Tumor Biol. DOI 10.1007/s13277-014-2039-1

RESEARCH ARTICLE

Elevated expression of Cripto-1 correlates with poor prognosis in non-small cell lung cancer Chun-Hua Xu & Zhi-Hong Sheng & Hui-Di Hu & Ke-Ke Hao & Qing-Bo Wang & Li-Ke Yu

Received: 7 April 2014 / Accepted: 28 April 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract Human Cripto-1 (CR-1) plays an important oncogenic role during tumorigenesis and is overexpressed in a wide range of carcinomas, yet little is known about CR-1 in non-small cell lung cancer (NSCLC). The aims of this study were to detect CR-1 expression in NSCLC and to analyze its association with prognosis of NSCLC patients. The expression of CR-1 messenger RNA (mRNA) and protein in 35 cases of NSCLC and corresponding noncancerous tissue samples was examined by quantitative real-time reverse transcription–polymerase chain reaction (qRT–PCR) and Western blotting. Immunohistochemistry was performed to detect the expression of CR-1 in 128 NSCLC tissues. The expression levels of CR-1 mRNA and protein in NSCLC tissues were significantly higher than those in corresponding noncancerous tissues (P<0.001). A high level of CR-1 expression was correlated with poor tumor differentiation (P=0.002), tumor–node–metastasis (TNM) stage (P=0.004), and lymph node metastasis (P=0.001). The results of the Kaplan–Meier C.
analysis indicated that a high expression level of CR-1 resulted in a significantly poor prognosis of NSCLC patients. Multivariate Cox regression analysis revealed that CR-1 expression level was an independent prognostic parameter for the overall survival rate of NSCLC patients. Our data suggest that the high expression of CR-1 may play an important role in the progression of NSCLC, and CR-1 expression may offer a valuable marker for predicting the outcome of patients with NSCLC. Keywords Cripto-1 . Non-small cell lung cancer . Biomarker . Prognosis

Introduction Lung cancer has been one of the most common cancers, and it is also the leading cause of cancer-related deaths worldwide [1]. Non-small cell lung cancer (NSCLC) accounts for 80– 85 % of total lung malignancies [2]. Despite advances in multidisciplinary treatment approaches, the 5-year survival in patients with NSCLC remains only about 15 % [3, 4]. Several independent prognostic factors for survival have been identified: performance status, disease stage, and the amount of weight loss [5]. However, the discriminate value of most potential prognostic biomarkers is insufficient to predict the optimal therapeutic course for an individual. Thus, it is urgent to explore novel prognostic markers and therapeutic targets, both of which may be realized through a more sophisticated understanding of the molecular mechanisms involved in lung carcinogenesis. Human CR-1 is a member of the epidermal growth factorcripto FRL1 cryptic (EGF-CFC) family, which is indispensable for early embryonic development [6, 7]. CR-1 has been shown to have an important role in vertebrate development. Evidence suggests that CR-1 is also involved in the

Tumor Biol.

pathogenesis and progression of human carcinoma [7–17]. Recently, it has been reported that plasma CR-1 might represent a novel biomarker for the detection of breast and colon carcinomas [18]. In gastric cancer, CR-1 overexpression is associated with poor prognosis [19]. To the best of our knowledge, little has been uncovered regarding the involvement of CR-1 genes in NSCLC. In this study, we investigated CR-1 expression in NSCLC tissues and its correlation with clinicopathological characteristics, including the survival of patients with NSCLC.

Materials and methods Patients and tissue samples A total of 128 primary lung cancer specimens were collected from patients with NSCLC undergoing surgery at the Department of Thoracic Surgery of Nanjing Chest Hospital between May 2004 and August 2006. In addition, 35 self-pairs of NSCLC specimens and adjacent noncancerous lung tissues were snap-frozen in liquid nitrogen and stored at −80 °C following surgery for quantitative real-time reverse transcription–polymerase chain reaction (qRT–PCR) and Western blot analysis. None of the patients received any chemotherapy or radiotherapy before surgery. Clinicopathological data were obtained by medical records in the archives room. The data included patient age, gender, smoking condition, tumor size, tumor differentiation, lymph nodal status, and pathological stage. The postoperative pathological staging was determined according to the seventh edition of the tumor–node–metastasis (TNM) classification [20]. Histological type was determined according to the classification by the World Health Organization. Inclusion criteria for this study were surgical complete resection of the tumor, patient survival for more than 3 months after surgery, and the patient did not die of causes other than lung cancer within 5 years following surgery. Follow-up information was obtained by phone investigations. The median follow-up of surviving patients at the time of analysis was 24 months (range, 3–84 months). The date of the last followup was December 28, 2012. Patient characteristics are summarized in Table 1. The study was approved by the Research Ethics Committee of The Nanjing Chest Hospital, Nanjing, China. Informed consent was obtained from all patients. All specimens were handled and made anonymous according to accepted ethical and legal standards.

