EGR1 Predicts PTEN and Survival in Patients With Non–Small-Cell Lung Cancer
http://www.100md.com
《临床肿瘤学》
the Thoracic Oncology Program and Biostatistics Core, H. Lee Moffitt Cancer Center and Research Institute
Departments of Interdisciplinary Oncology, University of South Florida College of Medicine, Tampa, FL
ABSTRACT
PURPOSE: The zinc finger transcription factor early growth response gene 1 (EGR1) is underexpressed in non–small-cell lung cancer (NSCLC) compared with normal lung. EGR1 expression has been linked to tumor suppression as a result of cell cycle arrest and apoptosis through regulation of tumor suppressor pathways including PTEN. For these reasons, we hypothesized that reduced levels of EGR1 would correlate with inferior outcome in patients with NSCLC.
PATIENTS AND METHODS: Patients who underwent surgical resection for NSCLC had RNA extracted from tumor tissue and EGR1 gene expression was quantified by real-time quantitative polymerase chain reaction. The levels of EGR1 expression were examined in relationship to patient characteristics, histology, tumor stage, PTEN expression, and overall and disease-free survival.
RESULTS: EGR1 expression strongly correlated with PTEN expression (P < .0001). No correlation of EGR1 with histology or stage was detected. Patients with high levels of EGR1 had better overall and disease-free survival compared with patients with low levels of EGR1 (P = .040 and P = .096, respectively). In a stratified log-rank test, low EGR1 expression was predictive of poor survival independent of tumor stage.
CONCLUSION: EGR1 gene expression predicts PTEN levels and survival after surgical resection of NSCLC. Consistent with its known tumor suppressor properties, lower levels of EGR1 are associated with poor outcome. Identification of patients with low EGR1 therefore may identify patients at high risk for disease recurrence and may also identify patients who have tumors resistant to therapy secondary to loss of pathways such as PTEN.
INTRODUCTION
Tumor stage continues to be the most important predictor for outcome after surgical resection for early stage non–small-cell lung cancer (NSCLC).1 Patients who undergo curative surgical resection for apparently localized disease have 5-year survivals ranging between 25% and 70%, suggesting the need for better identification of occult metastatic disease. A better understanding of molecular pathways that influence the lung cancer phenotype may lead to the identification of patients at high risk for recurrence and thus interventions can be directed at those who derive maximum benefit.
Increasing use of gene expression profiling technology is allowing the identification of differential gene expression in lung cancers compared with normal lung tissue. One gene product consistently identified as underexpressed in NSCLC tumors is the zinc finger transcription factor early growth response gene 1 (EGR1). Previous studies from the Lung Cancer Study Group found decreased coordinate expression of EGR1, along with other early response genes such as C-JUN and C-FOS, in 72% (73 of 101) of NSCLC tumor samples compared with adjacent normal lung tissue.2 More recent studies investigating gene expression patterns in NSCLC using cDNA arrays have shown downregulation of EGR1 in lung tumors compared with normal lung tissue.3-5 EGR1, also known as zif268, NGFI-A, Krox24, and TIS8, has been linked to diverse cellular pathways including differentiation and tumor suppression through cell cycle arrest and apoptosis (reviewed by Thiel and Cibelli,6 and Adamson and Mercola7). EGR1 is linked to a number of tumor suppressor pathways including the regulation of PTEN, p53, p21, transforming growth factor beta, and TOE1.8-11 EGR1 gene expression is elicited by multiple stimuli, including growth factors, cytokines, UV and gamma irradiation, and hypoxia.6,7 The functions of EGR1 are diverse and appear to be dependent on tissue type. In prostate cancer, EGR1 promotes growth and survival of prostate cancer cells and EGR1 levels are correlated with increased Gleason score.11-13 This contrasts to studies in lung, brain, and breast tissues, in which decreased EGR1 is associated with tumor formation.14,15 Studies with glioma and sarcoma cell lines have found that EGR1 can suppress transformation and tumorigenicity through diverse mechanisms including inhibition of cell growth, counteraction of apoptosis, and enhanced cell adhesion.16-21 No studies have shown direct evidence of tumor suppressor properties of EGR1 in lung cancer cells but the reduced levels of EGR1 in lung cancer tissues compared with normal lung tissues does suggest a tumor suppressor function for EGR1 in NSCLC.
