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Prognostic significance of expression of eukaryotic initiation factor 4E and 4E binding protein 1 in patients with pathological stage I invasive lung adenocarcinoma
Lung Cancer 70 (2010) 329–334
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Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
Prognostic significance of expression of eukaryotic initiation factor 4E and 4E binding protein 1 in patients with pathological stage I invasive lung adenocarcinoma Nobuhiko Seki a,∗ , Tasaburo Takasu b , Shigeki Sawada c , Masao Nakata d , Rieko Nishimura e , Yoshihiko Segawa f , Reishi Shibakuki g , Toshiaki Hanafusa b , Kenji Eguchi a a
Division of Medical Oncology, Department of Internal Medicine, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan First Department of Internal Medicine, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan c Department of Surgery, National Hospital Organization Shikoku Cancer Center, Matsuyama, Ehime 791-0288, Japan d Division of Thoracic and Cardiovascular Surgery, Department of Surgery, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan e Department of Pathology, National Hospital Organization Shikoku Cancer Center, Matsuyama, Ehime 791-0288, Japan f Department of Medicine and Thoracic Oncology, National Hospital Organization Shikoku Cancer Center, Matsuyama, Ehime 791-0288, Japan g Department of Pathology, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan b
a r t i c l e
i n f o
Article history: Received 24 October 2009 Received in revised form 13 January 2010 Accepted 11 March 2010 Keywords: Translational control eIF4E eIF4E binding protein 1 Prognosis Lung adenocarcinoma
a b s t r a c t Background: Both eukaryotic initiation factor 4E (eIF4E) and eIF4E binding protein 1 (4E-BP1) are involved in the malignant progression of human cancers. However, the role of eIF4E and 4E-BP1 expression as prognostic markers has not been evaluated concurrently in any human cancers. Methods: The expression of eIF4E and 4E-BP1 was semiquantitatively examined with immunohistochemical staining in 80 patients with pathological stage I invasive lung adenocarcinoma. Results: The 10-year survival rate was significantly lower for patients with high eIF4E expression (64.0% [n = 36]) than for patients with low eIF4E expression (89.9% [n = 44], P = 0.024), and in patients with high eIF4E expression the 10-year survival rate was lower for patients with low 4E-BP1 expression (39.0% [n = 12]) than for patients with high 4E-BP1 expression (85.2% [n = 24], P = 0.036). In patients with low eIF4E expression, the 10-year survival rate was lower for patients with low 4E-BP1 expression (87.6% [n = 36]) than for patients with high 4E-BP1 expression (100% [n = 8]), but statistical analysis was impossible because all patients with high 4E-BP1 expression were censored. Unfavorable prognostic factors for survival were age greater than 65 years (P = 0.015), pathological stage IB disease (P = 0.045), high eIF4E expression (P = 0.008), and low 4E-BP1 expression (P = 0.007). Conclusions: Both eIF4E and 4E-BP1 are potential new prognostic factors for survival and stratification in patients with pathological stage I lung adenocarcinoma. The eIF4E and 4E-BP1 status may provide a basis for individualized therapy. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer is a leading cause of cancer death in most industrialized countries. Among the various histological types of lung cancer, adenocarcinoma is rapidly becoming more prevalent and now accounts for more than 50% of all lung cancers [1]. However, its poor survival rate has not improved significantly in the past two decades. Even in pathological stage IA lung adenocarcinoma, the 5-year survival rate is at most 80% [2]. To further improve survival rates in patients with adenocarcinoma, prognostic classifications based on tumor biology will be crucial. Such classifications might
∗ Corresponding author. Tel.: +81 3 3964 1211x1587; fax: +81 3 3964 7094. E-mail address: [email protected] (N. Seki). 0169-5002/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2010.03.006
help physicians select the most appropriate treatment for each subset of patients. Translational control is an integral part of the regulation of gene expression [3–5]. Changes in the efficiencies of mRNA translation influence gene expression at the protein level, resulting in alterations of cell function and phenotype [5–10]. In eukaryotes, almost all mRNAs are modified at their 5 -termini with an m7 GpppN cap structure (where N is any nucleotide) [11]. Translation of such mRNAs typically requires cap-dependent unwinding of the secondary structure in the 5 -untranslated regions by the initiation complex eIF4F. This complex consists of the cap-binding protein eIF4E, the adaptor protein eIF4G, and the ATP-dependent RNA helicase eIF4A [4–6]. Because eIF4E is the least abundant of the various translation initiation factors [12] and is, therefore, the rate-limiting component for cap-dependent translation
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[13], changes in the levels of eIF4E profoundly affect translation rates. Recent in vitro studies have shown that experimental overexpression of eIF4E preferentially enhances the selective synthesis of malignancy-related proteins, although their translation is repressed under normal cellular conditions [5,9,14,15]. These upregulated key proteins promote tumorigenesis (c-myc, cyclin D1), angiogenesis (fibroblast growth factor 2, vascular endothelial growth factor), and metastasis (heparanase, matrix metalloproteinase 9, and CD44v6) [14]. An in vivo study has revealed that ectopic eIF4E expression in transgenic mice increases the incidence of multiple cancers, including lung adenocarcinoma (up to 21%), lymphoma, hepatoma, and angiosarcoma [16]. A major signaling pathway through which eIF4E activity controls cancer is the phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, via the phosphorylation of eIF4E binding proteins (4E-BPs) [17]. The 4E-BPs, of which 3 isoforms are present in mammalian cells, are suppressors of eIF4E activity [18]. When 4E-BPs are active (nonphosphorylated), they bind to eIF4E and impede formation of the initiation complex; translation is then blocked and apoptosis is favored. However, when 4E-BPs are inactive (phosphorylated), their affinity for eIF4E is reduced, eIF4E is released, and cap-dependent translation can start. Thus, induction of the constitutively active 4E-BPs leads to cell cycle arrest, which correlates with the loss of cyclin D1 expression and increased levels of the cyclin-dependent kinase inhibitor p27Kip1 [19]. In contrast, constitutive activation of the PI3K/Akt/mTOR signaling pathway leads to inactivation of the tumor-suppressor activity of 4E-BPs, and in turn, contributes to cancer development [17]. In this regard, patients in whom 4E-BPs are highly phosphorylated or are expressed at low levels regardless of their phosphorylation status exhibit tumor progression and have an unfavorable prognosis [20]. During the last decade, ubiquitous eIF4E expression or 4E-BP1 expression has been linked to a variety of human cancers, such as those of the breast, esophagus, stomach, colon, head and neck, lung, cervix, ovary, prostate, and bladder, and to lymphoma [21–24]. However, to our knowledge, the roles of eIF4E expression and 4EBP1 expression as prognostic markers have not been concurrently evaluated in any human cancers. In this study, we immunohistochemically investigated eIF4E expression and 4E-BP1 expression to evaluate their prognostic significance in 80 patients with pathological stage I invasive lung adenocarcinoma.
