Comparative Analysis of Cancer-Associated Genes and Deve

癌症相关基因的比较分析及开发

• 批准号: 7291785
• 负责人: VLADIMIR LARIONOV
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7291785
• 关键词: Comparative; Analysis; Cancer; Associated; Genes

Recent studies have revealed that diversifying selection may have also acted on tumor suppressor genes. Because most tumors are formed after reproductive age, tumor promotion or suppression itself is not likely to be subject to natural selection, and selective pressures acting on the genes are most likely related to another more physiological role for these proteins, specifically in the developing embryo. The hypothesis of developmental evolution suggests that genes that form the basis of our adaptive evolution and have multiple functions may also be involved in disease predisposition. Our lab identified two new human cancer-associated genes that have evolved under pressure of diversifying selection, ASPM encoding the microcephaly gene, and SPANX genes encoding cancer-testis antigens. We also demonstrated that not only exon 11, but entire coding sequence of the breast cancer BRCA1 gene has rapidly evolved with evidence of Darwinian selection. During last year we concentrated on studying these genes to elucidate their possible role in carcinogenesis.1). Previous studies in mouse suggest that ASPM is preferentially expressed in the developing brain. Our analysis revealed that ASPM is widely expressed in fetal and adult tissues and up-regulated in malignant cells. Several alternatively spliced variants encoding putative ASPM isoforms with different numbers of putative calmodulin-binding IQ motifs were identified. The major ASPM transcript contains 81 IQ domains, most of which are organized into a higher order repeat structure (HOR). Another prominent spliced form contains an in-frame deletion of exon 18 and encodes 14 IQ domains not organized into a HOR. This variant is conserved in mouse. Other spliced variants lacking both putative N-terminal calponin-homology CH domains and a part of the IQ motifs were also detected, suggesting the existence of isoforms with potentially different functions. To elucidate the biochemical function of human ASPM, we developed peptide specific antibodies to the N- and C-termini of ASPM. In a Western analysis of proteins from cultured human and mouse cells, the antibodies detected bands with mobilities corresponding to the predicted ASPM isoforms. Immunostaining of cultured human cells with antibodies revealed that ASPM is localized in the centrosomes defects of which have been found in numerous forms of cancer. Therefore, further studies of ASPM polymorphism and isoform function will not only clarify the molecular mechanisms of microcephaly, but also may link this centrosomal protein with the predisposition to cancer.2). Comparison of primate BRCA1 gene homologues allowed us to reconstruct an evolutionary history of the BRCA1 locus. The impact of Alu repeats, CpG dinucleotides, and a mixture of positive selection and conservation of the coding sequences were the main factors that shaped BRCA1 evolution in primates. These comparisons also provided a basis for identification of conservative amino-acid residues in BRCA1 and prediction of missense changes that compromise BRCA1 function. Missense mutations that confer the highest predisposition to breast and ovarian cancers are located in the evolutionarily conserved regions, phosporylated residues, and in specific protein-binding domains. 3). Genetic linkage studies indicate that 750 kb genomic region on chromosome Xq27 containing five SPANX genes (SPANX-A1, -A2, -B, -C, and -D) is implicated in prostate carcinogenesis. SPANX genes are >95% homologous and reside within large segmental duplications (SDs) with a high level of similarity which confounds mutational analysis of this gene family by routine PCR methods. We applied transformation-associated recombination cloning (TAR) in yeast in order to characterize individual SPANX genes from prostate cancer patients showing linkage to Xq27-28 and unaffected controls. Analysis of genomic TAR clones revealed a dynamic nature of the region of linkage.

最近的研究表明，多样化选择也可能对肿瘤抑制基因起作用。由于大多数肿瘤是在生育年龄后形成的，因此肿瘤促进或抑制本身不太可能受到自然选择的影响，作用于基因的选择压力很可能与这些蛋白质的另一个更生理作用有关，特别是在发育中的胚胎中。发育进化假说表明，形成我们适应性进化基础并具有多种功能的基因也可能参与疾病易感性。我们的实验室确定了两个新的人类癌症相关基因，它们是在多样化选择的压力下进化而来的，ASPM编码小头畸形基因，而SPAN X基因编码癌症睾丸抗原。我们还证明，不仅外显子11，但乳腺癌BRCA 1基因的整个编码序列已经迅速进化的达尔文选择的证据。在去年，我们集中研究这些基因，以阐明它们在致癌作用中的可能作用。之前对小鼠的研究表明ASPM优先在发育中的大脑中表达。我们的分析表明，ASPM在胎儿和成人组织中广泛表达，并在恶性细胞中上调。几个选择性剪接变体编码推定ASPM亚型与不同数量的推定钙调蛋白结合IQ基序进行了鉴定。主要ASPM转录本包含81个IQ结构域，其中大部分被组织成高阶重复结构（HOR）。另一个突出的剪接形式包含外显子18的框内缺失，并编码14个未组织成HOR的IQ结构域。该变体在小鼠中是保守的。也检测到其他剪接的变体，缺乏推定的N-末端钙调蛋白同源CH结构域和IQ基序的一部分，这表明存在具有潜在不同功能的异构体。为了阐明人ASPM的生化功能，我们开发了针对ASPM的N-和C-末端的肽特异性抗体。在对来自培养的人类和小鼠细胞的蛋白质的Western分析中，抗体检测到具有对应于预测的ASPM同种型的迁移率的条带。用抗体对培养的人细胞进行免疫染色，结果显示ASPM位于中心体中，在许多癌症中发现了中心体缺陷。因此，对ASPM基因多态性和亚型功能的进一步研究，不仅可以阐明小头畸形的分子机制，而且可能将该中心体蛋白与癌症易感性联系起来。灵长类动物BRCA 1基因同源物的比较使我们能够重建BRCA 1基因座的进化历史。Alu重复序列、CpG二核苷酸的影响以及编码序列的正选择和保守的混合是塑造灵长类动物BRCA 1进化的主要因素。这些比较也为鉴定BRCA 1中的保守氨基酸残基和预测损害BRCA 1功能的错义变化提供了基础。赋予乳腺癌和卵巢癌最高易感性的错义突变位于进化上保守的区域、磷酸化残基和特定的蛋白质结合结构域中。3）。遗传连锁研究表明，染色体Xq 27上的750 kb基因组区域含有五个SPAN基因（SPAN-A1，-A2，-B，-C和-D），涉及前列腺癌的发生。SPAN基因是>95%同源的，并且存在于具有高水平相似性的大片段重复（SD）内，这混淆了通过常规PCR方法对该基因家族的突变分析。我们在酵母中应用了转化相关重组克隆（TAR），以表征前列腺癌患者和未受影响的对照组中显示与Xq 27 -28连锁的单个SPAN基因。基因组TAR克隆的分析揭示了连锁区域的动态性质。