Functional Genetic Variations In HSP27 And HSP70 Genes Were Associated With DNA Damage Level, Lung Cancer Risk And Prognosis

Lung cancer is the most frequently occurring malignancy with increasing incidence and is the leading cause of mortality in cancer-related deaths in China and world-wide. Individual's genetic background and the environmental pollutant both contribute to lung carcinogenesis, which is a multiple factors related multi-step process. The abundant polycyclic-aromatic-hydrocarbons (PAHs) in coke-oven emission (COE) may cause DNA damage for coke-oven workers. DNA damage is a crucial early biological event for lung carcinogenesis and the main cause for lung cancer of coke oven workers. Numerous occupational epidemiological studies have revealed that, coke oven workers with a similar environmental PAHs exposure level had dramatically different DNA damage levels. Furthermore, only a small number of those exposed develop lung cancer with distinct latency periods, suggesting that genetic heterogeneity may play a role in determining the individual's genetic toxicity and lung cancer susceptibility when exposing to carcinogenic PAHs.Most lung cancer patients are often diagnosed at an advanced stage with only a 15% five-year survival rate. The TNM (tumor node metastasis) stage and therapy strategies are the most important clinical prognostic factors for lung cancer patients. However, lung cancer patients with similar therapy strategies and the same stages had dramatically different responses to anticancer therapies and had distinct survival outcomes, likely due to the genetic heterogeneity of gene/protein expression profiles.Heat shock proteins (HSPs) are a group of stress inducible cyto-protective proteins that can be stimulated by heat shock, oxidative stress, cytotoxic pollutant and etc, which can initiate the cellular programmed heat shock response and follow with the synthesis of HSPs. HSPs function as molecular chaperones, binding to the nascent polypeptides to assist them proper refolding, assembling and translocation, and facilitating the misfolding peptides to repair or targeting damaged polypeptides for degradation. Hsp27 and Hsp70 were the widely studied and crucial members of the HSPs super-family, and can both participate in cancer development, proliferation and prognosis through the following pathways. First, Hsp27 and Hsp70 can protect and enable the cell to recover and survive from harmful stress stimili. Second, Hsp27 and Hsp70 can keep the correct structure and biological activities of proteins and prohibit the irreversible aggregation of degenerate proteins, which help to maintain the cellular homeostasis. Third, Hsp27 and Hsp70 can interact with many key apoptosis-related proteins and exhibit anti-apoptotic functions in both intrinsic and extrinsic apoptosis pathways. Finally, Hsp27 and Hsp70 may also participate in the DNA repair pathways by interacting with Uracil-DNA glycosylase (UDG), Human AP endonuclease 1 (HAP1), human mutL homolog 1 (hMLH1) and human mutL homolog 2 (hMLH2) proteins.The heterogeneity of lung cancer susceptibility and prognosis may be due to the genetic variants of the key process genes. As a result, we hypothesized that the single nucleotide polymorphisms (SNPs) which exist in the HSPB1 genes and HSPA1A-HSPA1B-HSPA1L gene cluster may affect the expression levels and/or functional activities of Hsp27 and Hsp70 proteins and contribute to lung cancer susceptibility and survival. In the present study, we carried out the population based epidemiological studies and both the in vivo and in vivo biological researches, including gene resequencing, tagSNPs identification, case-control and case-only survival studies, genotype-phenotype correlation study, and the functional analyses. This study aimed to identify the associations between functional genetic variants of the hsp27 and hsp70 genes and the DNA damage levels of coke-oven workers, lung cancer susceptibility and survival, which would provide scientific research methods and rules for identifying genetic biomarkers for lung cancer risk and prognosis.PartⅠAssociations of genetic variations in the HSPB1 gene with lung cancer risk and prognosis.Heat shock protein 27 (Hsp27) is the key member of the small HSPs family, which can be elevated by various environmental and pathophysiologic stumili. Previous studies have confirmed the anti-apoptotic properties of Hsp27 in both intrinsic and extrinsic apoptosis pathways. Hsp27 has been reported to be an independent prognosis biomarker for many cancers, such as