Our research program combines molecular cytogenetics and molecular biology approaches to study the genetic alterations in solid tumors, hematological malignancies and in transformed cells. The major objective of this project is to identify recurrent chromosomal alterations and to isolate new genes relevant both to neoplastic development, and as potential targets for therapy. In the past year,progress has been made in molecular genetics and cytogenetics of several solid tumors and hematological malignancies, as well as of tumors developed in transgenic mice. This resulted in the identification of new recurrent genomic alterations, and isolation of new genes, relevant to both initiation and progression of neoplasia and as possible useful markers in prognosis and diagnosis of the disease. Novel genomic regions of recurrent DNA amplification and deletions, as well as chromosome translocations in cancer cells were identified by fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH) and spectral karyotyping (SKY). Deletion of the short arm of chromosome 8 is one of the most common alterations in several human cancers including hepatocellular carcinoma (HCC). From this critical region of deletion and loss of heterozygosity, long suspected to harbor tumor suppressor genes, we identified and isolated the DLC-1 gene, a regulator of the Rho family small GTPases that control actin cytoskeleton organization, membrane trafficking, gene expression, cell proliferation, malignant transformation, metastasis. In the past year, compelling evidence was provided that DLC-1 acts as a tumor suppressor gene. Restoration of DLC-1 expression in cell lines derived from metastatic breast adenocarcinomas lacking endogenous gene expression caused significant growth inhibition and prevented the development of tumors in athymic nude mice. In two isogenic breast cancer cell lines with diametrically opposite metastatic capabilities, DLC-1 was found to be down-regulated in the metastatic relative to the non-metastatic cells. Similarly, in a different model, DLC-1 was down-regulated in breast cancer cell populations highly metastatic to bone and not in populations weakly metastatic to adrenal gland.The ability of DLC-1 to prevent the growth of cells derived from metastatic tumors shows that this gene has tumor suppressive activity in breast cancer and may be involved in the process of metastasis. Also, the findings suggest a potential clinical application in the treatment of aggressive metastatic breast cancer or prevention of metastasis. Downregulation or inactivation of the DLC-1 gene during the tumor development occurs either due to the copy-number deletion at the genomic level, or to aberrant promoter methylation at the transcriptional level, and in certain cancers may be associated with cell dissemination and metastasis, the major cause of cancer death. A different degree of promoter hypermethylation was detected in all cell lines derived from of liver, breast, colon and prostate tumors with aberrant DLC-1 expression. The hypermethylation status was reversed by treatment with a demethylating agent. Altered DNA methylation is one of the most promising markers for early detection, prediction of cancer risk and prognosis of disease and the profile of DLC-1 methylation may serve as a useful marker. Point mutation is another common mechanism responsible for gene inactivation. Exonic missense mutations and intronic insertions/deletions of DLC-1 were found only in subset of HCC and other solid tumors. Meanhwile, several polymorphisms have been identified that could be used for determining whether DLC-1 is linked to cancer susceptibility loci. The mouse DLC-1 gene was also isolated, and the exon/intron organization was characterized. An intragenic polymorphic microsatellite marker was identified that was useful for linkage mapping and LOH analysis. To provide an animal model system for investigating the biological functions of DLC-1 in vivo, we successfully used homologous recombination in embryonic stem cells to generate mice with a disrupted DLC-1 gene. The phenotype of the mice was characterized and the data will be reported soon. Identification of two regions of high-level DNA amplification in HCC led us to examine alterations of two known genes, EMS1 and SMAD5. Over-representation of 11q13 chromosomal region was detected by CGH and a fragment of the EMS1 oncogene was isolated and subsequently found to be amplified in primary HCC and over expressed in HCC cell lines in the absence of gene amplification. The EMS1 gene encodes cortactin, a cortical actin-associated protein that is a substrate for Src kinase and is involved in cytoskeleton organization. Alterations of the EMS1 gene that lead to overexpression of cortactin may be associated with the development of HCC. EMS1 amplification and overexpression is indicative of an unfavorable prognosis in several cancers and may have similar prognostic implications in liver cancer. The minimal region of DNA copy-number gains at 5q31 overlaps with the location of the common fragile site (FS), FRA5C, and the locus of the SMAD5 gene. Deletions and unbalanced translocations with breakpoints near the SMAD5 locus, recurrent formation of isochromosome 5q leading to selective loss of 5p and gain of 5q, and intrachromosomal amplification of SMAD5 gene at the fragile site FRA5C were detected in HCC, indicating that this locus is a frequent target in liver cancer. SMAD high-level amplification at FRA5C is one of the few examples of gene amplification at a common FS through breakage-fusion-bridge cycles. These results show that SMAD5 undergoes copy number gain, high level amplification and increased expression rather than loss of expression, suggesting SMAD5 does not function as a tumor suppressor gene in HCC. The consequences of SMAD5 overexpression on function of other genes, such bone morphogenetic protein genes might be important in hepatocarcinogenesis. A new constitutive translocation involving a FS was also characterized. Members of a family in which multifocal clear renal cell carcinoma (RCC) segregates with a balanced constitutional chromosome t (2; 3) translocation, YAC and BAC contigs encompassing the 2q and 3q breakpoints were constructed, and BACs crossing the breakpoints were partially sequenced. By FISH, the 3q breakpoint was placed at 3q13, possibly near the border with 3q21 and the 2q breakpoint was localized closely telomeric to fragile site FRA2G on chromosome 2.Based on the genomic map of the 2q breakpoint an EST was identified near the translocation, and the full length c DNA of novel gene, DIRC1 which is disrupted by the balanced translocation t (2; 3) was isolated. Low expression of this gene was detected in several tissues and a GFP- DIRC1 fusion protein was expressed in vitro. Lack of mutations and rare polymorphism in tumors and tumor cell lines obscure the role of DIRC1 gene in RCC. The genetic changes in tumors spontaneously developing in certain transgenic mice closely evoke those that occur during the progression of human cancers. The cytogenetic analyses of spontaneous HCC and acute promyelocytic leukemia (APL) in transgenic mice resulted in identification of novel genomic alterations and cloning of new genes. Our SKY analysis of HCC in MYC transgenic mice identified recurrent gains of chromosome 19 and double minute chromosomes (DM) derived from chromosome 19. DMs are cytological manifestations of high level DNA amplification. By screening a cDNA library of mouse chromosome 19, a new putative mouse ortholog of the human Rho GTPase activating protein 8, ARHGAP8 was cloned. The open reading frame encodes a peptide of 387 amino acids with high homology to human ARHGAP8 in its N-terminal
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