Overexpression of eukaryotic initiation factor 4E (eIF4E) and its clinical significance in lung adenocarcinoma

Lung Cancer 66 (2009) 237–244

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Overexpression of eukaryotic initiation factor 4E (eIF4E) and its clinical significance in lung adenocarcinoma Rui Wang, Jian Geng, Jing-hua Wang, Xiao-yuan Chu, Huai-cheng Geng, Long-bang Chen ∗ Department of Medical Oncology, Nanjing General Hospital of Nanjing Military Command, PLA, Nanjing 210002, China

a r t i c l e

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Article history: Received 27 September 2008 Received in revised form 31 January 2009 Accepted 4 February 2009 Keywords: Eukaryotic initiation factor 4E (eIF4E) Lung adenocarcinoma p53 Immunohistostaining Prognosis

a b s t r a c t Background: Eukaryotic initiation factor 4E (eIF4E), an important regulator of translation, plays important roles in tumor transformation, progression and metastasis. However, the clinical significance of eIF4E expression in lung adenocarcinoma (AdC) remains unclear. The aim of this study was to explore the expression of eIF4E gene in lung adenocarcinoma cell lines and tissues, and to investigate its relationship with clinical characteristics and prognosis of patients with lung adenocarcinoma in combination with p53 status. Methods: Semi-quantitative RT-PCR and Western blotting assays were performed to detect the expression of eIF4E mRNA and protein in normal human lung epithelial cell line, immortalized lung epithelial cell line and lung adenocarcinoma cell lines. Additionally, the expression of eIF4E gene was also detected in 32 cases of lung adenocarcinoma tissues, tumor adjacent tissues and tumor surrounding normal tissues by the same methods. Moreover, expression of eIF4E and the status of p53 in specimens from 76 patients with lung adenocarcinoma were examined by immunohistochemical staining. Correlations between eIF4E expression and clinicopathological features, and the effect of eIF4E on prognosis of patients with lung adenocarcinoma were evaluated by statistical analysis. Results: The levels of eIF4E mRNA and protein expression were higher in lung adenocarcinoma cell lines and in telomerase-immortalized lung epithelial cell line than in the normal lung epithelial cell line. The expression of eIF4E gene showed statistical difference between tumor tissues, tumor adjacent tissues and tumor surrounding normal tissues (P < 0.05). Moreover, the higher levels of eIF4E expression were correlated with poorer differentiation (P = 0.012), higher pathological stage (P < 0.0001) and clinical stage (P = 0.002), a higher incidence of hematogenous metastasis (P = 0.007) and cancer-related death (P = 0.036). The 5-year survival rate of patients with higher eIF4E expression was significantly lower than that of patients with lower eIF4E expression (P = 0.0045). Furthermore, in a multivariate analysis by Cox regression model, high eIF4E expression was confirmed to be an independent prognostic factor (HR: 2.258; unfavorable, P = 0.0056), while lymph node (HR: 2.033; unfavorable, P = 0.0440) and hematogenous metastasis (HR: 3.489; unfavorable, P < 0.0001) were also significant prognostic factors. Conclusion: High eIF4E expression was correlated with poorer overall survival in lung adenocarcinoma patients. eIF4E might be a better clinical marker predicting the prognosis for lung adenocarcinoma patients in combination with p53 status. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Lung cancer is the leading cause of cancer death in men and women worldwide. There are about 85% of lung cancer cases diagnosed with non-small cell lung cancer (NSCLC), among which adenocarcinoma and squamous cell carcinoma are the two major histological subtypes, although adenocarcinoma is the most predominant histologic subtype in many parts of the world [1,2].

∗ Corresponding author. Tel.: +86 25 80860123. E-mail address: chenlb [email protected] (L.-b. Chen). 0169-5002/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2009.02.001

As for its poor prognosis, it is important to improve the survival by employing a state-of-art molecular technique to diagnose an early lung adenocarcinoma [3,4]. Presently, although many clinical pathological factors have been testified to be associated with prognosis, it is still crucial of exploiting better prognostic markers to improve clinical treatment of patients with lung adenocarcinoma. Eukaryotic initiation factor 4E (eIF4E), a 25-kDa cap binding protein, delivers cellular mRNAs to the eIF4F translation initiation complex by binding the 5 -cap structure of these mRNAs [5,6]. This protein is the limiting component of the eukaryotic protein synthesis initiation complex, therefore, the upregulated levels of eIF4E expression can selectively affect transport of specific transcripts and increase translation of mRNAs encoding some proteins

