In the past year significant progress has been made in the molecular cytogenetics and molecular biology of cancer cells. We obtained information on chromosome alterations in several novel cancer model systems, provided further evidence regarding the role of DLC family genes in cancer development, and identified TMEM7 as a potential tumor suppressor in HCC. The heat shock transcription factor Hsf1 regulates expression of heat shock proteins and, as such, protects cells against apoptosis triggered by protein damaging agents. To understand the role of Hsf1 in tumorigenesis, hsf1-/- mice were crossed with p53-/- mice to analyze p53-mediated tumorigenesis in the presence or absence of the hsf1 gene. While p53-/- mice develop mostly lymphomas, hsf1-/-p53-/- mice develop mostly testicular cancers and sarcomas - yet both succumb to their respective tumors by the age of 35 weeks. We demonstrated by spectral karyotyping analysis (SKY) that loss of the hsf1 gene increased genomic instability in mouse embryo fibroblasts from hsf1-/-p53-/- animals. This suggests that the loss of Hsf1 in mice already deficient for p53 gene promotes an increase in genomic instability that triggers a change in the program of neoplastic transformation that favors the formation of solid tumors over lymphomas. In addition to its major function removal of topological stress in DNA associated with replication, transcription, repair and recombination - human topoisomerase 1 (Top1) has been proposed to play a role in many other important processes. Since total elimination of Top1 is lethal, stable Top1 small interfering RNA derivatives (siTop1) of colon and breast carcinoma cell lines were generated. Reduction of Top1 availability resulted in altered expression of 55 different genes, increased genomic instability (as demonstrated by SKY analysis) and decreased nucleolus organizing region (NOR) activity (demonstrated by controlled chromosomal silver-staining assay) and formation of histone H2A.X foci associated with replication. Taken together, these findings point to a non-classical role of Top1 in maintenance of genomic integrity, gene-specific regulation of transcription, and the response to various anticancer agents. The most compelling characteristic of cells acting as self-renewing mammary gland stem cells (MGSC) is their ability to reconstitute a differentiated mammary gland in vivo following implantation into cleared mammary gland fat pads. In recent years the ability of such cells to grow as nonadherent, free-floating-mammospheres has become an in vitro test of their MGSC function. Using specific immunostaining and FACS, several fractions of mouse mammary gland cells with specific surface marker signatures were isolated and showed to be able to both form mammospheres and to regenerate mammary gland on implantation into a mammary fat pad. Culturing of those cells changes their immunophenotype and also affects their genomic stability, as evidenced by SKY analysis of freshly isolated and precultured samples, opening the possibility that accumulation of somatic alterations in MGSC may be a factor in tumorigenesis. We have continued our studies on DLC1, a Rho GTPase activating protein and tumor suppressor originally isolated in the Molecular Cytogenetics Section. To learn more about the molecular mechanisms by which DLC1 influences cell behavior, we entered into a partnership with Myriad Genetics to use yeast two-hybrid screening to identify proteins that interact with DLC1. A number of potential DLC1 binding partners were found, including 3 members of the tensin family of focal adhesion proteins (tensin1, tensin2, and tensin3), which act as a link between the actin cytoskeleton and the cytoplasmic tails of integrins. The tensin1 binding site of DLC1 was characterized by Dr. Douglas Lowygroup, who demonstrated that mutation of a critical residue (Y442) abolished tensin1 binding, eliminated the focal adhesion localization of DLC1, and reduced its ability to inhibit cell migration and growth in soft agar We had used gene targeting to generate DLC1 knockout mice and found that gene is essential for normal development, as Dlc1-/- embryos died at midgestation. Since the embryonic lethality precluded attempts at studying the role of DLC1 at later stages of development and during tumorigenesis in adult tissues, we set out to produce a conditional DLC1 knockout strain to allow tissue-specific inactivation of the gene using Cre-LoxP technology. In collaboration with Dr. Snorri Thorgeirsson, Dr. Douglas Lowy, and the Gene Targeting Facility at NCI-Frederick, we have successfully obtained mouse embryonic stem cells that carried the correctly targeted conditional allele and were able to generate chimeric mice. Breeding of the chimeras to identify offspring with germline transmission of the conditional allele is currently underway. DLC1 is the founding member of a family of closely related Rho GTPase activating proteins that have tumor suppressor properties. The DLC2 gene on chromosome 13q13 had been shown by others to be down-regulated in HCC, and we demonstrated that its expression is also reduced in several other common human cancers. We also characterized a novel gene on chromosome Xq13 that encodes DLC3, a third member of the DLC family that is around 50% identical to DLC-1 and DLC-2. The DLC3 mRNA was widely expressed in normal tissues, but was down-regulated in a number of human cancer cell lines and in primary tumors from kidney, lung, ovary, breast, and prostate. Transfection of human breast and prostate cancer cells with a DLC3 expression vector inhibited cell proliferation, colony formation, and growth in soft agar, indicating that DLC3 may act as a tumor suppressor in these tissues. In addition, DLC3 was found to stimulate GTP hydrolysis by RhoA and to bind tensin1. Both genetic and environmental factors are important in the development and prevention of breast cancer. Among such external factors, diet is thought to play an important role. A low incidence of breast cancer in Asia has been attributed in part to a high intake of flavonoids. Flavone treatment restored DLC1 expression in aggressive breast and colon carcinoma cell lines that were DLC-1- negative due to promoter hypermethylation and also inhibited cell proliferation, induced cell cycle arrest and apoptosis, and lead to deregulation of several proto-oncogene and tumor suppressor genes. The results obtained with cells from aggressive or metastatic breast tumors suggest the possibility that administration of flavone, alone or in combination with DNA methyltransferase and histone deacetylase inhibitors, may be an effective adjunct to chemotherapy. The TMEM7 gene, which encodes a transmembrane protein, is among the candidate tumor suppressor genes located at a region of deletion 3p21.3 in various cancers. This gene is expressed specifically in the liver, and the Tmem7 protein shares substantial sequence homology with human and mouse 28-kDa interferon-alpha (IFN-α) responsive protein. We investigated the role of TMEM7 in the development of hepatocellular carcinoma (HCC) and demonstrated down-regulation or silencing of the gene is due aberrant DNA methylation and histone deacetylation, in the absence of genomic deletion and mutation. Ectopic expression of TMEM7 in HCC lines suppressed cell proliferation, colony formation, and cell migration in vitro and reduced tumor formation in nude mice. Treatment of two highly invasive HCC cell lines with IFN-α for 7 days significantly increased TMEM7 expression and inhibited cell migration. These observations implicate loss of TMEM7 expression in hepatocarcinogenesis and suggest that modification of TMEM7 expression by IFN-α may have potential therapeutic relevance in a subset of HCC
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