| Objective:This study aimed to explore the expression pattern and clinical significance of tumor suppressor gene runt-related transcription factor 3(RUNX3)in non-small cell lung cancer and to investigate the roles that epigenetic marks like histone modification and DNA methylation might play in the regulation of RUNX3expression and possible mechanisms.Methods:A total of 208 NSCLC patients and block samples who had never received other therapies before were collected and built into tissue microarrays(TMA).Protein level of RUNX3,trimethylated histone H3 at lysine 27(H3K27me3),enhancer of zeste homolog 2(EZH2),c-myc,cyclinD1,transforming growth factor beta 1(TGF-?1),Ki-67,DNA methyltransferase 1/3b(DNMT1/3b)were firstly detected and evaluated with immunohistochemistry,western blotting and even immunofluorescence to determine their expressions and mutual correlations.TdT mediated dUTP-biotin nick end labelling(TUNEL)was used to assess the degree of cancerous apoptosis.After DNA was extracted from the formalin-fixed specimens,pyrosequencing was used to evaluate the methylation level of RUNX3 gene promoter.Results:1.In the first part of our study,archival formalin-fixed,paraffin-embedded tissue sections from a series of 5 normal lung tissue and 208 patients(as a result of tissue dropping in tissue microarray,only 188 cases left;128 males and 60 females;age range:36-78 years old)undergone surgery for NSCLC at the Department of Thoracic Surgery of Fujian Cancer Hospital&Fujian Medical University Cancer Hospital during 2010-2011 were selected.None had received chemo-or radio-therapy prior to tissue collection.The histopathologic features of cancerous specimens and TNM staging was determined according to the 8th AJCC guidelines for NSCLC.By means of immunohistochemistry and TdT mediated dUTP-biotin nick end labelling(TUNEL),expression of RUNX3 protein,index of cellular proliferation like Ki-67 and apoptotic index were determined.Semi-quantification and X-tile software were utilized to determine the IHC staining score and its cutoff point.Then all patients were dichotomized into groups of high and low expression of RUNX3,or nuclear RUNX3expression(positive in the nucleus and positive or negative in the cytoplasm)and non-nuclear RUNX3 expression(positive in the cytoplasm or negative in the whole cell).Expression patterns in NSCLC consisted of 4 categories,i.e.,negative,nucleus,cytoplasm and whole-cell expression,exactly as what it was in gastric,colorectal and breast cancer.The RUNX3 IHC score in normal lung tissue,sqamous cell carcinoma and adenocarcinoma was 12.00,6.93±0.55 and 6.38±0.48,respectively(P<0.01).After dichotomizing into high and low RUNX3 expression subgroups in accordance with the result of X-tile software,it's found of 55 cases(29%)with negative expression,133 cases(71%)with positive expression.Of the 133 positive expression case,10(7.5%)were nuclear expression,52(39.1%)were cytoplasmic expression,and 71(53.4%)were pancellular expression.Then correlation of different RUNX3expression patterns(high and low or nuclear and non-nuclear)and clinicopathologic parameters were determined by means of?~2 analysis,firstly it's found that higher RUNX3 expression was significantly associated with patients with advanced age(P=0.025),lower ECOG PS(P=0.019)as well as absence of postoperative metastasis(P=0.002).In addition,higher RUNX3 expression was observed to have a trend to correlate with absence of LN metastasis(P=0.062)as well as absence of distant metastasis at diagnosis(P=0.083).Eventually,no association had been discovered between expression of RUNX3 and gender(P=0.196),histology(P=0.536),smoking status(P=0.995),cellular differentiation(SCC only,P=1.000),lymphatic vessels invasion(P=0.223),nerve invasion(P=0.492),pleural