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Introduction

Lung cancer is a major cause of mortality from malignant disease, due to its high incidence, malignant behavior and the lack of major advancements in treatment strategy (1). A great deal of progress has been made in the target therapy for non-small cell lung cancer (NSCLC), largely owing to the development of small molecular inhibitors, including epidermal growth factor receptor (EGFR) (2–6) and ALK (7). By contrast, patients with the principal subtype of NSCLC, squamous cell lung cancer, rarely respond to these agents. Mutations in the discoidin domain receptor (DDR) kinase gene have been identified in squamous cell lung cancer from large-scale Sanger sequencing (8). DDRs have been shown to exhibit altered expression patterns in multiple types of human cancer, including lung cancers (9,10). The mechanism by which the receptors may contribute to oncogenesis is not well known; however, given the important role of the receptors in transmitting signals from the extracellular matrix (ECM), it is possible that the receptors act as regulators of cell proliferation, adhesion, migration and tumor metastasis (9). The downregulation of DDR2 mRNA expression has been reported in NSCLCs (10). Davies et al screened for mutations in patients with lung cancer by comprehensively sequencing 518 kinases in the human genome and described a novel DDR2 gene mutation (R105S) in the discoidin domain (11). However, the mutation status of the DDR2 gene in the Japanese population has not been well reported.

In this study, the mutation status of DDR2 at the discoidin and kinase domains in lung squamous histology tumors was investigated. This study also investigated DDR2 mRNA levels via real-time PCR using LightCycler. The findings were compared with the clinicopathological features of lung cancer.

Patients and methods

Patients

The study group included patients with lung cancer who had undergone surgery at the Department of Surgery II, Nagoya City University Hospital (Nagoya, Japan). Patient consent was obtained from the patients or a family member. The study wa approved by the ethics committee of the hospital. Tumor samples were immediately frozen and stored at −80°C until they were assayed. Hammerman et al demonstrated that the DDR2 mutations were present within the squamous histology of lung cancer (8), therefore this study focused on squamous cell carcinomas. The clinical and pathological characteristics of the 166 patients with lung cancer for DDR2 gene analyses were as follows: 143 (86.1%) were male and 23 were female; 143 (86.1%) were diagnosed with squamous cell carcinomas and 22 were adenosquamous cell carcinomas; 153 (92.2%) were smokers and 13 were non-smokers. The clinical and pathological characteristics of the 92 patients with lung cancer for DDR2 gene mRNA expression analyses were as follows: 80 (87%) were male and 12 were female; 87 (94.6%) were diagnosed with squamous cell carcinomas and four were adenosquamous cell carcinomas; five (5.4%) were light-smokers (Brinkman index <100) and 52 (56.5%) were of pathological stage I.

PCR assays for DDR2 mRNA expression

Total RNA was extracted from lung cancer tissues using an Isogen kit (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. The RNA concentration was determined using a spectrophotometer and adjusted to a concentration of 200 ng/ml. Approximately 10 cases were excluded for each assay as there were not enough tumor cells to extract sufficient tumor RNA. RNA (1 μg) was reverse transcribed using Superscript II enzyme (Gibco-BRL, Gaithersburg, MD, USA) with 0.5 μg oligo (dT)12–16 (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). The reaction mixture was incubated at 42°C for 50 min and then at 72°C for 15 min. PCR analyses were performed using 1 μl of each DNA sample. The PCR was performed using LA-Taq kit (Takara Bio Inc., Shiga, Japan) in a 25-μl reaction vessel. The primer sequences for the discoidin domain of the DDR2 gene were as follows: forward: 5′-CAGCTTCCAGTCAGTGGTCA-3′ and reverse: 5′-GCCAGCCCACATAGTCATAG-3′ (643 bp, exons 3–7). The cycling conditions were as follows: initial denaturation at 94°C for 5 min, followed by 40 cycles at 94°C for 45 sec, 60°C for 45 sec and 72°C for 45 sec. The primer sequences for the DDR2 gene kinase domain were as follows: forward: 5′-TTTGGGGAGGTTCATCTCTG-3′ and reverse: 5′-GTCAGGACAAATGGCTGGTT-3′ (747 bp, exons 9–12). The cycling conditions were as follows: initial denaturation at 94°C for 5 min, followed by 40 cycles at 94°C for 45 sec, 60°C for 45 sec and 72°C for 45 sec. The products were purified by a Qiagen PCR purification kit (Qiagen, Valencia, CA, USA). The samples were sequenced using the ABI prism 3100 analyzer (Applied Biosystems Japan Ltd., Tokyo, Japan) and analyzed by BLAST and chromatograms via manual review.