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contributing to angiogenesis and proliferation such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor 2 (FGF-2), cell-cycle regulatory protein (cyclin D1) and pro-oncogenic protein (c-Myc) which are correlated with malignant transformation, progression and metastasis [7–10]. eIF4E has been considered to be an oncogene in vivo which overexpresses in a variety of malignancies including head and neck squamous carcinoma, colon carcinoma, lymphomas and cervix carcinoma [11–14]. Although some data indicate that eIF4E is a good biomarker for clinical diagnosis and prognosis evaluation, the clinical significance of eIF4E expression and its correlations with p53 status in lung adenocarcinoma remain unclear. In the present study, therefore, RT-PCR and Western blot assays were performed to detect the expression of eIF4E mRNA and protein in normal lung epithelial cell line, telomerase-immortalized lung epithelial cell line and lung adenocarcinoma cell lines. In addition, we also evaluated the difference of eIF4E expression between lung adenocarcinoma tissues, tumor adjacent tissues and tumor surrounding normal tissues at both transcriptional and translational levels. Moreover, we retrospectively examined 76 primary lung adenocarcinoma samples by immunohistochemical staining, and the correlations between eIF4E expression and clinicopathological characteristics including p53 status were investigated. Furthermore, combining follow-up data, the effect of eIF4E alone or along with p53 status on clinical prognosis of patients with lung adenocarcinoma was explored. 2. Materials and methods 2.1. Cell lines and cell culture Four human lung adenocarcinoma cell lines (SPCA-1, A549, Calu6 and Lu-165) were preserved in our laboratory. A normal human lung epithelial cell line (L132) was purchased from the Institute of Cell Biology (Shanghai, China). A telomerase-immortalized lung epithelial cell line (hTERT-c) was established and preserved by our laboratory. All cell lines were maintained in RPMI 1640 medium (Invitrogen, USA) supplemented with 10% fetal–bovine–serum (FBS, JRH Biosciences). Cells were incubated at 37 ◦ C in a humidified atmosphere of 5% CO2 . Experiments were performed with exponentially growing cells. 2.2. Patients and tissue samples During the period from 1998 to 2000, consecutively 108 patients with lung adenocarcinoma who received surgery at the Department of Chest Surgery of Nanjing Hospital were followed up by phone. Thirty-two cases of lung adenocarcinoma tissues, corresponding tumor adjacent tissues (about 1.5-cm distance from tumor edge) and surrounding normal tissues (about 5.0-cm distance from tumor edge) were collected. Moreover, 77 cases of primary lung adenocarcinoma samples were embedded in paraffin. Clinical, operative, histopathological and follow-up data were obtained by medical records in a computerized registry database including patient age, gender, course, smoking condition, tumor site, pathological and clinical stage, histological differentiation, nodal status, and follow-up information. Patients who received preoperative chemotherapy or radiotherapy were excluded from this research. Follow-up focused specifically on recurrence (local and distant), disease-free survival and overall survival. The data collected were entered prospectively into the registry database. This research was approved by the Institutional Review Board and Human Ethics Committee of the Regional Cancer Center. Tumors were classified and graded according to World Health Organization (WHO) criteria. A written informed consent was

Table 1 Patient features (2001–2004). Patients characteristics

No. of patients (%)

Median age (range)

60.3 (36–68)

Gender Male Female

48 (63.2%) 28 (26.7%)

Age (years) ≤55 >55

22 (28.9%) 54 (71.1%)

Smoking condition Non-smokers Smokers

40 (52.6%) 36 (47.4%)

Tumor differentiation Well Moderate Poor

17 (22.5%) 28 (36.8%) 31 (40.7%)

Pathological stage (pT) T1–2 T3–4

36 (47.4%) 40 (52.6%)

Lymph node metastasis (pN) N0 N1 /N2 /N3

26 (34.2%) 50 (65.8%)

Hematogenous metastasis (pM) Absent Present

35 (46.1%) 41 (53.9%)

Clinical stage (p-stage) I–II III

31 (40.8%) 45 (59.2%)