Table 1 Correlation between clinicopathological characteristics and CR1 expression Characteristics

Total

CR-1 protein expression Low

High

36 22

36 34

Gender Male Female Age <60 ≥60 Tumor size (cm) ≤3 >3 Smoking condition Nonsmoker Smoker Histological type Squamous cell carcinoma Adenocarcinoma Tumor differentiation Well-moderate Poor TNM stage I–II III–IV Lymph node metastasis N0 N1–3

P 0.283

72 56

0.858 73 55

34 24

39 31

79 49

32 26

47 23

0.202

0.478 71 57

30 28

41 29

74 54

36 22

38 32

0.472

0.002* 62 66

37 21

25 45

70 58

40 18

30 40

66 62

40 18

26 44

0.004*

0.001*

*P<0.05 (statistically significant difference)

sample was reverse transcribed using Superscript RT kit. Primer sequences used for CR-1 are forward 5′-GATACA GCACAGTAAGGAGC-3′ and reverse 5′-TAGTTCTGGA GTCCTGGAAG-3′, and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are forward 5′-CACCCAGCACAA TGAAGAT-3′ and reverse 5′-CAAATAAAGCCATGCC AAT-3′. We used the SYBR Green kit (Invitrogen Life Technologies, Carlsbad, CA, USA) to execute the amplification of the cDNA. The RT-PCR cycling parameters were performed as follows: denaturation at 95 °C for 15 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. The expression of GAPDH was used to normalize that of the target genes. Each assay was done in triplicate, and the average was calculated. The expression level of CR-1 was expressed as 2−ΔΔCt, where ΔCt = Ct (CR-1) − Ct (GAPDH).

Quantitative real-time reverse transcription–polymerase chain reaction

Western blots

Total RNAs were purified from tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), and 2 μg RNA of each

A subset of patients in the cohort had adequate frozen tissue for Western blot analysis. We cut 4-μm slices from frozen

Tumor Biol. Fig. 1 qRT–PCR analysis of CR1 in 35 pairs of fresh frozen NSCLC tissues and corresponding noncancerous tissues. The CR-1 mRNA levels were significantly higher in NSCLC tissues than those in paired noncancerous tissues (P<0.001)

tissue samples and extracted them in 400 μl of lysis 250 solution (0.05 M Tris, pH 7.4, 0.25 M NaCl, 0.005 M EDTA, and 0.1 % NP-40) supplemented with a cocktail of protease inhibitors. Samples were sonicated using an ultrasonic dismembrator (Fisher Scientific, Pittsburgh, PA) at setting 7 for seven to eight strokes, followed by centrifugation at 4 °C for 20 min. The protein concentration was measured by the Bradford protein assay (Bio-Rad, Hercules, CA). Twenty micrograms of total protein for each sample was resolved by 7.5 % SDS-PAGE. The proteins were transferred to a polyvinylidine fluoride membrane, and binding was blocked with 5 % nonfat dry milk in 0.02 M Tris (pH 7.5), 0.15 M NaCl, and 0.1 % Tween 20 (TBST) for 1 h at ambient temperature. The blot was incubated with mouse monoclonal antihuman CR-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in a 1:500 dilution in fresh TBST and 5 % milk, and the bound antibody was detected using horseradish peroxidase conjugated secondary antibody and chemiluminescence. The other primary antibody used was mouse monoclonal anti-β-actin at a dilution of 1:10,000. Immunohistochemistry CR-1 protein expression in 128 tumor tissue samples was confirmed by immunohistochemistry analysis, which was performed on formalin-fixed, paraffin-embedded, 4-μm thick tissue sections using the avidin–biotin–peroxidase complex method. The sections were deparaffinized and dehydrated using a graded series of ethanol solutions. Endogenous peroxidase activity was halted through the administration of 0.3 % hydrogen peroxidase and methanol for 20 min. After having been rinsed in phosphatebuffered saline, the tissue sections were processed in a 0.01-M citrate buffer (pH 6.0) inside a heat-resistant plastic container. Sections were then irradiated in a domestic