invasion(P=0.364),vascular invasion(P=0.437),T-staging(P=0.488),mediastinal LN involvement(P=0.464),TNM-staging(P=0.230 or P=0.579),degree of resectibility(P=0.788),depth of invasion(P=0.757),serum CEA level(P=0.160),postoperative regional relapse(P=0.148)or Ki-67 expression(P=0.701).Secondly it's demonstrated that non-nuclear localization was significantly associated with ADE histology(P=0.009),lymphatic vessels invasion(P=0.050),lymph node involvement(P=0.034)and postoperative metastasis(P=0.035).No correlation had been determined between localization of RUNX3 expression and age(P=0.911),gender(P=0.125),ECOG PS(P=0.592),smoking status(P=0.360),cellular differentiation(SCC only,P=1.000),nerve invasion(P=0.431),pleural invasion(P=0.236),vascular invasion(P=0.496),T-staging(P=0.137),mediastinal LN involvement(P=0.652),M-staging(P=0.701),TNM-staging(P=0.774 or P=0.602),degree of resectibility(P=0.868),depth of invasion(P=0.240),serum CEA level(P=0.790),postoperative regional relapse(P=0.671)or Ki-67 expression(P=0.377).Survival analysis by Kaplan-Meier method with log-rank test indicated that patients with high level of RUNX3 exhibited much better outcome and longer OS than those with low RUNX3 expression(P=0.0142),regardless of various localizations of RUNX3 expression.However,when focusing on the effect that different localization of RUNX3 expression might have on OS via Kaplan-Meier analysis,we found that no statistical significance had been discovered between patient groups of nuclear and non-nuclear localization of RUNX3 expression(P=0.3781).It had to be noticed that when categorized into 4 subgroups,i.e.,negative,nuclear,cytoplasmic and whole-cell expression of RUNX3,patients with negative expression in both nuclear and cytoplasm showed the worst outcome and shortest OS,while the other 3 subgroups had no difference in OS with one other.In addition,the proliferative and apoptotic indices of NSCLC patients with different expression patterns were also evaluated and the results indicated that no difference had been shown in either subgroups of different expression levels or different localization[(23.78±2.79),%vs.(18.44±2.36)%,P=0.20;(21.14±2.14)%vs.(19.88±3.36)%,P=0.876,respectively].As for the apoptotic index,no difference had been demonstrated within subgroups of different RUNX3 expression localization(1.62±0.85 vs.1.58±0.72,P=0.73)while obvious statistical significance existed between subgroups of different RUNX3 expression levels,that's,in comparison to low level of RUNX3 expression in NSCLC patients,those with higher RUNX3 level demonstrated much higher cancerous apoptotic index(6.34±1.03 vs.2.02±0.75,P<0.001).2.In the second part of our research,in consideration of the important roles that H3K27me3,EZH2 and c-myc might play on the up-and down-stream signal regulation of RUNX3,we evaluated the expression of H3K27me3,EZH2 and c-myc in the 188 NSCLC patients by means of immunohistochemistry,immunofluorescent co-staining as well as Western blotting to further analyze the effect of their expressions or co-expressions might have on the significance as well as their correlations with other clinicopathologic parameters.IHC score of H3K27me3 and EZH2 in normal lung tissue,adenocarcinoma and sqamous cell carcinoma was 12.0,5.01±0.46,4.36±0.52 and 0,1.19±0.23,2.96±0.47,respectively.Of 188NSCLC patients,116(62%)were high H3K27me3 expression while 72(38%)were low H3K27me3 expression.As for EZH2 expression,the number of high and low was63(34%)and 125(66%),respectively.As for the expression of several biomarkers like RUNX3,H3K27me3,EZH2 and c-myc in squamous cell carcinoma with different levels of differentiation(well,moderate and poor),their IHC scores were8.00±2.83,6.79±0.64,7.21±1.27(P=0.865);10.00±2.00,4.19±0.58,3.43±1.16(P=0.03);3.75±1.93,3.21±0.56,1.71±0.88(P=0.436)and 5.75±2.84,4.89±0.52,3.36±0.95(P=0.368),respectively.And the expression of several biomarkers like RUNX3,H3K27me3,EZH2 and c-myc in adenocarcinoma with different predominant growth patterns(lepidic,acinar,pappilary,micropappilary and solid),their IHC scores were 5.60±1.71,6.74±0.57,4.67±1.94,4.00±2.31,6.59±1.33(P=0.683);6.00±1.84,5.57±0.57,3.44±1.69,1.00±0.58,3.53±1.02(P=0.207);0.20±0.20,1.27±0.30,0.78±0.66,0.67±0.67,1.76±0.78(P=0.575);1.30±0.42,1.22±0.27,0.78±0.40,0.00,1.47±0.58(P=0.817),respectively.Using?