Advances in Brief Correlation between Insulin-like Growth Factor-binding Protein-3 Promoter Methylation and Prognosis of Patients with Stage I Non-Small Cell Lung Cancer

Purpose: The activities of insulin-like growth factors (IGFs) in regulating cell proliferation, differentiation, and apoptosis are modulated by a family of high-affinity specific IGF-binding proteins (IGFBPs), especially IGFBP-3, the most abundant IGFBP in circulation. Hypermethylation of the promoter represses the expression of the IGFBP-3 gene. The purpose of this study was to determine whether the methylation status of IGFBP-3 promoter influences the prognosis of non-small cell lung cancer (NSCLC). Experimental Design: Eighty-three patients with pathological stage I NSCLC who had undergone curative surgery were investigated for promoter hypermethylation of IGFBP-3 by methylation-specific PCR. Statistical analyses, all two-sided, were performed to determine the prognostic effect of methylation status of the IGFBP-3 promoter on various clinical parameters. IGFBP-3 was the only molecular parameter tested on these tissues in this study. Results: Hypermethylation of the IGFBP-3 promoter was found in 51 (61.5%) of the 83 tumors. The clinicopathological factors, such as age, histological type, histological grade, gender, and smoking status, of corresponding patients, did not exhibit statistically significant association with the methylation status of IGFBP-3 promoter. However, patients with a hypermethylated IGFBP-3 promoter had a significantly lower 5-year disease-specific, disease-free, and overall survival rate than did those without a methylated IGFBP-3 promoter (53.1% versus 86.1%, P 0.006; 36.5% versus 76.2%, P 0.007; and 38.9% versus 64.0%, P 0.022, respectively). Moreover, multivariate analysis indicated that hypermethylation of the IGFBP-3 promoter was the only independent predictor for disease-free and diseasespecific survival among the clinical and histological parameters tested. Conclusions: Hypermethylation of the IGFBP-3 promoter, as measured by methylation-specific PCR, is a frequent phenomenon and strongly associated with poor prognosis among patients with stage I NSCLC. Introduction NSCLC accounts for 75–80% of lung cancer cases, and its dismal survival rate has improved only marginally in the past 2 decades. Surgical resection is the treatment of choice for patients with early-stage disease. Patients with advanced disease are treated with surgery, chemotherapy, radiation therapy, or combinations of the above, all of which have substantial side effects. Despite these treatments, the 5-year survival rate of patients with NSCLC remains low (1), illustrating the need for more effective strategies for early diagnosis and chemoprevention. To identify useful biomarkers for early detection and chemoprevention strategies in lung cancer, it is important to understand the genetic abnormalities that occur during lung tumorigenesis. Tumorigenesis is a multistep process that results from the accumulation and interplay of genetic and epigenetic changes (2). Overexpression of dominant oncogenes and inactivation of tumor-suppressor genes by genetic changes, such as mutations in p53, occur frequently in lung cancer patients and generally are poor prognostic factors for this disease. Epigenetic changes, including aberrant methylation of normally unmethylated CpG islands in the promoter region, are also implicated in lung tumorigenesis (reviewed in Ref. 3). Aberrant methylation of CpG islands in the promoter region, the covalent addition of a methyl group to the 5 position of cytosine, has been associated with transcriptional inactivation of gene expression by alterations of chromatin structure that directly prevent the binding of transcription factors (4). An increasing number of genes have been found to play important roles in cell-cycle control, DNA damage repair, the inhibition of apoptosis, the invasion of tumors, and growth factor response (3, 5). Thus, methylation of DNA involved in these pathways may have a critical role in lung tumor progression (5–7). Received 5/31/02; revised 7/18/02; accepted 7/28/02. 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. 1 Supported in part by M. D. Anderson Cancer Center Institutional Grant RP33763 (to H-Y. L.), National Cancer Institute Grant U19 CA 68437 (to W. K. H.), and the Tobacco Research Fund from the State of Texas (to M. D. Anderson Cancer Center). W. K. H. is an American Cancer Society Clinical Research Professor. 2 To whom requests for reprints should be addressed, at Box 432, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030. Phone: (713) 792-6363; Fax: (713) 796-8655; E-mail: [email protected]. 3 The abbreviations used are: NSCLC, non-small cell lung cancer; IGFBP-3, insulin-like growth factor-binding protein 3; MSP, methylationspecific PCR; DAP, death-associated protein; CI, confidence interval. 