Yongli Guo

A six-nucleotide insertion-deletion polymorphism in the CASP8 promoter is associated with susceptibility to multiple cancers

Caspases are important in the life and death of immune cells and therefore influence immune surveillance of malignancies. We tested whether genetic variants in CASP8, CASP10 and CFLAR, three genes important for death receptor–induced cell killing residing in tandem order on chromosome 2q33, are associated with cancer susceptibility. Using a haplotype-tagging SNP approach, we identified a six-nucleotide deletion (−652 6N del) variant in the CASP8 promoter associated with decreased risk of lung cancer. The deletion destroys a stimulatory protein 1 binding site and decreases CASP8 transcription. Biochemical analyses showed that T lymphocytes with the deletion variant had lower caspase-8 activity and activation-induced cell death upon stimulation with cancer cell antigens. Case-control analyses of 4,995 individuals with cancer and 4,972 controls in a Chinese population showed that this genetic variant is associated with reduced susceptibility to multiple cancers, including lung, esophageal, gastric, colorectal, cervical and breast cancers, acting in an allele dose–dependent manner. These results support the hypothesis that genetic variants influencing immune status modify cancer susceptibility.

The role of P53 and MDM2 polymorphisms in the risk of esophageal squamous cell carcinoma.

The tumor suppressor P53 pathway plays a crucial role in preventing carcinogenesis and genetic variations of this pathway may be associated with cancer susceptibility. We tested this hypothesis by examining the contribution of functional polymorphisms in P53 and MDM2 to risk of esophageal squamous cell carcinoma (ESCC). DNA from 758 ESCC patients and 1,420 controls were genotyped for P53 codon 72Arg>Pro and MDM2 309T>G polymorphisms. Odds ratios (OR) and 95% confidence intervals (CI) of ESCC were estimated by logistic regression. We observed an increased risk of ESCC associated with the P53 Pro/Pro (OR, 1.83; 95% CI, 1.43-2.35; P < 0.001) or MDM2 GG (OR, 1.49; 95% CI, 1.16-1.91; P = 0.002) genotype, compared with the P53 Arg/Arg or MDM2 TT genotype, respectively. Interaction between these P53 and MDM2 polymorphisms increased risk of ESCC in a multiplicative manner, with the OR being 3.10 (95% CI, 2.07-4.69) for subjects carrying both P53 Pro/Pro and MDM2 GG genotypes. Significant interactions were observed between these polymorphisms and smoking, with risk being the highest (OR, 5.29; 95% CI, 2.91-9.61) in smokers having both P53 Pro/Pro and MDM2 GG genotypes. The MDM2 GG genotype was also associated with risk of developing poorly differentiated and advanced ESCC compared with the GT or TT genotype (OR for high-grade and stages III-IV versus low-grade and stages I-II = 1.60; 95% CI, 1.00-2.64; P = 0.049). The P53 and MDM2 polymorphisms may be genetic determinants for the development of ESCC.

Functional Genetic Variations in Cytotoxic T-Lymphocyte Antigen 4 and Susceptibility to Multiple Types of Cancer

Antitumor T lymphocytes play a pivotal role in immunosurveillance of malignancy. The CTL antigen 4 (CTLA-4) is a vital negative regulator of T-cell activation and proliferation. This study examined whether genetic polymorphisms in CTLA-4 are associated with cancer susceptibility. A two-stage investigation using haplotype-tagging single nucleotide polymorphism approach and multiple independent case-control analyses was performed to assess the association between CTLA-4 genotypes and cancer risk. Functional relevance of the polymorphisms was examined by biochemical assays. We found that the 49G>A polymorphism in the CTLA-4 leading sequence causing (17)Ala to (17)Thr amino acid substitution is associated with increased susceptibility to multiple cancers, including lung, breast, esophagus, and gastric cardia cancers. Genotyping in 5,832 individuals with cancer and 5,831 control subjects in northern and southern Chinese populations showed that the CTLA-4 49AA genotype had an odds ratio of 1.72 (95% confidence interval, 1.50-2.10; P = 3.4 x 10(-7)) for developing cancer compared with the 49GG genotype. Biochemical analyses showed that CTLA-4-(17)Thr had higher capability to bind B7.1 and stronger inhibitory effect on T-cell activation compared with CTLA-4-(17)Ala. T cells carrying the 49AA genotype had significantly lower activation and proliferation rates compared with T cells carrying the 49GG genotype upon stimulation. These results are consistent with our hypothesis and indicate that genetic polymorphisms influencing T-cell activation modify cancer susceptibility.

