CCND1 Messenger RNA Expression Is Correlated with EGFR Mutation Status in Lung Cancer

original contribution CCND1 Messenger RNA Expression Is Correlated with EGFR Mutation Status in Lung Cancer Hidefumi Sasaki, Katsuhiro Okuda, Katsuhiko Endo, Osamu Kawano, Haruhiro Yukiue, Tomoki Yokoyama, Motoki Yano, Yoshitaka Fujii Abstract BACKGROUND: Epidermal growth factor receptor (EGFR) stimulation markedly increases cyclin D1 protein expression. Recently, it has been reported that cyclin D1 expression was increased in EGFR mutant cell lines; however, the expression status of CCND1 in EGFR mutant lung cancer tissues has not been reported. PATIENTS AND METHODS: We have investigated the CCND1 messenger RNA (mRNA) levels and other clinicopathologic data in 74 lung cancers. The CCND1 mRNA levels were quantified by real-time reverse-transcriptase polymerase chain reaction using LightCycler®. RESULTS: The CCND1/GAPDH mRNA levels were significantly higher in adenocarcinoma (35.125 ± 37.387) than in non-adenocarcinoma (15.2 ± 24.699; P = .0158), and CCND1/GAPDH mRNA levels were not significantly different among smoking status, sex, or pathologic stage. The CCND1/GAPDH mRNA levels were significantly higher in lung cancer with EGFR mutation (39.713 ± 41.265) than in lung cancer without EGFR mutation (21.805 ± 29.152; P = .0338). CCND/GAPDH mRNA expression did not correlate with prognosis of lung cancer. CONCLUSION: Using the LightCycler® real-time reverse-transcriptase polymerase chain reaction assay, CCND1 gene expression might correlate with EGFR mutation in lung cancer. However, further studies are needed to confirm the impact of cyclin D1 for a molecular target of lung cancer. Clinical Lung Cancer, Vol. 8, No. 8, 493-496, 2007

Key words: Cyclin D1, Polymerase chain reaction, Prognostic factors

Introduction

Phase II/III trials have shown partial responses in 8%-12% of unselected patients with progressive NSCLC after chemotherapy,7,8 with especially higher response in never-smokers, women, and those of Asian ethnicity (> 20%).7,9,10 Originally, 2 reports showed that EGFR mutation status at adenosine triphosphate–binding pockets in patients with NSCLC were correlated with the clinicopathologic features related to good response to gefitinib.11,12 It has been reported that EGFR stimulation markedly increased cyclin D1 protein expression.13 Recently, it has been reported that cyclin D1 expression was increased in EGFR mutant cell lines.14 Additionally, flavopiridol might have therapeutic benefit in NSCLCs. Flavopiridol directly inhibits CDK4 and CDK6 and reduces the transcription of cyclin D1.15 The reduction of cells in response to flavopiridol was more evident in EGFR mutant cell lines.14 Because many tumor markers have been sought out in lung carcinoma for screening and diagnostic purposes, we questioned whether CCND1 messenger RNA (mRNA) levels could serve as a marker for patients with lung cancer. In this report, we investigated CCND1 mRNA levels by means of the real-time reverse-transcriptase polymerase chain reaction (PCR; RT-PCR) assay using LightCycler®.16 The findings were compared with the clinicopathologic features and with the EGFR mutation status of lung cancer.

In mammalian cells, the progression of replicating cells through the cell cycle is controlled by the sequential formation, activation, and subsequent inactivation of a series of specific cyclin-dependent kinase (CDK) complexes. There is now abundant evidence demonstrating that disturbances in specific cyclins, CDKs, or the inhibitory proteins play an important role in several types of human cancer, including lung cancer.1-3 Lung cancer is a major cause of death from malignant diseases as a result of its high incidence, malignant behavior, and lack of major advancements in treatment strategy.4 The epidermal growth factor receptor (EGFR) autocrine loop is important in regulating epithelial carcinogenesis.5,6 The EGFR tyrosine kinase inhibitor gefitinib has been approved in Japan for the treatment of non–small-cell lung cancer (NSCLC) since 2002. Department of Surgery II, Nagoya City University Medical School, Nagoya, Japan Submitted: May 18, 2007; Revised: Jul 17, 2007; Accepted: Aug 13, 2007 Address for correspondence: Hidefumi Sasaki, MD, PhD, Department of Surgery II, Nagoya City University Medical School, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan Fax: 81-52-853-6440; e-mail: [email protected]

