged relatively little from 1.3 to 4.9 ng/ml (Table 1). As we selected NSCLC cell lines resistant to TKI therapy for this study, the IC50 values for erlotinib (8.6 –115 M) in these cell lines were considerably higher than the upper value indicative of clinical sensitivity (2.5 M).22 For the combined treatment, the NSCLC cell lines were treated with varying concentrations (0.01–250 M) of erlotinib either in the absence or presence of 1 ng/ml romidepsin. MTS assays were performed to determine the cell viability and pairs of cell viability curves for each cell line are shown in Figure 1A. We found that in the presence of 1 ng/ml romidepsin, the IC50 values of erlotinib in six of nine NSCLC cell lines examined decreased to less than 2.5 M. The CI has been calculated, and ⬍1 is considered synergism. The CI values ranged from 0.25 to 0.77 in these cell lines (Table 1) indicating that romidepsin and erlotinib showed synergism in all the NSCLC cell lines examined. A dose-normalized IC50 isobologram was also made and shown in Figure 1B, illustrating the synergism geometrically.20
Combination of Romidepsin and Erlotinib Induces Apoptosis in NSCLC Cell Lines We next examined whether inducing apoptosis played a role in the ability of romidepsin to sensitize lung cancer cells to erlotinib. NCI-H1299 and NCI-H23 were treated with either 5 M erlotinib or 1 ng/ml romidepsin, or the combination of these two drugs. After 72 hours, apoptosis was determined using the cell death detection enzyme-linked immunosorbent assay kit (Roche Applied Science). The mono- and oligonucleosomes indicating apoptosis were detected by antibodies against DNA and histones, and quantified by densitometry. As shown in Figure 2, while 5 M of erlotinib and 1 ng/ml of romidepsin did not induce obvious apoptosis, cotreatment of erlotinib and romidepsin caused a 2.3 (p ⫽ 0.01) and 2.85 (p ⫽ 0.002) fold increase in apoptosis in NCI-H1299 and NCI-H23 cells, respectively.
Coadministration of Romidepsin and Erlotinib Shows Inhibition on NCI-H1299 Cell Line Xenografts We further studied the combined effect of romidepsin and erlotinib on tumor growth in vivo. We injected 5 ⫻ 106 NCI-H1299 cells subcutaneously into 20 female BALB/c athymic nude mice. After visible tumors were seen at day 7, the mice were divided into four groups (five mice per group). One group was used as a phosphate-buffered saline treated control. The other three groups were intraperitoneally in-
Synergistic Effects of Erlotinib and Romidepsin
jected with either romidepsin alone, erlotinib alone or the combination of both agents. Tumor size was measured at the indicated days. After 20 days, the mice were killed. As shown in Figure 3, at day 20, although erlotinib and romidepsin treatment alone suppressed NCI-H1299 cell line xenograft growth to 72% (p ⫽ 0.47) and 43% (p ⫽ 0.08) respectively compared with the phosphate-buffered saline treated control group, these were not statistically significant. Only the combined treatment inhibited NCI-H1299 xenograft growth to 28%, causing a significant decrease on tumor growth (p ⫽ 0.04).
DISCUSSION EGFR TKIs such as erlotinib and gefitinib have been found to be efficient in the treatment of NSCLCs that express mutant EGFR.3,4 However, resistance to TKIs develops in some EGFR mutant NSCLCs. Furthermore, the majority of NSCLCs containing wild type EGFR are resistant to EGFR TKIs.22 To solve this problem, several new TKIs that have a broader spectrum of kinase activities have been developed to treat NSCLCs. For example, EXEL-7647 (XL647), a novel spectrum-selective kinase inhibitor with potent activity against the EGF and vascular endothelial growth factor receptor tyrosine kinase families, has shown efficiency in therapy for both wild type and mutant EGFR in vitro and in vivo.23 In addition, combinational therapies have been used to overcome NSCLC resistance to EGFR TKIs. For example, the combined use of erlotinib and the humanized vascular endothelial growth factor receptor monoclonal antibody bevacizumab in advanced, chemotherapy-refractory NSCLCs has shown promising results.24 Our data indicated that the HDAC inhibitor romidepsin decreased the IC50 values of erlotinib in NSCLC cell lines with wild type EGFR, including KRAS mutant cell lines which are considered to be particularly resistant to EGFR TKIs. These cell lines are composed of different histologic types including adenocarcinoma, squamous carcinoma and large cell types. Since most of EGFR mutant cell lines are very sensitive to erlotinib, we examined three which exhibit erlotinib resistance: NCI-H1975 and NCI-H820 cell lines have a secondary T790M mutation, which is responsible for the resistance5; NCI-H1650 cell line has a homozygous deletion of phosphatase and tensin homolog deleted on