To ensure the fidelity of mRNA extraction and reverse transcription, the samples were subjected to PCR amplification with oligonucleotide primers specific for the constitutively expressed gene β-actin and normalized using a β-actin detection kit (Nihon Gene Research Laboratories, Miyagi, Japan). The primer sequences for the DDR2 gene were as follows: forward: 5′-CCACTATGCAGAGGCTGACA-3′ and reverse: 5′-CAGAGATGAACCTCCCCAAA-3′ to amplify a 183-bp fragment. The cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 50 cycles at 95°C for 5 sec, 60°C for 5 sec and 72°C for 8 sec. PCR reactions were performed using a LightCycler-FastStart DNA Master SYBR-Green I kit (Roche Molecular Biochemicals, Mannheim, Germany) and quantified.

Statistical analysis

Statistical analysis was performed using the Mann-Whitney U test for unpaired samples and Wilcoxon’s signed-rank test for paired samples. Linear correlations between the variables were determined by means of a simple linear regression. Correlation coefficients were determined by rank correlation using the Spearman’s test and ξ2 test. The overall survival rate of patients with lung cancer was examined using the Kaplan-Meier method and differences were examined using the log-rank test. Analyses were performed using the Stat-View software package (Abacus Concepts Inc., Berkeley, CA, USA). P<0.05 was considered to indicate a statistically significant result.

Results

DDR2 gene mutation status in Japanese patients with lung cancer

The current study sequenced the kinase domain of DDR2 gene for 173 squamous histology NSCLC samples. Of 173 patients, from direct sequencing using cDNA samples, no mutations were identified. This study also sequenced the discoidin domain of the DDR2 gene for 166 squamous histology NSCLC samples, where 148 samples overlapped. Of 166 patients, from direct sequencing using cDNA samples, no mutations were identified. However, 14 DDR2 polymorphism cases (8.4%) were identified (Fig. 1). The nucleotide T at 408 was changed to C. The amino acid was not converted from histidine (CAT>CAC, His>His). This DDR2 polymorphism status was not correlated with gender (male 11/143 vs. female; 3/23, p=0.3914), age (age ≤65, 7/71 vs. >65, 7/104; p=0.5724) or smoking status (smoker 14/153 vs. non-smoker 0/13; p=0.2544). The polymorphism cases tended to be higher in squamous cell carcinomas (p=0.0796) when compared with the adenosquamous carcinomas. The polymorphism cases were predominantly observed in advanced stages (stage II–IV, 11/74 vs. stage I, 3/92; p=0.0075).

Figure 1 DDR2 polymorphism at the discoidin domain, histidine136 to histidine, at nucleotide 408 (T to C change). Left panel, forward sequence from lung cancer samples. Right panel, reverse sequence. DDR2, discoidin domain receptor 2.

DDR2 gene expression status in Japanese patients with lung cancer

In the 92 tissues from histologically confirmed lung squamous cell carcinoma, the mean value for DDR2 mRNA level as standardized by the mRNA level of β-actin (10.915±1.546, mean ± standard deviation) was significantly lower than the tissues from non-malignant lung tissue (22.790±3.382, p=0.0013). The T/N ratios of the DDR2 mRNA levels for each sample were: stage I, 0.978±0.187; stage II, 1.027±0.369; stage III, 1.532±0.611; and stage IV, 0.17 (Table I). The DDR2 mRNA T/N ratios in squamous cell carcinoma (1.113±1.765) and adenosquamous cell carcinomas (0.676±0.423) were not significantly different (p=0.5839). No significant difference in DDR2 mRNA levels T/N ratio was observed between gender and age. The patient groups were further stratified according to clinicopathological factors. The T/N ratio was not significantly different between the light-smokers (Brinkman index <100; 0.386±0.368) and smokers (>100; 1.129±1.765; p=0.1479). The T/N ratio of DDR2 mRNA levels in each sample were: T1, 0.884±0.240; T2, 0.990±0.232; T3, 1.295±0.406; and T4, 1.975±1.048. Although there was no significant difference, DDR2 mRNA levels were higher in advanced T stages (Table I).

Table I Clinicopathological data of 92 patients with lung cancer.

Table I

Clinicopathological data of 92 patients with lung cancer.

	DDR2
Factors	No. of patients n (%)	T-N ratio of DDR2/β-actin mRNA levels	P-value
Mean age (years)	66.7±9.0	92
Stage
I	52 (56.5)	0.978±0.187	NS
II	21 (22.8)	1.027±0.369
III	18 (19.6)	1.532±0.611
IV	1 (1.1)	0.17
Tumor status
T1	30 (32.6)	0.884±0.240	NS
T2	34 (37.0)	0.990±0.232
T3	8 (8.7)	1.295±0.406
T4	10 (10.8)	1.975±1.048
Lymph node metastasis
N0	65 (70.7)	1.025±0.162	NS
N1	15 (16.3)	1.682±0.835
N2	12 (13.0)	0.753±0.272
Pathological subtype
Adenosquamous	5 (5.4)	0.676±0.423	0.5839
SCC	87 (94.6)	1.113±1.765
DDR2 polymorphism
T408C	7 (8.2)	3.301±3.972	0.0465
T408T	78 (91.8)	0.929±1.717
Smoking
BI<100	5 (5.4)	0.386±0.368	0.1479
BI>100	78 (94.6)	1.129±1.760
Age (years)
≤65	35 (38.0)	1.318±1.716	0.3204
>65	57 (62.0)	0.948±1.724
Gender
Male	80 (87.0)	1.115±1.823	0.3272
Female	12 (13.0)	0.916±0.765

[i] T-N, tumor-normal; NS, not significant; SCC, squamous cell carcinoma; BI, Brinkman index; DDR2, discoidin domain receptor 2.