~2 test to evaluate the correlation between H3K27me3 expression and other clinicopathologic parameters,the results demonstrated that low H3K27me3expression was significantly correlated with male gender(P=0.010),high ECOG PS(P=0.000),smokers(P=0.000),non-pTNM-?disease(P=0.030),postoperative relapse(P=0.006),low RUNX3 expression(P=0.001),non-nuclear RUNX3localization(P=0.003)and low EZH2 expression(P=0.002).In addition,low H3K27me3 expression inclined to closely associated with lymphatic vessel involvement(P=0.094),vascular involvement(P=0.069)and lymph node involvement(P=0.079),and probably their real correlation would be further demonstrated in the future study with larger sample size.No correlation had been indicated within H3K27me3 expression and age(P=0.640),body mass index(P=0.841),differentiation level of SCC(P=1.000),nerve involvement(P=0.383),pleural involvement(P=0.521),T staging(P=0.159),mediastinal lymph node involvement(P=0.401),M staging(P=0.409),resectibility(P=0.584),depth of invasion(P=0.416),serum CEA level(P=0.305)or postoperative distant metastasis(P=0.252).As for the correlation of EZH2 with other clinicopathologic parameters,it's found that low EZH2 expression was significantly correlated with ADE histology(P=0.005),low RUNX3 expression(P=0.018),non-nuclear RUNX3 localization(P=0.001)and low H3K27me3 expression(P=0.002).While no association had been determined between EZH2 expression and age(P=0.638),gender(P=0.714),ECOG PS(P=0.341),BMI(P=0.386),smoking status(P=0.720),differentiation level of SCC(P=0.423),lymphatic vessels involvement(P=0.881),nerve involvement(P=0.335),pleural involvement(P=0.843),vascular involvement(P=0.483),T staging(P=0.663),lymph node involvement(P=0.642),mediastinal lymph node involvement(P=0.939),M staging(P=0.665),TNM staging(P=0.882),resectibility(P=0.341),depth of invasion(P=0.344),serum CEA level(P=0.376),postoperative regional relapse(P=0.129)or postoperative distant metastasis(P=0.639).Kaplan-Meier survival analysis with log-rank test showed that in comparison with patients with low H3K27me3 expression,NSCLC patients with higher H3K27me3 expression had better prognosis and longer overall survival(P=0.0037),while no survival difference had been demonstrated in patients with different expression levels of EZH2(P>0.05).Furthermore,we also analyzed the significance of co-expression of some biomarkers,among which co-expression of RUNX3 and H3K27me3 stood a vital significance in the prediction of better outcome and longer OS in early-stage NSCLC,especially pTNM stage-?(P=0.0124).Correlation analysis demonstrated that non-coexpression of RUNX3 and H3K27me3 was significantly associated with male sex(P=0.037),high ECOG PS(P=0.000),smoker(P=0.001),postoperative relapse(P=0.000),postoperative distant metastasis(P=0.048),non-nuclear expression of RUNX3(P=0.000)and low EZH2 expression(P=0.016)while had nothing to do with age(P=0.918),histology(P=0.145),BMI(P=0.300),lymphatic vessels involvement(P=0.246),nerve involvement(P=0.314),pleural involvement(P=0.811),vascular involvement(P=0.144),T staging(P=0.189),lymph node involvement(P=0.152),mediastinal lymph node involvement(P=0.608),M staging(P=0.667),TNM staging(P=0.227?P=0.231),resectibility(P=0.850),depth of invasion(P=0.245),serum CEA level(P=0.174)or Ki-67 expression(P=0.947).So coexpression of RUNX3 and H3K27me3 could be used as a good marker as survival gain,so as to discriminiate whether or not TNM stage-?NSCLC patients would have a better survival.3.In the third part of our research,a total of 188 NSCLC specimens were analyzed of their DNMT1/3b as well as its downstream protein TGF-?1 expression,and their association with other clinicopathologic parameters.No correlation had been demonstrated within either of the three proteins'expression and survival(P>0.05),while?