Synchronous Alterations of Wnt and Epidermal Growth Factor ...

Imaging, Diagnosis, Prognosis

Synchronous Alterations of Wnt and Epidermal Growth Factor Receptor Signaling Pathways through Aberrant Methylation and Mutation in Non ^ Small Cell Lung Cancer Makoto Suzuki,1 Hisayuki Shigematsu,3 Takahiro Nakajima,1 Rieko Kubo,1 Shinichiro Motohashi,1 Yasuo Sekine,1 Kiyoshi Shibuya,1 Toshihiko Iizasa,1 Kenzo Hiroshima,2 Yukio Nakatani,2 Adi F. Gazdar,3 and Takehiko Fujisawa1

Abstract

Purpose: The Wnt and epidermal growth factor receptor (EGFR) signaling pathways play crucial roles in the pathogenesis of a variety of malignant tumors. Although the details of each cascade are understood, very little is known about their collective effects in non ^ small cell lung cancer (NSCLC). Experimental Design: A total of 238 NSCLC samples were examined for methylation of Wnt antagonists [secreted frizzled-related protein (sFRP)-1, sFRP-2, sFRP-5, Wnt inhibitory factor-1, and Dickkopf-3] and for EGFR and KRAS mutations. Protein expression levels of h-catenin were assayed in 91of the 238 NSCLCs. Results: We found that (a) aberrant methylation of Wnt antagonists is common in NSCLCs; (b) methylation of sFRP-2 is more prevalent in females, nonsmokers, and adenocarcinoma cases; (c) Dickkopf-3 methylation is significantly associated with a poor prognosis in adenocarcinomas; (d) there is a positive correlation between activated EGFR mutation and nuclear accumulation of h-catenin; (e) KRAS mutation and aberrant methylation of Wnt antagonists are positively correlated; and (f) EGFR mutation is significantly associated with a good prognosis in tumors lacking methylated Wnt antagonist genes. Conclusions: These results contribute to a better understanding of the cross-talk between the Wnt and EGFR signaling pathways and help foster development of chemotherapeutic treatments in NSCLCs.

The Wingless-type (Wnt) signaling cascade is a major regulator of embryonic development. Activation of the Wnt signal transduction pathway results in an increase in the cytoplasmic pool of h-catenin, which is subsequently translocated to the cell nucleus where it interacts with members of the T-cell factor/ lymphocyte enhancer factor family. These downstream gene targets include developmental regulators and other genes involved in cell proliferation and cancer progression (1, 2). Aberrant activation of the Wnt signal transduction pathway has

Authors’ Affiliations: Departments of 1Thoracic Surgery and 2Basic Pathology, Graduate School of Medicine, Chiba University, Chiba, Japan and 3Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas,Texas Received 3/12/07; revised 7/5/07; accepted 7/16/07. Grant support: Grant-in Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology of Japan; Smoking Research Foundation; Inohana Foundation, Chiba University; and Chiba Foundation for Health Promotion and Disease Prevention (all to M. Suzuki). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Makoto Suzuki, Department ofThoracic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. Phone: 81-42-222-7171; Fax: 81-43-226-2172; E-mail: smakoto528@ yahoo.co.jp. F 2007 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-0591

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been closely linked to tumorigenesis in a variety of human tumors (3). Two functional classes of extracellular Wnt antagonists have been identified (4). The first class includes the secreted frizzledrelated protein (sFRP) family (sFRP1-sFRP5) and Wnt inhibitory factor-1 (Wif-1). These proteins inhibit Wnt signaling by directly binding to Wnt molecules (4). The second class is the Dickkopf (Dkk) family of proteins (Dkk1-Dkk4) that inhibit Wnt signaling by binding to the LRP5/LRP6 component of the Wnt receptor complex. These antagonists control Wnt signaling in normal conditions. Down-regulation of Wnt antagonists, such as sFRP and Dkk, through aberrant methylation results in anomalous Wnt signaling and has been identified in several human malignancies, including non – small cell lung cancer (NSCLC; refs. 5 – 10). Although aberrant methylation of a Wnt antagonist gene (sFRP-1 or Wif-1) has been reported in lung cancer (6, 8, 11, 12), the methylation status of these genes in NSCLC has not been comprehensively analyzed. In addition, a detailed mechanism for h-catenin transport and accumulation in the nucleus as a late step of Wnt signaling has not yet been elucidated for many types of cancer (13, 14). Dysregulation of the epidermal growth factor receptor (EGFR) signaling pathway has also been implicated in a variety of cancers. NSCLCs bearing an EGFR mutation have generated considerable interest because such mutations are associated with an increased sensitivity to gefitinib, an EGFR inhibitor