microwave oven for 20 min. After microwave irradiation, the slides were allowed to cool at room temperature. The following antibody was applied as the primary antibody: rabbit antibody specific to CR-1 (1:100, MAB2771; R&D Systems, Minneapolis, MN, USA). The sections were incubated with the primary antibody overnight at 4 °C followed by the secondary antibody. The results were visualized with diaminobenzidine. In each immunohistochemistry run, the negative controls were stained without a primary antibody. Two independent observers particularly experienced in immunohistochemistry evaluated the slides. Both readers were blinded to clinicopathological data and patient outcomes. The expression of CR-1 was quantified using a visual grading system based on the extent of staining (percentage of positive tumor cells graded on a scale from 0 to 3: 0<1 %, 1=1–33 %, 2=33–67 %, and 3>67 %) and the intensity of staining (graded on a scale of 0–3: 0= none, 1 = weak staining, 2 = moderate staining, and 3 = strong staining). The combination of extent (E) and intensity (I) of staining was obtained by the product of E × I called EI, varying from 0 to 9 for each spot. The mean EI score was calculated for each NSCLC specimen. EI scores of 0–3 were considered a low expression, and EI scores >3 were considered a high expression [21]. In all of the samples, the evaluations of the two observers were identical, the remaining slides were reevaluated, and consensus decisions were made. Statistical analysis All statistical analyses were carried out using the SPSS 17.0 statistical software package. The expression CR-1 messenger RNA (mRNA) and protein between NSCLC and adjacent noncancerous tissue was analyzed by the t test. The χ2 and

Tumor Biol.

Fig. 2 Western blotting analysis of CR-1 protein expression in NSCLC (T) and adjacent noncancerous (N) tissues. The protein expression of CR-1 in NSCLC tissues was significantly higher than that in noncancerous tissues (P<0.001)

Fisher exact tests were used to analyze the relationship between CR-1 expression and the clinicopathological characteristics. Survival curves were plotted by the Kaplan–Meier method and compared using the log-rank test between the high and low expression of CR-1 cases. Survival data were evaluated using the Cox proportional hazards model. Independent prognostic factors were determined by multivariate analysis. P<0.05 was considered to be statistically significant.

Results CR-1 mRNA expression in NSCLC tissue and noncancerous tissue We examined the CR-1 mRNA expression in 35 pairs of NSCLC and adjacent noncancerous tissues using qRT–PCR. Results showed that CR-1 mRNA level was significantly higher in NSCLC tissue compared to that in adjacent noncancerous tissue (1.46±0.32 vs. 0.37±0.15, P<0.001). Thus, CR1 may play important roles in the progression of NSCLC (Fig. 1). CR-1 protein expression in NSCLC tissue and noncancerous tissue To investigate whether CR-1 was also elevated at the protein level, Western blotting was performed on the same specimens

Fig. 3 Immunohistochemical analysis of CR-1 in NSCLC patients. a Low expression level of CR-1. b High expression level of CR-1. a, b Original magnification×100

that were used in the detection of CR-1 mRNA. The Western blot analysis of CR-1 protein also showed that the expression of CR-1 protein was significantly higher in NSCLC tissue compared to that in adjacent noncancerous tissue (1.58±0.43 vs. 0.38±0.17, P<0.001) (Fig. 2). Relationship between CR-1 expression and clinicopathological characteristics of NSCLC patients To further determine whether CR-1 protein upregulation is linked to the clinical characteristics of NSCLC patients, we examined the expression of CR-1 protein in 128 NSCLC tissue samples by immunohistochemistry. The positive immunoreactivity of CR-1 was localized in the membrane and the intracytoplasm of the lung cancer. According to the CR-1 immunoreactive intensity, 70 (54.7 %) patients were classified as high CR-1 group, and 58 (45.3 %) patients were classified as low CR-1 group (Fig. 3). Table 1 summarizes the relationships between CR-1 expression and clinicopathological characteristics of NSCLC patients. We found that the expression of CR-1 protein was significantly correlated with poor tumor differentiation, TNM stage, and lymph node metastasis of NSCLC patients (P= 0.002, 0.004, and 0.001, respectively). However, statistical analysis revealed no significant correlations between CR-1 expression and age, gender, smoking condition, histological type, and tumor size.

Tumor Biol.

stage, lymph node metastasis, and CR-1 protein expression were significantly associated with overall survival of NSCLC patients. By multivariate analysis, we showed that CR-1 protein expression and TNM stage were independent prognostic factors for overall survival of NSCLC patients.