Electronic forwarding or copying is a violation of US and International Copyright Laws. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by CIG Media Group, LP, ISSN #1525-7304, provided the appropriate fee is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA 978-750-8400.

Clinical Lung Cancer September 2007

493

CCND1 mRNA and EGFR Mutation in Lung Cancer Table 1 Clinicopathologic Data of 74 Patients with Lung Cancer CCND1 Characteristic Total

No. of CCND1/GAPDH Patients (%) mRNA Levels 74

12.7 ± 20.495

P Value .7181 r = 0.02†

Disease Stage I

41 (55.4 %)

32.55 ± 37.131

II

4 (5.4%)

23.194 ± 25.299

29 (39.2%)

21.86 ± 31.547

Non-smoker

27 (30.6%)

37.993 ± 38.317

Smoker

47 (69.4%)

22.031 ± 30.077

Adenocarcinoma

47 (69.4%)

35.125 ± 37.387

Others

27 (30.6%)

15.2 ± 24.699

Positive

25 (33.8%)

39.713 ± 41.265

Negative

49 (66.2%)

21.805 ± 29.152

≤ 60

26 (35.1%)

33.048 ± 41.282

> 60

48 (64.9%)

25.042 ± 30.346

Male

50 (67.5%)

25.037 ± 35.132

Female

27 (36.4%)

33.725 ± 33.145

III/IV

NS

Smoking Status .0549

Pathologic Subtype .0158

EGFR Mutation Status .0338

Age (Years)* .3444

Sex .3141

*The

mean age for all patients was 63.8 years ± 9 years. with CCND1 levels for all 74 patients. Abbreviation: NS = not significant †Correlation

Patients and Methods The study group included 74 patients with lung cancer who had undergone surgery at the Department of Surgery II, Nagoya City University Medical School, Japan, between 1997 and 2002. The lung tumors were classified according to the general rule for clinical and pathologic record of lung cancer in Japan. The institutional review board of Nagoya City University Medical School approved the research. All tumor samples were immediately frozen and stored at –80°C until assayed. The clinical and pathologic characteristics of the 74 patients with lung cancer are shown in Table 1; these include 41 cases of stage I disease, 4 at stage II, and 28 at stage III/IV. The mean age was 63.8 years (range, 42-88 years). Among the patients with lung cancer, 47 (63.5%) were diagnosed as having adenocarcinoma, 50 (67.5%) were men, and 27 (36.4%) were never-smokers. Reverse-Transcriptase Polymerase Chain Reaction Assay for CCND1 Total RNA was extracted from lung cancer tissues and adjacent non-malignant lung tissues using the Isogen® kit according to the manufacturers’ instructions. Total RNA was also extracted from the lung cancer cell line, CCL185 (A549, adeno-

494

Clinical Lung Cancer September 2007

carcinoma cell line). This RNA was used as a positive control. RNA concentration was determined by spectrophotometer and adjusted to a concentration of 200 ng/mL. RNA (1 μg) was reverse transcribed by the Superscript II enzyme with 0.5 μg oligo (dT)12-16. The reaction mixture was incubated at 42°C for 50 minutes and then at 72°C for 15 minutes. To ensure the fidelity of mRNA extraction and reverse transcription, all samples were subjected to PCR amplification with oligonucleotide primers specific for the constitutively expressed gene glyceraldehyde3-phosphate dehydrogenase (GAPDH) and normalized. The quantitative PCR reactions were performed using LightCycler® FastStart Master Sybr Green I kit. RT-PCR assay for the cyclin D1 gene was performed using the Human Cyclin D1 kit. The gene expression levels were stated as the relative intensity of the fluorescent dye to the A549 cell line. Epidermal growth factor receptor mutation status in patients with lung cancer was evaluated from genomic DNA sequence11 or direct sequence.18 These data are already published elsewhere.11,18,19