3669 Vol. 8, 3669–3675, December 2002 Clinical Cancer Research Research. on April 15, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from IGFBP-3, the most abundant IGFBP in circulation, regulates the role of IGFs in cellular growth, differentiation, transformation, and apoptosis (8–11). The finding of a negative correlation between serum IGFBP-3 levels and lung cancer risk (12) suggests that IGFBP-3 protects against the effects of systemic IGF-I. IGFBP-3 also has IGF-I-independent antiproliferative and proapoptotic effects, as shown by the finding that IGFBP-3 overexpression inhibits the growth of fibroblasts that are IGF-1 receptor (IGF-1R) null (13, 14). Overexpression of IGFBP-3 has significant growth-inhibitory effects on NSCLC in vitro and in vivo, indicating the potential of IGFBP-3 as a tumor suppressor in lung cancer (15). Expression of IGFBP-3 is induced by other growth-inhibitory (and apoptosis-inducing) agents, such as tumor growth factor1 (16), tumor necrosis factor(17), retinoic acid (18), anti-estrogen ICI 182780 (19), vitamin D and its analogues EB1089 and CB1093 (20, 21), and transcription factor p53 (22), raising the possibility that these agents mediate their cellular effects through IGFBP-3. It has been shown that the expression of IGFBP-3 was repressed by the polymorphism or hypermethylation in the promoter region of the gene (23, 24). In the present study, we sought to elucidate whether hypermethylation of the IGFBP-3 promoter is associated with the prognosis of patients. We analyzed surgically resected primary tumor specimens from 83 patients with pathological stage I NSCLC for the methylation status of CpG sites in the 5 end of the IGFBP-3 gene. Other molecular parameters have been tested in previous study using the same tissues (e.g., the expression of PTEN, cyclooxygenase-2, cyclin B1, RAR , Fhit, and Ecadherin and type IV collagenase, the hypermethylation of the DAP kinase promoter, and microsatellite alteration profiles; Refs. 25–32). In this study, we have tested IGFBP-3 as a molecular parameter specifically. In addition, statistical analysis was performed to determine the prognostic effect of the hypermethylation status of IGFBP-3 on various clinical parameters. Materials and Methods Clinical Samples. Formalin-fixed, paraffin-embedded tissue blocks of lung cancer were obtained from surgical specimens from 83 patients who were diagnosed with pathological stage I NSCLC and who had undergone curative surgical removal of a primary lesion at The University of Texas M. D. Anderson Cancer Center from 1975 through 1998. Tissue sections (4 m thick) were obtained from each block, stained with H&E, and reviewed by a pathologist to confirm the diagnosis and the presence or absence of tumor cells in these sections. All of the information, including clinical, pathological, and follow-up data, was based on reports from our tumor registry service. The study was reviewed and approved by the institution’s surveillance committee to allow us to obtain tissue blocks and all pertinent information. Microdissection and DNA Extraction. DNA was extracted from microdissected tumor specimens as described previously (33, 34). Briefly, tumor parts in sections from formalin-fixed and paraffin-embedded tissue blocks were dissected under a stereomicroscope. Dissected tissues were digested in 200 l of digestion buffer containing 50 mM Tris-HCl (pH 8.0), 1% SDS, and proteinase K (0.5 mg/ml) at 42°C for 36 h. The purification of digested products was performed by phenol/chloroform extraction. DNA was then precipitated by the ethanol precipitation method in the presence of glycogen (Boehringer Mannheim Biochemicals, Indianapolis, IN) and recovered in distillated water. Bisulfite Modification and MSP. The entire DNA from the microdissected tumor samples was mixed with 1 g of salmon sperm DNA (Life Technologies, Inc., Gaithersburg, MD) and submitted for chemical modification as described by Herman et al. (35). Briefly, DNA was denatured with 2 M NaOH, followed by treatment with 10 mM hydroquinone and 3 M sodium bisulfite (Sigma Chemical Co., St. Louis, MO). After it was purified in a Wizard SV Plus kit column (Promega, Madison, WI), the DNA was treated with 3 M NaOH and precipitated with three volumes of 100% ethanol, and a onethird volume of 10 M NH4OAc at room temperature over 30 min. The precipitated DNA was washed with 70% ethanol and dissolved in 20 l of distilled water. PCR was conducted with primers specific for either the methylated or the unmethylated sequence of the IGFBP-3 promoter. Methylated primers consisted of BP-3 sense 5 -CGAAGTACGGGTTTCGTAGTCG-3 and BP-3 antisense 5 CGACCCGAACGCGCCGACC-3 ; unmethylated primers consisted of BP-3 sense 5 TTGGTTGTTTAGGGTGAAGTATGGGT-3 and BP-3 antisense 5 -CACCCAACCACAATACTCACATC-3 . The 25l total-reaction volume contained 2 l of modified DNA, 1% DMSO, all four deoxynucleoside triphosphates (each at 200 M), 1.5 mM MgCl2, 0.4 M PCR primers, and 0.25 units of HotStar TaqDNA polymerase (Qiagen, Valencia, CA). Negative control samples without DNA were included for each set of PCR. For methylated PCR, DNA was amplified by an initial cycle at 95°C for 15 min as required for enzyme activation, followed by 40 cycles of 94°C for 30 s, 66°C for 1 min, and 72°C for 1 min and ending with a 5-min extension at 72°C for 1 min in a thermocycler (Applied Biosystems, Foster City, CA). For unmethylated PCR, the annealing temperature was 64°C. PCR products were separated on 2.5% agarose gels and visualized after staining with ethidium bromide. Statistical Analysis. In univariate analysis, independent sample t and 2 tests were used to analyze continuous and categorical variables, respectively. Survival probability as a function of time was computed by the Kaplan-Meier estimator. The log-rank test was used to compare patient survival time between groups. Overall survival, disease-specific survival (from the date of diagnosis to date of death specifically from cancer-related causes), and disease-free survival time (from the date of completion of surgery to the date of relapse or death of cancer-related causes) were analyzed. Cox regression was used to model the influence of promoter hypermethylation on survival time, with adjustment for clinical and histopathological parameters (age, sex, tumor histology subgroup, and smoking status). The two-sided test was used to test equal proportion between groups in two-way contingency tables. All of the statistical tests were two-sided. P 0.05 was considered to be statistically significant.