Identification of Single Nucleotide Polymorphisms in Cancer-Related Genes

一个人的遗传背景在他们对癌症的易感性、疾病的进展和对治疗的反应方面起着重要的作用。基因变异可以被用作了解包括癌症和艾滋病在内的复杂疾病的标记。我们与纪念斯隆·凯特琳癌症研究所的科学家合作，完成了对有家族病史的德系犹太人乳腺癌的全基因组关联研究。最初的扫描确定了一些与潜在显著关联的基因座。从这些数据中，我们选择了在重复队列中进行后续研究的基因座。<i>TRIM5</i>基因编码一种参与保护细胞免受艾滋病毒感染的蛋白质。我们已经确定了人类<I>TRIM5</I>基因的某些区域似乎经过了选择，可能是对以前的感染做出了反应。人类基因组中的其他<I>TRIM</I>家族序列似乎也已被感染，可能参与了对其他病毒感染的保护。通过研究补体B和C2(CFH和C2)基因的变异，我们确定了该基因与年龄相关性黄斑变性(AMD)患者的相关性。AMD是导致老年人失明的主要原因，据估计，多达1000万美国人患有AMD。通过检测BF和C2基因中的遗传变异，确定了具有显著保护作用的单倍型。补体途径在对致癌病原体的反应中很重要。进一步研究这一先天免疫反应的重要组成部分可能有助于深入了解人类疾病。去除CFH相邻的CFHR1基因的缺失对AMD具有高度的保护作用。我们已经从年龄相关眼病研究的受试者那里获得了3000多个DNA，并正在对它们进行分型，以寻找相关标记，以了解关联的细节并确定其他基因座。HH/PTCH通路已被证明在几乎所有的基底细胞癌和部分髓母细胞瘤中都发生了突变。此外，许多肿瘤表现出配体依赖的HH/PTCH通路的激活，包括胰腺、前列腺、胃肠道和小细胞肺癌。类固醇的天然产物分子环多巴胺与HH/PTCH途径的下游调节因子SMO特异性结合。环多巴胺已被证明可以抑制HH/PTCH激活的细胞系和异种移植瘤的生长。为了证明环丙胺是否抑制HH/PTCH靶基因的表达，我们用前列腺癌(DU-145，LNCaP)、胃癌(AGS)和乳腺癌(SK-BR3)细胞系进行处理，证实环丙胺抑制增殖。从处理和未处理的DU-145细胞中提取的RNA进行定量RT-PCR，观察到PTCH、SMO、GLI1和GLI2的表达降低。为了进一步开发HH/PTCH抑制剂，我们设计了针对SMO蛋白的TM结构域和细胞内环的多肽。SMO是G蛋白偶联受体(GPCR超家族)的成员之一，其他GPCRs也是这种方法的靶点。虽然针对几个TM结构域的多肽对细胞增殖没有影响，但针对细胞内环2和3的多肽是有效的抑制因子。与环多巴胺一样，活性多肽导致HH/PTCH靶基因表达减少。TUNEL检测显示，环多巴胺和SMO多肽均通过诱导细胞凋亡发挥作用。

A persons genetic background plays an important role in their susceptibility to cancer, disease progression, and response to therapy. Genetic variations can be used as markers to understand complex diseases including cancer and AIDS. In collaboration with scientists at the Memorial Sloan Kettering Cancer Institute we have completed a whole genome association study of breast cancer in Ashkenazi Jewish subjects with a family history of disease. The initial scan identified a number of loci with potentially significant associations. From this data we have selected loci for follow up studies in a replication cohort. The <I>TRIM5</I> gene encodes a protein involved in the protection of cells from HIV infection. We have identified regions of the human <I>TRIM5</I> gene that appear to have undergone selection, perhaps in response to previous infection. Other <I>TRIM</I> family sequences in the human genome also appear to have been infected and may be involved in the protection to infection by other viruses. By studying variants in the complement B and C2 (<I>CFH</I>, <I>C2</I>) genes we have identified association in this gene in patients with age-related macular degeneration (AMD). AMD is the leading cause of blindness in the elderly and is estimated to effect as many as of 10 million Americans. By examining the genetic variants in the <I>BF</I> and <I>C2</I> genes significantly protective haplotypes were identified. The complement pathway is important in the response to pathogens causative for cancer. Further study of this important component of the innate immune response could lead to insight into human disease. A deletion that removes the <I>CFHR1</I> gene adjacent to <I>CFH</I> is highly protective for AMD. We have obtained over 3000 DNAs from subjects in the Age-Related Eye Disease Study, and are typing them for the associated markers to understand the details of the association and identify additional loci. The HH/PTCH pathway has been demonstrated to be mutated in virtually all basal cell carcinomas and a portion of medulloblastomas. In addition, many tumors display ligand-dependent activation of the HH/PTCH pathway including pancreas, prostate, gastrointestinal tract, and small cell lung tumors. The steroid-like, natural product molecule cyclopamine specifically binds to SMO, the downstream regulator of the HH/PTCH pathway. Cyclopamine has been shown to inhibit the growth of HH/PTCH-activated cell lines and xenografts. To demonstrate whether cyclopamine inhibits the expression of HH/PTCH target genes we treated prostate (DU-145, LnCaP), gastric (AGS), and breast cancer (SK-BR3) cell lines and confirmed that cyclopamine inhibits proliferation. RNA isolated from treated and untreated DU-145 cells was subjected to quantitative RT-PCR and a reduction in expression of PTCH, SMO, and GLI1, and GLI2 was observed. To further the development of HH/PTCH inhibitors we designed peptides against TM domains and intracellular loops of the SMO protein. SMO is a member of the G-protein-coupled receptor (GPCR) superfamily, and other GPCRs have been targeted by this approach. While peptides against several TM domains failed to have an effect on cell proliferation, peptides against intracellular loops 2 and 3 were potent inhibitors. As with cyclopamine, the active peptides result in a decrease in expression of HH/PTCH target genes. Both cyclopamine and SMO peptides act by eliciting apoptosis, as measured by the TUNEL assay.