chromosome 10 and therefore no detectible phosphatase and tensin homolog deleted on chromosome 10 protein level, which is associated with resistance to TKIs in human tumors.25 We found that addition of romidepsin also increased the sensitivity of erlotinib in TKI resistant EGFR mutant NSCLC cell lines. Our data indicated that the HDAC inhibitor romidepsin and TKI erlotinib showed synergy in all nine NSCLC cell lines examined. The underlying mechanisms of this synergistic effect are not known. Besides the secondary T790M mutation, several other mechanisms have been proposed to explain the resistance of NSCLCs to EGFR TKIs. It is reported that epithelial to mesenchymal transition is a determinant of sensitivity of NSCLCs to EGFR inhibition.26 Epithelial to mesenchymal transition is characterized by the combined loss of epithelial cell junction proteins such as E-cadherin and the
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FIGURE 1. Romidepsin synergistically increased the sensitivity of erlotinib in non-small cell lung cancer (NSCLC) cell lines. A, Nine NSCLC cell lines were treated with varying concentrations of erlotinib either in the absence or presence of romidepsin (1 ng/ml) for 72 hours. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and representative pairs of cell viability curves are shown. B, The dose-normalized isobologram for erlotinib and romidepsin with normalization of dose with IC50 to unity on both x and y axis. All the points represent the drug combination that yield 50% cell reduction. If the point falls on the lower left part of the graph, synergism is indicated.
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Synergistic Effects of Erlotinib and Romidepsin
FIGURE 2. Erlotinib and romidepsin induced apoptosis in non-small cell lung cancer (NSCLC) cell lines. NCI-H1299 cell line (A) and NCI-H23 cell line (B) treated with 5 M erlotinib or/and 1 ng/ml romidepsin were analyzed for apoptosis using a cell death detection kit (Roche). Results represent as means ⫾ SD of three independent experiments. *p ⬍ 0.05 versus control group (Student’s t test).
FIGURE 3. Growth inhibition of NCI-H1299 cell line xenografts by erlotinib and romidepsin coadministration. Five ⫻ 106 NCI-H1299 cells were injected subcutaneously into each of twenty female BALB/c athymic nude mice. These mice were divided into four groups at day 7 after tumor development. They were injected with either 1 ⫻ phosphate-buffered saline (PBS), romidepsin alone, erlotinib alone or the combination. Romidepsin was administered 3 times at 4-day intervals (1.2 mg/kg body weight). Erlotinib was administered 5 days a week (50 mg/kg body weight). Tumor sizes were measured at the indicated days. Results are shown as means ⫹ SEM of groups of 5 mice. Statistical significance was determined by Student’s t test (*p ⬍0.05 versus control).
gain of mesenchymal markers such as vimentin or fibronectin.27 Restoring E-cadherin expression has been shown to increase sensitivity to EGFR inhibitors in NSCLCs.13 Persistent activity of MAPK or/and AKT pathway has also been related to EGFR TKI resistance. Treatment of specific inhibitors of MAPK or AKT pathway can increase the sensitivity of NSCLC cell lines to TKIs.6 Romidepsin can regulate expression of many genes. It was reported that romidepsin treatment led to altered expression of cyclin A, cyclin E, and p21, reduced protein levels of ErbB1, ErbB2, and Raf-1.28 Furthermore, romidepsin inhibited ERK1/2 MAPK activities.28 Modification of important players in signal transduction pathways, or other important cellular events by romidepsin may account for the synergy. In our studies, in the presence of romidepsin at the concentration of 1 ng/ml, the IC50 values of erlotinib in six out of nine NSCLC cell lines decreased to ⬍2.5 M, which is considered to be sensitive to erlotinib in vitro,22 and approximately corresponds to the plasma steady-state con-
centration of erlotinib in patients treated with a dose of 150 mg daily.29 The concentration of romidepsin used for our in vitro combination treatment is far below 25 ng/ml that corresponds to 50% of the free drug concentration in plasma from lung cancer patients receiving this drug at the maximum tolerated dose.28 This suggested that the combined treatment of romidepsin and erlotinib will be beneficial in clinical trials. A recent study indicated that although romidepsin exhibited modest clinical efficacy in lung cancer patients at the tested dose and schedule, the biologic effects mediated by romidepsin supported the evaluation of this HDAC inhibitor in combination with other targeted agents in lung cancer patients.18 Our data indicates that combination of erlotinib with romidepsin may benefit NSCLC patients not predicted to respond to TKI therapy.
