|Year : 2018 | Volume
| Issue : 8 | Page : 65-71
Death-associated protein kinase promoter methylation correlates with clinicopathological and prognostic features in nonsmall cell lung cancer patients: A cohort study
Xiao-Yu Yang1, Jun Zhang1, Xiao-Ling Yu1, Guo-Feng Zheng1, Fei Zhao1, Xiao-Jing Jia2
1 Department of Pathology, Xinxiang Medical University, Xinxiang 453003, P.R, China
2 Department of Radiation Therapy, The Second Hospital of Jilin University, Changchun 130000, Jilin, P.R. China
|Date of Web Publication||26-Mar-2018|
Department of Radiation Therapy, The Second Hospital of Jilin University, No. 218 Ziqiang Road, Nanguan District, Changchun 130041, Jilin
Source of Support: None, Conflict of Interest: None
Objective: The objective was to study the correlation between death-associated protein kinase (DAPK) promoter methylation and the clinicopathological and prognostic features in nonsmall cell lung cancer (NSCLC) patients.
Materials and Methods: A total of 117 NSCLC patients were recruited into our study between December 2012 and December 2014. Methylation-specific polymerase chain reaction was employed to detect the methylation status of DAPK in cancer tissues, peficancerous tissues, and serum samples of 117 NSCLC patients. In addition, serum samples of 115 healthy subjects were analyzed as controls. A literature search of English and Chinese databases, based on predefined criteria, identified published studies closely related to this study. Data were extracted, and meta-analysis was performed using STATA 12.0 software (STATA Corporation, College Station, TX, USA).
Results: Our study results showed that DAPK promoter methylation frequency was significantly higher in NSCLC tissues compared to peficancerous normal tissues (58.1% vs. 12.8%, χ2 = 52.45, P < 0.001). When serum samples were compared, DAPK methylation frequency in NSCLC patients was higher than the control group (27.4% vs. 0, χ2 = 37.07, P < 0.001). Our meta-analysis results demonstrated that DAPK methylation frequency was lower in tumor node metastasis (TNM) stage I-II compared to TNM stage III-IV (relative risk [RR] =0.87, 95% confidence interval [CI] =0.76–0.99, P = 0.041). DAPK promoter methylation frequency in NSCLC patients with lymph node metastasis was significantly higher compared to the patients with no metastases (RR = 1.26, 95% CI = 1.04–1.52, P = 0.020). Finally, the 5-year survival rate was lower in NSCLC patient group with high frequency of DAPK methylation, compared to the patient group with unmethylated DAPK (RR = 0.71, 95% CI = 0.56–0.89, P = 0.004).
Conclusion: Our results showed that DAPK promoter methylation is tightly correlated with clinicopathological features of NSCLC and is associated with poor prognosis in patients.
Keywords: 5-year survival rate, clinicopathological features, cohort study, death-associated protein kinase, methylation, nonsmall cell lung cancer, prognosis
|How to cite this article:|
Yang XY, Zhang J, Yu XL, Zheng GF, Zhao F, Jia XJ. Death-associated protein kinase promoter methylation correlates with clinicopathological and prognostic features in nonsmall cell lung cancer patients: A cohort study. J Can Res Ther 2018;14, Suppl S1:65-71
|How to cite this URL:|
Yang XY, Zhang J, Yu XL, Zheng GF, Zhao F, Jia XJ. Death-associated protein kinase promoter methylation correlates with clinicopathological and prognostic features in nonsmall cell lung cancer patients: A cohort study. J Can Res Ther [serial online] 2018 [cited 2019 Jun 19];14:65-71. Available from: http://www.cancerjournal.net/text.asp?2018/14/8/65/158197
| > Introduction|| |
Nonsmall cell lung cancer (NSCLC) is one of the most common malignancies and remains the leading cause of cancer-related mortality worldwide. NSCLC is the major histological subtype among lung cancers, accounting for 80–85% of all lung neoplasms, and is further subdivided into large cell carcinoma, squamous cell carcinoma, and adenocarcinoma. NSCLC is diagnosed with equal frequency in men and women, and approximately 1.2 million people are diagnosed with NSCLC annually worldwide, with 2 out of 3 cases diagnosed in adults over the age of 65. The pathogenesis of NSCLC is multi-factorial and involves genetic factors as well as environmental and occupational factors. Important risk factors for NSCLC include active cigarette smoking, second-hand smoking, asbestos exposure, radon gas exposure, other airborne chemicals and particles, and malnutrition or poor diet., Unfortunately, at first diagnosis, majority of NSCLC patients present advanced stages of the disease, severely impacting the 5-year survival rates, which is currently at <18% in females and 14% in males. DNA methylation is an important epigenetic modification, and methylation-associated gene silencing is a prominent mechanism related to the loss of tumor suppressor gene (TSG) expression in multiple cancers. Previous studies have shown that NSCLC pathogenesis includes altered methylation patterns in multiple genes, and these epigenetic changes serve as important biomarkers for NSCLC detection. The prominent DNA methylation changes relevant to cancers involve genes critical for maintaining epithelial phenotype, apoptosis and DNA repair such as breast cancer 1, cadherin 13, adenomatous polyposis coli, and death-associated protein kinase 1 (DAPK).,
