| Lung cancer is the leading cause of cancer-related death both worldwide and in China, principlely categorized into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC occupies around 80% of total lung cancers. It is estimated that in 2000 in China, the mortalityrate of lung cancer was 19.44 per 100,000, with 27.16 per 100,000 in males and 11.31 per 100,000 in females. About 80% of lung cancer cases were diagnosed at advanced disease stage. Though there is improved multidisciplinary comprehensive management with surgery, chemotherapy and radiotherapy,5-year survival rate of lung cancer is still poor as 10% in China and a little better in developed regions.Survival of patients with the same disease stage, histology and regimens could vary much in clinic as well as in terms of response to the same treatment regimens, which may be attributable to the different molecular characteristics of each tumor. In this circumstances, "personalized medicine" has been raised to tailor the treatment for each patient according to the moleculat biomarkers validated or to be validated which can predicte the response, survival and staging as well. Molecular targeted therapy could benefic in specific sub-group of patients, which highlighted the importance of molecular subtyping for lung cancers. Some targeted therapies and response prediction have been established such as:(1) For EGFR mutant lung cancer patients, small molecular inhibitors targeting the tyrosine kinase domain of epidermal growth factor receptor (EGFR-TKI), gefitinib or erlotinib, could significantly prolong the progression-free survival (PFS) of advanced disease. (2) Small molecule inhibitors tyrosine kinase domain of against anaplastic lymphoma kinase (ALK-TKI) could also greatly benefit in lung cancer patients with ALK gene fusion. (3) KRAS mutation has consistently been shown as a predictive biomarker for the resistance to these TKIs, and how to conquer KRAS mutant cancers is still under intensive investigation. Yet for each targeted therapy, due to the redundant survival pathways in tumor cells, duration of efficacy of TKI was limited and relapse of diease was a definite event during the disease course. To understand the mechanisms and develop treatments against the drug resistance is one of the major tasks of modern tumor biology. The detailed catalog of mutation in lung cancers are being intensively studied and will be helpful for future more developed targeted therapy.In addition, how to treat the lung cancer patients with wild type EGFR and KRAS and ALK remains to be improved. Present standard care of these patients in the first line setting is boublet regimen based on plantinum with limited response of nearly 30% in clinic. So how to improve the efficacy in these sub-population is of great interests in this study."Resisting cell death" is one of the hallmarks of malignant cancer cells. Functioning of the molecules in the apoptosis pathway may play roles during the entire process of cancer development and evolution. Molecules in the apoptotic pathway can be classified as regulators and effectors. The process of apotosis can be divided into stages of activation and execution. To promote the pro-apototic activation process is one of the strategies of cancer treatment. In this study, we try to analyse the status of apoptotic pathways in clinical specimens with different somatic mutations in EGFR, KRAS, and ALK. EGFRwtKRASwtALKwt (Triple negative, TriNeg) tumors were taken as the research focus. Cell models with different EGFR mutation, KRAS mutation and ALK gene fusion were used to evaluate the efficacy of caspase 3 activator, procaspase-activating compound 1 (PAC-1), and the possible synergistic anti-cancer effects in combination with cic-plantinum.Chapter 1 Profiling of genome-wide and apoptosis-related gene expression patterns (GEP) in EGFRmut, KRASmut, ALKmut and EGFRwtKRASwtALKwt lung cancersMethodsWith informed consent, cancer tissues of 145 cases of lung cancers receiving radical surgery during 2003 through 2006 were collected. DNAs were extracted using QIAamp DNA blood kit, EGFR and KRAS were tested for potential mutations. Total RNAs were also extracted and RACE-PCR-sequencing technology was adopted for ALK gene fusion testing. Then lung cancers were divided into four sub-groups