On the basis of the potential for tumor suppressor properties of EGR1 in lung tissues and reports of reduced levels of EGR1 in lung cancer, we examined the prognostic role of EGR1 in NSCLC. Given the ability of EGR1 to regulate the PTEN tumor suppressor pathway and our previous results demonstrating the prognostic value of PTEN, it was our hypothesis that reduced levels of EGR1 would correlate with inferior outcome.11,22 We found that EGR1 is a significant and strong predictor of PTEN, and reduced levels of EGR1 are associated with poor overall survival (OS) and disease-free survival (DFS) in NSCLC.
PATIENTS AND METHODS
Patient Population
Fresh-frozen tumor specimens from patients with resected NSCLC were collected from 1991 to 2001. Specimens were macrodissected to enrich for tumor tissue. Specimens were microscopically examined to ensure adequate tumor quality and confirm the histologic diagnosis. Only specimens with 60% of cells consisting of tumor were processed for analysis. Use of the tumor specimens and review of clinical data were approved by the institutional review board. During this time interval, patient's symptoms, prior medical history, use of tobacco products, demographics, and other relevant parameters were prospectively collected on hard-copy questionnaires. Information obtained from the questionnaire was verified in a face-to-face interview by the treating surgeon.
Tumor staging was based on computed tomography of the chest and upper abdomen, mediastinal lymph node dissection, and gross and microscopic evaluation of the resected lung tissue. Other staging studies, including brain imaging and bone scans, were performed at the discretion of the surgeon. The date of first pathologic verification of malignancy was used as the date of diagnosis. After surgery, patients were observed for 3 months by the surgeon. They were then given a choice of follow-up at regular intervals (every 3 to 12 months) by the referring physician or at our institution, with standard radiographs and/or computed tomography of the chest. The first date of unequivocal clinical evidence of disease or histologic confirmation, in patients with questionable status, was recorded as the recurrence date. The date of death was obtained through family or care provider contact, and it was verified by review of public records. Overall survival was defined as the time elapsed from histologic diagnosis to death. Disease-free survival was defined as the time elapsed from surgery to recurrence or death.
Quantitative Reverse Transcriptase Polymerase Chain Reaction Analysis
RNA was isolated from tumor tissue as previously described and reverse transcribed into cDNA using established techniques.22 Quantitative real-time polymerase chain reaction analysis was performed for each sample using 5 ng of RNA, and reactions were run in triplicate on 96-well plates. Reverse transcription reactions were also done on serial dilutions of total human RNA (Applied Biosystems Inc [ABI], Foster City, CA) to generate a standard curve for EGR1, PTEN, and 18S. TaqMan probes and primers for 18S rRNA (part No. 4319413E; ABI) were purchased as a predeveloped assay system from ABI. EGR1 and PTEN primers were designed to span intron-exon boundaries to reduce the possibility of genomic DNA amplification. The EGR1 probe was labeled with 6-FAM on the 5' end and a minor groove binder nonfluorescent quencher on the 3' end. The PTEN probe was labeled with 6-FAM on the 5' end and TAMRA was used as a quencher on the 3' end. Probe sets are listed in Table 1. Fluorescent emission was recorded in real-time (ABI prism 7700; Perkin-Elmer, Foster City, CA). A linear equation was derived from the standard curve cycle threshold values and used to determine the relative amount of RNA in the sample for EGR1, PTEN, and 18S rRNA. The relative amount of RNA in each sample was normalized to that sample's 18S rRNA expression levels.
Statistical Analysis
Correlation coefficients between the expression of EGR1 and demographic data were calculated according to Spearman. The Wilcoxon rank sum test was used to test for significant associations between dichotomous variables and EGR1 expression, and the Kruskal-Wallis test was used for variables with more than two categories. OS and DFS probabilities were estimated using the Kaplan-Meier method, and log-rank testing was used to determine the level of significance between survival curves. The stratified log-rank test was done to compare groups while adjusting for a potential confounder such as disease stage.
RESULTS
Fresh tumor specimens during a 10-year period were collected from pathologically staged patients undergoing complete resection for NSCLC. Table 2 lists the demographic and clinical data on the patients included in this study. There were 49 women and 76 men. The median age of the study cohort was 68.5 years and median follow-up time for the study cohort was 101 months (range, 39 to 161 months). The histopathology included adenocarcinoma (n = 53), squamous cell carcinoma (n = 50), bronchioloalveolar carcinoma (n = 8), and large-cell carcinoma (n = 14). Forty-five patients had stage IA disease (tumor size < 3.0 cm), 56 had stage IB disease (tumor size 3.0 cm), and 24 patients had either stage II or III disease. Eleven patients were lifelong nonsmokers (< 100 cigarettes), 29 were active smokers, and 74 were former smokers. The smoking history was not disclosed by 11 patients. At the time of the analysis, 64 patients had died and 61 patients remained alive.