ACKNOWLEDGMENTS Supported by grants from the Specialized Program of Research Excellence in Lung Cancer (P50CA70907), National Cancer Institute, Bethesda, MD. REFERENCES 1. Olayioye MA, Neve RM, Lane HA, Hynes NE. The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J 2000;19:3159 –3167. 2. Johnston JB, Navaratnam S, Pitz MW, et al. Targeting the EGFR pathway for cancer therapy. Curr Med Chem 2006;13:3483–3492. 3. Haber DA, Bell DW, Sordella R, et al. Molecular targeted therapy of lung cancer: EGFR mutations and response to EGFR inhibitors. Cold Spring Harb Symp Quant Biol 2005;70:419 – 426. 4. Janne PA, Johnson BE. Effect of epidermal growth factor receptor tyrosine kinase domain mutations on the outcome of patients with non-small cell lung cancer treated with epidermal growth factor receptor tyrosine kinase inhibitors. Clin Cancer Res 2006;12:4416s– 4420s. 5. Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2005;2:e73. 6. Janmaat ML, Kruyt FA, Rodriguez JA, Giaccone G. Response to epidermal growth factor receptor inhibitors in non-small cell lung cancer cells: limited antiproliferative effects and absence of apoptosis associated with persistent activity of extracellular signal-regulated kinase or Akt kinase pathways. Clin Cancer Res 2003;9:2316 –2326. 7. Pao W, Wang TY, Riely GJ, et al. KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med 2005;2:e17. 8. Verdone L, Caserta M, Di Mauro E. Role of histone acetylation in the control of gene expression. Biochem Cell Biol 2005;83:344 –353. 9. Kouzarides T. Histone acetylases and deacetylases in cell proliferation. Curr Opin Genet Dev 1999;9:40 – 48.
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10. Glozak MA, Sengupta N, Zhang X, et al. Acetylation and deacetylation of non-histone proteins. Gene 2005;363:15–23. 11. Fouladi M. Histone deacetylase inhibitors in cancer therapy. Cancer Invest 2006;24:521–527. 12. Marks PA, Dokmanovic M. Histone deacetylase inhibitors: discovery and development as anticancer agents. Expert Opin Investig Drugs 2005;14:1497–1511. 13. Witta SE, Gemmill RM, Hirsch FR, et al. Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines. Cancer Res 2006;66:944 –950. 14. Konstantinopoulos PA, Vandoros GP, Papavassiliou AG. FK228 (depsipeptide): a HDAC inhibitor with pleiotropic antitumor activities. Cancer Chemother Pharmacol 2006;58:711–715. 15. Ueda H, Nakajima H, Hori Y, Goto T, Okuhara M. Action of FR901228, a novel antitumor bicyclic depsipeptide produced by Chromobacterium violaceum no. 968, on Ha-ras transformed NIH3T3 cells. Biosci Biotechnol Biochem 1994;58:1579 –1583. 16. Kosugi H, Ito M, Yamamoto Y, et al. In vivo effects of a histone deacetylase inhibitor, FK228, on human acute promyelocytic leukemia in NOD/Shi-scid/scid mice. Jpn J Cancer Res 2001;92:529 –536. 17. Sasakawa Y, Naoe Y, Inoue T, et al. Effects of FK228, a novel histone deacetylase inhibitor, on human lymphoma U-937 cells in vitro and in vivo. Biochem Pharmacol 2002;64:1079 –1090. 18. Schrump DS, Fischette MR, Nguyen DM, et al. Clinical and molecular responses in lung cancer patients receiving Romidepsin. Clin Cancer Res 2008;14:188 –198. 19. Shigematsu H, Lin L, Takahashi T, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 2005;97:339 –346. 20. Chou TC. Theoretical basis, experimental design, and computerized
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simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006;58:621– 681. Lee JJ, Kong M, Ayers GD, Lotan R. Interaction index and different methods for determining drug interaction in combination therapy. J Biopharm Stat 2007;17:461– 480. Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 2007;7:169 –181. Gendreau SB, Ventura R, Keast P, et al. Inhibition of the T790M gatekeeper mutant of the epidermal growth factor receptor by EXEL7647. Clin Cancer Res 2007;13:3713–3723. Byers LA, Heymach JV. Dual targeting of the vascular endothelial growth factor and epidermal growth factor receptor pathways: rationale and clinical applications for non-small-cell lung cancer. Clin Lung Cancer 2007;8(Suppl 2):S79 –S85. Guo A, Villen J, Kornhauser J, et al. Signaling networks assembled by oncogenic EGFR and c-Met. Proc Natl Acad Sci USA 2008;105:692– 697. Thomson S, Buck E, Petti F, et al. Epithelial to mesenchymal transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. Cancer Res 2005;65:9455–9462. Thiery JP. Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol 2003;15:740 –746. Yu X, Guo ZS, Marcu MG, et al. Modulation of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells by depsipeptide FR901228. J Natl Cancer Inst 2002;94:504 –513. Hidalgo M, Siu LL, Nemunaitis J, et al. Phase I and pharmacologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies. J Clin Oncol 2001; 19:3267–3279.