Death-associated protein kinase, also called death-associated protein 2, is a calcium/calmodulin-dependent serine/threonine kinase originally identified as a mediator of interferon-c-induced cell death, but recently, its role as a metastasis suppressor has been described. Human DAPK gene is located on chromosome 9p34 and contains ankyrin repeat domain, a kinase domain, and a death domain.DAPK mediates apoptosis upon activation by a variety of apoptotic stimuli such as tumor necrosis factor-α, interferon gamma, or anti-Fas antibodies. In addition, DAPK is also involved in the regulation of other cellular processes, including autophagy, cell migration, apoptosis and membrane blebbing. Dysfunction of DAPK signaling pathway is linked to various diseases such as stroke, inflammation, cancer, and atherosclerosis. Moreover, abnormal methylation of the CpG island in DAPK promoter is an important mechanism for loss of DAPK gene expression in tumor cells. One clinical study showed that the loss of DAPK expression is associated with aggressive cancer phenotypes and advanced tumor stages, including metastasis. Multiple studies supported this view and showed that DAPK promoter methylation correlates with the clinicopathologic features in NSCLC patients and is associated with poor prognosis in patients., However, other studies contradicted these findings. In order to address this issue, we conducted a cohort study to obtain a correlation between DAPK promoter methylation and the clinicopathological features and prognosis in NSCLC patients.
| > Materials and Methods|| |
The study was performed with the approval of the Institutional Review Board, Department of Pathology, Xinxiang Medical University. Informed written consent was obtained from all eligible patients, and all procedures were conducted according to the Declaration of Helsinki.
A total of 117 NSCLC patients who were admitted to the Department of Pathology, Xinxiang Medical University between December 2012 and December 2014 were selected for this study. The patients included 75 males and 42 females (age range, 41–78 years; mean age, 60.1 ± 5.6 years), with a body mass index (BMI) of (18.5 ± 2.2) kg/cm 2. Diagnosis of NSCLC was confirmed in all patients by histological or cytological examination and included 56 cases of squamous cell carcinoma and 61 cases of adenocarcinoma. According to the tumor node metastasis (TNM) staging criteria formulated by the Union Internationale Contre le Cancer in 2009 for lung cancers: 20 patients were in stage I, 25 in stage II, 32 in stage III, and 40 in stage IV. A total of 69 patients were lymph node positive, and 48 patients were lymph node negative. NSCLC patients' fasting blood samples were drawn before surgery, and 5 ml blood was placed into ethylenediaminetetraacetic acid (EDTA)-containing anticoagulant tube. Blood samples were immediately centrifuged at 3000 r/min for 15 min to separate plasma and cryopreserved at − 20°C until further use. During the same period, a group of 113 healthy subjects without respiratory diseases or tumors of any kind were selected as the control group. The healthy subjects included 67 males and 46 females (age range 40–77 years; mean age, 58.9 ± 6.2 years) with BMI of (19.2 ± 2.5) kg/cm 2. Approximately, 1 cm 3 of cancer tissues (resected lung tumor tissues by surgery) and peficancerous tissues (normal tissues at a distance of 3–5 cm from the tumor front) were obtained and stored in liquid nitrogen for cryopreservation. There was no significant difference in age, gender, BMI, and smoking history between NSCLC patients and healthy subjects (all P > 0.05).
The main equipment and reagents
Genomic DNA extraction was performed using QIAamp DNA Blood Midi Kit (QIAGEN Company, Germany). Methylation assay kit was purchased from Qiagen Company and DNA molecular markers were purchased from Huamei Biological Engineering Co., Ltd. DAPK gene methylation and nonmethylation primers were synthesized by Shanghai Sangon Company. Agarose was a product of Sigma Company, with other routine reagents from domestic companies manufacturing analytical reagents. The main equipment in this study included: PTC-100 polymerase chain reaction (PCR) machine, DYY-10 electrophoresis apparatus (Beijing six-one instrument factory), TXT-20M gel imaging system (UVP Bioimaging System), hema 8000 gene amplification instrument (Red Maple Co., Ltd), Wizard DNA purification system (Promega, USA).