Fresh tissues were used to perform quantitative polymerase chain reaction analysis for both EGR1 and PTEN. We explored differences in EGR1 expression among women and men; age; smoking status; stages I, II, and III; performance status 0 and more than 0; absence or presence of weight loss; age; and histopathology. Table 3 lists correlations between EGR1 and patient characteristics, and no significant correlations were found with sex, histology, stage, and quantitative smoking history (measured in pack-years). We also examined for differences in EGR1 in active smokers and patients who had quit smoking tobacco. We identified a trend toward higher EGR1 levels in patients who had quit compared with those patients who were active smokers at the time of diagnosis (P = .09). Finally, we identified a highly significant correlation between the levels of EGR1 and PTEN (Spearman correlation coefficient, 0.42; P < .0001) consistent with biochemical studies showing direct regulation of PTEN by EGR1.11
Kaplan-Meier OS and DFS curves for EGR1 and PTEN expression dichotomized by the median level for each RNA are shown in Figure 1. EGR1 levels below the median predicted worse OS and DFS compared with patients with EGR1 levels above the median (P = .040 and P = .096, respectively). Similarly, levels of PTEN below the median predict poor survival in this patient cohort (P = .026 and P = .048, respectively). Because tumor stage is a critical prognostic variable for outcome after surgical resection of NSCLC, we next performed a stratified log-rank test to assess whether EGR1 and PTEN are prognostic of survival independent of stage. Results are listed in Table 4. We found that EGR1 and PTEN are independent predictors of outcome after surgical resection of NSCLC.
DISCUSSION
Our results show that expression levels of EGR1 are strongly associated with expression levels of PTE, and low levels of EGR1 predict poor outcome after surgical resection of NSCLC. These data are consistent with a model in which EGR1 functions as a tumor suppressor gene in lung tissues through regulation of PTEN, and loss of this pathway results in more aggressive disease.22 Previous reports have shown rare genetic alterations of PTEN but point toward promoter methylation as one mechanism of PTEN loss in NSCLC.23 On the basis of previous biochemical studies as well as our own results, it appears that EGR1 is an important modulator of PTEN. EGR1 has been found to directly regulate the PTEN gene promoter and upregulate transcription of the PTEN gene.11 The virulence associated with loss of PTEN expression in early-stage NSCLC could result from enhanced tumor invasion and metastasis. PTEN is linked to control of invasion and metastasis through direct negative regulation of focal adhesion kinase.24-26 Our previous studies demonstrated that PTEN reduced phosphorylation of focal adhesion kinase, suppressed invasion and metastasis formation, and increased survival in an animal model.27 PTEN is also thought to function by negatively regulating the action of phosphatidylinositol 3-kinase and its downstream substrate AKT. Loss of PTEN function either through mutations or reduced expression can result in increased AKT activity and suppression of apoptosis.28-31 Published data suggest that AKT is constitutively active in NSCLC cells and is involved in resistance to a wide array of apoptotic stimuli including cytotoxic chemotherapy, radiation therapy, tumor necrosis factor–related apoptosis-inducing ligand, and EGFR tyrosine kinase inhibitors.32-34
Although our results show a strong correlation between EGR1 and PTEN, EGR1 may function as a tumor suppressor in lung cancer by regulating additional tumor suppressor genes including p53, p21, TOE1, and TGF. Loss of EGR1-driven expression of these genes would be predicted to allow for enhanced tumor cell growth and reduced levels of apoptosis. Given the ability of EGR1 to regulate these apoptotic pathways, it is reasonable to assume that EGR1 might affect chemotherapy and/or radiation sensitivity of tumors. This is supported by biochemical studies showing cells devoid of EGR1 are resistant to UV light–induced apoptosis.11
Our observations of reduced EGR1 levels correlating with reduced PTEN and poor outcome in NSCLC suggest a potential value for therapeutic strategies that enhance EGR1 in tumors. It is currently unclear why EGR1 is reduced in lung cancer compared with normal lung tissue. Possible mechanisms include chromosomal abnormalities resulting in allele loss of EGR1, epigenetic mechanisms such as promoter methylation, or deregulation of signaling pathways that maintain EGR1 transcription in normal tissues. The EGR1 promoter contains a number of serum response elements that respond to activation of the Ras-Raf-MEK-ERK signaling pathway. Interestingly, one recent study has found reduction in ERK activity in lung cancers compared with normal lung tissue.35 This suggests that reduced ERK activity in lung cancers may be responsible for corresponding reductions in EGR1.