gastric, liver, and prostate carcinoma. In this part, we resequenced the full-length HSPB1 gene in 60 randomly selected healthy Han Chinese, constructed the haploblock structure of the HSPB1 gene, and selected 3 tagSNPs (-1271G>C,-1230G>A,-870T>C) by the Haploview4.0 software. We genotyped the three selected tagSNPs in 1152 paired Chinese lung cancer cases and controls from Wuhan city. The multivariate logistic regression analysis, after adjusted for age, sex, smoking status, pack-years and family history of cancer, revealed that, when compare with the-1271GG genotype, subjects with the-1271GC and-1271CC genotypes had a 1.26-fold and a marginal 1.27-fold risk of developing lung cancer (P=0.013,0.073, respectively), and there was a dose-response effect of the-1271C allele in increasing lung cancer risk (Ptrend=0.024). However, there were no associations between the-1230G>A and-870T>C polymorphisms and lung cancer risk (P>0.05). Another 500 paired cases and controls from Shanghai and Nanjing city were used for replication. Also in this panel, subjects with-1271GC or-1271CC genotypes also had a significantly increased risk of lung cancer (adjusted odds ratio [OR]=1.44,1.65, and P=0.011,0.017, respectively), and there was also a dose-response effect of the-1271C allele in increasing lung cancer risk (Ptrend=0.004).We further analyzed the association between the 3 tagSNPs and the prognosis of 248 advanced NSCLC patients from Wuhan city. The Kaplain-Meier method and log-rank test showed that, patients with-1271GC or-1271CC genotype have a longer median survival time (MST) than those with-1271GG genotype (Log-rank P=0.004). After adjusted for age, sex, smoking status, histology, TNM stage, surgery, chemotherapy, and radiotherapy status, the multivariate Cox regression model revealed that, compared with the-1271GG genotype, patients with the-1271GC or-1271CC genotypes had a 29% or a 45% decreased death risk (adjusted hazard ratio [HR]=0.71,0.55,P=0.045,0.012, respectively). There was a dose-response effect of the-1271C allele in reducing death risk (Ptrend=0.007). However, there were no associations between the-1230G>A,-870T>C polymorphisms and the survival of advanced NSCLC patients (P>0.05). The effect of-1271C allele was then validated in 335 advanced NSCLC patients from Nanjing city. The Kaplain-Meier method and log-rank test showed that, advanced NSCLC patients with-1271GC or-1271CC genotype have a longer MST than those with-1271 GG genotype (Log-rank P=0.029). The multivariate Cox regression model revealed that, compared with the-1271GG genotype, patients with-1271GC genotype had a 27% decreased death risk (adjusted HR=0.73, P=0.029), and patients with-1271GC+CC genotype have a longer MST and decrease death risk (Log-rank P=0.009, adjusted HR=0.75, P=0.032).In this part, we constructed the haploblock structure of HSPB1 gene in Han Chinese population, selected 3 tagSNPs, and found the-1271C allele in the-1271G>C polymorphism was associated with increased lung cancer susceptibility and decreased death risk for advanced NSCLC patients in two independent populations respectively. The results indicated that the HSPB1 gene-1271G>C polymorphism may be used as a genetic biomarker for lung cancer risk and prognosis.PartⅡAssociations of SNPs in the HSPA1L-HSPA1A-HSPA1B gene cluster with lung cancer risk and prognosis.The HSPA1L-HSPA1A-HSPA1B gene cluster was located on the HLA-III region in chromosome 6p21.3, encoding the inducible Hsp70 protein. Similar with Hsp27, the Hsp70 protein was normally low-expressed in the cytosol while over-expressed in the nucleus and exhibited cyto-protective role for cell DNA and proteins under stress conditions. Researches indicated that Hsp70 was over-expressed in numerous cancer tissues. The serum and/or plasma Hsp70 antigen and antibody may be important biomarkers for cancer diagnosis and prognosis.According to the Han Chinese Beijing data in the HapMap project and our previous resequencing results of HSPA1L-HSPA1A-HSPA1B gene cluster, we selected and genotyped 6 tagSNPs in the region in 1152 paired Chinese lung cancer cases and controls. The multivariate logistic regression revealed that, when compare with the HSPA1B gene +39CC genotype, subjects with the+39CT and+39CT+TT genotypes had an increased risk of lung cancer development (adjusted OR=1.39, P=0.013,0.073, respectively). There was a dose-response effect of the+39T allele in increasing lung cancer risk (Ptrend=0.026). The HSPAIB gene-912CT polymorphism were located in the same haploblock. The haplotype analysis