Copyright © 2009 by the International Association for the Study of Lung Cancer
Histone Deacetylase Inhibitor Romidepsin Enhances Anti-Tumor Effect of Erlotinib in Non-small Cell Lung Cancer (NSCLC) Cell Lines Wei Zhang, PhD,* Michael Peyton, PhD,* Yang Xie, PhD,†‡ Junichi Soh, MD,* John D. Minna, MD,*†§储 Adi F. Gazdar, MD,*†¶ and Eugene P. Frenkel, MD†§
Introduction: Most epidermal growth factor receptor (EGFR) mutant non-small cell lung cancers (NSCLCs) are sensitive to EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib or gefitinib, but many EGFR wild type NSCLCs are resistant to TKIs. In this study, we examined the effects of the histone deacetylase inhibitor, romidepsin, in combination with erlotinib, in NSCLC cell lines and xenografts. Methods: For in vitro studies, nine NSCLC cell lines with varying mutation status and histology were treated with erlotinib and romidepsin alone or in combination. 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays were performed to determine the concentration that inhibits 50% (IC50) value of each drug or the combination. For in vivo studies, NCIH1299 xenografts were inoculated subcutaneously into athymic nude mice. Romidepsin and/or erlotinib were injected intraperitoneally after tumors developed and tumor sizes were measured. Results: We found that romidepsin increased the sensitivity of erlotinib synergistically in all nine NSCLC cell lines including EGFR and KRAS wild type cell lines, KRAS mutant cell lines, and TKI resistant EGFR mutant cell lines. This effect was partially due to enhanced apoptosis. Furthermore, cotreatment of erlotinib and romidepsin inhibited NCIH1299 xenograft growth in athymic nude mice. Conclusions: These observations support a role for the combination of a histone deacetylase inhibitor and a TKI in the treatment of NSCLCs. Key Words: Erlotinib, Romidepsin, Non-small cell lung cancer, Epidermal growth factor receptor, KRAS. (J Thorac Oncol. 2009;4: 161–166)
*Hamon Center for Therapeutic Oncology Research, †Harold C. Simmons Comprehensive Cancer Center, ‡Department of Clinical Sciences, §Division of Hematology/Oncology, Department of Internal Medicine, Departments of 储Pharmacology, and ¶Pathology, University of Texas Southwestern Medical Center, Dallas, Texas. Disclosure: The authors declare no conflicts of interest. Address for correspondence: Eugene P. Frenkel, MD, Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75235. E-mail: [email protected] Copyright © 2009 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/09/0402-0161
T
he epidermal growth factor receptor (EGFR) belongs to the EGFR family of tyrosine kinase receptors.1 Upon activation, EGFR can promote cell proliferation and survival through Ras/MEK/MAPK and/or PI3K/AKT signaling pathways.2 In recent years, non-small cell lung cancers (NSCLCs) containing EGFR mutations have been found to be sensitive to EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib (Tarceva) or gefitinib (Iressa), and these drugs have been successfully used in the therapy for such patients.3,4 Unfortunately, some patients with cancers containing EGFR mutations develop resistance to TKIs after a period of time. Although the mechanisms of resistance are not completely understood, the existence of the secondary T790M mutation activity appears to be the major mechanism.5 Furthermore, most of the NSCLCs containing wild type EGFR receptor are resistant to therapy with EGFR TKIs.6 In addition, the presence of KRAS mutations is associated with primary resistance to TKIs.7 Thus, the role of erlotinib and gefitinib for the treatment of NSCLC is limited. Acetylation of histones plays an important role in chromatin organization and gene expression.8 Acetylation is determined by both histone acetyltransferases and histone deacetylases (HDACs).9 HDACs remove the acetyl groups from the lysine residues of the histone tail leading to compaction of chromatin and resultant repression of gene transcription.9 Many nonhistone proteins can be HDAC substrates such as p53, cMyc, Stat3, and