Genomic DNA extraction from tumor tissue was done using phenol-chloroform method. After grinding the tissue in liquid nitrogen, tissues were immersed in 10 volumes of lysis buffer and incubated at 37°C for 1 h. Proteinase K was added and incubated at 50°C for 3 h. An equal volume of phenol was added after the solution cooled to room temperature, mixed thoroughly, and centrifuged at 5000 r/min for 15 min. The upper aqueous phase was transferred to a fresh tube. The aqueous phase was similarly transferred after the extraction was repeated 2 times (chloroform was used the 3rd time). To the aqueous phase, 0.2 volume of 10 mol/L ammonium acetate and 2 volumes of alcohol were added to precipitate DNA. After pelleting the precipitated DNA at 5000 r/min for 5 min, the supernatant was removed, and the DNA was washed with 70% ethanol and dried. The DNA was finally dissolved in Tris-EDTA (TE) buffer and stored at −20°C.
Genomic DNA extraction from the serum was performed using QIAamp DNA Blood Midi Kit following the protocol listed in “blood and blood fluids” of the manufacturer's user manual. Briefly, the protocol was as follows:First, 400 μL blood, 400 μL amoebocyte lysate, and 40 μL protease were added into 2 ml centrifuge tub, mixed and incubated at 56°C for 10 min; second, the lyate was transferred into QIAamp centrifuge tube and centrifuged at 6000 r/min for 1 min; third, 400 μL of 70% ethyl alcohol was added to wash and centrifuged at the same speed; finally, 500 μL elution buffer AW2 was added and centrifuged at 20,000 r/min for 3 min and the eluted DNA was cryopreserved at −20°C.
Detection of gene methylation
DNA modification and DNA purification: Purified genomic DNA in a volume of 50 μL was used for this assay. DNA was mixed with 5.5 μL of 3 mol/L sodium hydroxide incubated in a water bath at 42°C for 30 min. A 30 μL volume of 10 mmol/L hydroquinol and 510 μL of 3.9 mol/L sodium bisulfite were added, along with two drops of paraffin oil and the sample was placed in the water bath in dark at 55°C for 16 h. After the treatment, DNA was purified using Wizard DNA purification system and finally dissolved in 50 μL TE buffer.
Polymerase chain reaction primer design: The sequence of the upstream primer of methylated DAPK was: 5'-GGATAGTCGGATCGAGTTAACGTC-3', and downstream primer was: 5'-CCCTCCCAAACGCCGA-3'. The amplification product was 98 bp, with an annealing temperature of 61°C. The sequence of the upstream primer for nonmethylated DAPK was: 5'-GGAGGATAGTTGGATTGAGTTAAGTT-3', and the downstream primer was: 5'-CAAATCCCTCCCAAACACCAA-3'. The amplification product was 106 bp, with an annealing temperature of 60°C. The sequence of the upstream primer of internal control was: 5'-CAACTTCATCCACGTTCACC-3', and the downstream primer was: 5'-GAAGAGCCAAGGACAGGTAC-3'. The amplification product was 268 bp, with an annealing temperature of 59°C.
Polymerase chain reaction steps and analysis: PCR reaction system (25 μL): 5.0 μL buffer solution (Qiagen), 2.5 μL buffer, 0.5 μL 10 mm/L dNTPs, 0.5 μL 20 μM/L primers, 0.75 μL HotstarTap enzyme and 5.0 μL modified DNA. Purified DNA isolated from peripheral whole blood and treated with or without Sss I methylase in healthy subjects was used as a positive control of methylation and nonmethylation. The amplification conditions were: Predenaturation at 95°C for 15 min, denaturation 94°C for 50s, annealing for 50s and extension at 72°C for 50s, for a total of 35 cycles. Final extension was at 72°C for 7 min. The PCR products were electrophoresed using 2.0% agarose gels, and the PCR product was visualized after ethidium bromide staining and observed under ultraviolet lamp. The standard criteria for methylation were: The PCR bands of expected size as amplified using methylation-specific primers showed the positive promoter methylation status of DAPK in samples.