2. Materials and methods 2.1. Patients and tissue samples Approval for this study was obtained from the Institutional Review Board. Specimens of pathological stage I invasive lung adenocarcinoma from 80 patients who had undergone curative surgical resection without prior chemotherapy or radiotherapy from January 1985 through December 1997 were retrieved from the archives of the Department of Pathology of the National Hospital Organization Shikoku Cancer Center, Ehime, Japan. On the basis of the World Health Organization lung tumor classification system, tissue samples were subclassified into the following histological groups: papillary adenocarcinoma, acinar adenocarcinoma, and solid adenocarcinoma with mucin formation [25]. Mixed adenocarcinomas with bronchioloalveolar features were excluded from this study because of their histological and prognostic heterogeneity [26]. The medical charts of all patients were reviewed, and the clinicopathological characteristics are summarized in Table 1. None of the patients received postoperative adjuvant chemotherapy. The median duration of follow-up was 7.8 years (range, 1.1–16.2 years). 2.2. Immunohistochemical studies Immunohistochemical studies were performed as described previously [27]. To detect eIF4E and 4E-BP1, a mouse monoclonal anti-human eIF4E primary antibody (Transduction Laboratories, Lexington, KY) and a rabbit polyclonal anti-human 4E-BP1 primary antibody (New England Biolabs, Beverly, MA), respectively, were used. On the basis of previous investigations [27,28], the intensity in bronchial epithelial cells was defined as an internal control to evaluate eIF4E staining intensity in tumor cells. The staining intensity in tumor cells was designated as 4Elow when it was less than this internal control and was otherwise designated as 4Ehigh . Because considerable degree of heterogeneity in 4E-BP1 staining was observed in stromal cells, bronchial epithelial cells, and interstitial lymphocytes as well as tumor cells, and because the levels of 4E-BP1 staining in these nontumor cells were relatively weak compared with those of eIF4E staining, at least 1000 tumor cells in representative fields were manually counted in each section to evaluate 4E-BP1 staining intensity in tumor cells. Tumor cells showing either cytoplasmic or nuclear 4E-BP1 expression were considered 4E-BP1-positive tumor cells as described previ-
Table 1 Characteristics of patients with pathological stage I invasive lung adenocarcinoma according to eIF4E and 4E-BP1 status. Total
Pa
eIF4E expression Low (n = 44)
High (n = 36)
Age (years) ≤65 >65
39 41
24 20
15 21
Sex Male Female
30 50
13 31
17 19
Smoking Nonsmoker Smoker
48 32
31 13
17 19
Histological subtype Papillary Acinar Solid
64 15 1
35 8 1
29 7 0
Pathological stage IA IB
59 21
35 9
24 12
a
Chi-square test.
Pa
4E-BP1 expression Low (n = 48)
High (n = 32)
27 21
12 20
18 30
12 20
30 18
18 14
38 9 1
26 6 0
38 10
21 11
0.36
0.16
0.16
0.99
0.03
0.58
0.66
0.71
0.30
0.28
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Fig. 1. Expression of eIF4E and 4E-BP1 in invasive lung adenocarcinoma. (A) Anti-eIF4E antibody. (B) Anti-4E-BP1 antibody. Cells expressing eIF4E and 4E-BP1 show brown staining (original magnification 200×).
ously [29–31]. On the basis of the histogram of 4E-BP1-positive tumor cells, cases were divided into 2 subgroups using the median value (45%) as a cutoff point. Cases were considered 4E-BP1low if the percentage of 4E-BP1-positive tumor cells was <45% but were otherwise considered 4E-BPhigh . All sections were evaluated by 2 independent investigators (N.S. and R.N.) in a blinded manner without any information regarding clinicopathological characteristics. 2.3. Statistical analysis The postoperative period was measured from the date of surgery to the date of the last follow-up or death. Patients who died of other causes were censored. The Chi-square test was used to evaluate the association between the patient characteristics (listed in Table 1) and the levels of either eIF4E expression or 4E-BP1 expression. Survival curves were constructed with the Kaplan–Meier method, and the statistical significance of differences in survival rates between groups of patients was evaluated with the log-rank test. Multivariate Cox proportional hazards model analysis was performed to identify significant independent prognostic factors for survival. All calculations were performed with StatView 5.0J software (SAS Institute Inc., Cary, NC). Differences were considered significant when P < 0.05.
3. Results 3.1. Immunohistochemical analysis for eIF4E and 4E-BP1 The typical immunostaining patterns of eIF4E and 4E-BP1 in invasive lung adenocarcinomas are shown in Fig. 1. eIF4E expression was homogeneously positive in the cytoplasm of tumor cells, stromal cells (mainly consisting of fibroblasts), bronchial epithelial cells, and normal interstitial lymphocytes, whereas 4E-BP1 expression was heterogeneously positive in the cytoplasm of these cells. Only a few cells that had intense cytoplasmic staining showed nuclear staining. In contrast, both eIF4E expression and 4E-BP1 expression were negative in alveolar epithelial cells and fibroblasts in the normal interstitium.
3.2. Patient characteristics Characteristics of patients with pathological stage I invasive lung adenocarcinoma according to eIF4E and 4E-BP1 status are shown in Table 1. With respect to the 4 categorical variables of age, sex, histological subtype, and pathological stage, there were no significant differences between patients with low levels of eIF4E (4Elow group) and those with high levels of eIF4E (4Ehigh group).
Fig. 2. Overall survival of patients with pathological stage I invasive lung adenocarcinoma. (A) Comparison of survival between patients expressing low levels of eIF4E (4Elow group) and those expressing high levels of eIF4E (4Ehigh group). (B) Comparison of survival between patients expressing low levels of 4E-BP1 (4E-BP1low group) and those expressing high levels of 4E-BP1 (4E-BPhigh group). (C) Comparison of the combined effects of eIF4E and 4E-BP1 on survival.
