Introduction EGFR and IGF1R play important roles in the tumori- genesis and development of lung cancers, and they have preferred therapeutic molecular targets. Through the ERK/MAPK and PI3K/AKT cell signaling path- ways respectively, EGFR and IGF1R promote cell proliferation, drug resistance and metastasis of tumors (Mukohara et al., 1999; LeRoith and Roberts, 2003). EGR1 (early growth response gene 1) is a zinc finger transcription factor, subjecting to immediate early gene family, which regulates early cell growth. EGR1 over- expression is detected in the adenocarcinoma cells with K-RAS mutation-positive and EGFR mutation-positive (Ferraro et al., 2005). Recent study suggests that EGR1 is possibly a signaling crosstalk between the Erk pathway and Akt pathway. EGR1 may play a compen- satory role in the EGFR- or IGF1R-targeted therapy, by reducing drug resistance to the targeted drugs. EGFR overexpression is reported to be correlated with poor survival (Hirsch et al., 2003). The relationship between IGF1R expression and prognosis is not deter- mined (Nakagawa et al., 2012; Kim et al., 2014; Kim et al., 2012), and EGR1 expression seems to be a fine predictor in non-small cell lung cancer (NSCLC) patients (Ferraro et al., 2005). The present study intends to investigate the activated state of both the EGFR and IGF1R signaling pathways in NSCLC, and the relationships among EGR1, EGFR, IGF1R and clinical data. Materials and Methods Patients and specimens Patients included in the present study underwent surgi- A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2016; 11: S123-S130 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index; ISSN: 1991-0088 Abstract The purpose of this study was to investigate the incidence and clinical significance of alterations in EGFR, IGF1R and the cell signaling pathway activities induced by them, as well as EGR1 expression in resected non-small cell lung cancer (NSCLC). The protein expressions of biomarker were evaluated by Western blotting in tissues from 19 surgically resected NSCLCs. High expressions of EGR1, EGFR and IGF1R were detected in more than 30% tumor tissues. High expressions of pErk and pAkt were detected in more than 50% paracancer tissues. There were significant correlations between the NSCLC target factors detected (p<0.05). Alterations of protein expressions of target factor detected in NSCLC were significantly associated with altera- tions in pathological subtype, differentiation, pathological stage, and smoking history. Positive EGR1 might be associated with good survival, while positive pErk might be associated with poor prognosis. Article Info Received: 16 January 2016 Accepted: 30 January 2016 Available Online: 9 March 2016 DOI: 10.3329/bjp.v11iS1.26413 Cite this article: Yu D, Tan P, Yu M, Shi S, Wang X. EGR1, EGFR and IGF1R protein ex- pressions in non-small cell lung can- cer and their clinical significances. Bangladesh J Pharmacol. 2016; 11: S123-S130. EGR1, EGFR and IGF1R protein expressions in non-small cell lung cancer and their clinical significances Dujuan Yu1, Ping Tan1, Min Yu2, Shaomin Shi1 and Xue Wang3 1The Department of Respiration, China Japan Union Hospital of Jilin University, Changchun, Jilin, 130 033, China; 2The Department of Cardiology, Affiliated Hospital of Beihua University, Jilin, Jilin, 1340 00, China; 3The Center of Physical Examination, China Japan Union Hospital of Jilin University, Changchun, Jilin, 130 033, China. This paper was presented in the 3rd International Conference on Biomedicine and Pharmaceutics in Zhuhai, China, on December 11-13, 2015. http://www.bioxbio.com/if/html/BANGL-J-PHARMACOL.html http://www.asiajol.info/index.php/record/view/50670 cal resections for NSCLC from 2010 to 2011 in China Japan Union Hospital of Jilin University. The patients’ clinical and histopathological information was presen- ted in Table I. No patients underwent antineoplastic therapy prior to surgery. Totally 38 resected tissue specimens were obtained from 19 patients. All the patients donated their primary lung squamous cell carcinoma (SCC) tissues (S1-S10), corresponding para- cancer tissues (SP1–SP10), primary lung adenocarci- noma tissues (A1-A9) and the corresponding para- cancer tissues (AP1–AP9). HE staining was obtained (Figure 1). All the patients gave the informed consent. Western blotting Cells were separated from tumor mass and paired paracancer tissues, then lysed in the lysis buffer in the presence of protease and phosphatase inhibitors. The protein concentration was determined by the BCA method (Bio-Rad, USA). Proteins were separated on 15% gels by SDS-PAGE and transblot to PVDF mem- branes (Millipore, China). The membranes were incuba -ted in TBS-Tween 20 (TBST) containing 5% BSA 2 hours at room temperature. Monoclonal primary anti- bodies against EGR1, EGFR, IGF1R β subunit, pErk