demonstrated that subjects with T.912-T+39 haplotype had an increased lung cancer risk compared to subjects with the C.912-C+39 haplotype (adjusted OR=1.37, P=0.008). However, there were no associations between the HSPA1L gene+2763C>T, HSPA1L gene+2437A>G, HSPA1A gene-110A>C, HSPA1A gene+190G>C, and HSPAIB gene-912C>T polymorphisms and lung cancer risk (P>0.05). Another 1000 paired cases and controls from Beijing city were used for replication. Also in this panel, multivariate logistic regression analysis indicated that, when compared with the HSPA1B gene+39CC genotype, subjects with+39CT or +39CT+TT genotype had significantly increased risk of lung cancer (adjusted OR=1.30, 1.28, and P=0.038,0.048, respectively).We further analyzed the associations between the 6 tagSNPs and the prognosis of 281 advanced NSCLC patients from Wuhan city. The Kaplain-Meier method and log-rank test showed that, patients with-912CT or-912TT genotype have shorter MST than those with-912CC genotype (Log-rank P=0.005). Multivariate Cox regression model revealed that, when compared with the-912CC genotype, the-912CT or-912TT genotype was associated with increased death risk for advanced NSCLC patients (adjusted HR=1.62,1.92, P=0.001,0.017, respectively); when compared with the+39CC genotype, advanced NSCLC patients with the+39CT+TT genotypes had a 51% higher death risk (adjusted HR=1.51, P=0.006). The haplotype analyses indicated that,-912C-+39T haplotype was associated with increased death risk for advanced NSCLC patients compared to the.912C-+39C haplotype (adjusted HR=1.51, P=5.9×10-4). In addition, the death risk of advanced NSCLC patients with CC/TC or CC/TT diplotype were significantly increased when compared with patients with the CC/CC diplotype (adjusted HR=1.57,1.78, P=0.004,0.033, respectively).In this part, we selected 6 tagSNPs in the HSPA1L-HSPA1A-HSPA1B gene cluster, and found consistently that the+39T allele in the HSPA1B gene+39C>T polymorphism was associated with increased lung cancer risk in two independent lung cancer case-control studies. In addition, advanced NSCLC patients with the-912T allele in the promoter of HSPA1B and the+39T allele in the 5'UTR of HSPA1B had increased death risk. These results should be validated in another independent population, and the biological functions of the positive SNPs need further investigations.Part III Associations of genetic variations in the BCL2, BAG1,APEX1 gene and lung with cancer risk and prognosis.Hsp70 facilitated its cyto-protection role by interacted with numerous proteins. B cell lymphoma-2 (Bcl-2) and Bcl-2-associated athanogene 1 (BAG-1) were important anti-apoptotic proteins. The BAG-1 protein was initially identified as a binding partner of the anti-cell death protein Bcl-2 and was shown to be involved in the regulation of apoptosis. BAG-1 can bind with Bcl-2 and enhance its anti-apoptotic activity. It is now widely accepted that BAG-1 was the co-chaperone of Hsp70 through binding to the ATP-binding domain of Hsp70 and promoting the interaction between Hsp70 and Bcl-2, and then enhance the structure and activities of Bcl-2. The Hsp70, Bcl-2, and BAG-1 can form a protein complex and took parts in the inhibition of apoptosis pathways. In addition, Hsp70 could interact with DNA repair protein HAP-1 and enhance its endonuclease activities.According to the Han Chinese Beijing data in the HapMap project, we selected the tagSNPs located in the functional region of BCL2, BAG1, and APEX1 gene. We genotyped the selected tagSNPs in 1152 paired Chinese lung cancer cases and controls, and found that, when compared with the BCL2 gene+21 AA genotype, subjects with the +21AG or+21AG+GG genotypes had a significantly decreased risk of lung cancer development (adjusted OR=0.76,0.77,P=0.025,0.027, respectively). There was a dose-response effect of the+21G allele in decreasing lung cancer risk (Ptrend=0.039). Compared to the BCL2 gene c* 1204GG genotype, the c* 1204GA and AA genotype were associated with marginal decrease risk of lung cancer development (adjusted OR=0.83,0.79, P=0.050,0.087, respectively), while the c.* 1204GA+AA genotype was associated with a significant decrease risk of lung cancer (adjusted OR=0.82, P=0.029). There was also a dose-response effect of the c.*1204A allele in decreasing lung cancer susceptibility (Ptrend=0.039). However, there were no associations between the other tagSNPs (BCL2 gene-938C/A, BAG1 gene c.*1612G/C, and APEX1 gene Ile64Val, Asp148Glu polymorphisms) and lung cancer susceptibility (P>0.05).Combination analysis of the HSPA1B gene+39CT+TT genotype, the BCL2 gene +21AA genotype and c.