Discussion

Fig. 4 Overall survival rate of NSCLC patients estimated according to the CR-1 expression level in NSCLC tissue with immunohistochemical staining. The 5-year survival rate was 29.3 % in the low CR-1 expression group, whereas it was only 14.3 % in the high CR-1 expression group (P=0.038)

Correlation between CR-1 expression levels and patient survival The 5-year survival rate of 128 patients was 21.1 %. From Kaplan–Meier survival curves, we observed that patients with high CR-1 expression survived a shorter survival time than patients with low CR-1 levels (5-year survival rates, 14.3 and 29.3 %, respectively), and the result was statistically significant (χ2 =4.302, P=0.038, log-rank test) (Fig. 4). Thus, the expression of CR-1 protein could affect the prognosis of NSCLC patients. To evaluate the possibility of CR-1 used as an independent risk factor for poor prognosis, conventional clinicopathological factors and CR-1 protein levels were assessed by Cox’s univariate and multivariate hazard regression model (Table 2). Univariate analysis indicated that tumor differentiation, TNM

Human CR-1 is a member of the EGF-CFC family, regulates essential steps in early embryogenesis, and has a key role in cell migration, angiogenesis, and stem cell maintenance [6, 7]. Numerous studies have demonstrated high expression levels of CR-1 to correlate with malignant transformation, tumor invasiveness, metastatic spreading, and poor prognosis [19, 22, 23]. However, the prognostic significance of CR-1 in NSCLC is still unclear. In this study, we investigated CR-1 expression in NSCLC and its correlation with clinicopathological characteristics, including the survival of patients with NSCLC. In the present study, we showed that CR-1 expression determined by qRT–PCR and Western blot was significantly higher in NSCLC tissues than those in adjacent noncancerous tissues. These observations support the hypothesis that CR-1 may function as an oncogene in NSCLC and also suggest that CR-1 may play an important role in the tumorigenesis of NSCLC. We further assessed the CR-1 protein in NSCLC tumor specimens by immunohistochemistry and analyzed its correlation to clinicopathological characteristics. We found that CR-1 expression was significantly higher in cancerous than in noncancerous tissues, which was consistent with previous findings in other cancer types [23]. Additionally, CR-1 positively correlates with poor tumor differentiation, TNM stage, and lymph node metastasis in NSCLC patients. It

Table 2 Univariate and multivariate analysis of prognostic factors in 128 NSCLC patients Variables

CR-1 protein (high vs. low) Age (<60 vs. ≥60) Gender (male vs. female) Smoking condition (nonsmoker vs. smoker) Histological type (squamous cell carcinoma vs. adenocarcinoma) Tumor size (≤3 cm vs. >3 cm) Tumor differentiation (Well-moderate vs. poor) Lymph node metastasis (N0 vs. N1–3) TNM stage (I–II vs. III–IV) HR hazard ratio, CI confidence interval

Univariate analysis

Multivariate analysis

HR

95 % CI

P

HR

95 % CI

P

1.325 1.214 1.363 1.090 1.109 1.192 1.414 1.291 1.324

0.647–2.214 0.683–2.028 0.868–2.140 0.753–1.578 0.721–1.708 0.693–1.982 1.063–1.879 0.672–2.084 0.678–2.324

<0.001 0.693 0.178 0.647 0.637 0.673 0.017 0.007 0.006

1.245 1.135 1.640 0.271 1.355 1.342 1.321 1.231 1.345

0.563–2.256 0.456–2.135 0.849–3.169 0.731–0.418 0.754–2.437 0.686–2.109 0.684–2.093 0.568–1.873 0.784–2.134

<0.001 0.542 0.141 0.271 0.310 0.149 0.628 0.412 0.002

Tumor Biol.

agrees with the fact that CR-1 plays a role in enhancing cell proliferation of human lung cancer cells and may thereby contribute to the early progression of carcinoma tumors. In the Kaplan–Meier survival analysis, the overall survival of patients with a high CR-1 expression was significantly shorter than that of patients with low CR-1 expression. Univariate analyses showed that increased CR-1 expression in NSCLC tissues is significantly associated with the overall survival rate. Moreover, multivariate analysis demonstrated that CR-1 expression is an independent risk factor in the prognosis of NSCLC patients. Our results not only suggest a potentially promising use of CR-1 as a valuable prognostic marker, but also imply a possible link between the biological function of CR-1 and the pathogenesis of NSCLC. Nonetheless, further studies are needed to elucidate the mechanisms by which CR-1 participates in the development and progression of lung cancer and to address whether CR-1 could be used as a target for therapeutic approaches. In summary, our results indicate that CR-1 is highly expressed in NSCLC and associated with poor tumor differentiation, TNM stage, and lymph node metastasis, as well as poor prognosis of NSCLC patients. Furthermore, CR-1 levels appear to be an independent predictor of survival for patients with NSCLC. Acknowledgments This work was supported in part by a grant from “Twelve-Five Plan” the Major Program of Nanjing Medical Science and Technique Development Foundation (Molecular Mechanism Study on Metastasis and Clinical Efficacy Prediction of Non-small Cell Lung Cancer) (LK-Yu). Conflicts of interest None

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