We conclude that low expression of EGR1 correlates with low expression of PTEN and is predictive of poor survival in patients undergoing surgical resection for NSCLC. Identification of patients with low EGR, therefore, may identify patients at high risk for disease recurrence and may additionally identify patients who have tumors resistant to therapy secondary to loss of pathways such as PTEN. Additional study of mechanisms of EGR1 downregulation in lung cancers is warranted because reversal of EGR1 deregulation in tumors may have therapeutic value.
Authors' Disclosures of Potential Conflicts of Interest
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Honoraria: Gerold Bepler, AstraZeneca, Eli Lilly, Genentech. Research funding: Gerold Bepler, Eli Lilly. For a detailed description of these categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and Disclosures of Potential Conflicts of Interest found in Information for Contributors in the front of each issue.
Acknowledgment
We thank Rebecca Alexander for administrative assistance.
NOTES
Supported in part by NIH grants R01 CA 102726, NIH U01 CA101222, and by the Molecular Biology Core Facility and the Molecular Imaging Core at the H. Lee Moffitt Cancer Center & Research Institute.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Levin WJ, Press MF, Gaynor RB, et al: Expression patterns of immediate early transcription factors in human non-small cell lung cancer: The Lung Cancer Study Group. Oncogene 11:1261-1269, 1995
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McDoniels-Silvers AL, Nimri CF, Stoner GD, et al: Differential gene expression in human lung adenocarcinomas and squamous cell carcinomas. Clin Cancer Res 8:1127-1138, 2002
Yamagata N, Shyr Y, Yanagisawa K, et al: A training-testing approach to the molecular classification of resected non-small cell lung cancer. Clin Cancer Res 9:4695-4704, 2003
Thiel G, Cibelli G: Regulation of life and death by the zinc finger transcription factor Egr-1. J Cell Physiol 193:287-292, 2002
Adamson ED, Mercola D: Egr1 transcription factor: Multiple roles in prostate tumor cell growth and survival. Tumour Biol 23:93-102, 2002
Adamson E, de Belle I, Mittal S, et al: Egr1 signaling in prostate cancer. Cancer Biol Ther 2:617-622, 2003
Krones-Herzig A, Adamson E, Mercola D: Early growth response 1 protein, an upstream gatekeeper of the p53 tumor suppressor, controls replicative senescence. Proc Natl Acad Sci U S A 100:3233-3238, 2003
De Belle I, Wu JX, Sperandio S, et al: In vivo cloning and characterization of a new growth suppressor protein TOE1 as a direct target gene of Egr1. J Biol Chem 278:14306-14312, 2003
Virolle T, Adamson ED, Baron V, et al: The Egr-1 transcription factor directly activates PTEN during irradiation-induced signalling. Nat Cell Biol 3:1124-1128, 2001
Abdulkadir SA, Qu Z, Garabedian E, et al: Impaired prostate tumorigenesis in Egr1-deficient mice. Nat Med 7:101-107, 2001
Svaren J, Ehrig T, Abdulkadir SA, et al: EGR1 target genes in prostate carcinoma cells identified by microarray analysis. J Biol Chem 275:38524-38531, 2000
Calogero A, Arcella A, De Gregorio G, et al: The early growth response gene EGR-1 behaves as a suppressor gene that is down-regulated independent of ARF/Mdm2 but not p53 alterations in fresh human gliomas. Clin Cancer Res 7:2788-2796, 2001
Huang RP, Fan Y, de Belle I, et al: Decreased Egr-1 expression in human, mouse and rat mammary cells and tissues correlates with tumor formation. Int J Cancer 72:102-109, 1997
Huang RP, Darland T, Okamura D, et al: Suppression of v-sis-dependent transformation by the transcription factor, Egr-1. Oncogene 9:1367-1377, 1994
Huang RP, Liu C, Fan Y, et al: Egr-1 negatively regulates human tumor cell growth via the DNA-binding domain. Cancer Res 55:5054-5062, 1995
Liu C, Yao J, de Belle I, et al: The transcription factor EGR-1 suppresses transformation of human fibrosarcoma HT1080 cells by coordinated induction of transforming growth factor-beta1, fibronectin, and plasminogen activator inhibitor-1. J Biol Chem 274:4400-4411, 1999