Hsp90.10 Such potential targets have provided a new strategy for the role of HDAC inhibitors in cancer therapy. HDAC inhibitors can induce growth arrest, differentiation, and apoptosis in tumor cells.11 Several HDAC inhibitors have been used in clinical trials.12 HDAC inhibitors are also excellent candidates for combination therapies. The HDAC inhibitor MS-275 was shown to increase the sensitivity of gefitinib in NSCLC cell lines.13 Romidepsin (previously termed FK228), also known as depsipeptide, is a natural cyclic peptide HDAC inhibitor. It bears the molecular structure of depsipeptides, namely sequences of alternating amino- and hydroxy-carboxylic acid residues.14 It has shown important inhibition of in vitro growth of several tumor cell types including lung and prostate cancers, lymphomas and leukemias.14 –17 Romidepsin has recently been studied in a clinical trial for treatment of lung cancer.18 In this article, we investigated the effects of combining
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TABLE 1. The IC50 Values of Romidepsin and Erlotinib in NSCLC Cell Lines Mutation Statusa
NCI-H1299 NCI-2882 HCC95 NCI-H23 NCI-H157 NCI-H460 NCI-H1975 NCI-H820 NCI-H1650
Histology
EGFR
KRAS
Romidepsinb IC50 ⴞ SD (ng/ml)
LC AD SQ AD SQ LC AD AD AD
wt wt wt wt wt wt mut mut mut
wt wt wt mut mut mut wt wt wt
4.6 ⫾ 0.2 1.6 ⫾ 0.04 2.5 ⫾ 0.05 2.9 ⫾ 0.2 1.6 ⫾ 0.02 2.1 ⫾ 0.07 1.3 ⫾ 0.04 2.4 ⫾ 0.1 4.9 ⫾ 0.3
Erlotinib IC50 ⴞ SD (M)b No Romidepsin
Romidepsin (1 ng/ml)
CIc
90.0 ⫾ 2.2 115.0 ⫾ 3.7 107.0 ⫾ 2.2 52.8 ⫾ 4.7 75.0 ⫾ 3.0 105.3 ⫾ 4.4 14.4 ⫾ 0.1 8.6 ⫾ 0.6 28.3 ⫾ 1.4
2.6 ⫾ 0.01 1.9 ⫾ 0.4 5.7 ⫾ 0.9 0.07 ⫾ 0.01 0.3 ⫾ 0.03 1.7 ⫾ 0.2 0.01 ⫾ 0.02 1.8 ⫾ 0.02 4.8 ⫾ 0.8
0.25 0.65 0.45 0.34 0.63 0.50 0.77 0.63 0.37
a
The mutation status of NSCLC cell lines were determined as described in Materials and Methods. The IC50 values of erlotinib and romidepsin were defined as the concentration needed for a 50% reduction in the absorbance calculated based on the cell viability curves. CI combination index. Values ⬍1 are interpreted as synergism. (see Materials and Methods). EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer; LC, large cell; AD, adenocarcinoma; SQ, squamous carcinoma; wt, wild type; mut, mutant; IC50, concentration that inhibits 50%. b c
romidepsin and erlotinib on the sensitivity of EGFR wild type and mutant NSCLC cell lines resistant to TKI therapy.
MATERIALS AND METHODS Cell Lines and Drugs Nine NSCLC cell lines (Table 1) were cultured in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 5% fetal bovine serum and incubated in humidified air and 5% CO2 at 37°C. The cell lines were established by us, DNA fingerprinted, and free of mycoplasma by molecular tests (iNtRON Biotechnology, Korea). Romidepsin was provided by Fujisawa Pharmaceutical Co. (Japan) (This drug is now the property of Gloucester Co., MA). Erlotinib (Tarceva) (OSI Pharmaceuticals, Inc., NY) was purchased from UT Southwestern pharmacy.
DNA Isolation and Polymerase Chain Reaction DNA was isolated from NSCLC cell lines and polymerase chain reaction was performed to determine the KRAS and EGFR mutation status of each cell line according to previously reported methods.19
MTS Assay Drug sensitivity was measured by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTS) assay (Promega, Madison, WI). Two ⫻ 103 cells were seeded into each well of a 96-well microtiter plate. After 24 hours, the cells were treated with varying concentrations (0.01–250 M) of erlotinib either in the absence or presence of 1 ng/ml romidepsin for 72 hours at 37°C. MTS solution was added to each well, and the plates were incubated for 1 hour at 37°C. Optical density was measured at 490 nm using a SpectraMax 190 spectrophotometer (Molecular Devices, Sunnywale, CA). Each experiment was carried out in eight replicate wells for each drug concentration and repeated twice. The concentration that inhibits 50% (IC50) value was defined as the concentration needed for a 50% reduction in the absorbance calculated based on the cell viability curves.