FASL –844C polymorphism is associated with increased activation-induced T cell death and risk of cervical cancer

The FAS receptor–ligand system plays a key role in regulating apoptotic cell death, and corruption of this signaling pathway has been shown to participate in tumor-immune escape and carcinogenesis. We have recently demonstrated (Sun, T., X. Miao, X. Zhang, W. Tan, P. Xiong, and D. Lin. 2004. J. Natl. Cancer Inst. 96:1030–1036; Zhang, X., X. Miao, T. Sun, W. Tan, S. Qu, P. Xiong, Y. Zhou, and D. Lin. 2005. J. Med. Genet. 42:479–484) that functional polymorphisms in FAS and FAS ligand (FASL) are associated with susceptibility to lung cancer and esophageal cancer; however, the mechanisms underlying this association have not been elucidated. We show that the FAS –1377G, FAS –670A, and FASL –844T variants are expressed more highly on ex vivo–stimulated T cells than the FAS –1377A, FAS –670G, and FASL –844C variants. Moreover, activation-induced cell death (AICD) of T cells carrying the FASL –844C allele was increased. We also found a threefold increased risk of cervical cancer among subjects with the FASL –844CC genotype compared with those with the –844TT genotype in a case-control study in Chinese women. Together, these observations suggest that genetic polymorphisms in the FAS–FASL pathway confer host susceptibility to cervical cancers, which might be caused by immune escape of tumor cells because of enhanced AICD of tumor-specific T cells.

Characterization of functional excision repair cross-complementation group 1 variants and their association with lung cancer risk and prognosis.

Purpose: The excision repair cross-complementation group 1 (ERCC1) plays a pivotal role in DNA repair and has been linked to protection against carcinogenesis and resistance to platinum-based anticancer drugs. We tested whether genetic variants in the ERCC1 gene are associated with susceptibility to lung cancer and efficacy of platinum-chemotherapy in patients with small cell lung cancer (SCLC). Experimental Design: Thirty individual DNA samples were sequenced to search for single-nucleotide polymorphisms, and the functions of the variants were investigated by a series of biochemical assays. A case-control study was done in 988 patients with lung cancer and 986 control subjects. According to the genotypes, a comparison of chemotherapy outcome in 162 SCLC patients was executed. Overall survival was computed by Cox model adjusted for clinical factors. Results: We identified two functional variants in the ERCC1 5′-flanking region, −433T>C and 262G>T, which cooperatively influence transcriptional regulation of ERCC1. The 262G allele had significantly lower affinity to bind nuclear protein(s) and was associated with decreased ERCC1 RNA expression. The case-control analysis showed that the −433C and 262G alleles are associated with an increased susceptibility to lung cancer, alone and in a gene-smoking joint effect manner. In contrast, the analysis of chemotherapy outcome of SCLC patients revealed that the 262G allele is associated with better drug response and longer survival time compared with the 262T allele. Conclusions: These findings are consistent with the notion that DNA repair is a double-edged sword in cancer and suggest that functional single-nucleotide polymorphisms in ERCC1 might serve as simple and less invasive biomarkers for personalized chemotherapy of platinum-based anticancer drugs.