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Table 2 Multivariate Cox proportional hazards model analysis for prognostic factors. Factors (favorable vs. unfavorable)
Hazards ratio
Age (years, ≤65 vs. >65) Sex (female vs. male) Smoking (nonsmoker vs. smoker) Histological subtype (papillary vs. acinar/solid) Pathological stage (I A vs. I B) eIF4E expression (low vs. high) 4E-BP1 expression (high vs. low)
6.02 8.33 3.45 2.43 3.37 5.38 5.26
However, the percentage of smokers was significantly higher in the 4Ehigh group than in the 4Elow group (P = 0.03). On the other hand, these 5 variables showed no significant differences between patients with low levels of 4E-BP1 (4E-BP1low group) and those with high levels of 4E-BP1 (4E-BPhigh group). 3.3. Prognostic significance of eIF4E and 4E-BP1 in invasive lung adenocarcinoma Overall survival rates of patients with pathological stage I invasive lung adenocarcinoma stratified by eIF4E and/or 4E-BP1 status are shown in Fig. 2. The 10-year overall survival rate of the 4Ehigh group (64.0% [n = 36]) was significantly lower than that of the 4Elow group (89.9% [n = 44]; log-rank test, P = 0.024; Fig. 2A). The 10-year overall survival rate in the 4E-BP1low group (70.8% [n = 48]) was slightly but not significantly lower than that in the 4E-BPhigh group (85.8% [n = 32]; log-rank test, P = 0.308; Fig. 2B). In patients of the 4Elow group, the 10-year overall survival rate was lower in the 4EBP1low subgroup (87.6% [n = 36]) than in the 4E-BPhigh subgroup (100% [n = 8]; Fig. 2C), but statistical analysis was not possible owing to a lack of uncensored cases in the 4E-BPhigh subgroup. In patients of the 4E high group, the 10-year overall survival rate was significantly lower in the 4E-BP1low subgroup (39.0% [n = 12]) than in the 4E-BPhigh subgroup (85.2% [n = 24]; log-rank test, P = 0.036; Fig. 2C). 3.4. Prognostic factor analysis Of the 7 variables listed in Table 2, those shown by the multivariate Cox proportional hazards model analysis to be unfavorable prognostic factors were age greater than 65 years (P = 0.015), pathological stage IB (P = 0.045), high levels of eIF4E expression (P = 0.008), and low levels of 4E-BP1 expression (P = 0.007). 4. Discussion The present study in a homogeneous population of 80 patients with pathological stage I invasive lung adenocarcinoma has demonstrated, using multivariate analysis, that both eIF4E expression and 4E-BP1 expression are significant independent prognostic factors. The fact that all of the patients in this study were treated at a single institution and received lengthy follow-up care after surgery increases the reliability of our results. This study is, to our knowledge, the first to show that both eIF4E and 4E-BP1 affect survival in patients with lung cancer. When the effects of both eIF4E expression and 4E-BP1 expression were examined, the 4Elow /4E-BPhigh subgroup had the highest 10-year survival rate, whereas 4Ehigh /4E-BP1low subgroup had the lowest 10-year survival rate. These results strongly suggest the imbalance between eIF4E expression and 4E-BP1 expression determines the prognosis of invasive lung adenocarcinoma. This hypothesis is supported by several previous studies that evaluated the total amounts of eIF4E and 4E-BP1 expressed regardless of phosphorylation status. Martin et al. have reported that nodenegative human gastrointestinal carcinomas have a significantly higher 4E-BP1/eIF4E ratio than do node-positive carcinomas [23].
95% Confidence interval 1.42–25.57 0.61–100.00 0.24–47.62 0.59–9.99 1.03–11.11 1.55–18.52 1.43–16.66
P 0.015 0.117 0.368 0.219 0.045 0.008 0.007
Similarly, Salehi and Mashayekhi have demonstrated that a higher 4E-BP1/eIF4E ratio is associated with a less advanced stage, including lymph node involvement, in human esophageal cancer [24]. Several targeted agents have recently been evaluated in clinical trials of lung cancer. Among the possible targets for cancer therapy, the mTOR pathway is one of the most promising. The mTOR pathway, which is responsible for the phosphorylation of 4E-BP1, plays an important role in cell proliferation by coupling cell growth with the G1-to-S progression. Compounds targeting the mTOR pathway include rapamycin and its derivatives, cell cycle inhibitor 779 (temsirolimus), RAD001 (everolimus), and AP23573 [32]. Several phase I and phase II trials with mTOR inhibitors in non-small cell lung cancer have been registered [32]. Furthermore, the most recent studies have now shown the possibility of developing novel therapeutic agents that directly target eIF4E by means of an eIF4E-specific, second-generation antisense oligonucleotide (ASO) [33,34]. In fact, data from recent phase I trials in patients with prostate cancer have shown that clusterin-specific, second-generation ASOs effectively reach tumor tissue and, in a dose-dependent manner, inhibit expression of the target RNA and protein and, thereby, provide a rationale for advancing eIF4E-specific ASOs to phase I trials for the treatment of human cancers [35]. However, in patients treated with these compounds, potential molecular predictors of survival, such as epidermal growth factor receptor (EGFR) mutation and copy number for EGFR inhibitors (gefitinib, erlotinib, and cetuximab) [36–38], have not been identified. If reliable predictive factors are identified, methods to optimize the therapeutic effects of mTOR inhibitors and eIF4E-specific ASOs in lung cancer might be developed. Several in vitro studies support this proposal. First, Feigenblum and Schneider have shown that eIF4E and 4E-BP1 can act either in concert or in opposition to independently regulate cap-dependent translation [39]. Second, Dilling et al. have shown that a higher 4E-BP1/eIF4E ratio is an important determinant of rapamycin sensitivity [40]. Third, Sun et al. have demonstrated that rapamycin resistance by eIF4E activity through the PI3K/Akt pathway is suppressed by the concurrent use of the PI3K inhibitor LY294002, providing a mechanistic basis for enhancing mTOR-targeted cancer therapy by combining an mTOR inhibitor with a PI3K or Akt inhibitor in a specific patient population [41]. Thus, molecular and pharmacological intervention targeting components of the capdependent translation machinery, especially eIF4E and 4E-BP1, may provide a new paradigm for individualized therapy for lung adenocarcinoma. To date, there have been no reports regarding the effect on survival of 4E-BP1 in patients with lung cancer. However, two recent studies have used immunohistochemical analysis to examine the combined effects on survival of eIF4E and factors other than 4EBP1 in patients with lung cancer. Wang et al. have investigated the effects on survival of eIF4E and p53 in patients with lung adenocarcinoma [42], whereas Khoury et al. have examined the effects on survival of eIF4E and cyclin D1 in patients with non-small cell lung cancer [43]. When the combined effects of both factors were examined with multivariate Cox proportional hazards model analysis, Wang et al. found that only eIF4E was a significant prognostic