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Apoptosis Assay Apoptosis assay was preformed using cell death detection enzyme-linked immunosorbent assay (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s protocol.
Xenograft Animal Models Five ⫻ 106 NCI-H1299 cells were resuspended in 200 l of RPMI 1640 and inoculated into the subcutaneous tissue of the flanks of 25-week-old female Bagg Albino (BALB/c) athymic nude mice (Charles river Laboratories, Wilmington, MA). After tumors developed, romidepsin was injected intraperitoneally into the mice three times at 4 day intervals (1.2 mg/kg body weight). Erlotinib was injected daily 5 days a week (50 mg/kg body weight). The 1 ⫻ phosphate-buffered saline solution was used as a control solution. Tumor sizes were measured and calculated from the following formula: tumor size ⫽ L ⫻ W2/2, where L and W represent the length and the width of the tumor mass respectively.
Statistical Analysis The Loewe additivity model was used to characterize drug interaction between erlotinib and romidepsin. In this model, the combination index (CI) was defined as CI ⫽ d1/Dy,1 ⫹ d2/Dy,2, where d1 and d2 are the doses of drug 1 and drug 2 in mixture, which produces an effect y, Dy,1 and Dy,2 are the doses that produce the same effect y when using alone.20,21 If the CI is equal, less than, or greater than 1, the combination dose (d1, d2) is called as additive, synergistic or antagonistic, respectively. We also used isobologram to illustrate the drug interaction geometrically.20 The Student’s t test was used to determine the potential differences between the control group and the drug treatment groups. p value ⬍0.05 was considered statistically significant.
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Journal of Thoracic Oncology • Volume 4, Number 2, February 2009
RESULTS Romidepsin Decreases the IC50 Values of Erlotinib in NSCLC Cell Lines Nine NSCLC cell lines of varying EGFR and KRAS mutation status were chosen to study, including three cell lines containing wild type EGFR and KRAS, three containing mutant KRAS and three containing mutant EGFR but resistant to TKIs. These NSCLC cell lines also had various histologies including adenocarcinoma, squamous carcinoma and large cell types (Table 1). We first examined the cytotoxicity of romidepsin in these NSCLC cell lines. The IC50 values of romidepsin ranged relatively little from 1.3 to 4.9 ng/ml (Table 1). As we selected NSCLC cell lines resistant to TKI therapy for this study, the IC50 values for erlotinib (8.6 –115 M) in these cell lines were considerably higher than the upper value indicative of clinical sensitivity (2.5 M).22 For the combined treatment, the NSCLC cell lines were treated with varying concentrations (0.01–250 M) of erlotinib either in the absence or presence of 1 ng/ml romidepsin. MTS assays were performed to determine the cell viability and pairs of cell viability curves for each cell line are shown in Figure 1A. We found that in the presence of 1 ng/ml romidepsin, the IC50 values of erlotinib in six of nine NSCLC cell lines examined decreased to less than 2.5 M. The CI has been calculated, and ⬍1 is considered synergism. The CI values ranged from 0.25 to 0.77 in these cell lines (Table 1) indicating that romidepsin and erlotinib showed synergism in all the NSCLC cell lines examined. A dose-normalized IC50 isobologram was also made and shown in Figure 1B, illustrating the synergism geometrically.20
Combination of Romidepsin and Erlotinib Induces Apoptosis in NSCLC Cell Lines We next examined whether inducing apoptosis played a role in the ability of romidepsin to sensitize lung cancer cells to erlotinib. NCI-H1299 and NCI-H23 were treated with either 5 M erlotinib or 1 ng/ml romidepsin, or the combination of these two drugs. After 72 hours, apoptosis was determined using the cell death detection enzyme-linked immunosorbent assay kit (Roche Applied Science). The mono- and oligonucleosomes indicating apoptosis were detected by antibodies against DNA and histones, and quantified by densitometry. As shown in Figure 2, while 5 M of erlotinib and 1 ng/ml of romidepsin did not induce obvious apoptosis, cotreatment of erlotinib and romidepsin caused a 2.3 (p ⫽ 0.01) and 2.85 (p ⫽ 0.002) fold increase in apoptosis in NCI-H1299 and NCI-H23 cells, respectively.