*1204GG genotype assessed by logistic regression models revealed that there was a dose-response effect of the risky genotype in increasing the risk of lung cancer development (Ptrend<0.001).We further analyzed the associations between the selected tagSNPs and the prognosis of 281 advanced NSCLC patients. The Kaplain-Meier method and log-rank test showed that, patients with c* 1204GA or AA genotype have higher median MST than those with the c* 1204GG genotype (Log-rank P=0.027). Multivariate Cox regression model revealed that, when compared with the c.*1204GG genotype, the c.*1204GA or AA genotype genotypes was associated with decreased death risk for advanced NSCLC patients (adjusted HR=0.69,0.67, P=0.013,0.068, respectively). There was a dose-response effect of the c.*1204A allele in reducing death risk (Ptrend=0.018).Combination analysis of the HSPAIB gene-912CT+TT and+39CT+TT genotype, the BCL2 gene c* 1204GG genotype assessed by multivariate Cox regression models revealed that there was a dose-response effect of the risky genotype in decreasing MST and increasing death risk for advanced NSCLC patients (Ptrend<0.001).The results of this part demonstrated that the+21G allele of+21 A>G polymorphism and the c.*1204A allele of the c.*1204G>A polymorphism in the BCL2 gene were associated with decreased lung cancer susceptibility and decreased death risk for advanced NSCLC patients. The risky genotypes of positive SNPs in HSPAIB and BCL2 gene combinatorially contributed to increase the risk of lung cancer development and death of advanced NSCLC patients. The epidemiological results of this part warrant validation by larger populations, and the biological functions of the risk SNPs need further investigations.PartⅣAssociations of risk SNPs in the HSPB1, HSPAIB, and BCL2 genes with DNA damage levels caused by PAHs.The abundant PAHs in coke-oven emission (COE) may cause DNA damage, which was an important early biological event in carcinogenesis and the main cause for lung cancer of coke oven workers. Determination of the genetic background and DNA damage levels of the occupational PAHs exposed coke-oven workers will provide us with a good model for analyzing the "genotype-phenotype" relationship.A total of 309 male subjects were recruited from a steel plant in Taiyuan, northern China, of whom 207 workers had been occupationally exposed to COE and were defined as the exposed group. The other 102 workers from the same factories without COE exposure in their workplaces were used as the control group. We genotyped the above positive SNPs (HSPB1 gene-1271G>C, HSPA1B gene-912C>T and+39C>T,BCL2 gene+21A>G and c.*1204G/A polymorphism) and evaluated their associations with the DNA damage levels of 309 workers. The results showed that the Olive Tail Moment (OTM) values in the exposed groups (1.22±1.10) were significantly higher than those in the controls (0.60±0.92) (P<0.001). In the coke-oven works (exposed group), subjects with the-1271GC,-1271CC, or-1271GC+CC genotype had marginally or significantly higher OTM values than subjects with the-1271GG genotype (P=0.033,0.061,0.027, respectively). There was a dose-response effect of the-1271C allele in increasing OTM values in the exposed group (Ptrend=0.031). In the control group, there was also a dose-response effect of the-912T allele in HSPA1B gene in increasing OTM values (Ptrend=0.038). However, there were no relationships between genotypes of the other polymorphisms and the DNA damage levels in both the exposed and control group (all P>0.05).The results of this part indicated that subjects carrying the-1271C allele of HSPB1 gene had higher DNA damage levels and lower DNA repair capacities (DRC) when exposed to exogenous cytotoxic factors like PAHs. Considering the results of Part I, we can hypothesize that the lower DRC induced by-1271C allele may be an important reason for the increase risk for lung carcinogenesis. Furthermore, the decreased DRC may render the advanced NSCLC patients more sensitive to chemo-and radio-therapy with improved prognosis.PartⅤFunctional analysis of the HSPB1 gene-1271G>C polymorphism and HSPA1B gene-912C>T and+39C>T polymorphismThe promoter region and 5'untranslated region (5'UTR) were located above the translation start coden. The cis-transcription elements in the promoter can interact with the corresponding transcription factors and then initiate the transcription of target genes, while the secondary structure of the 5'UTR may stabilize the mRNA. Therefore, genetic variations