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Imaging, Diagnosis, Prognosis

Fig. 1. Representative examples of the methylation-specific PCR assay. The methylated forms of sFRP-2 and Wif-1are illustrated. M, methylated form; U, unmethylated form; MA, marker; P, positive control; N, negative control.

(15, 16). We have also shown the clinical importance of EGFR and KRAS mutations in resected NSCLCs (17, 18). Primary NSCLCs were first examined for alteration of the Wnt signaling pathway, including aberrant methylation of Wnt antagonists and abnormal expression of h-catenin. Next, because these two pathways have critical roles in the pathogenesis of NSCLC through cellular proliferation and transformation, the results of aberrant Wnt signaling were correlated with mutations in EGFR signaling.

Materials and Methods Patients. Surgically resected samples were obtained from 238 unselected patients with NSCLC at the Chiba University Hospital (Chiba, Japan) from 1995 to 2000. This study was approved by the Institutional Review Board and written informed consent was obtained from all participants. All patients received curative intent surgery, but none had received any treatment before resection. Resected samples were immediately frozen and stored at -80jC until used. Each sample

was used for methylation and mutation assays, whereas 91 of the 238 cases were analyzed by immunohistochemistry. DNA extraction and methylation-specific PCR. Genomic DNA was obtained from primary tumors and nonmalignant tissues by digestion with proteinase K (Life Technologies, Inc.) followed by phenol/ chloroform (1:1) extraction (19). The DNA was treated with sodium bisulfite as described previously (20). PCR amplification of sFRP-1, sFRP-2, sFRP-5, Wif-1, and Dkk-3 gene targets was done using bisulfitetreated DNA as the template and specific primer sequences for the methylated and unmethylated forms of the genes (5). DNA methylation patterns in the CpG island of these genes were determined with methylation-specific PCR as reported by Herman et al. (21). Bisulfitetreated universal methylated DNA (Chemicon) was used as a positive control for the methylated alleles. DNAs from lymphocytes (n = 14) of healthy nonsmoking volunteers were used as negative controls for methylation-specific assays. Nine microliters of each PCR product were loaded on 2% agarose gels stained with ethidium bromide. Results were confirmed by repeating the bisulfite treatment and methylation-specific PCR for all samples. Mutation assay. Sequences of the first four exons (18 – 21) of the EGFR tyrosine kinase domains and exon 2 of KRAS were analyzed (17). All PCR products were incubated with exonuclease I and shrimp alkaline phosphatase (Amersham Biosciences Corp.) and sequenced directly using a dye terminator cycle sequencing kit from Applied Biosystems (Perkin-Elmer Corp.). All sequence variants were confirmed by independent PCR amplifications and sequenced in both directions. Immunohistochemistry. Immunostaining was done on 5-Am-thick sections using h-catenin mouse monoclonal antibody (clone 14; BD Transduction Laboratories) at a 1:400 dilution for 12 h. The slides were prepared with antigen retrieval using citrate buffer [10 mmol/L (pH 6.0)] before incubation with primary antibody. Nonimmune serum was used instead of the primary antibody in negative controls. 3,3¶-Diaminobenzidine (Sigma-Aldrich) was used as the chromogen, with hematoxylin as the counterstain. h-Catenin expression was detected at the plasma membrane of normal bronchial epithelial cells, gland cells, and pneumocytes, which served as internal controls. Cytoplasmic and/or nuclear staining was regarded as positive. The positive cells were counted and divided into three categories (65 (122) Smoking Smoker (171) Never (67) Histology Adenocarcinoma (135) Squamous cell carcinoma (87) Large cell carcinoma (13) Adenosquamous carcinoma (3) p-Stage I (85) II, III, IV (153)