Cancer-related death Alive Death

31 (40.8%) 46 (59.2%)

obtained from all subjects prior to the subjects’ participation (Table 1). 2.3. Reverse transcriptase (RT)-PCR analysis of eIF4E mRNA expression Total RNAs were extracted from lung adenocarcinoma cell lines, immortalized lung epithelial cell line and normal lung epithelial cell line using TRIzol reagent (Invitrogen, USA). RNA was reverse transcribed into cDNA in a 20-␮L reaction system using Superscript First-Strand Synthesis Kit for RT-PCR (Promega Inc.) under conditions described by the supplier. First-strand cDNAs were synthesized and analyzed by PCR to detect the expression of ␤-actin and eIF4E. The primers were as follows: eIF4E, forward: 5 -ATGGCGACTGTCGAACCGG-3 , reverse: 5 -GCTATCTTATCACCTTTAGC-3 ; ␤-actin, forward: 5 -ATGGATGATGATGATATCGCC3 , reverse: 5 -GTGAT-GACCTGGCCGTCAGG-3 . PCR products were visualized by ethidium bromide staining of 1.5% agarose gel. 2.4. Western blot analysis of eIF4E protein expression Total protein extracts from cells or tissues were separated on SDS (10–12%) polyacrylamide gel electrophoresis (20–50 g/lane), and electro-transferred to a PVDF (polyvinylidene fluoride) membrane. Anti-eIF4E (1:10,00) and anti-actin (1:5000) antibody (Santa Cruz Biotechnology, Heidelberg, Germany) were diluted in TBST (Tris-buffered saline/Tween) (5% milk powder) and incubated at 4 ◦ C overnight. The appropriate secondary antibody was applied (1:2000; horseradish peroxidase anti-mouse and horseradish peroxidase anti-rabbit) at room temperature for 1 h. Visualization was performed by enhanced chemiluminescence (ECL; Amersham).

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Fig. 1. RT-PCR analysis of eIF4E mRNA expression in normal lung epithelial cell line (L132), an immortalized cervical epithelial cell lines (hTERT-c) and lung adenocarcinoma cell lines (SPCA-1, A549, Calu-6 and Lu-165). ␤-Actin was used to normalize for any differences in mRNA loading between lanes.

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The immunoreactivity was evaluated in five areas of each slide for correlation and confirmation of the tissue diagnosis, and the intensity of the immunoreactivity (intensity score) according to the positive cells was stratified into four categories: ≤25% of cells staining positive, 1 score; 26–50% of cells staining positive, 2 score; 51–75% of cells staining positive, 3 score; >75% of cells staining positive, 4 score; as previously described [15,16]. An intensity score of ≥2 with at least 50% of tumor cells with positive eIF4E staining was used to classify tumor patients with high expression group and <2 intensity score with <50% of tumor cells with positive eIF4E staining was used to classify tumor patients with low expression group. In the tumor, positive p53 staining was the presence of an unequivocal brown stain in the nucleus of 10% or more of tumor cells. All slides were scored in the absence of any clinical data simultaneously assessed by two observers (J.-G., and H.C.-G.) who were blinded to the clinical features and outcomes. Each observer estimated the percentage of cells stained and graded the intensity of immunostaining based on a visual assessment of the intensity of brown reaction product within the cell cytoplasm. In a final discussion round all slides were reviewed and the results were confirmed by a third observer (R.-W.). The final immunostaining score reported was the average of three observers. 2.6. Statistical analysis

Fig. 2. Western blot analysis of eIF4E mRNA expression in normal lung epithelial cell line (L132), an immortalized cervical epithelial cell lines (hTERT-c) and lung adenocarcinoma cell lines (SPCA-1, A549, Calu-6 and Lu-165). ␤-Actin was used to normalize for any differences in protein loading between lanes.

2.5. Immunohistochemical staining Immunohistochemical analysis was performed retrospectively. Resected lung adenocarcinoma tissues were fixed in 10% formaldehyde and embedded in paraffin. Sections (4 ␮m), cut from the original paraffin blocks, were deparaffinized in xylene and rehydrated in graded alcohols and distilled water. After inhibition of endogenous peroxidase activity for 30 min with methanol containing 0.3% H2 O2 , the sections were blocked with 10% normal goat serum (Invitrogen, USA) for 20 min and incubated overnight with rabbit anti-human eIF4E (diluted 1:500) or anti-human p53 polyclonal antibody (diluted 1:300, Santa Cruz Biotechnology, CA) at 4 ◦ C. The sections were then incubated with biotinylated anti-rabbit IgG for 30 min at room temperature, followed by incubation with peroxidase-conjugated avidin/biotin complexes and stained with 3,3-diaminobenzidine tetrahydrochloride (DAB). Finally, the sections were counter stained with hematoxylin. Normal rabbit serum was used as a negative control for the staining reactions.