Haplotypes in Matrix Metalloproteinase Gene Cluster on Chromosome 11q22 Contribute to the Risk of Lung Cancer Development and Progression

Purpose: Matrix metalloproteinases (MMP) play important roles in cancer development and single nucleotide polymorphisms (SNP) in some MMP genes were shown to confer susceptibility to certain cancers. This study examined the association between genotypes and haplotypes in the MMP1-MMP3-MMP12 gene cluster and risk of lung cancer development and metastasis. Experimental Design: A two-stage investigation was conducted. First, 35 SNPs covering these genes were selected and validated in 190 patients and 190 controls. Twenty-two validated SNPs were then analyzed in an entire case-control panel consisting of 711 patients and 716 controls. Associations with the risk of lung cancer were estimated by logistic regression. Results: The investigated MMP gene region could be partitioned into two major haplotype blocks. One common haplotype in the block composed of major part of MMP1 transcription region was significantly associated with increased risk for the development [odds ratio (OR), 1.35; 95% confidence interval (95% CI), 1.11-1.63; P = 0.01; permutated P = 0.134] and distant metastasis of lung cancer (ORs for stage IV versus stages I-III, 1.67; 95% CI, 1.12-2.50; P = 0.009; permutated P = 0.048) and the other showed a protective effect against metastasis (ORs for stage IV versus stages I-III, 0.22; 95% CI, 0.07-0.62; P = 0.001; permutated P = 0.011). Another common haplotype in the block across MMP3 was significantly associated with decreased risk for developing lung cancer (OR, 0.71; 95% CI, 0.59-0.86; P = 0.003; permutated P = 0.027). Conclusions: The observed multiple cancer-associated genetic variants suggested that the MMP1-MMP3-MMP12 gene cluster plays important roles in lung cancer development and progression.

Role of CD14 Promoter Polymorphisms in Helicobacter pylori Infection–Related Gastric Carcinoma

Purpose: Genetic variation in CD14 may affect CD14 expression and susceptibility to Helicobacter pylori infection–related cancers. This study examined functional single nucleotide polymorphisms (SNP) in the CD14 promoter and their associations with risk of developing gastric carcinoma in relation to H. pylori infection. Experimental Design: Thirty individual DNAs were sequenced to identify variants, and the function of the variants was examined by reporter gene assays. Genotypes and haplotypes were analyzed in 470 patients and 470 controls, and odds ratios (OR) and 95% confidence intervals (95% CI) were estimated by logistic regression. Serologic H. pylori antibody and soluble CD14 (sCD14) levels were measured by ELISA. Results: Two SNPs (−651C>T and −260C>T) were identified, of which the −260CT and −260TT genotypes were associated with elevated risk of gastric carcinoma (OR, 1.77; 95% CI, 1.09-2.85 and OR, 1.95; 95% CI, 1.20-3.16, respectively). Haplotype analysis suggested a synergistic effect of the two SNPs (OR for the T−651-T−260 haplotype, 3.39 versus OR for the C−651-T−260 haplotype, 1.45; P = 0.02), which is consistent with reporter gene assays. A multiplicative joint effect between H. pylori infection and −260C>T polymorphism was observed (OR for the presence of both −260TT genotype and H. pylori infection, 4.03; 95% CI, 1.80-9.04). Patients had significantly higher sCD14 than controls (1,866 ± 2,535 ng/mL versus 1,343 ± 2,119 ng/mL; P < 0.001), and this difference was associated with the CD14 −260 polymorphism and H. pylori infection. Conclusions: Functional polymorphism in CD14 is associated with greater risk of H. pylori–related gastric carcinoma, which might be mediated by elevated sCD14.

Suppression of CYP2C9 by MicroRNA hsa-miR-128-3p in Human Liver Cells and Association with Hepatocellular Carcinoma

Published studies have identified genetic variants, somatic mutations, and changes in gene expression profiles that are associated with hepatocellular carcinoma (HCC), particularly involving genes that encode drug metabolizing enzymes (DMEs). CYP2C9, one of the most abundant and important DMEs, is involved in the metabolism of many carcinogens and drugs and is down-regulated in HCC. To investigate the molecular mechanisms that control CYP2C9 expression, we applied integrative approaches including in silico, in vitro, and in vivo analyses to elucidate the role of microRNA hsa-miR-128-3p in the regulation of CYP2C9 expression and translation. RNA electrophoresis mobility shift assays demonstrated a direct interaction between hsa-miR-128-3p and its cognate target, the CYP2C9 transcript. Furthermore, the expression of a luciferase reporter gene containing the 3′-UTR of CYP2C9 and the endogenous expression of CYP2C9 were suppressed by transfection of hsa-miR-128-3p. Importantly, chemically-induced up- or down-regulation of hsa-miR-128-3p correlated inversely with the expression of CYP2C9. Finally, an association analysis revealed that the expression of hsa-miR-128-3p is inversely correlated with the expression of CYP2C9 in HCC tumor tissues. Altogether, the study helped to elucidate the mechanism of CYP2C9 regulation by hsa-miR-128-3p, and the inverse association in HCC.