Coadministration of Romidepsin and Erlotinib Shows Inhibition on NCI-H1299 Cell Line Xenografts We further studied the combined effect of romidepsin and erlotinib on tumor growth in vivo. We injected 5 ⫻ 106 NCI-H1299 cells subcutaneously into 20 female BALB/c athymic nude mice. After visible tumors were seen at day 7, the mice were divided into four groups (five mice per group). One group was used as a phosphate-buffered saline treated control. The other three groups were intraperitoneally in-
Synergistic Effects of Erlotinib and Romidepsin
jected with either romidepsin alone, erlotinib alone or the combination of both agents. Tumor size was measured at the indicated days. After 20 days, the mice were killed. As shown in Figure 3, at day 20, although erlotinib and romidepsin treatment alone suppressed NCI-H1299 cell line xenograft growth to 72% (p ⫽ 0.47) and 43% (p ⫽ 0.08) respectively compared with the phosphate-buffered saline treated control group, these were not statistically significant. Only the combined treatment inhibited NCI-H1299 xenograft growth to 28%, causing a significant decrease on tumor growth (p ⫽ 0.04).
DISCUSSION EGFR TKIs such as erlotinib and gefitinib have been found to be efficient in the treatment of NSCLCs that express mutant EGFR.3,4 However, resistance to TKIs develops in some EGFR mutant NSCLCs. Furthermore, the majority of NSCLCs containing wild type EGFR are resistant to EGFR TKIs.22 To solve this problem, several new TKIs that have a broader spectrum of kinase activities have been developed to treat NSCLCs. For example, EXEL-7647 (XL647), a novel spectrum-selective kinase inhibitor with potent activity against the EGF and vascular endothelial growth factor receptor tyrosine kinase families, has shown efficiency in therapy for both wild type and mutant EGFR in vitro and in vivo.23 In addition, combinational therapies have been used to overcome NSCLC resistance to EGFR TKIs. For example, the combined use of erlotinib and the humanized vascular endothelial growth factor receptor monoclonal antibody bevacizumab in advanced, chemotherapy-refractory NSCLCs has shown promising results.24 Our data indicated that the HDAC inhibitor romidepsin decreased the IC50 values of erlotinib in NSCLC cell lines with wild type EGFR, including KRAS mutant cell lines which are considered to be particularly resistant to EGFR TKIs. These cell lines are composed of different histologic types including adenocarcinoma, squamous carcinoma and large cell types. Since most of EGFR mutant cell lines are very sensitive to erlotinib, we examined three which exhibit erlotinib resistance: NCI-H1975 and NCI-H820 cell lines have a secondary T790M mutation, which is responsible for the resistance5; NCI-H1650 cell line has a homozygous deletion of phosphatase and tensin homolog deleted on chromosome 10 and therefore no detectible phosphatase and tensin homolog deleted on chromosome 10 protein level, which is associated with resistance to TKIs in human tumors.25 We found that addition of romidepsin also increased the sensitivity of erlotinib in TKI resistant EGFR mutant NSCLC cell lines. Our data indicated that the HDAC inhibitor romidepsin and TKI erlotinib showed synergy in all nine NSCLC cell lines examined. The underlying mechanisms of this synergistic effect are not known. Besides the secondary T790M mutation, several other mechanisms have been proposed to explain the resistance of NSCLCs to EGFR TKIs. It is reported that epithelial to mesenchymal transition is a determinant of sensitivity of NSCLCs to EGFR inhibition.26 Epithelial to mesenchymal transition is characterized by the combined loss of epithelial cell junction proteins such as E-cadherin and the
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FIGURE 1. Romidepsin synergistically increased the sensitivity of erlotinib in non-small cell lung cancer (NSCLC) cell lines. A, Nine NSCLC cell lines were treated with varying concentrations of erlotinib either in the absence or presence of romidepsin (1 ng/ml) for 72 hours. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and representative pairs of cell viability curves are shown. B, The dose-normalized isobologram for erlotinib and romidepsin with normalization of dose with IC50 to unity on both x and y axis. All the points represent the drug combination that yield 50% cell reduction. If the point falls on the lower left part of the graph, synergism is indicated.
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Journal of Thoracic Oncology • Volume 4, Number 2, February 2009
Synergistic Effects of Erlotinib and Romidepsin
FIGURE 2. Erlotinib and romidepsin induced apoptosis in non-small cell lung cancer (NSCLC) cell lines. NCI-H1299 cell line (A) and NCI-H23 cell line (B) treated with 5 M erlotinib or/and 1 ng/ml romidepsin were analyzed for apoptosis using a cell death detection kit (Roche). Results represent as means ⫾ SD of three independent experiments. *p ⬍ 0.05 versus control group (Student’s t test).