Statistical analyses were performed using SPSS 10.0 statistical software (SPSS Inc., Chicago, IL). The difference was evaluated by Student’s t-test and chi-square test. Mann–Whitney U-test was used to analyze the relationship between eIF4E expression and clinicopathologic characteristics. The postoperative survival curves were calculated using the Kaplan–Meier method and differences in the survival rates were analyzed using the log-rank test. Univariate and multivariate data analysis of the prognostic factors was performed with Cox’s regression model. P < 0.05 was considered to indicate a significant difference.

3. Results 3.1. Detection of eIF4E mRNA and protein expression in cell lines To detect the levels of eIF4E mRNA and protein expression in lung epithelial cell lines, semi-quantitative RT-PCR and Western blotting assays were performed in the following cell lines: a normal lung epithelial cell line (L132), an immortalized lung epithelial cell line (hTERT-c), and four lung adenocarcinoma cell lines (SPCA1, A549, Calu-6 and Lu-165). As shown in Figs. 1 and 2, the levels of eIF4E mRNA and protein expression were comparatively higher in lung adenocarcinoma cell lines, intermediate in immortalized lung epithelial cells, and lower in normal lung epithelial cells.

Fig. 3. Representative results of eIF4E mRNA expression in different lung tissues by RT-PCR (Cases 7, 13, and 22). Lane T: tumor tissues; Lane A: tumor adjacent tissues; Lane S: tumor surrounding tissues.

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Fig. 4. Representative results of eIF4E protein expression in different lung tissues by Western blotting (Cases 7, 13, and 22). Lane T: tumor tissues; Lane A: tumor adjacent tissues; Lane S: tumor surrounding tissues.

3.2. Detection of eIF4E mRNA and protein expression in tumor tissues

3.3. Immunohistochemical staining of eIF4E or p53 protein in tumor tissues

Next, we detected the levels of eIF4E mRNA and protein expression in lung adenocarcinoma tissues, correspondingly tumor adjacent tissues and surrounding normal tissues, respectively. As shown in Figs. 3 and 4, the levels of eIF4E mRNA and protein expression showed significantly difference between lung adenocarcinoma tissues (mRNA: 0.801 ± 0.078; protein: 0.745 ± 0.056), tumor adjacent tissues (mRNA: 0.324 ± 0.047; protein: 0.338 ± 0.026) and surrounding normal lung tissues (mRNA: 0.125 ± 0.026; protein: 0.062 ± 0.008).

In this report, immunohistochemistry was also performed to determine expression and subcelluar localization of eIF4E or p53 protein in the 76 paraffin-embedded lung adenocarcinoma specimens. We observed that the eIF4E staining was located in the cytoplasm of lung adenocarcinoma cells and the p53 staining was located in the nucleus of tumor cells. Thirty-five specimens showed low eIF4E expression (46.1%; Fig. 5A and B), and 41 specimens showed high eIF4E expression (53.9%; Fig. 5C and D). Forty-one specimens showed negative expression of p53 protein (53.9%;

Table 2 Relationship between eIF4E protein expression and clinicopathological factors. Clinicaopathological factors

High eIF4E protein expression (n = 41)

Low eIF4E protein expression (n = 35)

P-value

N

%

N

%

Gender Male Female

26 15

63.4 36.6

22 13

62.8 37.2

Age at surgery Mean ± S.D. ≤55 >55

61.3 ± 3.12 12 29

29.3 70.7

59.6 ± 5.34 10 25

28.6 71.4

Smoking condition Non-smokers Smokers

22 19

53.7 46.2

18 17

51.4 48.6

0.846

Tumor differentiation Well Moderate Poor

4 16 21

9.8 39.0 51.2

13 12 10

37.1 34.2 28.5

0.012*

pT factor T1–2 T3–4

8 33

19.5 80.5

28 7

80.0 20.0

<0.0001*

12 29

29.3 70.7

14 21

48.8 51.2

0.326

13 28

31.7 68.3

22 13

62.9 37.1

0.007*

Clinical stage I–II III

10 31

24.4 75.6

21 14

60.0 40.0

0.002*

Cancer-related death Alive Death

12 29

29.2 70.8

19 17

51.4 48.6

0.036*

pN factor N0 N1 /N2 /N3 pM factor Absent Present

N, number; S.D., standard deviation. * Statistically significant difference (P < 0.05).