A database was constructed using Epi-Data software (EpiData Association, Odense, Denmark), and statistical analysis was performed using STATA version 12.0 software (STATA Corporation, College Station, TX, USA). The methylation rate was calculated as follows: Number of DAPK methylations detected in specimens/total number of samples and data was reported as percentages. The comparison of methylation rates was performed by χ2 test, with P < 0.05 suggesting a significant difference. The following electronic databases were searched to identify relevant articles or data collection: PubMed and China National Knowledge Infrastructure (last updated search in October, 2014). Based on the combination principle of keywords and free words, the search terms included: Carcinoma, nonsmall cell lung, NSCLC, NSCLC, DAPK, DAPK, and DAPK. Meta-analysis was conducted using STATA version 12.0 software (STATA Corporation, College Station, TX, USA). The relative risk (RR) and its corresponding 95% confidence interval (95% CI) were used to evaluate DAPK promoter methylation and clinicopathological features and prognosis in NSCLC patients by applying random-effects model or fixed-effect model. Z test was adopted for the significance of pooled RRs.
| > Results|| |
Methylation frequencies of death-associated protein kinase
Our study results showed that DAPK methylation frequencies in NSCLC tumor tissues and peficancerous tissues were 58.1% (68/117) and 12.8% (15/117), respectively, showing a statistically significant difference between the two groups (χ2 = 52.45, P < 0.001) [Figure 1]. Methylation frequency of DAPK in the serum of NSCLC patients was 27.4% (32/117), and no DAPK methylation was detected from serum samples of healthy controls, indicating significant differences between the two groups in relation to DAPK methylation status (χ2 = 37.07, P < 0.001) [Figure 2].
|Figure 1: Methylation frequencies of death-associated protein kinase in nonsmall cell lung cancer tissues and adjacent normal tissues|
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|Figure 2: Methylation frequencies of death-associated protein kinase in serum of nonsmall cell lung cancer patients and healthy controls|
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Correlation of death-associated protein kinase methylation with clinicopathological features of nonsmall cell lung cancer
The following results are related to analysis from NSCLC tissue samples. DAPK methylation frequencies in adenocarcinoma and squamous cell carcinoma were 57.4% (35/61) and 59.8% (33/56), respectively, indicating no significant differences between the two groups (P = 0.085). DAPK methylation frequencies in TNM stage I-II and TNM stage III-IV of NSCLC were 51.1% (23/45) and 62.5% (45/72), with no statistical significance between the two groups (P = 0.225). In NSCLC with lymph node metastasis, DAPK methylation frequency was 59.4% (41/69), compared to 52.1% (25/48) in NSCLC without metastasis, revealing no significant differences between the two groups (P = 0.431) [Table 1].
|Table 1: DAPK methylation frequency in the tissue from patients with different clinical types of NSCLC|
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The following results are related to analysis from NSCLC blood samples. DAPK methylation frequencies in adenocarcinoma and squamous cell carcinoma patients were 27.9% (17/61) and 26.8% (15/56), respectively, with no evidence of statistical significance between the two groups (P = 0.900). DAPK methylation frequencies in TNM I-II and TNM III-IV were 22.2% (10/45) and 27.8% (20/72), respectively, with no statistical significance (P = 0.503). DAPK methylation frequencies in patients with lymph node metastasis were 33.3% (23/69) and it was 25.0% (12/48) in patients without metastases, also showing no statistical differences (P = 0.333) [Table 2].
|Table 2: DAPK methylation frequency in the plasma from patients with different clinical types of NSCLC|
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Correlation of death-associated protein kinase methylation with prognosis of nonsmall cell lung cancer
The 5-year survival rate in NSCLC patients with DAPK methylation was 76.5% (52/68), while it was 91.8% (45/49) in patients with unmethylated DAPK promoter, showing statistical significance (χ2 = 4.75, P= 0.029) [Figure 3].
|Figure 3: The 5-year survival rates of nonsmall cell lung cancer patients with death-associated protein kinase (DAPK) methylation or nonmethylated DAPK|
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In our meta-analysis, 14 published cohort studies were enrolled, and these 14 studies contained a combined total of 1238 NSCLC patients.,,,,,,,,,,,,, A total of 15 studies (including the present study) examined the association between DAPK promoter methylation and histological classification of NSCLC patients. No heterogeneity was detected among the studies (I2 = 0.0%, Ph = 0.523), therefore a fixed-effect model was employed. Fourteen out of 15 studies (including our study) reported the correlation of DAPK promoter methylation in NSCLC patients with clinical TNM staging and no heterogeneity existed among the studies (I2 = 0.0%, Ph = 0.654), thus a fixed-effect model was used. Only 6 of the 15 studies reported the relation between DAPK promoter methylation and NSCLC metastasis (including this study), and no heterogeneity was found among these studies (I2 = 0.0%, Ph = 0.654), therefore fixed-effect model was used. Only 5 out of 15 studies (including the current study) investigated the association between DAPK promoter methylation and NSCLC patient prognosis. No heterogeneity was detected (I2 = 65.2%, Ph = 0.022), hence, a random-effect model was applied. The meta-analysis results showed that DAPK promoter methylation frequency in NSCLC patients with TNM stage I-II was lower than the frequency observed in TNM stage III-IV (RR = 0.87, 95% CI = 0.76–0.99, P= 0.041) [Figure 4]. Further, DAPK promoter methylation frequency in NSCLC patients with lymph node metastasis was significantly higher compared to the frequency observed in patients without metastases (RR = 1.26, 95% CI = 1.04–1.52, P= 0.020) [Figure 5]. Accordingly, the 5-year survival rate of NSCLC patients with DAPK methylation was lower than the patients with unmethylated DAPK (RR = 0.71, 95% CI = 0.56–0.89, P= 0.004) [Figure 6]. However, no significant correlation existed between DAPK methylation and the NSCLC histological type (RR = 1.04, 95% CI = 0.92–1.17, P= 0.530) [Figure 7].