~2 test indicated that high expression of DNMT1 was significantly correlated with high BMI(P=0.009),high expression of RUNX3(P=0.005),non-coexpression of RUNX3 and H3K27me3(P=0.048),high expression of EZH2(P=0.002),non-coexpression of EZH2 and H3K27me3(P=0.043),high expression of DNMT3b(P=0.000)and high expression of Ki-67(P=0.012).High DNMT3b expression was closely associated with low EZH2 expression(P=0.026)and nuclear expression of RUNX3(P=0.015),and marginally associated with high Ki-67 expression(P=0.053).Low expression of TGF-?1 was obviously correlated with non-lymphatic invasion(P=0.032),abscense of LN metastasis(P=0.046)and low RUNX3 expression(P=0.036).Furthermore,a total of 40 cases with different localization of RUNX3protein,i.e.,negative,cytoplasmic,nuclear and whole-cell expression,10 cases for each pattern,were extracted of their DNA and further used for pyrosequencing to assess their methylation degree at the promoter region of RUNX3 gene.As a result,3CpG islands were evaluated of their methylation level,together with their sum value,the number went 4,i.e.,MET-1/2/4/Total.By means of Spearman rank correlation analysis,the correlation coefficients of RUNX3 expression and MET-1,MET-2,MET-4 and MET-Total were-0.589(P=0.000),-0.589(P=0.000),-0.577(P=0.000)and-0.555(P=0.000),respectively,indicating a significant correlation between low RUNX3 expression and hypermethylation of the promoter of RUNX3 gene.The correlation coefficients of EZH2 expression and MET-1,MET-2,MET-4 and MET-Total were-0.422(P=0.007),-0.316(P=0.047),-0.258(P=0.108)and-0.353(P=0.026),respectively.The correlation coefficients of H3K27me3 expression and MET-1,MET-2,MET-4 and MET-Total were-0.089(P=0.585),-0.022(P=0.891),0(P=1.000)and-0.011(P=0.946),respectively.Chi-square analyses demonstrated that hypomethylation at MET-1 was significantly associated with high RUNX3 expression(P=0.001),nuclear RUNX3 expression(P=0.024),high EZH2 expression(P=0.020),hypomethylation at MET-2(P=0.000),hypomethylation at MET-4(P=0.000),and hypomethylation of MET-total(P=0.000),while wasn't correlated with histological types(P=0.896),H3K27me3 level(P=0.833),parallel expression of EZH2 and H3K27me3(P=0.133),coexpression of EZH2 and H3K27me3(P=0.626),smoking status(P=0.292),DNMT1 expression(P=0.197)and DNMT3b expression(P=0.809).Conclusions:Expression level of RUNX3 is significantly associated with the prognosis of NSCLC patients and patients with high level of RUNX3 expression would have better outcome and longer OS while those with low RUNX3 expression showed worse outcome and shorter OS.Quite different from the pattern in GC,CRC or BC,no correlation of nuclear/non-nuclear RUNX3 expression and prognosis had been demonstrated in NSCLC.Coexpression of RUNX3 and H3K27me3 in early NSCLC patients demonstrated much better prognosis,especially in pTNM stage-?patients and could be used as a good marker in the prediction of outcome of early NSCLC patients.The reduction of RUNX3 expression in NSCLC largely resulted from hypermethylation at the promoter of RUNX3 gene,and expression of epigenetic marks like EZH2 or H3K27me3 was positively correlated with the expression of RUNX3. |
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