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factor in patients with lung adenocarcinoma [42], and Khoury et al. found that neither eIF4E nor cyclin D1 was a significant prognostic factor in patients with non-small cell lung cancer [43]. Therefore, to date no marker other than 4E-BP1 has been identified as a clinically useful prognostic marker for survival in combination with eIF4E in patients with lung cancer. The present study, however, provides the first evidence that eIF4E and 4E-BP1 status might be used to stratify patients and to select targeted agents for individualized therapy for lung adenocarcinoma. Our study has several limitations. First, the number of patients was small. Additional studies involving greater numbers of patients will be necessary to confirm our findings. However, because heterogeneity in the pathological stage and the histological subclassification of adenocarcinomas are associated with prognostic heterogeneity [26], we included only patients with pathological stage I invasive lung adenocarcinoma and excluded patients with mixed adenocarcinomas having bronchioloalveolar features, despite our sample size becoming small, to evaluate more precisely the prognostic significance of eIF4E and 4E-BP1 expression. Therefore, we believe that our adherence to an extremely homogenous population with lengthy follow-up care increases the accuracy of our results. Second, although adjustment with multivariate Cox proportional hazards model analysis suggested the prognostic significance of 4E-BP1 expression, the overall survival rates did not differ significantly between the 4E-BPhigh group and the 4EBP1low group (Fig. 2B). In view of the baseline characteristics of patients (Table 1), there seemed to be a large imbalance in age and pathological stage, which also turned out to be prognostic factors, although they showed no significant difference in 4E-BP1 status. In fact, the number of patients 65 years or younger in the 4EBPhigh group (n = 12) was approximately half that in the 4E-BP1low group (n = 27). Similarly, the number of patients with pathological stage IA invasive lung adenocarcinoma in the 4E-BPhigh group (n = 21) was approximately half that in the 4E-BP1low group (n = 38). No such large imbalance was observed between the 4Ehigh group and the 4E low group. Therefore, the small number of patients due to our desire for a homogenous population might have increased the effects of several prognostic factors on survival, resulting in a smaller difference in survival between the 4E-BPhigh group and the 4E-BP1low group. Third, the expression of phosphorylated 4E-BP1 was not examined in our study although phosphorylation of 4E-BP1 is very important to activate the PI3K/Akt/mTOR pathway. Additional studies to analyze the expression of phosphorylated 4E-BP1 and compare it with the expression of eIF4E and total 4E-BP1 will be necessary. However, it is well known that the expression of phosphorylated and total 4E-BP1 strongly correlate with the survival of patients with ovarian, breast, prostate cancers, and rhabdomyosarcoma [20,22]. Furthermore, it is also well known that the expression of eIF4E and total 4E-BP1 regardless of phosphorylation status are associated with the tumor progression of patients with gastrointestinal and esophageal cancers [23,24]. Therefore, in the current study, we investigated the expression of only eIF4E and total 4EBP1 to evaluate their prognostic significance in patients with lung adenocarcinoma. Fourth, in our study, the presence or absence of an imbalance in the expression of eIF4E and of 4E-BP1 could not be precisely determined because expression was not quantified with Western blot analysis. Instead we performed only semiquantitative analysis with immunohistochemical staining. Although we have previously shown that the results of immunohistochemical studies are generally consistent with those of Western blotting for eIF4E expression [27], we have not performed validation studies to confirm the consistency of results between immunohistochemistry and Western blotting for 4E-BP1 expression. Thus, such additional studies will be needed. On the other hand, because immunohistochemical studies are more widely available in clinical practice than is Western blotting, we believe the focus should be placed
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on immunohistochemical studies for evaluating eIF4E and 4E-BP1 expression. Therefore, larger, more comprehensive studies involving immunohistochemical analysis of both eIF4E expression and 4E-BP1 expression will be necessary to confirm their prognostic significance. In conclusion, we have provided evidence that both eIF4E and 4E-BP1 are potential new prognostic factors for survival in patients with lung adenocarcinoma. The stratification of patients with pathological stage I lung adenocarcinoma on the basis of eIF4E and 4E-BP1 status may provide a new paradigm for individualized therapy. Conflict of Interest statement The authors indicated no potential conflicts of interest. Acknowledgments This work was supported in part by a Grant-in-Aid for Cancer Research [17–2] from the Ministry of Health, Labour and Welfare of Japan, and supported in part by a Grant-in-Aid for the Third-Term Comprehensive 10-Year Strategy for Cancer Control (Category: Japanese General Screening Study for Asbestos-Related Diseases) from the Ministry of Health, Labour and Welfare of Japan. The authors thank Dr. James R. Jett for his critical review of our manuscript. References [1] Devesa SS, Bray F, Vizcaino AP, Parkin DM. International lung cancer trends by histologic type: male:female differences diminishing and adenocarcinoma rates rising. Int J Cancer 2005;117:294–9. [2] Naruke T, Tsuchiya R, Kondo H, Asamura H. Prognosis and survival after resection for bronchogenic carcinoma based on the 1997 TNM-staging classification: the Japanese experience. Ann Thorac Surg 2001;71:1759–64. [3] Hershey JW. Translational control in mammalian cells. Annu Rev Biochem 1991;60:717–55. [4] Gallie DR. A tale of two termini: a functional interaction between the termini of an mRNA is a prerequisite for efficient translation initiation. Gene 1998;216:1–11. [5] Clemens MJ. Targets and mechanisms for the regulation of translation in malignant transformation. Oncogene 2004;23:3180–8. [6] Raught B, Gingras AC. eIF4E activity is regulated at multiple levels. Int J Biochem Cell Biol 1999;31:43–57. [7] Lazaris-Karatzas A, Montine KS, Sonenberg N. Malignant transformation by a eukaryotic initiation factor subunit that binds to mRNA 5 cap. Nature (Lond) 1990;345:544–7. [8] De Benedetti A, Rhoads RE. Overexpression of eukaryotic protein synthesis initiation factor 4E in HeLa cells results in aberrant growth and morphology. Proc Natl Acad Sci USA 1990;87:8212–6. [9] Polunovsky VA, Rosenwald IB, Tan AT, et al. Translational control of programmed cell death: eukaryotic translation initiation factor 4E blocks apoptosis in growth-factor-restricted fibroblasts with physiologically expressed or deregulated Myc. Mol Cell Biol 1996;16:6573–81. [10] Kevil CG, De Benedetti A, Payne DK, Coe LL, Laroux FS, Alexander JS. Translational regulation of vascular permeability factor by eukaryotic initiation factor 4E: implications for tumor angiogenesis. Int J Cancer 1996;65:785–90. [11] Shatkin AJ. Capping of eucaryotic mRNAs. Cell 1976;9:645–53. [12] Duncan R, Milburn SC, Hershey JW. Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in translational control. Heat shock effects on eIF-4F. J Biol Chem 1987;262:380–8. [13] Hiremath LS, Webb NR, Rhoads RE. Immunological detection of the messenger RNA cap-binding protein. J Biol Chem 1985;260:7843–9. [14] De Benedetti A, Graff JR. eIF-4E expression and its role in malignancies and metastases. Oncogene 2004;23:3189–99. [15] Li S, Takasu T, Perlman DM, et al. Translation factor eIF4E rescues cells from Myc-dependent apoptosis by inhibiting cytochrome c release. J Biol Chem 2003;278:3015–22. [16] Ruggero D, Montanaro L, Ma L, et al. The translation factor eIF-4E promotes tumor formation and cooperates with c-Myc in lymphomagenesis. Nat Med 2004;10:484–6. [17] Jacobson BA, Alter MD, Kratzke MG, et al. Repression of cap-dependent translation attenuates the transformed phenotype in non-small cell lung cancer both in vitro and in vivo. Cancer Res 2006;66:4256–62. [18] Poulin F, Gingras AC, Olsen H, Chevalier S, Sonenberg N. 4E-BP3, a new member of the eukaryotic initiation factor 4E-binding protein family. J Biol Chem 1998;273:14002–7.