Statistical Methods Statistical analyses were done using the Mann-Whitney U test for unpaired samples and the Wilcoxon signed-rank test for paired samples. Linear relationships between variables were determined by means of simple linear regression. Correlation coefficients were determined by rank correlation using Spearman’s test. Differences among the means of the stage and pathologic subtypes in the patients were examined using the Kruskal-Wallis test and Fisher’s PLSD test. The overall survival of patients with lung cancer was examined by the Kaplan-Meier method, and differences were examined using the log-rank test, Breslow-Gehan-Wilcoxon test, and Cox proportional hazard regression model. All analyses were done using the StatView® software package and were considered significant when the P value was < .05.

Results CCND1 Expression in Lung Cancer From the 74 lung cancer tissues, the mean value for CCND1 mRNA level was standardized by the mRNA level of GAPDH in lung cancer tissues (mean ± standard deviation: 27.855 ± 34.516) not related to age. No significant difference in CCND1/GAPDH mRNA level was found between sexes (male: 25.037 ± 35.132; female: 33.725 ± 33.145; P = .3141). There was a tendency toward a higher CCND1/GAPDH mRNA level in non-smokers (37.993 ± 38.317) than in smokers (22.031 ± 30.077; P = .0549). The CCND1/GAPDH mRNA levels from 74 lung cancers were as follows: stage I, 32.55 ± 37.131; stage II, 23.194 ± 25.299; and stage III/IV, 21.86 ± 31.547. The CCND1/GAPDH level in stage I lung cancer was higher than the level in stage II or III/IV lung cancers; however, the levels were not significantly different. The CCND1/GAPDH mRNA levels in pathologic subtypes were also evaluated. CCND1/ GAPDH mRNA level in adenocarcinoma (35.125 ± 37.387) was significantly higher than the level in non-adenocarcinoma lung cancers (15.2 ± 24.699; P = .0158; Table 1). The overall survival (terminated December 31, 2006) of 74 patients with

Hidefumi Sasaki et al Figure 1 CCND1/GAPDH mRNA Level and EGFR Mutations in Lung Cancer 180

P = .0338

Cyclin D1/GAPDH mRNA levels

160 140 120 100 80 60 40 20 0 EGFR Mutant

Wild-Type

EGFR mutant lung cancer had significantly higher CCND1 mRNA levels (mean ± standard deviation: 39.713 ± 41.265) than EGFR wild-type lung cancer (21.805 ± 29.152; P = .0338).

lung cancer was studied in reference to the CCND1/GAPDH mRNA levels. The prognosis between the group with high CCND1/GAPDH levels (> 15.111, median levels; n = 37; 13 were dead) and the group with low CCND1/GAPDH levels (< 15.111; n = 37; 13 were dead) was not significantly different (log-rank test, P = .9714; Breslow-Gehan-Wilcoxon test, P = .7736). Using multi-variate analysis, pathologic stage (P < .0001) was the only prognostic factor of lung cancers.

Relationship Between CCND1 Expression and EGFR Mutations Epidermal growth factor receptor mutation status was also evaluated.18,19 Of 74 lung cancers studied, 25 had EGFR mutations. Epidermal growth factor receptor mutation status was correlated with sex (male, 9 of 50 vs. female, 16 of 24; P < .0001), pathologic subtype (adenocarcinoma, 23 of 47 vs. non-adenocarcinoma, 2 of 27; P = .0003), and smoking status (smokers, 8 of 47 vs. non-smokers, 17 of 27; P < .0001). Epidermal growth factor receptor mutant lung cancers had significantly higher CCND1 mRNA levels (39.713 ± 41.265) than EGFR wild-type lung cancers (21.805 ± 29.152; P = .0338; Figure 1).