0.960

0.947

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Fig. 5. Immunohistochemical staining of eIF4E or p53 protein expression in lung adenocarcinoma tissues. eIF4E protein expression was mainly localized within cytoplasma, and p53 protein expression was mainly localized within nucleus (200×). (A) Low eIF4E expression (1+); (B) low eIF4E expression (2+); (C) high eIF4E expression (3+); (D) high eIF4E expression (4+); (E) negative expression of p53; (F) positive expression.

Fig. 5E), and 35 specimens showed positive expression of p53 protein (46.1%; Fig. 5F) 3.4. Relationship between eIF4E expression and clinicopathologic factors

carcinoma patients with higher eIF4E protein expression showed poorer differentiation, higher pathological and clinical stage, a higher incidence of hematogenous metastasis and cancer-related death. 3.5. Correlations of eIF4E expression with patient survival

The relationship between eIF4E expression in lung adenocarcinoma and clinicopathologic factors is summarized in Table 2. The difference by statistical analysis indicated that the level of eIF4E protein expression was correlated with poorer differentiation (P = 0.012), higher pathological stage (P < 0.0001) and clinical stage (P = 0.002), a higher incidence of hematogenous metastasis (P = 0.007) and cancer-related death (P = 0.036). However, there were no obvious differences in gender, age, smoking condition and lymph node metastasis between the higher and lower expression groups. Thus, these experimental data suggested that lung adeno-

For all patients, the 5-year overall survival rate of the higher eIF4E expression group was 37.3% compared with a rate of 59.8% for the lower eIF4E expression group. From Kaplan–Meier survival curves, we observed that lung adenocarcinoma patients with higher eIF4E expression showed significantly shorter survival than those with lower eIF4E expression, which showed a significantly poorer prognosis for patients with higher eIF4E expression (P = 0.024, log-rank test; Fig. 6A). Additionally, the 5-year overall survival rate of pStage I–II patients with high eIF4E expres-

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Fig. 6. Kaplan–Meier survival curve of lung adenocarcinoma patients stratified by eIF4E protein expression or pStage I–II. (A) Patients whose tumors had high eIF4E protein expression showed significantly shorter survival after surgery than patients whose tumors had low eIF4E protein expression (P = 0.024; log-rank test); (B) pStage I–II patients with high eIF4E expression showed significantly shorter survival after surgery than patients with pStage I–II patients with low eIF4E expression (P < 0.01; log-rank test).

sion group (47.8%) was significantly lower than that of pStage I–II patients with low eIF4E expression group (61.4%; P < 0.01, log-rank test; Fig. 6B). Furthermore, we also analyzed the mean disease-free survival between two eIF4E expression groups. The mean disease-free survival of the high eIF4E expression group [5.22 years (95% CI, 4.58–6.46)] was also significantly lower than that of the low eIF4E expression group [7.03 years (95% CI, 6.15–8.24)]. 3.6. Relationship between eIF4E and p53 expressions In 76 patients with lung adenocarcinoma, the positive rate of p53 expression was 46.1%. There were 41 cases with higher eIF4E expression, among which there were 20 cases with aberrantly positive expression of p53; there were 35 cases with lower eIF4E expression, among which there were 15 cases with positive

Fig. 7. Kaplan–Meier survival curve of lung adenocarcinoma patients according to the status of p53. (A) Patients whose tumors had positive p53 protein expression showed significantly shorter survival after surgery than patients whose tumors had negative p53 protein expression (P= 0.007; log-rank test). (B) eIF4E(H)/p53(+) group showed significantly shorter survival after surgery than other groups [eIF4E(L)/p53(−), P = 0.0015; eIF4E(H)/p53(−), P = 0.0033; eIF4E(L)/p53(+), P = 0.0126].

expression of p53. There was no obvious correlation between eIF4E expression and p53 status (P > 0.05). According to p53 status, we analyzed the postoperative survival of patients. As shown in Fig. 7A, we observed that the 5-year overall of the p53-positive group (71.4%) was significantly higher than that of the p53-negative group (31.7%) (P = 0.007). In addition, to further determine the prognostic value of eIF4E expression combined with p53 status, we divided all the patients into the following groups: high eIF4E expression and p53-positive group [eIF4E(H)/p53(+)]; high eIF4E expression and p53-negative group [eIF4E(H)/p53(−)]; low eIF4E expression and p53-positive group [eIF4E(L)/p53(+)]; low eIF4E expression and p53-negative group [eIF4E(L)/p53(−)]. Kaplan–Meier survival curves showed that the eIF4E (H)/p53(+) group had the poorest prognosis, with a 5-year overall survival rate of 27.6%, which was obviously lower than that of other groups (Fig. 7B; P < 0.05 for all groups).