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Prognostic significance of expression of eukaryotic initiation factor 4E and 4E binding protein 1 in patients with pathological stage I invasive lung adenocarcinoma Nobuhiko Seki a,∗ , Tasaburo Takasu b , Shigeki Sawada c , Masao Nakata d , Rieko Nishimura e , Yoshihiko Segawa f , Reishi Shibakuki g , Toshiaki Hanafusa b , Kenji Eguchi a a
Division of Medical Oncology, Department of Internal Medicine, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan First Department of Internal Medicine, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan c Department of Surgery, National Hospital Organization Shikoku Cancer Center, Matsuyama, Ehime 791-0288, Japan d Division of Thoracic and Cardiovascular Surgery, Department of Surgery, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan e Department of Pathology, National Hospital Organization Shikoku Cancer Center, Matsuyama, Ehime 791-0288, Japan f Department of Medicine and Thoracic Oncology, National Hospital Organization Shikoku Cancer Center, Matsuyama, Ehime 791-0288, Japan g Department of Pathology, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan b
a r t i c l e
i n f o
Article history: Received 24 October 2009 Received in revised form 13 January 2010 Accepted 11 March 2010 Keywords: Translational control eIF4E eIF4E binding protein 1 Prognosis Lung adenocarcinoma
a b s t r a c t Background: Both eukaryotic initiation factor 4E (eIF4E) and eIF4E binding protein 1 (4E-BP1) are involved in the malignant progression of human cancers. However, the role of eIF4E and 4E-BP1 expression as prognostic markers has not been evaluated concurrently in any human cancers. Methods: The expression of eIF4E and 4E-BP1 was semiquantitatively examined with immunohistochemical staining in 80 patients with pathological stage I invasive lung adenocarcinoma. Results: The 10-year survival rate was significantly lower for patients with high eIF4E expression (64.0% [n = 36]) than for patients with low eIF4E expression (89.9% [n = 44], P = 0.024), and in patients with high eIF4E expression the 10-year survival rate was lower for patients with low 4E-BP1 expression (39.0% [n = 12]) than for patients with high 4E-BP1 expression (85.2% [n = 24], P = 0.036). In patients with low eIF4E expression, the 10-year survival rate was lower for patients with low 4E-BP1 expression (87.6% [n = 36]) than for patients with high 4E-BP1 expression (100% [n = 8]), but statistical analysis was impossible because all patients with high 4E-BP1 expression were censored. Unfavorable prognostic factors for survival were age greater than 65 years (P = 0.015), pathological stage IB disease (P = 0.045), high eIF4E expression (P = 0.008), and low 4E-BP1 expression (P = 0.007). Conclusions: Both eIF4E and 4E-BP1 are potential new prognostic factors for survival and stratification in patients with pathological stage I lung adenocarcinoma. The eIF4E and 4E-BP1 status may provide a basis for individualized therapy. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer is a leading cause of cancer death in most industrialized countries. Among the various histological types of lung cancer, adenocarcinoma is rapidly becoming more prevalent and now accounts for more than 50% of all lung cancers [1]. However, its poor survival rate has not improved significantly in the past two decades. Even in pathological stage IA lung adenocarcinoma, the 5-year survival rate is at most 80% [2]. To further improve survival rates in patients with adenocarcinoma, prognostic classifications based on tumor biology will be crucial. Such classifications might
∗ Corresponding author. Tel.: +81 3 3964 1211x1587; fax: +81 3 3964 7094. E-mail address: [email protected] (N. Seki). 0169-5002/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2010.03.006
help physicians select the most appropriate treatment for each subset of patients. Translational control is an integral part of the regulation of gene expression [3–5]. Changes in the efficiencies of mRNA translation influence gene expression at the protein level, resulting in alterations of cell function and phenotype [5–10]. In eukaryotes, almost all mRNAs are modified at their 5 -termini with an m7 GpppN cap structure (where N is any nucleotide) [11]. Translation of such mRNAs typically requires cap-dependent unwinding of the secondary structure in the 5 -untranslated regions by the initiation complex eIF4F. This complex consists of the cap-binding protein eIF4E, the adaptor protein eIF4G, and the ATP-dependent RNA helicase eIF4A [4–6]. Because eIF4E is the least abundant of the various translation initiation factors [12] and is, therefore, the rate-limiting component for cap-dependent translation
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[13], changes in the levels of eIF4E profoundly affect translation rates. Recent in vitro studies have shown that experimental overexpression of eIF4E preferentially enhances the selective synthesis of malignancy-related proteins, although their translation is repressed under normal cellular conditions [5,9,14,15]. These upregulated key proteins promote tumorigenesis (c-myc, cyclin D1), angiogenesis (fibroblast growth factor 2, vascular endothelial growth factor), and metastasis (heparanase, matrix metalloproteinase 9, and CD44v6) [14]. An in vivo study has revealed that ectopic eIF4E expression in transgenic mice increases the incidence of multiple cancers, including lung adenocarcinoma (up to 21%), lymphoma, hepatoma, and angiosarcoma [16]. A major signaling pathway through which eIF4E activity controls cancer is the phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, via the phosphorylation of eIF4E binding proteins (4E-BPs) [17]. The 4E-BPs, of which 3 isoforms are present in mammalian cells, are suppressors of eIF4E activity [18]. When 4E-BPs are active (nonphosphorylated), they bind to eIF4E and impede formation of the initiation complex; translation is then blocked and apoptosis is favored. However, when 4E-BPs are inactive (phosphorylated), their affinity for eIF4E is reduced, eIF4E is released, and cap-dependent translation can start. Thus, induction of the constitutively active 4E-BPs leads to cell cycle arrest, which correlates with the loss of cyclin D1 expression and increased levels of the cyclin-dependent kinase inhibitor p27Kip1 [19]. In contrast, constitutive activation of the PI3K/Akt/mTOR signaling pathway leads to inactivation of the tumor-suppressor activity of 4E-BPs, and in turn, contributes to cancer development [17]. In this regard, patients in whom 4E-BPs are highly phosphorylated or are expressed at low levels regardless of their phosphorylation status exhibit tumor progression and have an unfavorable prognosis [20]. During the last decade, ubiquitous eIF4E expression or 4E-BP1 expression has been linked to a variety of human cancers, such as those of the breast, esophagus, stomach, colon, head and neck, lung, cervix, ovary, prostate, and bladder, and to lymphoma [21–24]. However, to our knowledge, the roles of eIF4E expression and 4EBP1 expression as prognostic markers have not been concurrently evaluated in any human cancers. In this study, we immunohistochemically investigated eIF4E expression and 4E-BP1 expression to evaluate their prognostic significance in 80 patients with pathological stage I invasive lung adenocarcinoma.