FIGURE 3. Growth inhibition of NCI-H1299 cell line xenografts by erlotinib and romidepsin coadministration. Five ⫻ 106 NCI-H1299 cells were injected subcutaneously into each of twenty female BALB/c athymic nude mice. These mice were divided into four groups at day 7 after tumor development. They were injected with either 1 ⫻ phosphate-buffered saline (PBS), romidepsin alone, erlotinib alone or the combination. Romidepsin was administered 3 times at 4-day intervals (1.2 mg/kg body weight). Erlotinib was administered 5 days a week (50 mg/kg body weight). Tumor sizes were measured at the indicated days. Results are shown as means ⫹ SEM of groups of 5 mice. Statistical significance was determined by Student’s t test (*p ⬍0.05 versus control).
gain of mesenchymal markers such as vimentin or fibronectin.27 Restoring E-cadherin expression has been shown to increase sensitivity to EGFR inhibitors in NSCLCs.13 Persistent activity of MAPK or/and AKT pathway has also been related to EGFR TKI resistance. Treatment of specific inhibitors of MAPK or AKT pathway can increase the sensitivity of NSCLC cell lines to TKIs.6 Romidepsin can regulate expression of many genes. It was reported that romidepsin treatment led to altered expression of cyclin A, cyclin E, and p21, reduced protein levels of ErbB1, ErbB2, and Raf-1.28 Furthermore, romidepsin inhibited ERK1/2 MAPK activities.28 Modification of important players in signal transduction pathways, or other important cellular events by romidepsin may account for the synergy. In our studies, in the presence of romidepsin at the concentration of 1 ng/ml, the IC50 values of erlotinib in six out of nine NSCLC cell lines decreased to ⬍2.5 M, which is considered to be sensitive to erlotinib in vitro,22 and approximately corresponds to the plasma steady-state con-
centration of erlotinib in patients treated with a dose of 150 mg daily.29 The concentration of romidepsin used for our in vitro combination treatment is far below 25 ng/ml that corresponds to 50% of the free drug concentration in plasma from lung cancer patients receiving this drug at the maximum tolerated dose.28 This suggested that the combined treatment of romidepsin and erlotinib will be beneficial in clinical trials. A recent study indicated that although romidepsin exhibited modest clinical efficacy in lung cancer patients at the tested dose and schedule, the biologic effects mediated by romidepsin supported the evaluation of this HDAC inhibitor in combination with other targeted agents in lung cancer patients.18 Our data indicates that combination of erlotinib with romidepsin may benefit NSCLC patients not predicted to respond to TKI therapy.
ACKNOWLEDGMENTS Supported by grants from the Specialized Program of Research Excellence in Lung Cancer (P50CA70907), National Cancer Institute, Bethesda, MD. REFERENCES 1. Olayioye MA, Neve RM, Lane HA, Hynes NE. The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J 2000;19:3159 –3167. 2. Johnston JB, Navaratnam S, Pitz MW, et al. Targeting the EGFR pathway for cancer therapy. Curr Med Chem 2006;13:3483–3492. 3. Haber DA, Bell DW, Sordella R, et al. Molecular targeted therapy of lung cancer: EGFR mutations and response to EGFR inhibitors. Cold Spring Harb Symp Quant Biol 2005;70:419 – 426. 4. Janne PA, Johnson BE. Effect of epidermal growth factor receptor tyrosine kinase domain mutations on the outcome of patients with non-small cell lung cancer treated with epidermal growth factor receptor tyrosine kinase inhibitors. Clin Cancer Res 2006;12:4416s– 4420s. 5. Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2005;2:e73. 6. Janmaat ML, Kruyt FA, Rodriguez JA, Giaccone G. Response to epidermal growth factor receptor inhibitors in non-small cell lung cancer cells: limited antiproliferative effects and absence of apoptosis associated with persistent activity of extracellular signal-regulated kinase or Akt kinase pathways. Clin Cancer Res 2003;9:2316 –2326. 7. Pao W, Wang TY, Riely GJ, et al. KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med 2005;2:e17. 8. Verdone L, Caserta M, Di Mauro E. Role of histone acetylation in the control of gene expression. Biochem Cell Biol 2005;83:344 –353. 9. Kouzarides T. Histone acetylases and deacetylases in cell proliferation. Curr Opin Genet Dev 1999;9:40 – 48.
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