in the above two regions may change the regulation mechanism and affect the expression level of the target gene. We will analyze the biological function of the HSPB1 gene-1271G>C and the HSPA1B gene-912C>T and+39C>T polymorphisms in this part.Dual luciferase reporter assays were carried out to determinate the effects of the above three SNPs on the relative luciferase activities (RLA). Western blotting was also used to detect the expression levels of Hsp27 protein in the tumor and corresponding normal tissues of lung cancer patients. It was showed that the RLA driven by the promoter containing-1271C allele was significantly lower than that driven by the promoter containing-1271G allele in the 16HBE, H446, A549, and HepG2 cell lines (P < 0.05 for all). Hsp27 protein was over-expressed in lung cancer tissues when compared with that of corresponding normal tissues, and the Hsp27 expression levels in normal tissues of patients carrying the-1271CC genotype were marginally lower than those with the-1271GG genotype (P=0.057). However, the differences among three genotypes of the-1271G>C polymorphism in tumor tissues did not reach the significant level (P>0.05). The 16HBE, A549, and HepG2 cell lines were transfected with luciferase reporter plasmids that carried four different haplotypes of the HSPA1B gene, including-912C-+39C,-912C-+39T,-912T-+39C, and-912T-+39T haplotype. The results showed that the RLA driven by the promoter containing-912C-+39T,-912T-+39C, and-912T-+39T haplotype was significantly lower than that driven by the promoter containing-912C-+39C haplotype (all P<0.05). The RLA driven by the promoter containing-912T-+39T haplotype was significantly lower than that driven by the promoter containing-912C-+39T or-912T-+39C haplotype (all P<0.05); The RLA driven by the promoter containing-912C-+39T or-912T-+39C haplotype was significantly lower than that driven by the promoter containing-912C-+39C haplotype (all P<0.05). However, the RLA driven by the promoter containing-912C-+39T or-912T-+39C haplotype was not significantly different (P>0.05). The two reporter plasmids containing only the HSPAIB gene+39C>T polymorphism+39C or+39T allele were also transfected onto the 16HBE, A549, and HepG2 cell lines, and showed that the RLA driven by the promoter containing+39T allele was significantly lower than that driven by the promoter containing+39C allele (all P<0.05).The results of this part suggests that the-1271C allele may lead to a lower Hsp27 protein synthesis level than the-1271G allele, while lung cancer patients carried the-1271CC genotype had a marginally decreased Hsp27 expression level in the normal lung tissues. In addition, the-912T and+39T allele may also lead to a lower Hsp70 protein synthesis level than the-912C and+39C alleleIn summary, we selected tagSNPs in the HSPB1 genes and HSPA1L-HSPA1A-HSPAIB gene cluster and detected their associations with lung cancer risk and prognosis. The biological functions of the positive SNPs were also evaluated by the genotype-phenotype correlation study in occupationally PAHs exposed coke-oven workers as well as by luciferase reporter assay and western blotting analysis. This study has some advantages, first, to our knowledge, this study provided the first evidence that genetic variants in the HSPB1 and HSPA1B gene were associated with lung cancer risk and prognosis in Chinese populations; second, the epidemiological studies on the early events of lung cancer (DNA damage), lung cancer development and prognosis together with the biological functional studies make a plausible explanations for the functions of the positive SNPs; finally, the correlation between genetic variations of occupational PAHs exposed coke-oven workers and their DNA damage level will give a good study model for analyzing the genotype-phenotype relationship.There were several limitations in our study that needed to be addressed. First, the association between SNPs in the HSPA1B and BCL2 gene and lung cancer risk and prognosis should be validated by additional large population-based prospective studies and well-designed clinical investigations. Second, we should evaluate the biological mechanisms for COE induced DNA damage in the further studies. Finally, it is a complicate process from DNA damage to lung carcinogenesis. The lung cancer patients enrolled in our study can not represent the development of lung cancer of coke-oven workers. Thus associations between environmental risk factors and genetic variations lung cancer susceptibility in coke oven workers also need to be detected.