Functional evaluation of missense variations in the human MAD1L1 and MAD2L1 genes and their impact on susceptibility to lung cancer

Background Human MAD1 mitotic arrest deficient-like 1 (MAD1L1) and MAD2 mitotic arrest deficient-like 1 (MAD2L1) are two interactive proteins playing important roles in maintaining spindle checkpoint function. This study examined the functional relevance of missense coding single nucleotide polymorphisms (SNPs) in MAD1L1 and MAD2L1 and their association with susceptibility to lung cancer. Methods SNPs in the MAD2L1 coding region were discovered by sequencing and impact of MAD1L1 and MAD2L1 variants on spindle checkpoint function was examined by flow cytometry and mitotic index assay. The associations of MAD1L1 and MAD2L1 variants with lung cancer were analysed in a case–control cohort of 1000 patients and 1000 controls. ORs and 95% CIs were estimated by logistic regression. Results A novel C-to-A SNP at codon 84 of MAD2L1 (Leu84Met substitution) was discovered. Cells expressing MAD2L1-84Met and MAD1L1-558His had impaired spindle checkpoint function, with a lower 4N-DNA content and mitotic index when treated with nocodazole. Case–control analysis showed that the MAD2L1 Leu84Met SNP was associated with increased risk of lung cancer in an allele dose dependent manner, with the ORs being 2.55 (95% CI 1.95 to 3.33) for the Leu/Met and 2.68 (95% CI 2.05 to 3.48) for the Met/Met genotype compared with the Leu/Leu genotype. The MAD1L1 558 His/His genotype was also associated with 1.4-fold elevated lung cancer risk compared with the Arg/Arg genotype. Conclusion These results suggest that genetic variants in MAD1L1 and MAD2L1 confer susceptibility to lung cancer, which might result from reduced spindle checkpoint function due to attenuated function of MAD1L1 and/or MAD2L1.

Cigarette smoking strongly modifies the association of complement factor H variant and the risk of lung cancer.

The complement system is an important immunosurveillance mechanism against tumors, and complement factor H (CFH) is a key regulator for activation of the complement system. Expression of complement factor H has been demonstrated in cell lines from several malignancies. In this study we examined the contribution of the single-nucleotide polymorphism (SNP) Try402His (Y402H) (rs1061170) in the CFH gene to the risk of lung cancer in a case-control study with 1000 cases and 1000 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed by logistic regression. The frequencies for CFH Y402H genotypes among the cases were statistically significantly different from those among controls (χ(2)=8.66, P=0.003), with 402His/His or 402His/Try genotypes being over-represented among patients compared with controls (13.6% versus 9.4%, P<0.004). A multivariate regression analysis showed that a significantly increased risk of lung cancer for the 402His/His or 402His/Try carriers with OR (95% CI), 1.50 (1.12-2.00). When stratified by smoking status, the elevated risk of the cancer associated with variant CFH genotypes was observed among smokers, but not among non-smokers. When analyzed with cumulative smoking dose (pack-years), a super-multiplicative interaction was observed at different smoking levels. Among carriers with the 402Tyr/His or His/His genotype, the ORs of developing lung cancer for smoking<16, 16-28, or >28 pack-years were 0.98 (0.49-1.94), 2.36 (1.14-4.90), and 6.39 (3.49-11.68), respectively. These findings suggest that CFH Y402H polymorphism may interact with cigarette smoking to effect the development of lung cancer in the Chinese population.