Comparative Analysis of Cancer-Associated Genes and Deve

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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.