Liu C, Yao J, Mercola D, et al: The transcription factor EGR-1 directly transactivates the fibronectin gene and enhances attachment of human glioblastoma cell line U251. J Biol Chem 275:20315-20323, 2000
de Belle I, Huang RP, Fan Y, et al: p53 and Egr-1 additively suppress transformed growth in HT1080 cells but Egr-1 counteracts p53-dependent apoptosis. Oncogene 18:3633-3642, 1999
Calogero A, Lombari V, De Gregorio G, et al: Inhibition of cell growth by EGR-1 in human primary cultures from malignant glioma. Cancer Cell Int 4:1-12, 2004
Bepler G, Sharma S, Cantor A, et al: RRM1 and PTEN as prognostic parameters for overall and disease-free survival in patients with non–small-cell lung cancer. J Clin Oncol 22:1878-1885, 2004
Soria JC, Lee HY, Lee JI, et al: Lack of PTEN expression in non-small cell lung cancer could be related to promoter methylation. Clin Cancer Res 8:1178-1184, 2002
Tamura M, Gu J, Tran H, et al: PTEN gene and integrin signaling in cancer. J Natl Cancer Inst 91:1820-1828, 1999
Tamura M, Gu J, Takino T, et al: Tumor suppressor PTEN inhibition of cell invasion, migration, and growth: Differential involvement of focal adhesion kinase and p130Cas. Cancer Res 59:442-449, 1999
Tamura M, Gu J, Matsumoto K, et al: Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science 280:1614-1617, 1998
Gautam A, Li ZR, Bepler G: RRM1-induced metastasis suppression through PTEN-regulated pathways. Oncogene 22:2135-2142, 2003
Coffer PJ, Jin J, Woodgett JR: Protein kinase B (c-Akt): A multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem J 335:1-13, 1998 (pt 1)
Cantley LC, Neel BG: New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci U S A 96:4240-4245, 1999
Ramaswamy S, Nakamura N, Vazquez F, et al: Regulation of G1 progression by the PTEN tumor suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway. Proc Natl Acad Sci U S A 96:2110-2115, 1999
Stambolic V, Suzuki A, de la Pompa JL, et al: Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95:29-39, 1998
Brognard J, Clark AS, Ni Y, et al: Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res 61:3986-3997, 2001
Bianco R, Shin I, Ritter CA, et al: Loss of PTEN/MMAC1/TEP in EGF receptor-expressing tumor cells counteracts the antitumor action of EGFR tyrosine kinase inhibitors. Oncogene 22:2812-2822, 2003
Kandasamy K, Srivastava RK: Role of the phosphatidylinositol 3'-kinase/PTEN/Akt kinase pathway in tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in non-small cell lung cancer cells. Cancer Res 62:4929-4937, 2002
Keshamouni VG, Reddy RC, Arenberg DA, et al: Peroxisome proliferator-activated receptor-gamma activation inhibits tumor progression in non-small-cell lung cancer. Oncogene 23:100-108, 2004(Bernadette Ferraro, Gerol)
Departments of Interdisciplinary Oncology, University of South Florida College of Medicine, Tampa, FL
ABSTRACT
PURPOSE: The zinc finger transcription factor early growth response gene 1 (EGR1) is underexpressed in non–small-cell lung cancer (NSCLC) compared with normal lung. EGR1 expression has been linked to tumor suppression as a result of cell cycle arrest and apoptosis through regulation of tumor suppressor pathways including PTEN. For these reasons, we hypothesized that reduced levels of EGR1 would correlate with inferior outcome in patients with NSCLC.
PATIENTS AND METHODS: Patients who underwent surgical resection for NSCLC had RNA extracted from tumor tissue and EGR1 gene expression was quantified by real-time quantitative polymerase chain reaction. The levels of EGR1 expression were examined in relationship to patient characteristics, histology, tumor stage, PTEN expression, and overall and disease-free survival.
RESULTS: EGR1 expression strongly correlated with PTEN expression (P < .0001). No correlation of EGR1 with histology or stage was detected. Patients with high levels of EGR1 had better overall and disease-free survival compared with patients with low levels of EGR1 (P = .040 and P = .096, respectively). In a stratified log-rank test, low EGR1 expression was predictive of poor survival independent of tumor stage.