Table 3 Cox’s regression model analysis of prognostic factors in lung AdC patients. Variables

Unfavorable/favorable

Age (years) Smoking condition Tumor differentiation pT factor pN factor pM factor p53 expression eIF4E expression

≥55/<55 Smokers/non-smokers Poor/well + moderate T3–4 /T1–2 N1 + N2 + N3 /N0 Present/absent Positive/negative High/low

HR, hazard ratio; 95% CI, 95% confidence interval. * P < 0.0001.

Univariate analysis

Multivariate analysis

HR (95% CI)

P-value

HR (95% CI)

P-value

0.843 (0.43–1.86) 1.425 (0.63–3.49) 2.723 (1.85–4.00) 2.182 (1.33–3.88) 5.187 (1.46–9.36) 7.531 (4.17–13.65) 7.450 (2.53–12.45) 1.681 (1.06–2.67)

0.7451 0.4260 0.000* 0.0067 0.0188 0.000* 0.0080 0.0260

1.587 (0.05–1.82) 2.352 (0.31–4.23) 1.237 (0.74–4.09) 4.515 (0.17–9.18) 2.033 (1.02–4.06) 3.489 (2.03–8.35) 0.744 (0.39–1.42) 2.258 (1.76–5.15)

0.1244 0.7632 0.4010 0.2118 0.0440 0.000* 0.3710 0.0056

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3.7. Univariate and multivariate analyses for prognosis of lung adenocarcinoma patients Univariate and multivariate data analyses were performed using the Cox proportional hazards regression model to determine the prognostic value of eIF4E expression (Table 3). Tumor differentiation (poor/well + morderate), pathological stage (T3–4 /T1–2 ), lymph node (N1 + N2 + N3 /N0 ) or hematogenous (present/absent) metastasis, aberrant expression of p53 (positive/negative) and high eIF4E expression (score of ≥2/scores of <2) were the prognostic factors to predict a poor prognosis in univariate analysis (P= <0.0001, 0.0067, 0.0188, <0.0001, <0.0001, 0.0080 and 0.0260, respectively). By multivariate analysis of the prognostic factors, we confirmed that high eIF4E expression was an independent prognostic factor (unfavorable; risk ratio: 2.258; 95% confidence interval: 1.76–5.15; P = 0.0056), while lymph node or hematogenous metastasis were also independent prognostic factors (P = 0.0440 and <0.0001).

4. Discussion Lung adenocarcinoma (AdC) is the most common type of lung cancer, accounting for 30–35% of all cases. During the past 30 years, the frequency of adenocarcinomas has increased and squamous cell carcinomas (SCC) have decreased, which is due to a true change in the biological occurrence [17]. Despite improvements made in detection and treatments during the past two decades, the clinical behaviors of lung adenocarcinoma remain poor, with an unsatisfactory survival of clinical patients. Up to now, there have been many diagnostic and prognostic markers identified and investigated for lung adenocarcinoma, but a useful screening marker for patients with lung adenocarcinoma has not been clearly established [18]. Therefore, the identification of new molecular prognostic and predictive markers in lung adenocarcinoma helps to exactly evaluate the prognosis of patients and add further prognostic information, select high-risk patients for aggressive adjuvant treatments and set new anticancer therapies. Translation initiation factor 4E (eIF4E), a 25-kDa subunit of the cap binding complex eIF4F, is an important regulator and rare-limited molecule of translation. The most frequently aberrant change in the translational apparatus is the upregulated levels of eIF4E expression, which selectively affects transport of specific transcripts, increases cap-dependent translation, suppresses apoptosis and induces malignant transformation [19–21]. eIF4E gene has been reported to be considered as an oncogene in vivo, and its overexpression significantly promotes cell growth, proliferation, tumor invasion and metastasis [22]. Several groups have reported that the degree of eIF4E overexpression predicts cancer recurrence and outcome in breast cancer or head neck squamous cell carcinoma (HNSCC) patients [23,24]. Moreover, other studies also showed a significantly positive correlation between eIF4E overexpression and aggressive tumor behaviors such as poor differentiation and metastasis. Previously, our study indicated that the downregulation of eIF4E expression mediated by RNA interference (RNAi) could significantly inhibit cell growth, induce apoptosis and enhance chemosensitivity in breast carcinoma cells [25]. eIF4E is overexpressed in a variety of malignant cell lines and tissues, including lung cancer [26]. However, the actual role of eIF4E expression in lung adenocarcinoma still remains unclear. In this report, we firstly showed that the levels of eIF4E mRNA and protein expression were significantly higher in lung adenocarcinoma cell lines than in normal human lung epithelial cell line. Interestingly, the telomerase-immortalized lung epithelial cell also had a comparatively higher level of eIF4E mRNA and protein expression. Next, we evaluated the expression of eIF4E gene in lung adenocarcinoma tissues, tumor adjacent tissues and sur-