STAT3 promotes uncontrolled growth and survival through dysregulation of gene expression, including CCND1.24 In addition, the Ras/MAP/ERK kinase/ERK-dependent pathway is also implicated in the expression of the CCND1 gene.25,26 Previous study has also shown that downregulation of the expression and function of CDKs and cyclin D1 were candidates for the cell cycle regulator that was arrested by gefitinib treatment.27 Repression of cyclin D1 protein has been reported as a surrogate marker of response in aerodigestive tract cancers to erlotinib treatment.28 Rearrangement, amplification, and/or increased expression of the CCND1 gene and overexpression of its mRNA have been reported in several types of human cancers, including lung cancer.2,29,30 However, CCND1 expression was not associated with disease stage32 or histologic differentiation of lung cancers.33 Cyclin D1 was altered at a similar frequency in resectable and advanced lung tumors.32 In addition, alterations of other cell cycle regulators were also less frequent in advanced NSCLC than in resectable tumors.

Conclusion Our report shows that EGFR mutant lung cancer has significantly higher CCND1 mRNA levels. Therefore, the identification of critical effectors of mutant EGFR, such as cyclin D1, should have immediate clinical implications. The functional analyses were done for the mechanism to gefitinib sensitivity. The gefitinib-resistant H1975 cells remained sensitive to flavopiridol.14 Active inhibitors of the cyclin D1–CDK4/6 axis,30 alone or in combination with EGFR inhibitors, should be tested as a strategy to overcome or prevent resistance to EGFR inhibitors in NSCLCs. Using the LightCycler® RT-PCR assay described herein, the determination of CCND1 mRNA levels might correlate with EGFR mutation. Further studies and a longer follow-up time are needed to confirm the impact of cyclin D1 in the biologic behavior of EGFR mutant tumors.

Acknowledgements The authors thank Mrs. Yuri Yamamoto for her excellent technical assistance. This work was supported by Grants-in-Aid for Scientific Research, Japan Society for the Promotion of Science (No. 19390367, 18390381, and 18659407).

References Discussion We obtained findings that CCND1 mRNA expression levels were not significantly different with regard to sex, smoking status, or pathologic stage of lung cancer. In our analysis, higher CCND1 mRNA levels were significantly correlated with EGFR mutation status of lung cancers. Using chromatin immunoprecipitation assays, the nuclear EGFR was associated with the promoter region of cyclin D1.20 Amplification of the CCND1 gene occurred frequently in conjunction with amplification of the EGFR gene in NSCLC.21 It has been reported that mutant EGFR preferentially activated the AKT and STAT pathways compared with wild-type EGFR.22,23

1. Draetta GF. Mammalian G1 cyclins. Curr Opin Cell Biol 1994; 6:842-846. 2. Hunter T, Pines J. Cyclins and cancer. II: cyclin D and CDK inhibitors come of age. Cell 1994; 79:573-582. 3. Sherr CJ. G1 phase progression: cycling on cue. Cell 1994; 79:551-555. 4. Ginsberg RJ, Kris K, Armstrong G. Cancer of the lung. In: Principles and Practice of Oncology. 4th ed. Philadelphia, PA: Lippincott; 1993:673-682. 5. Herbst RS, Bunn PA. Targeting the epidermal growth factor receptor in non-small cell lung cancer. Clin Cancer Res 2003; 9:5813-5824. 6. Mendelsohn J, Baselga J. Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol 2003; 21:2787-2799. 7. Kris MG, Natale RB, Herbst RS, et al. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non small cell lung cancer: randomized trial. JAMA 2003; 290:2149-2158. 8. Thatcher N, Chang A, Purvish P, et al. Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: result from a randomized, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet 2005; 366:1527-1537. 9. Fukuoka M, Yano S, Giaccone G, et al. Multi-institutional randomized phase II

