Molecular Cytogenetics of Solid Tumors

实体瘤的分子细胞遗传学

• 批准号: 7592632
• 负责人: NICOLAE POPESCU
• 金额: $ 128.17万
• 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/7592632
• 关键词: 3p21.3; Aberrant DNA Methylation; Actins; Adult; Affect; Agar; Age; Alleles; Animals; Antineoplastic Agents; Apoptosis; Asia; Binding; Binding Sites; Biological Assay; Biological Models; Breast; Breast Cancer Prevention; Breast Carcinoma; Breast Fat Pad; Breeding; Cancer Model; Cancer cell line; Cell Cycle Arrest; Cell Line; Cell Nucleolus; Cell Proliferation; Cell physiology; Cells; Characteristics; Chimera organism; Chromosome abnormality; Chromosomes; Collaborations; Colon; Colon Carcinoma; Cytoplasmic Tail; Cytoskeleton; DLEC1 gene; DNA; DNA Methyltransferase; DNA Modification Methylases; Deletion Mutation; Detection; Development; Diet; Down-Regulation; Ectopic Expression; Embryo; Environmental Risk Factor; Excision; Family; Fatty acid glycerol esters; Fibroblasts; Flavones; Flavonoids; Focal Adhesions; Gene Family; Gene Silencing; Gene Targeting; Genes; Genetic; Genetic Transcription; Genome Stability; Genomic Instability; Genomics; Genus Cola; Growth; Guanosine Triphosphate; Heat shock proteins; Hepatocarcinogenesis; Histone Deacetylase Inhibitor; Histone Deacetylation; Human; Hydrolysis; Hypermethylation; Immunophenotyping; In Vitro; Incidence; Intake; Integral Membrane Protein; Integrins; Interferon-alpha; Invasive; Kidney; Knock-out; Knockout Mice; Laboratories; Lead; Learning; Link; Liver; Lung; Lymphoma; Maintenance; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of prostate; Malignant neoplasm of testis; Mammary Neoplasms; Mammary gland; Mediating; Messenger RNA; Modification; Molecular; Molecular Biology; Molecular Cytogenetics; Mus; Mutation; Natural regeneration; Neoplastic Cell Transformation; Normal tissue morphology; Nude Mice; Numbers; Ovary; Play; Primary Neoplasm; Primary carcinoma of the liver cells; Process; Property; Prostate; Proteins; Proto-Oncogenes; Recurrence; Research; Role; Sampling; Screening procedure; Sequence Homology; Silver Staining; Small Interfering RNA; Solid Neoplasm; Spectral Karyotyping; Staging; Stem cells; Stress; Surface; TP53 gene; Technology; Testing; Therapeutic; Thinking; Tissues; Topoisomerase; Transcriptional Regulation; Transfection; Tumor Suppressor Genes; Tumor Suppressor Proteins; Week; Xq13; Yeasts; base; cancer cell; cancer diagnosis; carcinogenesis; cell behavior; cell motility; chemotherapy; day; embryonic stem cell; expression vector; flavone; heat shock transcription factor; human DLC1 protein; human H2AX protein; implantation; in vivo; malignant breast neoplasm; member; novel; outcome forecast; programs; promoter; recombinational repair; reconstitution; response; rho GTPase-activating protein; sarcoma; tensin; transmission process; tumor; tumor progression; tumorigenesis; yeast two hybrid system