with EGFRmut, KRASmut, ALKmut and EGFRwtKRASwtALKwt separately. Qualified total RNAs extracted using mirVana miRNA isolation kit from Ambion incorporation were subject to genome wide gene expression profiling on the Affymetrix platform. Modules in the GeneSpring(?) software were used for the microarray data management, statistical analysis, pathway analysis and visualization, with focused attention on the apoptotic pathway.Results1. In the 145 cancer tissues, EGFR mutation was detected in 49 cases (33.6%,1 case with concurrent KRAS mutation), ALK gene fusion in 12 cases (8.2%), KRAS mutation in 10 cases (6.8%) and EGFRwtKRASwtALKwt in 75 cases (51.3%). EGFR mutation was significantly correlated to histological adenocarcinoma (P<0.001). In the 12 cases with ALK gene fusion,9 were adenocarcinoma and 1 was large cell carcinoma (P=0.093). In the 10 cases with KRAS mutation,9 were adenocarcinoma and lwas large cell carcinoma (P=0.093)o Alterations of EGFR, KRAS and ALK were shown as exclusive to each other (only one case with concomittent EGFR mutation and KRAS mutation. EGFR vs KRAS, P=0.016; EGFR vs ALK, P=0.060; ALK vs KRAS, P=1.000).2. After quality control of RNA (RIN>6.0), a total of 120 cases of cancer tissues and 53 cases of adjacent con-cancerous tissues were tested by Affymetrix human whole genome gene expression plus 2.0 microarray. In EGFRwtKRASwtALKwt cases, there were 69 cancer tissues and 36 adjacent tissues. In EGFRmut cases, there were 32 cancer tissues and 10 adjacent tissues. In KRASmut cases, there were 9 cancer tissues and 3 adjacent tissues. In ALKmut cases, there were 10 cancer tissues and 4 adjacent tissues.3. FDR (False Discovery Rate) P value was set at 0.0001,231 differentially expressed genes were found in EGFR mutant cancer tissues versus adjacent tissues, in which 94 genes were upregulated and 122 genes downregulated with fold change equal or greater than 2.0 (note:here the gene refer to a probe set on the macroarray chip). In KRAS muatant cases,20 differentially expressed genes in cancer tissues versus adjacent non-cancerous tissues were found, with 4 up-regulated and 16 down-regulated, all of which were equal or greater than 2.0 by fold change. In ALK fusion positive cases, only 3 differentially expressed genes in cancer tissues versus adjacent tissues were found, with 2 up-regulated genes with fold change equal or greater than 2.0. In EGFRwtKRASwtALKwt cases,7912 differentially expressed genes were found in cancer versus adjacent tissues, in which 2236 genes were upregulated and 3095 genes downregulated with fold change equal or greater than 2.0.Among all the four subtypes of lung cancers, CASP3 and PARP1 were found significantly upregulated only in the triple negative cases.4. When only gene list of apoptotic pathways were imported into the Genespring(?) software for statistical analysis, FDR P value was set at0.01, in EGFR mutant cancer cases,17 differentially expressed genes were found in EGFR mutant cancers in which 4 genes were upregulated and 6 genes downregulated with fold change equal or greater than 2.0. In KRAS muatant cases,2 differentially expressed genes in cancer tissues versus adjacent non-cancerous tissues were found, with 1 up-regulated and 1 down-regulated with fold change equal or greater than 2.0. In ALK fusion positive cases, only 1 differentially upregulated gene was found in cancer tissues versus adjacent tissues. In EGFRwtKRASwtALKwt cases,74 differentially expressed genes were found in cancer versus adjacent tissues, in which 12 genes were upregulated and 16 genes downregulated with fold change equal or greater than 2.0.Among all the four subtypes of lung cancers, CASP3 and PARP1 were found significantly upregulated only in the triple negative cases.5. In EGFRwtKRASwtALFwt cases, genes CAPN1, IRAK1, BIRC5, FADD, CASP3, PARP1, TRAF2, TNFRSF11A, CYCS, PIK3R2 and TNFRSF11A were all upreulgated with fold changes equal or greater than 2.0, whileas IL1R1, PRKAR2B, CSF2RB, TNFRSF10C, TNF, CHP, PIK3R1, IRAK3, TNFRSF10D, PIK3R5, CFLAR and AKT3 were down-regulated with fold changes equal or greater than 2.0.ConclusionsIn EGFRmut, KRASmut, ALKmut and EGFRwtKRAwtALKwt cases, microarray data analysis demonstrated only in EGFRwtKRAwtALKwt cases, CASP3 and PARP1 were significantly and differentially upregulated