Clinical Lung Cancer September 2007

495

CCND1 mRNA and EGFR Mutation in Lung Cancer 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

496

trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer. J Clin Oncol 2003; 21:2237-2246. Miller VA, Kris MG, Shah N, et al. Bronchioloalveolar pathologic subtype and smoking history predict sensitivity to gefitinib in advanced non-small-cell lung cancer. J Clin Oncol 2004; 22:1103-1109. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004; 304:1497-1500. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Eng J Med 2004; 350:2129-2139. Lonardo F, Dragnev KH, Freemantle SJ, et al. Evidence for the epidermal growth factor receptor as a target for lung cancer prevention. Clin Cancer Res 2002; 8:54-60. Kobayshi 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; 66:11389-11398. Shapiro GI. Preclinical and clinical development of the cyclin-dependent kinase inhibitor flavopiridol. Clin Cancer Res 2004; 10:4270-4275. Wittwer T, Ririe M, Andrew V, et al. The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques 1997; 22:176-181. Japan Lung Cancer Society, General Rule for Clinical and Pathological Record of Lung Cancer. 5th ed. The Japan Lung Cancer Society 1999; 5:1-177. Sasaki H, Shimizu S, Endo K, et al. EGFR and erbB2 mutation status in Japanese lung cancer patients. Int J Cancer 2005; 118:180-184. Sasaki H, Endo K, Konishi A, et al. EGFR mutation status in Japanese lung cancer patients: genotyping analysis using LightCycler. Clin Cancer Res 2005; 11:29242929. Lin SY, Makino K, Xia W, et al. Nuclear localization of EGF receptor and its potential new role as a transcription factor. Natl Cell Biol 2001; 3:802-808.

Clinical Lung Cancer September 2007

21. Reissmann PT, Koga H, Figlin RA, et al. Amplification and overexpression of the cyclin D1 and epidermal growth factor genes in non-small-cell lung cancer. Lung Cancer Study Group. J Cancer Res Clin Oncol 1999; 125:61-70. 22. Sordella R, Bell DW, Haber DA, et al. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 2004; 305:1163-1167. 23. Amann J, Kalyankrishna S, Massion PP, et al. Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancer. Cancer Res 2005; 65:226-235. 24. Turkson J. STAT proteins as novel targets for cancer drug discovery. Expert Opin Ther Targets 2004; 8:409-422. 25. Stacey DW. Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells. Curr Opin Cell Biol 2003; 15:158-163. 26. Naoki K, Chen TH, Richards WG, et al. Missense mutations of the BRAF gene in human lung adenocarcinoma. Cancer Res 2002; 62:7001-7003. 27. Chang GC, Hsu SL, Tsai JR, et al. Molecular mechanisms of ZD 1839-induced G1-cell cycle arrest and apoptosis in human lung adenocarcinoma A549 cells. Biochem Pharmacol 2004; 68:1453-1464. 28. Petty WJ, Dragnev KH, Memoli VA, et al. Epidermal growth factor receptor tyrosine kinase inhibition represses cyclin D1 in aerodigestive tract cancers. Clin Cancer Res 2004; 10:7547-7554. 29. Motokura T, Amold A. Cyclin D and oncogenesis. Curr Opin Genet Dev 1993; 3:5-10. 30. Sherr CJ. Mammalian G1 cyclins. Cell 1993; 73:1059-1065. 31. Gugger M, Kappeler A, Vonlanthen S, et al. Alterations of cell cycle regulators are less frequent in advanced non-small cell cancer than in resectable tumors. Lung Cancer 2001; 33:229-239. 32. Lingfei K, Pingzhang Y, Zhengguo L, et al. A study on p16, pRb, cdk4 and cyclinD1 expression in non-small cell lung cancers. Cancer Lett 1998; 130:93-101.