CONCLUSION: EGR1 gene expression predicts PTEN levels and survival after surgical resection of NSCLC. Consistent with its known tumor suppressor properties, lower levels of EGR1 are associated with poor outcome. Identification of patients with low EGR1 therefore may identify patients at high risk for disease recurrence and may also identify patients who have tumors resistant to therapy secondary to loss of pathways such as PTEN.
INTRODUCTION
Tumor stage continues to be the most important predictor for outcome after surgical resection for early stage non–small-cell lung cancer (NSCLC).1 Patients who undergo curative surgical resection for apparently localized disease have 5-year survivals ranging between 25% and 70%, suggesting the need for better identification of occult metastatic disease. A better understanding of molecular pathways that influence the lung cancer phenotype may lead to the identification of patients at high risk for recurrence and thus interventions can be directed at those who derive maximum benefit.
Increasing use of gene expression profiling technology is allowing the identification of differential gene expression in lung cancers compared with normal lung tissue. One gene product consistently identified as underexpressed in NSCLC tumors is the zinc finger transcription factor early growth response gene 1 (EGR1). Previous studies from the Lung Cancer Study Group found decreased coordinate expression of EGR1, along with other early response genes such as C-JUN and C-FOS, in 72% (73 of 101) of NSCLC tumor samples compared with adjacent normal lung tissue.2 More recent studies investigating gene expression patterns in NSCLC using cDNA arrays have shown downregulation of EGR1 in lung tumors compared with normal lung tissue.3-5 EGR1, also known as zif268, NGFI-A, Krox24, and TIS8, has been linked to diverse cellular pathways including differentiation and tumor suppression through cell cycle arrest and apoptosis (reviewed by Thiel and Cibelli,6 and Adamson and Mercola7). EGR1 is linked to a number of tumor suppressor pathways including the regulation of PTEN, p53, p21, transforming growth factor beta, and TOE1.8-11 EGR1 gene expression is elicited by multiple stimuli, including growth factors, cytokines, UV and gamma irradiation, and hypoxia.6,7 The functions of EGR1 are diverse and appear to be dependent on tissue type. In prostate cancer, EGR1 promotes growth and survival of prostate cancer cells and EGR1 levels are correlated with increased Gleason score.11-13 This contrasts to studies in lung, brain, and breast tissues, in which decreased EGR1 is associated with tumor formation.14,15 Studies with glioma and sarcoma cell lines have found that EGR1 can suppress transformation and tumorigenicity through diverse mechanisms including inhibition of cell growth, counteraction of apoptosis, and enhanced cell adhesion.16-21 No studies have shown direct evidence of tumor suppressor properties of EGR1 in lung cancer cells but the reduced levels of EGR1 in lung cancer tissues compared with normal lung tissues does suggest a tumor suppressor function for EGR1 in NSCLC.
On the basis of the potential for tumor suppressor properties of EGR1 in lung tissues and reports of reduced levels of EGR1 in lung cancer, we examined the prognostic role of EGR1 in NSCLC. Given the ability of EGR1 to regulate the PTEN tumor suppressor pathway and our previous results demonstrating the prognostic value of PTEN, it was our hypothesis that reduced levels of EGR1 would correlate with inferior outcome.11,22 We found that EGR1 is a significant and strong predictor of PTEN, and reduced levels of EGR1 are associated with poor overall survival (OS) and disease-free survival (DFS) in NSCLC.
PATIENTS AND METHODS
Patient Population
Fresh-frozen tumor specimens from patients with resected NSCLC were collected from 1991 to 2001. Specimens were macrodissected to enrich for tumor tissue. Specimens were microscopically examined to ensure adequate tumor quality and confirm the histologic diagnosis. Only specimens with 60% of cells consisting of tumor were processed for analysis. Use of the tumor specimens and review of clinical data were approved by the institutional review board. During this time interval, patient's symptoms, prior medical history, use of tobacco products, demographics, and other relevant parameters were prospectively collected on hard-copy questionnaires. Information obtained from the questionnaire was verified in a face-to-face interview by the treating surgeon.