and pAkt were purchased from Santa Cruz Biotech- nology Inc. (USA) and used by 1:1000 diluted. β-actin was the inner reference (Santa Cruz Bio.). Peroxidase- conjugated secondary antibodies against mouse IgG (Santa Cruz Bio.) were used to mark the blots. The enhanced chemiluminescence (ECL. Pierce Biotechno- 000 Bangladesh J Pharmacol 2016; 11: 000-000 000 Table I Patients’ clinical and histopathological data Clinical data n Clinical data n Clinical data n Cases 19 Tumor status Perineurium infiltration Mean age (60 years) T1 1 Yes 6 Gender T2 17 No 13 Male 11 T4 1 Pleura infiltration Female 8 Nodal status Yes 4 Smoking N0 7 No 5 Yes 11 N1 4 Bronchus infiltration No 8 N2 8 Yes 3 Pathology Pathological stage No 6 Squamous cell carcinoma 10 I 5 Total survival Adenocarcinoma 9 II 5 more than 4 years 3 Differentiation III 9 3-4 years 8 Poor 4 Vascular infiltration 1-3 years 7 Moderate 15 Yes 14 less than 1 year 1 No 5 Figure 1: HE staining fo lung cancer Note: A. Lung squamous cell carcinoma; B. Lung adenocarcinoma logy, Inc., USA) light system method was used for color development. Blots were scanned by a UVI Alliance 4.7 densitometer (Uvitec Inc., UK). The grayscale ratios of protein versus β-actin blots were quantified by UVI soft image quantification software (Uvitec Inc., UK). Statistical analysis The statistical software SPSS19.0 (SPSS, Inc., USA) was used for statistical analysis of the data. The Mann- Whitney U-test was used to analyze the relationships between the expression levels of biomarkers and clinic pathologic data. Spearman rank correlation coefficient was used to analyze the relationships among biomarker expression levels in tumor mass and/or paired para- cancer tissues. Kaplain-Meier test with log-rank test was used to evaluate the NSCLC survival impacted by the overexpression of biomarkers EGR1, EGFR and IGF1R in tumor tissues, or the overexpression of pErk and pAkt in paracancer tissues. Statistical significance was set at p<0.05. Results Western blotting analysis Overexpression of EGR1 protein was detected in 52.6% (10/19) NSCLC tissues. Overexpression of EGFR pro- tein was 36.8% (7/19) NSCLC tissues. Overexpression of IGF1R protein was 78.9% (15/19) NSCLC tissues. Overexpression of pErk protein was 63.2% (12/19) paracancer tissues. Overexpression of pAkt protein was detected in 52.6% (10/19) paracancer tissues (Figure 2). Protein expression comparison The difference for the expression of EGFR, IGF1R, pErk and pAkt between lung cancer tissues and paracancers were significant (p<0.05), while the difference for EGR1 expression between these two tissues was not signifi- cant ( Table II). Relationships among protein expressions of EGR1, EGFR, IGF1R, pErk, pAkt and clinical data in NSCLC patients EGR1 protein expression was correlated with differen- tiation, pathological subtype and smoking history. EGFR protein expression was correlated with the pathological subtype, pathological stage and smoking history. IGF1R protein expression was correlated with the pathological subtype. pErk protein expression was correlated with differentiation, pathological subtype, pathological stage and smoking history. pAkt protein expression was correlated with differentiation, patholo- Table II Protein expression comparison between lung cancer tissues and para-cancers Blot grayscale ratio (/actin) of lung cancer factors EGFR/ IGF1R/ EGR1/ pErk/ pAkt/ Cancer tissues 11.3 10.8 8.6 3.3 7.3 Paracancers 7.2 6.0 11.9 11.3 11.2 p value 0.036a 0.002a 1.000 0.001a 0.040a Mann-Whitney U-test is used to analyze the relationships between the expression levels of biomarkers; ap<0.05 indicates a statistically significant difference Figure 2: Western blot analysis of lung cancer biomarkers in NSCLC tissues and paired paracancers Note: Odd number lanes are cancer samples; even number lanes are paracancer samples gical subtype, pathological stage and smoking history (Table III). Survival analysis of factors EGR1 protein expression was not associated with prognosis. Neither EGFR protein expression nor IGF1R protein expression was associated with overall survival. Not only pErk protein expression but also pAkt protein expression were associated with poor prognosis (p<0.05) (Figure 3). Correlation analysis of protein factors EGFR, IGF1R, EGR1, pErk and pAkt protien expre- ssions were correlated with each other according to the analysis on the grayscale ratios (Table IV). Discussion In the present study, EGFR protein overexpression were detected in 30% NSCLC tissues. In NSCLC, the Table III Relationships among protein expression blot grayscale ratios (/β-actin) of EGFR, IGF1R, EGR1, pErk, pAkt and clinical data in NSCLC patients Clinical data Cases (n) EGFR/ IGF1R/ EGR1/ Paracancer pErk/ Paracancer pAkt/ Gender (0.152) (0.310) (0.152) (0.283) (0.069a) (p value) Male 11 8.3 8.8 8.4 8.8 8.0 Female 