2. Materials and methods 2.1. Patients and tissue samples Approval for this study was obtained from the Institutional Review Board. Specimens of pathological stage I invasive lung adenocarcinoma from 80 patients who had undergone curative surgical resection without prior chemotherapy or radiotherapy from January 1985 through December 1997 were retrieved from the archives of the Department of Pathology of the National Hospital Organization Shikoku Cancer Center, Ehime, Japan. On the basis of the World Health Organization lung tumor classification system, tissue samples were subclassified into the following histological groups: papillary adenocarcinoma, acinar adenocarcinoma, and solid adenocarcinoma with mucin formation [25]. Mixed adenocarcinomas with bronchioloalveolar features were excluded from this study because of their histological and prognostic heterogeneity [26]. The medical charts of all patients were reviewed, and the clinicopathological characteristics are summarized in Table 1. None of the patients received postoperative adjuvant chemotherapy. The median duration of follow-up was 7.8 years (range, 1.1–16.2 years). 2.2. Immunohistochemical studies Immunohistochemical studies were performed as described previously [27]. To detect eIF4E and 4E-BP1, a mouse monoclonal anti-human eIF4E primary antibody (Transduction Laboratories, Lexington, KY) and a rabbit polyclonal anti-human 4E-BP1 primary antibody (New England Biolabs, Beverly, MA), respectively, were used. On the basis of previous investigations [27,28], the intensity in bronchial epithelial cells was defined as an internal control to evaluate eIF4E staining intensity in tumor cells. The staining intensity in tumor cells was designated as 4Elow when it was less than this internal control and was otherwise designated as 4Ehigh . Because considerable degree of heterogeneity in 4E-BP1 staining was observed in stromal cells, bronchial epithelial cells, and interstitial lymphocytes as well as tumor cells, and because the levels of 4E-BP1 staining in these nontumor cells were relatively weak compared with those of eIF4E staining, at least 1000 tumor cells in representative fields were manually counted in each section to evaluate 4E-BP1 staining intensity in tumor cells. Tumor cells showing either cytoplasmic or nuclear 4E-BP1 expression were considered 4E-BP1-positive tumor cells as described previ-
Table 1 Characteristics of patients with pathological stage I invasive lung adenocarcinoma according to eIF4E and 4E-BP1 status. Total
Pa
eIF4E expression Low (n = 44)
High (n = 36)
Age (years) ≤65 >65
39 41
24 20
15 21
Sex Male Female
30 50
13 31
17 19
Smoking Nonsmoker Smoker
48 32
31 13
17 19
Histological subtype Papillary Acinar Solid
64 15 1
35 8 1
29 7 0
Pathological stage IA IB
59 21
35 9
24 12
a
Chi-square test.
Pa
4E-BP1 expression Low (n = 48)
High (n = 32)
27 21
12 20
18 30
12 20
30 18
18 14
38 9 1
26 6 0
38 10
21 11
0.36
0.16
0.16
0.99
0.03
0.58
0.66
0.71
0.30
0.28
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Fig. 1. Expression of eIF4E and 4E-BP1 in invasive lung adenocarcinoma. (A) Anti-eIF4E antibody. (B) Anti-4E-BP1 antibody. Cells expressing eIF4E and 4E-BP1 show brown staining (original magnification 200×).
ously [29–31]. On the basis of the histogram of 4E-BP1-positive tumor cells, cases were divided into 2 subgroups using the median value (45%) as a cutoff point. Cases were considered 4E-BP1low if the percentage of 4E-BP1-positive tumor cells was <45% but were otherwise considered 4E-BPhigh . All sections were evaluated by 2 independent investigators (N.S. and R.N.) in a blinded manner without any information regarding clinicopathological characteristics. 2.3. Statistical analysis The postoperative period was measured from the date of surgery to the date of the last follow-up or death. Patients who died of other causes were censored. The Chi-square test was used to evaluate the association between the patient characteristics (listed in Table 1) and the levels of either eIF4E expression or 4E-BP1 expression. Survival curves were constructed with the Kaplan–Meier method, and the statistical significance of differences in survival rates between groups of patients was evaluated with the log-rank test. Multivariate Cox proportional hazards model analysis was performed to identify significant independent prognostic factors for survival. All calculations were performed with StatView 5.0J software (SAS Institute Inc., Cary, NC). Differences were considered significant when P < 0.05.
3. Results 3.1. Immunohistochemical analysis for eIF4E and 4E-BP1 The typical immunostaining patterns of eIF4E and 4E-BP1 in invasive lung adenocarcinomas are shown in Fig. 1. eIF4E expression was homogeneously positive in the cytoplasm of tumor cells, stromal cells (mainly consisting of fibroblasts), bronchial epithelial cells, and normal interstitial lymphocytes, whereas 4E-BP1 expression was heterogeneously positive in the cytoplasm of these cells. Only a few cells that had intense cytoplasmic staining showed nuclear staining. In contrast, both eIF4E expression and 4E-BP1 expression were negative in alveolar epithelial cells and fibroblasts in the normal interstitium.
3.2. Patient characteristics Characteristics of patients with pathological stage I invasive lung adenocarcinoma according to eIF4E and 4E-BP1 status are shown in Table 1. With respect to the 4 categorical variables of age, sex, histological subtype, and pathological stage, there were no significant differences between patients with low levels of eIF4E (4Elow group) and those with high levels of eIF4E (4Ehigh group).
Fig. 2. Overall survival of patients with pathological stage I invasive lung adenocarcinoma. (A) Comparison of survival between patients expressing low levels of eIF4E (4Elow group) and those expressing high levels of eIF4E (4Ehigh group). (B) Comparison of survival between patients expressing low levels of 4E-BP1 (4E-BP1low group) and those expressing high levels of 4E-BP1 (4E-BPhigh group). (C) Comparison of the combined effects of eIF4E and 4E-BP1 on survival.