Tumor staging was based on computed tomography of the chest and upper abdomen, mediastinal lymph node dissection, and gross and microscopic evaluation of the resected lung tissue. Other staging studies, including brain imaging and bone scans, were performed at the discretion of the surgeon. The date of first pathologic verification of malignancy was used as the date of diagnosis. After surgery, patients were observed for 3 months by the surgeon. They were then given a choice of follow-up at regular intervals (every 3 to 12 months) by the referring physician or at our institution, with standard radiographs and/or computed tomography of the chest. The first date of unequivocal clinical evidence of disease or histologic confirmation, in patients with questionable status, was recorded as the recurrence date. The date of death was obtained through family or care provider contact, and it was verified by review of public records. Overall survival was defined as the time elapsed from histologic diagnosis to death. Disease-free survival was defined as the time elapsed from surgery to recurrence or death.
Quantitative Reverse Transcriptase Polymerase Chain Reaction Analysis
RNA was isolated from tumor tissue as previously described and reverse transcribed into cDNA using established techniques.22 Quantitative real-time polymerase chain reaction analysis was performed for each sample using 5 ng of RNA, and reactions were run in triplicate on 96-well plates. Reverse transcription reactions were also done on serial dilutions of total human RNA (Applied Biosystems Inc [ABI], Foster City, CA) to generate a standard curve for EGR1, PTEN, and 18S. TaqMan probes and primers for 18S rRNA (part No. 4319413E; ABI) were purchased as a predeveloped assay system from ABI. EGR1 and PTEN primers were designed to span intron-exon boundaries to reduce the possibility of genomic DNA amplification. The EGR1 probe was labeled with 6-FAM on the 5' end and a minor groove binder nonfluorescent quencher on the 3' end. The PTEN probe was labeled with 6-FAM on the 5' end and TAMRA was used as a quencher on the 3' end. Probe sets are listed in Table 1. Fluorescent emission was recorded in real-time (ABI prism 7700; Perkin-Elmer, Foster City, CA). A linear equation was derived from the standard curve cycle threshold values and used to determine the relative amount of RNA in the sample for EGR1, PTEN, and 18S rRNA. The relative amount of RNA in each sample was normalized to that sample's 18S rRNA expression levels.
Statistical Analysis
Correlation coefficients between the expression of EGR1 and demographic data were calculated according to Spearman. The Wilcoxon rank sum test was used to test for significant associations between dichotomous variables and EGR1 expression, and the Kruskal-Wallis test was used for variables with more than two categories. OS and DFS probabilities were estimated using the Kaplan-Meier method, and log-rank testing was used to determine the level of significance between survival curves. The stratified log-rank test was done to compare groups while adjusting for a potential confounder such as disease stage.
RESULTS
Fresh tumor specimens during a 10-year period were collected from pathologically staged patients undergoing complete resection for NSCLC. Table 2 lists the demographic and clinical data on the patients included in this study. There were 49 women and 76 men. The median age of the study cohort was 68.5 years and median follow-up time for the study cohort was 101 months (range, 39 to 161 months). The histopathology included adenocarcinoma (n = 53), squamous cell carcinoma (n = 50), bronchioloalveolar carcinoma (n = 8), and large-cell carcinoma (n = 14). Forty-five patients had stage IA disease (tumor size < 3.0 cm), 56 had stage IB disease (tumor size 3.0 cm), and 24 patients had either stage II or III disease. Eleven patients were lifelong nonsmokers (< 100 cigarettes), 29 were active smokers, and 74 were former smokers. The smoking history was not disclosed by 11 patients. At the time of the analysis, 64 patients had died and 61 patients remained alive.
Fresh tissues were used to perform quantitative polymerase chain reaction analysis for both EGR1 and PTEN. We explored differences in EGR1 expression among women and men; age; smoking status; stages I, II, and III; performance status 0 and more than 0; absence or presence of weight loss; age; and histopathology. Table 3 lists correlations between EGR1 and patient characteristics, and no significant correlations were found with sex, histology, stage, and quantitative smoking history (measured in pack-years). We also examined for differences in EGR1 in active smokers and patients who had quit smoking tobacco. We identified a trend toward higher EGR1 levels in patients who had quit compared with those patients who were active smokers at the time of diagnosis (P = .09). Finally, we identified a highly significant correlation between the levels of EGR1 and PTEN (Spearman correlation coefficient, 0.42; P < .0001) consistent with biochemical studies showing direct regulation of PTEN by EGR1.11
Kaplan-Meier OS and DFS curves for EGR1 and PTEN expression dichotomized by the median level for each RNA are shown in Figure 1. EGR1 levels below the median predicted worse OS and DFS compared with patients with EGR1 levels above the median (P = .040 and P = .096, respectively). Similarly, levels of PTEN below the median predict poor survival in this patient cohort (P = .026 and P = .048, respectively). Because tumor stage is a critical prognostic variable for outcome after surgical resection of NSCLC, we next performed a stratified log-rank test to assess whether EGR1 and PTEN are prognostic of survival independent of stage. Results are listed in Table 4. We found that EGR1 and PTEN are independent predictors of outcome after surgical resection of NSCLC.