Correlation between DDR2 and DDR2 polymorphisms

Of the 85 patients that overlapped, 7 polymorphism cases were found at the discoidin domain (C408T, H136H; Fig. 1). The DDR2 polymorphism in lung cancer had a significantly higher DDR2 mRNA level (T/N ratio, 3.301±3.972) than the wild-type DDR2 for lung cancer (T/N ratio, 0.929±1.717; p=0.0465). The overall survival rate of 162 patients with lung cancer from Nagoya City University, with follow-up until August 31, 2011, was studied with reference to the DDR2 gene status. The survival rate of patients with the DDR2 gene polymorphism (n=14, 5 mortalities) and the patients with the wild-type DDR2 (n=148, 41 mortalities) was not significantly different (log-rank test, p=0.7944; Fig. 2).

Figure 2 The overall survival rate of the 162 patients with lung cancer from Nagoya City University, with follow-up until August 31, 2011, was studied with reference to the DDR2 gene status. The survival of patients with the DDR2 gene polymorphism (n=14, 5 mortalities) and patients with the wild-type DDR2 (n=148, 41 mortalities) was not significantly different (log-rank test, p=0.7944). DDR2, discoidin domain receptor 2. Open circle, wild-type cases; closed circle, polymorphism cases.

Discussion

The present study has shown that DDR2 mRNA expression is significantly deregulated in NSCLC when compared with normal lung tissue. However, DDR2 mRNA levels were higher in the DDR2 polymorphism cases. The polymorphism cases (8.4%) were predominantly observed within the advanced lung cancers. The collagen-binding RTK DDRs have previously been linked to various human diseases, including cancers (12–15). Although the sample size was not large, no DDR2 mutations were observed in this cohort. However, the DDR2 expression pattern in lung cancer suggests that DDR2 contributes to the pathogenesis of lung cancer.

Previous studies have reported somatic mutations in the DDR2 gene at the discoidin or kinase domain (8,11), however, the present study did not confirm the existence of the DDR2 mutation. There are several explanations for this discrepancy. Lung cancer encompasses a broad range of clinical subtypes, in which the two cohorts differed. In addition, an ethnic difference between the studies on mutant DDR2 may exist, as in EGFR gene mutations (2–6). The present study cannot conclude that differences in the DDR2 sequence were due to methodology or PCR and sequencing methods. However, this study detected 8.4% of polymorphism cases at the discoidin domain in this cohort.

The mechanism by which DDRs may contribute to oncogenesis is not well known, however, given their role in transmitting signals from the ECM, it is likely that DDRs act as regulators of cell proliferation, adhesion, migration and subsequent tumor metastasis. Prolonged stimulation of DDR2 is associated with the upregulation of MMP-1 expression (16). DDR2 is also important in mediating fibroblast migration and proliferation via a MMP-2-dependent mechanism (17,18). Activated DDR2 has been noted to induce the expression of MMP-1, MMP-2 and MMP-13 (17,19). Similar to EGFR, it is conceivable that an altered expression of DDRs triggers abnormal activity, ultimately leading to enhanced proliferation and oncogenesis. In this study, the synonymous nucleotide change in DDR2 was present in approximately 10% of the present clinical cohort. This polymorphism was correlated with an increased DDR2 expression and advanced pathological stages of lung cancers.

It has been reported that the development of experimental liver metastasis using melanoma cells, which were stably transfected with a small interfering RNA for DDR2, was reduced compared with mock transfected clones (20). Findings of a previous study revealed that imatinib, nilotinib and dasatinib are potent inhibitors of the kinase activity of DDRs (21). The kinase profiles of the three compounds were addressed in two chemical proteomic studies which observed that the compounds also bind to the DDRs (21,22). Of these compounds, dasatinib was the most potent inhibitor of DDRs (17). Recently, it was observed that only dasatinib produced a response in mutant-DDR2 lung cancer cell lines (8). These findings may contribute to a greater understanding of the therapeutic potential of these inhibitor compounds with DDR2.

In conclusion, the DDR2 mutation in lung cancers of Japanese patients was observed to be extremely rare. However, the DDR2 polymorphism and/or its expression may be involved in the progression of lung cancer.

Acknowledgements

The authors thank Mrs. Miki Mochizuki for her technical assistance. This study was supported by Grants-in-Aid for Scientific Research, Japan Society for the Promotion of Science (JSPS; Nos. 23659674, 24592097, 21591820) and a grant for the Cancer Research Program for Developing the Supporting System for Upgrading Education and Research (2009) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References