in cancer tissues versus adjacent non-cancerous tissues, suggesting they could be taken as potential molecular targets for treatment.Chaper 2 Protein expression analysis of several apoptotic molecules in tissue microarray of NSCLCMethods144 cases of formalin-fixed paraffin-embedded tissues of NSCLC were collected during 2003 through 2006. These tissues were made into tissue micaroarray (TMA) with tissue microarayer of Beecher InstruMents. In the TMA block 26 cases of paired cancer versus adjacent non-cancerous tissues (lung parenchyma with 5～7 cm away from the edge of tumor mass) were included. Immunohistochemistry (IIHC) were performed to analyse the expression level of CASP3, CASP8, CASP9, PARP1, Cleaved CASP3 (C-CASP3), Cleaved PARP1 (C-PARP1), XIAP and BCL2. In each sample the staining intensity (negative=0, modest=1, intermediate=2 and strong=3) and positive celllproportion (0%=0, 1%～10%=1, 11%～50%=2,51%～80%=3, 81%～100%=4) were recoeded. IHC score for each sample was calculated by multiplying the intesnsity score with the proportion score, within a range of 0 through 12. The median value of all scores was used as cut-off value to separate high-expression from low-expression groups.Results1. In the evaluable 25 cases of paired cancer versus adjacent tissues, paired sample t test showed that C-PARP1 were not detected in apired samples, CASP8 was not significantly differentially expressed between cancer and adjacent tissues (P>0.05), other molecules CASP3, CASP9, PARP1, C-CASP3, XIAP and BCL2 were all expressed in cancers at a higher level than that in adjacent tissues (P<0.01).2. In the analysis of nonpaired 139 cases of cancers versus 25 adjacent tissues, non-parametric rank test showed that difference of C-PARP1 exrepssion between the two groups were not significant (P>0.05), CASP8 was significantly down-regulated in cancer tissues in comparison to adjacent tissues (P<0.05), other molecules CASP3, CASP9, PARP1, C-CASP3, XIAP and BCL2 were all expressed in cancers at a higher level than that in adjacent tissues (P<0.01).3. In the EGFRwtKRASwtALKwt cases, nonpaired 51 cases of cancers versus 12 adjacent tissues, non-parametric rank test showed that difference of C-PARP1 exrepssion between the two groups were not significant (P>0.05), CASP8 was significantly down-regulated in cancer tissues in comparison to adjacent tissues (P<0.05), other molecules CASP3, CASP9, PARP1, C-CASP3, XIAP and BCL2 were all expressed in cancers at a higher level than that in adjacent tissues (P<0.01).4. Relations of apoptotic molecules with clinical, pathological and survival factors were analyzed. In the bi-variate correlation analysis C-PARP1 was primarily found significantly correlated to overall survival (OS) (P<0.05). C-CASP3 and CASP8 were expressed in non-squamous cell carcinoma (non-SCC) at higher levels than that in SCC (P<0.05). BCL2 and PARP1 were expressed in squamous cell carcinoma (SCC) at higher levels than that in non-SCC (P<0.05). PARP1 also associated with male and smoking factors (P<0.05). Kaplan-Meir survival curve test showed that whether in all patients or in stage III-IV patients, C-PARP1 was significantly associated with OS (P<0.05), suggesting C-PARP1 might be a prognostic factor. In all patients with all stages, pN, Tumor size and disease stage were all statiscally associated with OS (P<0.05).In EGFRwtKRASwtALKwt (triple negative, TriNeg) patients, disease stage was significantly associated with OS (P<0.05). Expression of CASP3 showed a trend negative correlation with OS, with low expression of CASP3 favoring a prolonged OS in comparison with high expression of CASP3 (P=0.165); Expression of PARP1 showed a trend of positive correlation with OS, with high expression of PARP1 favoring a prolonged OS in comparison with low expression of PARP1 (P=0.198); Overall survivals between groups of low and high expression of C-PARP1 were not statistically significant (P=0.617).In non-EGFRwtKRASwtALKwt (non-triple negative, non-TriNeg) patients, tumor size and pN were both significantly associated with OS (P<0.05). Expression of CASP3 showed a trend of positive correlation with OS, with high expression of CASP3 favoring a prolonged OS in comparison with low expression of CASP3 (P=0.196); Expression of PARP1 showed a trend of negative correlation with