8 12.3 11.6 12.3 11.6 12.8 Differentiation (0.530) (0.460) (0.049a) (0.046a) (0.028a) (p value) Poor 4 8.3 8.0 5.0 5.0 4.5 Moderate 15 10.5 10.5 11.3 11.3 11.5 Pathology type (0.013a) (0.043a) (0.035a) (0.034a) (0.001a) (p value) SCC 10 7.0 7.5 7.4 7.4 6.0 AC 9 13.3 12.8 12.9 12.9 14.4 Pathological stage (0.018a) (0.120) (0.135) (0.015a) (0.015a) (p value) 6 4.7 6.2 7.0 5.8 5.8 4 11.5 12.8 8.5 7.5 7.5 9 12.9 11.3 12.7 13.9 13.9 Tumor status (0.111) (0.333) (0.556) (0.500) (0.500) (p value) T1 1 1.00 3.0 5.0 6.0 6.0 T2 17 10.00 9.9 9.8 9.7 9.7 Nodal status (0.086a) (0.164) (0.253) (0.083a) (0.083a) (p value) N0 7 6.3 6.9 8.0 7.6 7.6 N1 4 11.5 12.8 8.5 7.0 7.5 N2 8 12.5 11.4 12.5 13.4 13.4 Smoking (0.006a) (0.069a) (0.005a) (0.006a) (0.001a) (p value) Yes 11 7.0 8.0 6.9 7.0 6.3 No 8 14.1 12.8 14.3 14.1 15.1 Mann-Whitney U-test is used to analyze the relationships between the expression levels of lung cancer biomarkers and clinicopathologic data. ap< 0.05 indicates a statistically significant difference Table IV Correlation analysis of protein blot grayscale ratios (/β-actin) in NSCLC Tumor IGF1R/ Tumor EGR1/ Paracancer pErk/ Paracancer pAkt/ Tumor EGFR/ 0.689 (0.00*) 0.560 (0.013a) 0.665 (0.002a) 0.707 (0.001a) Tumor IGF1R/ - - 0.486 (0.035a) 0.649 (0.003a) 0.602(0.002a) Tumor EGR1/ - - - - 0.682 (0.001a) 0.735 (0.000a) Paracancer pErk/ - - - - - - 0.844 (0.000a) Spearman rank correlation coefficient is used to analyze the relationships among lung cancer biomarkers’ expression levels in tumor mass and/or paracancer tissues. Data are given as correlation coefficient (p value), and ap<0.05 indicates statistically significant difference Figure 3: Survival analysis of lung cancer biomarkers in NSCLC Note: Kaplain-Meier test and log-rank test was used to evaluate how the NSCLC survival was impacted by the overexpression of biomarkers EGR1, EGFR and IGF1R in tumor tissues, or the overexpression of pErk and pAkt in paracancer tissues EGFR protein expression was correlated with the pathological subtype, pathological stage and smoking history, but was not correlated with survival. Study has found that both EGFR gene copy number and its protein expression are over expression in NSCLC, and there is apparent correlation between them (Hirsch et al., 2003). In NSCLC patients, EGFR overexpression is related with fast metastasis, short disease free survival, high relapse rate, poor survival and lower chemo- radiation sensitivity (Ohsaki et al., 2000). These suggest that EGFR plays a biological role in NSCLC, affects deterioration, progression, lymph node metas- tasis and malignant proliferation, and may be involved in NSCLC pathogenesis. In the present study, IGF1R protein overexpression was detected in above 60% NSCLC tissues. In NSCLC, the IGF1R protein expression was correlated with the pathological subtype (Table III), not correlated with survival. High IGF1R gene copy number and protein overexpression are common in NSCLC, particularly in SCCs (Tran et al., 2014). The IGF1R signaling mecha- nism has been found to be associated with the resistance to a variety of anti-tumor therapies including EGFR inhibitors and cisplatin (LeRoith and Jr Roberts, 2003; Riedemann and Macaulay, 2006; Cortés- Sempere et al., 2013), acting as a potentially predictor for resistance to gefitinib in NSCLC patients (Peled et al., 2013; Chen et al., 2013). Although studies have presumed that IGF1R overexpression is related with tumor replace and poor disease-free survival, other studies consider it not to be prognostically relevant, and the clinical significance of IGF1R expression in NSCLC is not confirmed (Nakagawa et al., 2012; Kim et al., 2014; Kim et al., 2012). Study has reported that the IGF1R expression status is not correlated with either tumor pathological status, differentiation and history of smoking, or smoking index. Better sensitivities of chemotherapy are detected in patients with IGF1R negative (Ning et al., 2010). Patients with concomitant IGF1R/EGFR FISH+/IHC+ have poor disease-free survival and poor overall survival (p = 0.005 and p = 0.01, respectively) (Ludovini et al., 2013). IGF1R expre- ssion is associated with the smoking history, squamous- celled carcinoma histology, K-Ras mutations, wild-type EGFR and hyporesponsivenes to EGFR tyrosine kinase inhibitors (Kim et al., 2012). These suggest IGF1R play some biological role in NSCLC, and may be involved in NSCLC pathogenesis. In the present study, EGR1 protein overexpression was detected in more than 60% NSCLC tissues. In NSCLC, EGR1 protein expression was correlated with differen- tiation, pathological subtype and smoking history, but not associated with survival. These results suggested that EGR1 played biological roles in NSCLC, affected malignant transformation, malignant proliferation, and may be involved in NSCLC pathogenesis (0.05