Methylation (%)

Mutation (%)

SFRP-1

sFRP-2

sFRP-5

Wif-1

Dkk-3

Any sFRP Any other gene gene

57 (34) 24 (34)

77 (46) 46 (66)*

55 (33) 23 (33)

50 (30) 16 (23)

22 (13) 10 (14)

107 (64) 55 (79)*

115 (68) 58 (83)*

26 (15) 34 (49)*

42 (36) 39 (32)

63 (54) 60 (49)

40 (34) 38 (31)

36 (31) 30 (25)

12 (10) 20 (16)

79 (68) 83 (68)

83 (72) 90 (74)

34 (29) 26 (21)

7 (6) 8 (7)

57 (33) 24 (36)

78 (46) 45 (67)*

53 (31) 25 (37)

52 (30) 14 (21)

23 (13) 9 (13)

109 (64) 53 (79)*

120 (70) 53 (79)

21 (12) 39 (58)*

11 (6) 4 (6)

44 (33) 28 (32) 8 (62) 1 (33)

80 (59)c 32 (37) 8 (62) 3 (100)

43 30 5 0

30 28 7 1

22 7 2 1

100 (74) 60 (69) 10 (77) 3 (100)

56 (41)c 1 (1) 0 (0) 3 (100)

14 1 0 0

22 (26) 59 (39)

43 (51) 80 (52)

31 (36) 47 (31)

59 (69) 114 (75)

26 (31) 34 (22)

2 (2) 13 (8)

(32) (34) (38) (0)

(22) (32) (54) (33)

25 (29) 41 (27)

(16) (8) (15) (33)

12 (14) 20 (13)

96 53 10 3

(71) (61) (77) (100)

56 (66) 106 (69)

EGFR

KRAS

11 (7) 4 (6)

(10)c (1) (0) (0)

*The frequency of the group is significantly higher (P < 0.05) than the other group. cThe frequency of the group is significantly higher (P < 0.05) than the squamous cell carcinoma group.

Clin Cancer Res 2007;13(20) October 15, 2007

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Alterations of Wnt and EGFR Signaling in NSCLC

Fig. 2. Kaplan-Meier survival curves. A, survival of adenocarcinoma patients as a function of Dkk-3 methylation status. The 5-y overall survival rates of methylation (+) and methylation (-) cases were 37.0% and 67.1%, respectively. B, survival of patients with unmethylated Wnt antagonist genes as a function of EGFR mutation status. The 5-y overall survival rates of mutation (+) and mutation (-) cases were 77.9% and 50.0%, respectively.

25-75%, or >75%). In addition, the mean staining intensity in each specimen was categorized as 1, 2, or 3 (weak, moderate, or strong). The total immunostaining score was then divided into low, moderate, and high scores. This scoring method has been widely used to evaluate the results of immunohistochemical staining for h-catenin (10, 22, 23). Statistical analysis. The Fisher’s exact test and Mann-Whitney U test were applied to assess the association between categorical variables. Overall survival curves were calculated with the Kaplan-Meier method and compared by the log-rank test. The Cox proportional hazards regression model was used for multivariate analyses. The overall extent of methylation for the panel of genes was examined by comparing the methylation index (MI) for each case [MI = (total number of methylated genes) / (total number of analyzed genes)] and determining the mean for the different groups. Two MI values were calculated. The first was the MI for all genes examined and the second was the MI for three sFRP genes (sFRP-1, sFRP-2, and sFRP-5). The cases were also divided based on the presence or absence of sFRP gene methylation and the presence or absence of any other gene methylation (9). Statistical significance was defined as a P value of 63/V63)* 0.1 0.98 (0.55-1.76) 0.9 Stage (II, III, IV/I) 75% of tumor cells and strong staining intensity). B, adenocarcinoma with a moderate score (cytoplasmic and/or nuclear immunostaining in >75% of tumor cells and moderate staining intensity). C, adenocarcinoma with a low score (cytoplasmic and/or nuclear immunostaining in