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Table 2 Multivariate Cox proportional hazards model analysis for prognostic factors. Factors (favorable vs. unfavorable)
Hazards ratio
Age (years, ≤65 vs. >65) Sex (female vs. male) Smoking (nonsmoker vs. smoker) Histological subtype (papillary vs. acinar/solid) Pathological stage (I A vs. I B) eIF4E expression (low vs. high) 4E-BP1 expression (high vs. low)
6.02 8.33 3.45 2.43 3.37 5.38 5.26
However, the percentage of smokers was significantly higher in the 4Ehigh group than in the 4Elow group (P = 0.03). On the other hand, these 5 variables showed no significant differences between patients with low levels of 4E-BP1 (4E-BP1low group) and those with high levels of 4E-BP1 (4E-BPhigh group). 3.3. Prognostic significance of eIF4E and 4E-BP1 in invasive lung adenocarcinoma Overall survival rates of patients with pathological stage I invasive lung adenocarcinoma stratified by eIF4E and/or 4E-BP1 status are shown in Fig. 2. The 10-year overall survival rate of the 4Ehigh group (64.0% [n = 36]) was significantly lower than that of the 4Elow group (89.9% [n = 44]; log-rank test, P = 0.024; Fig. 2A). The 10-year overall survival rate in the 4E-BP1low group (70.8% [n = 48]) was slightly but not significantly lower than that in the 4E-BPhigh group (85.8% [n = 32]; log-rank test, P = 0.308; Fig. 2B). In patients of the 4Elow group, the 10-year overall survival rate was lower in the 4EBP1low subgroup (87.6% [n = 36]) than in the 4E-BPhigh subgroup (100% [n = 8]; Fig. 2C), but statistical analysis was not possible owing to a lack of uncensored cases in the 4E-BPhigh subgroup. In patients of the 4E high group, the 10-year overall survival rate was significantly lower in the 4E-BP1low subgroup (39.0% [n = 12]) than in the 4E-BPhigh subgroup (85.2% [n = 24]; log-rank test, P = 0.036; Fig. 2C). 3.4. Prognostic factor analysis Of the 7 variables listed in Table 2, those shown by the multivariate Cox proportional hazards model analysis to be unfavorable prognostic factors were age greater than 65 years (P = 0.015), pathological stage IB (P = 0.045), high levels of eIF4E expression (P = 0.008), and low levels of 4E-BP1 expression (P = 0.007). 4. Discussion The present study in a homogeneous population of 80 patients with pathological stage I invasive lung adenocarcinoma has demonstrated, using multivariate analysis, that both eIF4E expression and 4E-BP1 expression are significant independent prognostic factors. The fact that all of the patients in this study were treated at a single institution and received lengthy follow-up care after surgery increases the reliability of our results. This study is, to our knowledge, the first to show that both eIF4E and 4E-BP1 affect survival in patients with lung cancer. When the effects of both eIF4E expression and 4E-BP1 expression were examined, the 4Elow /4E-BPhigh subgroup had the highest 10-year survival rate, whereas 4Ehigh /4E-BP1low subgroup had the lowest 10-year survival rate. These results strongly suggest the imbalance between eIF4E expression and 4E-BP1 expression determines the prognosis of invasive lung adenocarcinoma. This hypothesis is supported by several previous studies that evaluated the total amounts of eIF4E and 4E-BP1 expressed regardless of phosphorylation status. Martin et al. have reported that nodenegative human gastrointestinal carcinomas have a significantly higher 4E-BP1/eIF4E ratio than do node-positive carcinomas [23].
95% Confidence interval 1.42–25.57 0.61–100.00 0.24–47.62 0.59–9.99 1.03–11.11 1.55–18.52 1.43–16.66
P 0.015 0.117 0.368 0.219 0.045 0.008 0.007
Similarly, Salehi and Mashayekhi have demonstrated that a higher 4E-BP1/eIF4E ratio is associated with a less advanced stage, including lymph node involvement, in human esophageal cancer [24]. Several targeted agents have recently been evaluated in clinical trials of lung cancer. Among the possible targets for cancer therapy, the mTOR pathway is one of the most promising. The mTOR pathway, which is responsible for the phosphorylation of 4E-BP1, plays an important role in cell proliferation by coupling cell growth with the G1-to-S progression. Compounds targeting the mTOR pathway include rapamycin and its derivatives, cell cycle inhibitor 779 (temsirolimus), RAD001 (everolimus), and AP23573 [32]. Several phase I and phase II trials with mTOR inhibitors in non-small cell lung cancer have been registered [32]. Furthermore, the most recent studies have now shown the possibility of developing novel therapeutic agents that directly target eIF4E by means of an eIF4E-specific, second-generation antisense oligonucleotide (ASO) [33,34]. In fact, data from recent phase I trials in patients with prostate cancer have shown that clusterin-specific, second-generation ASOs effectively reach tumor tissue and, in a dose-dependent manner, inhibit expression of the target RNA and protein and, thereby, provide a rationale for advancing eIF4E-specific ASOs to phase I trials for the treatment of human cancers [35]. However, in patients treated with these compounds, potential molecular predictors of survival, such as epidermal growth factor receptor (EGFR) mutation and copy number for EGFR inhibitors (gefitinib, erlotinib, and cetuximab) [36–38], have not been identified. If reliable predictive factors are identified, methods to optimize the therapeutic effects of mTOR inhibitors and eIF4E-specific ASOs in lung cancer might be developed. Several in vitro studies support this proposal. First, Feigenblum and Schneider have shown that eIF4E and 4E-BP1 can act either in concert or in opposition to independently regulate cap-dependent translation [39]. Second, Dilling et al. have shown that a higher 4E-BP1/eIF4E ratio is an important determinant of rapamycin sensitivity [40]. Third, Sun et al. have demonstrated that rapamycin resistance by eIF4E activity through the PI3K/Akt pathway is suppressed by the concurrent use of the PI3K inhibitor LY294002, providing a mechanistic basis for enhancing mTOR-targeted cancer therapy by combining an mTOR inhibitor with a PI3K or Akt inhibitor in a specific patient population [41]. Thus, molecular and pharmacological intervention targeting components of the capdependent translation machinery, especially eIF4E and 4E-BP1, may provide a new paradigm for individualized therapy for lung adenocarcinoma. To date, there have been no reports regarding the effect on survival of 4E-BP1 in patients with lung cancer. However, two recent studies have used immunohistochemical analysis to examine the combined effects on survival of eIF4E and factors other than 4EBP1 in patients with lung cancer. Wang et al. have investigated the effects on survival of eIF4E and p53 in patients with lung adenocarcinoma [42], whereas Khoury et al. have examined the effects on survival of eIF4E and cyclin D1 in patients with non-small cell lung cancer [43]. When the combined effects of both factors were examined with multivariate Cox proportional hazards model analysis, Wang et al. found that only eIF4E was a significant prognostic