OS, with low expression of PARP1 favoring a prolonged OS in comparison with high expression of PARP1 (P=0.198); Overall survivals between groups of low and high expression of C-PARP1 were statistically significant (P<0.001) with high C-PARP1 favoring a shorter OS.Notably, expressions of CASP3 and PARP1 were shown a trend of predicting the survival not in a same way in TriNeg and non-TriNeg patients, i.e., if high expression of these molecules in TriNeg patients favored a prolonged survival, then their high expression foavored a shorter OS in non-TriNeg patients.Multi-variate Cox regression model showed that disease stage, histology and C-PARP1 were significantly associated with OS, and C-PARP1 might server as a independent prognostic factor for these lung cancer patients. ConclusionsProtein expression analysis in paired and non-paired cancer versus adjacent tissues confirmed that apoptotic molecules CASP3, PARP1, CASP8, CASP9, XIAP and BCL2 were significantly upregulated in cancer tissues. CASP3 and PAPRl were also significantly upregulated in EGFBwtKRASwtALKwt cases, suggesting in these triple negative patients, CASP3 and PARP1 could be taken as potential treatment targets.BCL2 and PARP1 were expressed in squamous cell carcinoma (SCC) at higher levels than that in non-SCC. PARP1 also associated with male and smoking factors, suggesting that BCL2 and PARP1 might be potential treatment target for these sub-group of patients. Kaplan-Meir survival curve test and multivariate Cox ergression model both suggested that C-PARP1 might be a independent prognostic factor for NSCLC.Chaper 3 In vitro anti-cancer effects of cis-plantinum and/or PAC-1 in lung cancer cell lines with different genetic alterationsMethodsNon-small cacner cell lines of H1299 (EGFRwtKRASwtALKwt), A549 (KRASmut), PC9 (EGFRmut), H1650 (EGFRmut) and H1975 (EGFRmut) were taken as cell models, colorometric MTT (3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) tests were used to test in vitro anti-cancer effects of cis-plantinum (Cis), PAC-1 and Cis combined with PAC-1. Western blotting was performed to analyze the protein expression levels of CASP3, CASP9, C-CASP3, PARP1, C-PARP1, XIAP and BCL2, whereby ACTB used as internal control. Flow cytometry was perfromed to analyze the cell cycle changes in each experimental groups. Immunofluorescent cell chemistry was carried out to test the CASP3 and activated C-CASP3 in cells under fluorescent microscopy. Results1. By colorimetric MTT tests, it was found that single agent cis-plantinum could inhibit the growth of H1299, A549, PC9, H1650 and H1975 cells at a certain dose range with IC50 values of 1.9～11.2μM for these cells. PAC-1 could also inhibit the growth of H1299, A549, PC9, H1650 and H1975 cells at a certain dose range with IC50 values of 7.4～14.8μM for these cells. For combination tests, only in H1299 cells it was found that concurrent Cis+PAC-1 or Cis→PAC-1 sequential combination could have synergistic effects of inhibiting cell growth, while in other 4 cel lines, combination of the two drugs mostly presented antagonistic effects (CI> 1.0).2. Flow cytometry results showed that in all 5 cell lines, PAC-1 could block cellsat at G1 phase and Cis could block cells at G2/M phase. In H1299 cells which is EGFRwtKRAwtALLKwt, Cis→PAC-1 sequential combination had strong pro-apoptotic effects in H1299 cells.3. Western blotting results showed that in H1299 cells, PAC-1 and Cis+PAC-1 and Cis→PAC-1 could promote the activation of CASP3 into C-CASP3. Single agent PAC-1 could partially inhibit C-PARP1 prduction in H1299 and PC9 cells, but not in H1975 ells.4. Immunofluorescent cytochemistry showed that in all cell lines of H1299, A549, H1650, H1975 and PC9, PAC-1 coulod promote the scizzering of CASP3 into activative forms of C-CASP3, and it showed relatively stronger effects of inducing C-CASP3 formation in H1299 cells.ConclusionsIn vitro experiments preliminarily showed that in EGFRmut (PC9, H1650, H1975) or KRASmut (A549) cells, PAC-1 showed antagonistic effects when combined with cis-platinum. However, in EGFRwtKRASwtALLKwt cells (H1299), PAC-1 could play a synergistic role in combination with cis-platinum, which might result specifically from pomoting formations of active C-CASP33。...
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