【作者】 余科达；

【导师】 邵志敏； 狄根红； 胡震；

【作者基本信息】 复旦大学 ， 肿瘤学， 2009， 博士

【摘要】 第一部分:谷胱甘肽硫转移酶（GST）M1基因在乳腺癌发生中的复杂地位雌激素是引起乳腺癌的重要风险因素,雌激素醌类代谢产物是引起基因突变、导致乳腺癌发生的重要元凶。在正常乳腺组织中,部分Ⅱ期代谢酶承担着代谢雌醌或转化雌醌的作用。雌醌既能被醌氧化还原酶（NQO）还原形成半醌或其他物质后经由COMT途径被代谢;也可以通过谷胱甘肽硫转移酶（GST）途径被结合后降解清除。因此,NQO和GST这两类雌醌代谢酶与乳腺癌关系密切。很多研究针对GSTM1基因缺失多态与乳腺癌的发生风险开展了研究,但目前尚无一致性结论。不仅如此,此前的研究仅区分GSTM1有或无的状态,GSTM1基因剂量效应与乳腺癌发生的关系至今未被阐明。本研究第一部分中,我们首先开展了包括1920例研究对象的病例对照研究。结果显示GSTM1-null型（GSTM1-/-）相对于GSTM1-present型[包括纯合野生型（+/+）和杂合型（+/-）]提高了乳腺癌发生的风险。将我们的结果联合了40个相关研究进行Meta分析,得到总OR值为1.10（P＜0.001）。出乎意外的是,后续三分法研究提示,GSTM1+/+型相对于GSTM1+/-型也是风险型。为了深入研究GSTMl基因剂量与乳腺癌发生之间的关系,我们对GSTM1基因内的单核苷酸多态（SNP）进行了分析,发现一个位于GSTM1启动子区域的多态与乳腺癌发生显著相关。该多态能影响所在基因序列与转录因子AP-2α的结合活性,并最终影响启动子活性与GSTM1的mRNA表达水平。然而,该SNP低活性的等位基因对应于保护性作用。我们的结果提示GSTM1在乳腺癌发生中具有相对复杂的基因剂量关系,而非原先所预测的那样呈现乳腺癌风险与基因剂量是线性关系,而是U型曲线关系。从基因剂量效应来看,大概GSTM1单等位基因60-70%的表达即可满足正常生理对抗乳腺癌致病代谢物的需要。GSTM1功能完全缺失或者活性过强均是有害的。这一复杂的量效关系值得引起我们的注意。第二部分:GSTM4-GSTM2-GSTM1-GSTM5-GSTM3基因簇多态性和乳腺癌易感性的关联研究GSTMs基因簇串联排列于染色体lp13.3。目前虽然对GSTM1基因缺失多态予以了较多研究,对GSTM1-5基因簇多态与乳腺癌的关系尚缺乏报道。在第二部分研究中,我们针对GSTMs基因簇进行关联分析,对在此64 kb基因区域内的11个SNP进行大规模分型（847病例vs.676对照）。关联分析按GSTM1亚组进行分析。在GSTM1-/-组中,我们发现位于M3-like基因区的一个SNP在病例组和对照组中分布具有显著差异(P=2.0×10^-4,校正后P=0.001),变异型等位基因相对于野生型等位基因的OR值为1.75（95%CI:1.26-2.44）。相反,在GSTM1+/-组中,我们未能发现任何有意义的多态位点。流行病学的研究数据结合功能研究结果,提示保护型的基因型对应于高表达GSTM3的生物学效应。我们认为,M3-like基因多态与乳腺癌发生相关,GSTM3可能起到保护乳腺组织对抗致癌物质的作用,但是这种作用仅在GSTM1缺失时才显著。第三部分:NRH醌氧化还原酶2（NRH:Quinone Oxidoreductase 2,NQO2）与乳腺癌遗传易感性的关联研究和功能论证NRH醌氧化还原酶2（NQO2）不仅具有代谢雌醌的生物学活性,也具有稳定p53的功能,因此NQO2可作为乳腺癌易感候选基因。目前针对NQO2与乳腺癌的研究还非常少,在第三部分里,我们着重研究了乳腺癌和NQO2的关系。首先,我们通过病例对照研究（n=1,604）发现两个NQO2基因多态与散发性乳腺癌明显相关。这两个位点分别为29 bp-插入/缺失多态（29bp-I/D）与rs2071002（+237A＞C）多态,均位于NQO2启动子区。29bp-I/D的D等位基因与rs2071002的+237C等位基因能显著降低乳腺癌的发生风险（OR分别为0.76,P=0.0027;0.80,P=0.0031）。不仅如此,NQO2多态与野生型p53乳腺癌特异性相关(最显著的P值为3.3×10^-6)。这些一期研究中的发现,在第二期以家族性/散发性人群为主的病例对照研究中得到了进一步验证（n=1,442）。两期研究（n=3,046）的联合P值是3.8×10^-7（29bp-I/D）与2.3×10^-6（rs2071002）。我们进一步揭示了这两个具有潜在功能意义位点的作用机制。29bp-I/D通过29-Ⅰ等位基因引入了转录抑制因子Sp3的结合位点而起作用;rs2071002的+237A等位基因通过消除转录增强因子Sp1的结合位点而起作用。最后,我们在组织标本中验证了保护型多态位点所对应的NQO2表达水平更高。总之,我们的研究提示NQO2是一个乳腺癌易感基因。更多还原