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factor in patients with lung adenocarcinoma [42], and Khoury et al. found that neither eIF4E nor cyclin D1 was a significant prognostic factor in patients with non-small cell lung cancer [43]. Therefore, to date no marker other than 4E-BP1 has been identified as a clinically useful prognostic marker for survival in combination with eIF4E in patients with lung cancer. The present study, however, provides the first evidence that eIF4E and 4E-BP1 status might be used to stratify patients and to select targeted agents for individualized therapy for lung adenocarcinoma. Our study has several limitations. First, the number of patients was small. Additional studies involving greater numbers of patients will be necessary to confirm our findings. However, because heterogeneity in the pathological stage and the histological subclassification of adenocarcinomas are associated with prognostic heterogeneity [26], we included only patients with pathological stage I invasive lung adenocarcinoma and excluded patients with mixed adenocarcinomas having bronchioloalveolar features, despite our sample size becoming small, to evaluate more precisely the prognostic significance of eIF4E and 4E-BP1 expression. Therefore, we believe that our adherence to an extremely homogenous population with lengthy follow-up care increases the accuracy of our results. Second, although adjustment with multivariate Cox proportional hazards model analysis suggested the prognostic significance of 4E-BP1 expression, the overall survival rates did not differ significantly between the 4E-BPhigh group and the 4EBP1low group (Fig. 2B). In view of the baseline characteristics of patients (Table 1), there seemed to be a large imbalance in age and pathological stage, which also turned out to be prognostic factors, although they showed no significant difference in 4E-BP1 status. In fact, the number of patients 65 years or younger in the 4EBPhigh group (n = 12) was approximately half that in the 4E-BP1low group (n = 27). Similarly, the number of patients with pathological stage IA invasive lung adenocarcinoma in the 4E-BPhigh group (n = 21) was approximately half that in the 4E-BP1low group (n = 38). No such large imbalance was observed between the 4Ehigh group and the 4E low group. Therefore, the small number of patients due to our desire for a homogenous population might have increased the effects of several prognostic factors on survival, resulting in a smaller difference in survival between the 4E-BPhigh group and the 4E-BP1low group. Third, the expression of phosphorylated 4E-BP1 was not examined in our study although phosphorylation of 4E-BP1 is very important to activate the PI3K/Akt/mTOR pathway. Additional studies to analyze the expression of phosphorylated 4E-BP1 and compare it with the expression of eIF4E and total 4E-BP1 will be necessary. However, it is well known that the expression of phosphorylated and total 4E-BP1 strongly correlate with the survival of patients with ovarian, breast, prostate cancers, and rhabdomyosarcoma [20,22]. Furthermore, it is also well known that the expression of eIF4E and total 4E-BP1 regardless of phosphorylation status are associated with the tumor progression of patients with gastrointestinal and esophageal cancers [23,24]. Therefore, in the current study, we investigated the expression of only eIF4E and total 4EBP1 to evaluate their prognostic significance in patients with lung adenocarcinoma. Fourth, in our study, the presence or absence of an imbalance in the expression of eIF4E and of 4E-BP1 could not be precisely determined because expression was not quantified with Western blot analysis. Instead we performed only semiquantitative analysis with immunohistochemical staining. Although we have previously shown that the results of immunohistochemical studies are generally consistent with those of Western blotting for eIF4E expression [27], we have not performed validation studies to confirm the consistency of results between immunohistochemistry and Western blotting for 4E-BP1 expression. Thus, such additional studies will be needed. On the other hand, because immunohistochemical studies are more widely available in clinical practice than is Western blotting, we believe the focus should be placed
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on immunohistochemical studies for evaluating eIF4E and 4E-BP1 expression. Therefore, larger, more comprehensive studies involving immunohistochemical analysis of both eIF4E expression and 4E-BP1 expression will be necessary to confirm their prognostic significance. In conclusion, we have provided evidence that both eIF4E and 4E-BP1 are potential new prognostic factors for survival in patients with lung adenocarcinoma. The stratification of patients with pathological stage I lung adenocarcinoma on the basis of eIF4E and 4E-BP1 status may provide a new paradigm for individualized therapy. Conflict of Interest statement The authors indicated no potential conflicts of interest. Acknowledgments This work was supported in part by a Grant-in-Aid for Cancer Research [17–2] from the Ministry of Health, Labour and Welfare of Japan, and supported in part by a Grant-in-Aid for the Third-Term Comprehensive 10-Year Strategy for Cancer Control (Category: Japanese General Screening Study for Asbestos-Related Diseases) from the Ministry of Health, Labour and Welfare of Japan. The authors thank Dr. James R. Jett for his critical review of our manuscript. References [1] Devesa SS, Bray F, Vizcaino AP, Parkin DM. International lung cancer trends by histologic type: male:female differences diminishing and adenocarcinoma rates rising. Int J Cancer 2005;117:294–9. [2] Naruke T, Tsuchiya R, Kondo H, Asamura H. Prognosis and survival after resection for bronchogenic carcinoma based on the 1997 TNM-staging classification: the Japanese experience. Ann Thorac Surg 2001;71:1759–64. [3] Hershey JW. Translational control in mammalian cells. Annu Rev Biochem 1991;60:717–55. [4] Gallie DR. A tale of two termini: a functional interaction between the termini of an mRNA is a prerequisite for efficient translation initiation. Gene 1998;216:1–11. [5] Clemens MJ. Targets and mechanisms for the regulation of translation in malignant transformation. Oncogene 2004;23:3180–8. [6] Raught B, Gingras AC. eIF4E activity is regulated at multiple levels. Int J Biochem Cell Biol 1999;31:43–57. [7] Lazaris-Karatzas A, Montine KS, Sonenberg N. Malignant transformation by a eukaryotic initiation factor subunit that binds to mRNA 5 cap. Nature (Lond) 1990;345:544–7. [8] De Benedetti A, Rhoads RE. Overexpression of eukaryotic protein synthesis initiation factor 4E in HeLa cells results in aberrant growth and morphology. Proc Natl Acad Sci USA 1990;87:8212–6. [9] Polunovsky VA, Rosenwald IB, Tan AT, et al. Translational control of programmed cell death: eukaryotic translation initiation factor 4E blocks apoptosis in growth-factor-restricted fibroblasts with physiologically expressed or deregulated Myc. Mol Cell Biol 1996;16:6573–81. [10] Kevil CG, De Benedetti A, Payne DK, Coe LL, Laroux FS, Alexander JS. Translational regulation of vascular permeability factor by eukaryotic initiation factor 4E: implications for tumor angiogenesis. Int J Cancer 1996;65:785–90. [11] Shatkin AJ. Capping of eucaryotic mRNAs. Cell 1976;9:645–53. [12] Duncan R, Milburn SC, Hershey JW. Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in translational control. Heat shock effects on eIF-4F. J Biol Chem 1987;262:380–8. [13] Hiremath LS, Webb NR, Rhoads RE. Immunological detection of the messenger RNA cap-binding protein. J Biol Chem 1985;260:7843–9. [14] De Benedetti A, Graff JR. eIF-4E expression and its role in malignancies and metastases. Oncogene 2004;23:3189–99. [15] Li S, Takasu T, Perlman DM, et al. Translation factor eIF4E rescues cells from Myc-dependent apoptosis by inhibiting cytochrome c release. J Biol Chem 2003;278:3015–22. [16] Ruggero D, Montanaro L, Ma L, et al. The translation factor eIF-4E promotes tumor formation and cooperates with c-Myc in lymphomagenesis. Nat Med 2004;10:484–6. [17] Jacobson BA, Alter MD, Kratzke MG, et al. Repression of cap-dependent translation attenuates the transformed phenotype in non-small cell lung cancer both in vitro and in vivo. Cancer Res 2006;66:4256–62. [18] Poulin F, Gingras AC, Olsen H, Chevalier S, Sonenberg N. 4E-BP3, a new member of the eukaryotic initiation factor 4E-binding protein family. J Biol Chem 1998;273:14002–7.
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