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

在过去的一年里，癌细胞的分子细胞遗传学和分子生物学取得了重大进展。我们获得了几种新型癌症模型系统中染色体改变的信息，为DLC家族基因在癌症发展中的作用提供了进一步的证据，并确定了TMEM7在HCC中是一种潜在的肿瘤抑制因子。热休克转录因子Hsf1调节热休克蛋白的表达，从而保护细胞免受蛋白质损伤剂引发的凋亡。为了了解Hsf1在肿瘤发生中的作用，我们将Hsf1 -/-小鼠与p53-/-小鼠杂交，分析在存在或不存在Hsf1基因的情况下p53介导的肿瘤发生。p53-/-小鼠主要发展为淋巴瘤，hsf1-/-p53-/-小鼠主要发展为睾丸癌和肉瘤，但两者都在35周时死于各自的肿瘤。我们通过谱核型分析（SKY）证明，hsf1基因的缺失增加了hsf1-/-p53-/-动物小鼠胚胎成纤维细胞基因组的不稳定性。这表明，在已经缺乏p53基因的小鼠中，Hsf1的缺失促进了基因组不稳定性的增加，从而引发了肿瘤转化程序的变化，这种变化有利于实体瘤而不是淋巴瘤的形成。人类拓扑异构酶1 （Top1）除了具有去除DNA中与复制、转录、修复和重组相关的拓扑应力的主要功能外，还被认为在许多其他重要过程中发挥作用。由于Top1的完全消除是致命的，因此在结肠癌和乳腺癌细胞系中产生了稳定的Top1小干扰RNA衍生物（siTop1）。Top1可用性的降低导致55种不同基因的表达改变，基因组不稳定性增加（如SKY分析所示），核仁组织区（NOR）活性降低（如受控染色体银染色试验所示）和组蛋白H2A的形成。与复制相关的X焦点。综上所述，这些发现指出了Top1在维持基因组完整性、基因特异性转录调控和对各种抗癌药物的反应方面的非经典作用。作为自我更新乳腺干细胞（MGSC）的细胞最引人注目的特征是它们在植入清除的乳腺脂肪垫后能够在体内重建分化的乳腺。近年来，这种细胞以非黏附的、自由漂浮的乳房微球形式生长的能力已成为一种体外测试其MGSC功能的方法。利用特异性免疫染色和FACS，分离出具有特异性表面标记的小鼠乳腺细胞的几个部分，并显示出在植入乳腺脂肪垫后既能形成乳腺球，又能再生乳腺。这些细胞的培养改变了它们的免疫表型，也影响了它们的基因组稳定性，正如新鲜分离和预培养样本的SKY分析所证明的那样，这开启了MGSC中体细胞改变的积累可能是肿瘤发生的一个因素的可能性。我们继续对dcl1的研究，dcl1是一种Rho GTPase激活蛋白和肿瘤抑制因子，最初在分子细胞遗传学部分分离出来。为了更多地了解DLC1影响细胞行为的分子机制，我们与Myriad Genetics建立了合作伙伴关系，使用酵母双杂交筛选来鉴定与DLC1相互作用的蛋白质。许多潜在的DLC1结合伙伴被发现，包括局灶黏附蛋白tensin家族的3个成员（tensin1， tensin2和tensin3），它们作为肌动蛋白细胞骨架和整合素的细胞质尾部之间的联系。Douglas Lowygroup博士对DLC1的tensin1结合位点进行了鉴定，他证明了一个关键残基（Y442）的突变消除了tensin1结合，消除了DLC1的局点粘附定位，并降低了其在软琼脂中抑制细胞迁移和生长的能力。我们使用基因靶向技术产生了dcl1敲除小鼠，发现该基因对正常发育至关重要，因为dcl1 -/-胚胎在妊娠中期死亡。由于胚胎致死性阻碍了研究dcl1在发育后期和成人组织肿瘤发生过程中的作用，我们开始使用Cre-LoxP技术生产条件dcl1敲除菌株，以允许组织特异性失活该基因。通过与Snorri Thorgeirsson博士、Douglas Lowy博士和NCI-Frederick的基因靶向设施的合作，我们成功地获得了携带正确靶向条件等位基因的小鼠胚胎干细胞，并能够产生嵌合小鼠。目前正在对嵌合体进行育种，以鉴定具有条件等位基因生殖系传播的后代。DLC1是一个密切相关的Rho GTPase激活蛋白家族的创始成员，该家族具有肿瘤抑制特性。染色体13q13上的DLC2基因在HCC中被下调，我们证实其在其他几种常见人类癌症中的表达也降低。我们还在Xq13染色体上发现了一个编码DLC3的新基因，DLC3是DLC家族的第三个成员，与DLC-1和DLC-2相同约50%。DLC3 mRNA在正常组织中广泛表达，但在许多人类癌细胞系和肾、肺、卵巢、乳腺和前列腺的原发性肿瘤中表达下调。用DLC3表达载体转染人乳腺癌和前列腺癌细胞，可抑制软琼脂中细胞的增殖、集落形成和生长，表明DLC3可能在这些组织中起抑瘤作用。此外，DLC3被发现刺激GTP被RhoA水解并结合tensin1。遗传和环境因素在乳腺癌的发展和预防中都很重要。在这些外部因素中，饮食被认为起着重要作用。亚洲乳腺癌的低发病率部分归因于类黄酮的高摄入量。黄酮治疗可以恢复DLC-1在侵袭性乳腺癌和结肠癌细胞系中的表达，这些细胞系由于启动子超甲基化而呈DLC-1阴性，同时还可以抑制细胞增殖，诱导细胞周期阻滞和凋亡，并导致一些原癌基因和肿瘤抑制基因的失调。从侵袭性或转移性乳腺肿瘤细胞中获得的结果表明，单独或与DNA甲基转移酶和组蛋白去乙酰化酶抑制剂联合使用黄酮可能是化疗的有效辅助。编码跨膜蛋白的TMEM7基因是多种癌症中位于3p21.3缺失区域的候选肿瘤抑制基因之一。该基因在肝脏中特异性表达，Tmem7蛋白与人和小鼠28-kDa干扰素- α (IFN-α)应答蛋白具有大量序列同源性。我们研究了TMEM7在肝细胞癌（HCC）发展中的作用，并证明在没有基因组缺失和突变的情况下，该基因的下调或沉默是由于DNA甲基化和组蛋白去乙酰化异常。肝癌细胞系中TMEM7的异位表达抑制了细胞增殖、集落形成和细胞迁移，并减少了裸鼠肿瘤形成。用IFN-&#945；治疗两种高侵袭性HCC细胞系7天可显著增加TMEM7表达，抑制细胞迁移。这些观察结果暗示了TMEM7在肝癌发生过程中的表达缺失，并表明通过IFN-修饰TMEM7表达可能在HCC的一个亚群中具有潜在的治疗相关性