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rounding normal lung tissues. Results from RT-PCR and Western blotting assays indicated that the levels of eIF4E mRNA and protein expression were significantly higher in tumor tissues than in tumor adjacent tissues and surrounding normal lung tissues. With attention to the comparatively high levels of eIF4E expression in tumor adjacent tissues, we concluded that eIF4E might be correlated with invasion of lung adenocarcinomas. In addition, the results of immunohistochemical analysis confirmed that many adenocarcinoma cells exhibited low to high eIF4E staining mainly in the cytoplasm. By statistical analysis of the correlation between eIF4E expression and clinical features of patients with lung adenocarcinoma, we showed that higher eIF4E expression was closely correlated with poorer tumor differentiation, higher pathological or clinical stage, and hematogenous metastasis. Moreover, the most important point in this study was found that higher eIF4E expression was closely correlated with a higher incidence of cancerrelated death for patients with lung adenocarcinoma. By survival analysis, it was indicated that patients with high eIF4E expression showed shorter survival after surgery than patients with low eIF4E expression and high eIF4E expression was found as an independent prognostic predicator on 5-year postoperative survival. Mutations of p53, with frequencies approximately 50% in lung cancer, which can induce loss of tumor-suppressor function and cellular proliferation, has been reported to play a vital role in lung carcinogenesis [27]. Nathan et al. previously showed that the overexpression of eIF4E in the margins appeared to be a more sensitive indicator of recurrence in larynx cancers and might be an earlier event in the process of tumorigenesis than p53 [28], but there have no reports about the correlation between eIF4E expression and p53 status in lung adenocarcinoma. Immunohistochemical results showed that aberrant p53 staining was mainly located in the nucleus of lung adenocarcinoma cells. Moreover, the 5-year overall survival rate of lung adenocarcinoma patients was associated with the status of p53. Although the obvious correlation between eIF4E expression and p53 status was not found in our study, the eIF4E (H)/p53(+) group had the poorest prognosis, with a 5-year overall survival rate of 27.6%, which was significantly lower than that of other groups [eIF4E(H)/p53(−), eIF4E(L)/p53(+) and eIF4E(L)/p53(−)]. Thus, the prognostic importance of eIF4E overexpression in lung adenocarcinoma could be enhanced in combination with p53 status. Taken together, our findings showed that eIF4E was overexpressed in lung adenocarcinoma and higher eIF4E expression was associated with poorer tumor differentiation, higher pathological or clinical stage, regional lymph node or hematogenous metastasis, inducing a higher rate of cancer-related death. Additionally, those lung adenocarcinoma patients with higher eIF4E expression showed a shorter 5-year postoperative survival compared with those patients with lower eIF4E expression. Furthermore, eIF4E might be a better clinical marker predicting the prognosis for lung adenocarcinoma patients in combination with p53 status. Conflict of interest On behalf of all co-authors, we declare that there are no financial or other relationships that might lead to a conflict of interest of the present article. This manuscript has been read and approved by all the authors. References [1] Little AG, Gay EG, Gaspar LE, Stewart AK. National survey of non-small cell lung cancer in the United States: epidemiology, pathology and patterns of care. Lung Cancer 2007;57:253–60. [2] Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, et al. Cancer statistics, 2006. CA Cancer J Clin 2006;56:106–30.

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