【Abstract】 PartⅠ:A Functional Polymorphism in the Promoter Region of GSTM1 Implies a Novel Two-sided Role for GSTM1 in Breast Cancer SusceptibilityEstrogen-quinone can form unstable adducts with adenine and guanine in DNA,leading to depurination and mutation in vitro and in vivo.It seems that reactive equine estrogen metabolites mainly contribute to breast cancer.However,protective phaseⅡenzymes are active in breast tissue for protection against damage caused by reactive metabolites of endogenous and exogenous chemicals.The estrogen-quinone could be reduced by quinone oxidoreductases,or be conjugated by glutathione S-transferases.Therefore,quinone oxidoreductases and glutathione S-transferases are two kinds of important estrogen-quinone metabolizing related enzymes. Although a number of studies have been conducted to address the relationship between a gene deletion-polymorphism of glutathione-S-transferase M1（GSTM1） and breast cancer,no definite conclusion has been made and no clear risk pattern has yet to emerge for GSTM1.We first conducted case-control studies that included 1920 subjects using a true genotyping method.The results show that GSTM1-null（GSTM1-/-） confers an increased risk for breast cancer development compared with GSTM1-present individuals[homozygous wild-type（+/+） and heterozygous（+/-）],which was subsequently confirmed by a meta-analysis of all of the 41 relevant studies（odds ratio:1.10,P<0.001）.Unexpectedly,we found that GSTM1+/+ is also a risk genotype compared with GSTM1+/-.Furthermore,we identified a functional polymorphism in the GSTM1 promoter region associated with breast cancer.The variant allele modifies the DNA-binding to the AP-2αtranscription factor,resulting in reduced promoter activity and mRNA expression.However,this low-activity allele is associated with reduced breast cancer risk. It seems that approximately 60-70%expression from one allele of GSTM1 could suffice for protection against breast cancer;null- and over-activity of GSTM1 are both disadvantageous. These results indicate a U-shaped association of GSTM1 with breast cancer,which challenges the linear gene-dosage effect of GSTM1 that was previously proposed.We recommend that a more complicated role for GSTM1 should be considered in breast cancer risk prediction.PartⅡ:Genetic Variants in M3-like Gene within GSTM4-GSTM2-GSTM1-GSTM5-GSTM3 Cluster Influence Breast Cancer Susceptibility Depending on GSTM1Mu class GSTMs genes arrange in a tandem on chromosome 1p13.3.The previous single-variant-based findings warrant more comprehensive study to explore the relationship between genetic variants in GSTM1-5 gene cluster and breast cancer.In the present study, seventeen tagging single-nucleotide polymorphisms（SNPs） covering 64-kb of GSTMs cluster （excluding recombination-regions） are originally selected and genotyped in a pilot population. Eleven validated-SNPs enter large sample size genotyping in 847-cases and 676-controls.The association analyses are performed according to the absence or presence of GSTM1.In GSTM1-/- group,the allele frequency of one SNP in M3-like loci is significantly different between cases and controls(P=2.0×10^-4,corrected P=0.001),with odds ratio of 1.75（95% confidence interval:1.26-2.44） for variant genotype compared with wild-type genotype.Two haplotypes are also significantly linked with breast cancer（corrected P=0.044 and 0.010）.In contrast,no susceptible allele/haplotype is identified when GSTM1 is present.Based on epidemiological observations,we further conduct in vitro and ex vivo studies and identify two genetic variants in M3-like loci accounting for differential expression of GSTM3 in normal breast tissues by such means as altering binding of RNA-pol-Ⅱ.Protective genotypes demonstrated in association study stage are correlated with higher GSTM3 expression phenotypes.In conclusion,the variants/haplotypes in M3-like loci rather than in M1-like loci within GSTMs cluster are likely to contribute to breast cancer risk when GSTM1 is absent.Our study also indicates that increased GSTM3 catalyzing ability in normal breast tissue might protect against breast oncogenesis.PartⅢ:Functional Polymorphisms,Altered Gene Expression and Genetic Association Link NRH:Quinone Oxidoreductase 2 to Breast CancerWe hypothesized that NRH:quinone oxidoreductase 2（NQO2） is a candidate susceptibility gene for breast cancer because of its known enzymatic activity on estrogen-derived quinones and its ability to stabilize p53.We performed case-control studies to investigate the contributions of genetic variants/haplotypes of the NQO2 gene to breast cancer risk.In the first hospital-based study（n=1,604）,we observed significant associations between the incidence of breast cancer and a 29 bp-insertion/deletion polymorphism（29bp-I/D） and the rs2071002（+237A＞C） polymorphism,both of which are located within the NQO2 promoter region.Decreased risk was associated with the D-allele of 29bp-I/D（odds ratio（OR）,0.76;P=0.0027） and the +237C-allele of rs2071002（OR,0.80;P=0.0031）.Specifically,the susceptibility variants within NQO2 were notably associated with breast carcinomas with wild-type p53(the most significant P-value: 3.3×10^-6).The associations were successfully replicated in an independent population set （familial/early-onset breast cancer cases and community-based controls,n=1,442）.The combined P-values of the two studies（n=3,046） are 3.8×10^-7 for 29bp-I/D and 2.3×10^-6 for rs2071002. Furthermore,we revealed potential mechanisms of pathogenesis of the two susceptibility polymorphisms.Previous work has demonstrated that the risk-allele I-29 of 29bp-I/D introduces transcriptional-repressor Sp3 binding sites.Using promoter reporter-gene assays and electrophoretic-mobility-shift assays,our present work demonstrated that the other risk-allele, +237A-allele of rs2071002,abolishes a transcriptional-activator Sp1 binding site.Furthermore, an ex vivo study showed that normal breast tissues harboring protective genotypes expressed significantly higher levels of NQO2 mRNA than those in normal breast tissues harboring risk genotypes.Taken together,the data presented here strongly suggest that NQO2 is a susceptibility gene for breast carcinogenesis.更多还原