DISCUSSION
Our results show that expression levels of EGR1 are strongly associated with expression levels of PTE, and low levels of EGR1 predict poor outcome after surgical resection of NSCLC. These data are consistent with a model in which EGR1 functions as a tumor suppressor gene in lung tissues through regulation of PTEN, and loss of this pathway results in more aggressive disease.22 Previous reports have shown rare genetic alterations of PTEN but point toward promoter methylation as one mechanism of PTEN loss in NSCLC.23 On the basis of previous biochemical studies as well as our own results, it appears that EGR1 is an important modulator of PTEN. EGR1 has been found to directly regulate the PTEN gene promoter and upregulate transcription of the PTEN gene.11 The virulence associated with loss of PTEN expression in early-stage NSCLC could result from enhanced tumor invasion and metastasis. PTEN is linked to control of invasion and metastasis through direct negative regulation of focal adhesion kinase.24-26 Our previous studies demonstrated that PTEN reduced phosphorylation of focal adhesion kinase, suppressed invasion and metastasis formation, and increased survival in an animal model.27 PTEN is also thought to function by negatively regulating the action of phosphatidylinositol 3-kinase and its downstream substrate AKT. Loss of PTEN function either through mutations or reduced expression can result in increased AKT activity and suppression of apoptosis.28-31 Published data suggest that AKT is constitutively active in NSCLC cells and is involved in resistance to a wide array of apoptotic stimuli including cytotoxic chemotherapy, radiation therapy, tumor necrosis factor–related apoptosis-inducing ligand, and EGFR tyrosine kinase inhibitors.32-34
Although our results show a strong correlation between EGR1 and PTEN, EGR1 may function as a tumor suppressor in lung cancer by regulating additional tumor suppressor genes including p53, p21, TOE1, and TGF. Loss of EGR1-driven expression of these genes would be predicted to allow for enhanced tumor cell growth and reduced levels of apoptosis. Given the ability of EGR1 to regulate these apoptotic pathways, it is reasonable to assume that EGR1 might affect chemotherapy and/or radiation sensitivity of tumors. This is supported by biochemical studies showing cells devoid of EGR1 are resistant to UV light–induced apoptosis.11
Our observations of reduced EGR1 levels correlating with reduced PTEN and poor outcome in NSCLC suggest a potential value for therapeutic strategies that enhance EGR1 in tumors. It is currently unclear why EGR1 is reduced in lung cancer compared with normal lung tissue. Possible mechanisms include chromosomal abnormalities resulting in allele loss of EGR1, epigenetic mechanisms such as promoter methylation, or deregulation of signaling pathways that maintain EGR1 transcription in normal tissues. The EGR1 promoter contains a number of serum response elements that respond to activation of the Ras-Raf-MEK-ERK signaling pathway. Interestingly, one recent study has found reduction in ERK activity in lung cancers compared with normal lung tissue.35 This suggests that reduced ERK activity in lung cancers may be responsible for corresponding reductions in EGR1.
We conclude that low expression of EGR1 correlates with low expression of PTEN and is predictive of poor survival in patients undergoing surgical resection for NSCLC. Identification of patients with low EGR, therefore, may identify patients at high risk for disease recurrence and may additionally identify patients who have tumors resistant to therapy secondary to loss of pathways such as PTEN. Additional study of mechanisms of EGR1 downregulation in lung cancers is warranted because reversal of EGR1 deregulation in tumors may have therapeutic value.
Authors' Disclosures of Potential Conflicts of Interest
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Honoraria: Gerold Bepler, AstraZeneca, Eli Lilly, Genentech. Research funding: Gerold Bepler, Eli Lilly. For a detailed description of these categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and Disclosures of Potential Conflicts of Interest found in Information for Contributors in the front of each issue.
Acknowledgment
We thank Rebecca Alexander for administrative assistance.
NOTES
Supported in part by NIH grants R01 CA 102726, NIH U01 CA101222, and by the Molecular Biology Core Facility and the Molecular Imaging Core at the H. Lee Moffitt Cancer Center & Research Institute.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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