Comparative Analysis of Cancer-Associated Genes and Deve

最近的研究表明，多样化的选择可能也对肿瘤抑制基因产生了作用。由于大多数肿瘤是在生殖年龄之后形成的，肿瘤的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具生理性的作用有关，特别是在发育中的胚胎中。发育进化假说表明，构成我们适应性进化基础并具有多种功能的基因也可能与疾病易感性有关。我们的实验室发现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出来的，ASPM编码小头畸形基因，SPANX基因编码癌症-睾丸抗原。我们还证明，不仅是外显子11，而且乳腺癌BRCA1基因的整个编码序列都随着达尔文选择的证据而迅速进化。去年，我们集中精力研究这些基因，以阐明它们在癌症发生中的可能作用。以往对小鼠的研究表明，ASPM在发育中的大脑中优先表达。我们的分析表明，ASPM在胎儿和成人组织中广泛表达，在恶性细胞中上调。确定了几个编码假定的ASPM异构体的选择性剪接变异体，这些异构体具有不同数量的假定的钙调蛋白结合智商基序。主要的ASPM转录本包含81个IQ结构域，其中大部分被组织成更高顺序的重复结构(HOR)。另一种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。这个变异体在小鼠体内是保守的。还检测到其他剪接变异体，既没有推测的N端钙蛋白同源CH结构域，也没有部分IQ基序，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，我们研制了针对ASPM N-末端和C-末端的多肽抗体。在对培养的人类和小鼠细胞中的蛋白质进行的Western分析中，抗体检测到了迁移率与预测的ASPM亚型相对应的条带。用抗体对培养的人类细胞进行免疫染色显示，ASPM定位于中心体，其缺陷已在多种形式的癌症中被发现。因此，对ASPM基因的多态性和异构体功能的进一步研究不仅将阐明小头畸形的分子机制，而且可能将这种中心体蛋白与癌症的易感性联系起来。通过对灵长类BRCA1基因同源性的比较，我们可以重建BRCA1基因座的进化史。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性的混合是影响灵长类BRCA1进化的主要因素。这些比较也为鉴定BRCA1中保守的氨基酸残基和预测影响BRCA1功能的错义变化提供了基础。乳腺癌和卵巢癌的易感性最高的错义突变位于进化保守区、磷酸化残基和特定的蛋白结合区。3)。遗传连锁研究表明，染色体Xq27上750 kb的基因组区域包含5个SPANX基因(span x-A1、-A2、-B、-C和-D)，与前列腺癌的发生有关。SPANX基因有95%的同源性，并且存在于大片段重复(SDS)中，具有很高的相似性，这使得常规的PCR方法对该基因家族的突变分析产生了混乱。我们在酵母中应用了转化相关重组克隆(TAR)，以确定前列腺癌患者与Xq27-28连锁的单个SPANX基因和未受影响的对照的特征。对基因组焦油克隆的分析揭示了连锁区域的动态性质。

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.