|Figure 4: Meta-analysis on death-associated protein kinase promoter methylation frequencies of nonsmall cell lung cancer patients in tumor node metastasis (TNM) I-II and TNM III-IV|
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|Figure 5: Meta-analysis on death-associated protein kinase promoter methylation frequencies of nonsmall cell lung cancer patients with lymph node metastasis and without metastasis|
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|Figure 6: Meta-analysis on 5-year survival rate of nonsmall cell lung cancer patients with death-associated protein kinase (DAPK) methylation and nonmethylated DAPK|
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|Figure 7: Meta-analysis on correlation between death-associated protein kinase methylation and histology type of nonsmall cell lung cancer|
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| > Discussion|| |
Despite the development of novel diagnostic and therapeutic approaches for NSCLCs with primary histologies of adenocarcinoma and squamous cell carcinoma (constituting 60% and 25% of lung cancers, respectively), the outcomes continue to be dismal. Tumor growth, invasion, and metastasis of NSCLC are influenced by several factors and tumor angiogenesis, for instance, plays a significant role in tumor growth and metastasis of most tumors, including NSCLC.DAPK displays tumor suppressor activity by promoting apoptosis and autophagy.,, In this study, we examined the correlation between DAPK promoter methylation and the clinicopathological features and prognosis in NSCLC patients. Further, a meta-analysis was conducted to assess the overall results.
The findings in the present study revealed that DAPK methylation frequency was significantly higher in NSCLC tissues compared to peficancerous tissues. Moreover, DAPK methylation frequency in the serum of NSCLC patients was also higher compared to healthy controls. The 5-year survival rates of NSCLC patients with DAPK methylation was lower compared to the patients with unmethylated DAPK promoter. However, no significant correlation was found between DAPK methylation frequency and the clinicopathological features in NSCLC patients. Our study suggests that DAPK methylation correlates with NSCLC origin, and may be a crucial prognostic marker for NSCLC. Niklinska et al. suggest that promoter DNA methylation is a vital trigger in tumorigenesis through abolishing the expression of critical TSGs. DAPK and RASSF1A methylation is prominently linked to lung cancer. Interestingly, studies in cell lines derived from NSCLC demonstrated that DAPK expression is lost only in the highly metastatic clones and restoration of DAPK in these cells suppressed their ability to form lung metastases, suggesting that DAPK may have dual functions in suppressing early cell transformation and in preventing cancer metastasis at later stages., Previous studies have associated the tumor suppressor functions of DAPK with the promotion of anoikis, disruption of integrin signaling, suppression of cell motility, and regulation of metabolism through pyruvate kinase type M2 (PKM2)., PKM2 is a key enzyme in aerobic glycolysis in tumor cells and is a potential target for therapy in NSCLC., Our results show that DAPK methylation status may be reliable as an early biomarker for diagnosis and prognosis in NSCLC.
Our meta-analysis showed DAPK promoter methylation frequency in TNM stage I-II was lower than TNM stage III-IV. Moreover, DAPK promoter methylation frequency in NSCLC patients with lymph node metastasis was significantly higher compared to patients without metastasis. The 5-year survival rate of NSCLC patients with DAPK methylation was also lower compared to patients without DAPK methylation. These results suggest that DAPK methylation could be used as a biomarker for clinical diagnosis and prognosis in NSCLC. However, no significant correlation existed between DAPK methylation and NSCLC histology type.
| > Conclusion|| |
This study revealed that DAPK promoter methylation is an important indicator of tumor progression and is associated with poor prognosis in NSCLC patients, suggesting that it could be a valuable biomarker for early clinical diagnosis and prognosis in NSCLC patients.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2]