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

在过去的一年中，在癌细胞的分子细胞遗传学和分子生物学中取得了重大进展。我们获得了几种新型癌症模型系统中染色体改变的信息，提供了有关DLC家族基因在癌症发展中作用的进一步证据，并将TMEM7鉴定为HCC中潜在的肿瘤抑制剂。热休克转录因子HSF1调节热休克蛋白的表达，因此可以保护细胞免受蛋白质破坏剂触发的细胞凋亡。为了了解HSF1在肿瘤发生中的作用，将HSF1 - / - 小鼠与p53 - / - 小鼠交叉，以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，而HSF1 - / - p53 - / - 小鼠大多会出现睾丸癌和肉瘤 - 但到35周的年龄都屈服于各自的肿瘤。我们通过光谱核分型分析（SKY）证明了HSF1基因的丧失增加了HSF1 - / - p53 - / - 动物中小鼠胚胎成纤维细胞中基因组不稳定性。这表明，已经缺乏p53基因的小鼠中HSF1的丧失促进了基因组不稳定性的增加，这会触发肿瘤转化程序的变化，从而有利于淋巴瘤而不是实体瘤形成。除了其在与复制，转录，修复和重组相关的DNA中拓扑应力的主要功能外，还提出了人拓扑异构酶1（TOP1）在许多其他重要过程中发挥作用。由于TOP1的总消除是致命的，因此产生了结肠癌和乳腺癌细胞系的稳定的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（如天空分析所证明的）和核仁组织区域（NOR）活性（通过受控染色体银染色测定法）以及组蛋白H2A.X的形成与与复制相关的组蛋白H2A.x。综上所述，这些发现表明TOP1在维持基因组完整性，转录基因调控以及对各种抗癌剂的反应中的非经典作用。充当自我更新的乳腺干细胞（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中重新构造体内分化的乳腺的能力。近年来，此类细胞作为非粘合剂，自由浮动amammlophers的能力已成为对其MGSC功能的体外测试。使用特定的免疫染色和FACS，分离了具有特定表面标记特异性标记的小鼠乳腺细胞的几个部分，并证明能够在植入哺乳动物脂肪垫中形成哺乳动物和再生乳腺。这些细胞的培养改变了它们的免疫表型，也会影响其基因组稳定性，这可以通过对新鲜分离和预先培养的样品进行天空分析所证明，开放了MGSC中体细胞改变的可能性可能是肿瘤发生的因素。我们继续对DLC1（最初在分子细胞遗传学部分中分离出的Rho GTPase激活蛋白质和抑制肿瘤的Rho GTPase）的研究。为了了解有关DLC1影响细胞行为的分子机制的更多信息，我们与多种遗传学建立了伙伴关系，以使用酵母两杂交筛选来识别与DLC1相互作用的蛋白质。发现了许多潜在的DLC1结合伙伴，包括局灶性粘附蛋白的3个成员（tensin1，tensin2和tensin3），它们充当整联蛋白的肌动蛋白细胞骨架与细胞质尾巴的联系。 DOUGLAS LOWYGROUP博士的特征是DLC1的Tensin1结合位点的特征，他证明，关键残基的突变（Y442）废除了Tensin1的结合，消除了DLC1的局灶性粘附定位，并降低了基因在SoftAgar中的基本靶向ASET的基本靶标，并降低了其在基本上的迁移和生长的能力。 DLC1 - / - 胚胎在中间植物中死亡。由于胚胎致死率排除了研究DLC1在发育后期和成年组织中肿瘤发生期间的作用的尝试，因此我们着手使用CRE-LOXP技术产生有条件的DLC1敲除菌株以允许基因的组织特异性失活。与Snorri Thorgeirsson博士，Douglas Lowy博士以及NCI-Frederick的基因靶向设施合作，我们成功地获得了携带正确靶向有条件等位基因的小鼠胚胎干细胞，并能够生成嵌合小鼠。目前正在进行嵌合体的繁殖以鉴定有条件等位基因的种系传播的后代。 DLC1是与具有肿瘤抑制特性的密切相关的Rho GTPase激活蛋白质家族的创始成员。其他人在HCC中已证明13q13染色体上的DLC2基因在HCC中被下调，我们证明其表达在其他几种常见的人类癌症中也会降低。我们还表征了XQ13染色体XQ13上的一种新基因，该基因编码DLC3是DLC家族的第三个成员，该基因与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脏，肺，卵巢，乳房和前列腺的原发性肿瘤中被下调。用DLC3表达载体转染人乳腺癌和前列腺癌细胞，抑制了软琼脂的细胞增殖，菌落形成和生长，表明DLC3可以在这些组织中充当肿瘤抑制剂。另外，发现DLC3刺激RhoA刺激GTP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。亚洲乳腺癌的发病率很低，部分原因是类黄酮的摄入量很高。黄酮治疗恢复了由于启动子高甲基化而导致的DLC-1-阴性的侵袭性乳腺癌和结肠癌细胞系中的DLC1表达，还抑制了细胞增殖，诱导细胞周期停滞和凋亡，并导致几种原始癌和抑制肿瘤抑制基因的调节。用侵袭性或转移性乳腺肿瘤细胞获得的结果表明，单独或与DNA甲基转移酶和组蛋白脱乙酰基酶抑制剂的给药可能是化学疗法的有效辅助性的可能性。编码跨膜蛋白的TMEM7基因是位于各种癌症中缺失3p21.3区域的候选肿瘤抑制基因之一。该基因在肝脏中特别表达，TMEM7蛋白与人和小鼠28-kDa Interferon-Alpha（IFN-α）反应性蛋白具有实质序列同源性。我们研究了TMEM7在没有基因组缺失和突变的情况下，TMEM7在肝细胞癌（HCC）发展中的作用是由于异常的DNA甲基化和组蛋白脱乙酰化，证明了基因下调或沉默。 TMEM7在HCC系中的异位表达抑制了细胞增殖，菌落形成和细胞迁移，并在裸鼠中抑制了肿瘤形成。用IFN-α进行7天的两种高度侵入性HCC细胞系的处理显着增加了TMEM7表达并抑制细胞迁移。这些观察结果暗示了肝癌发生中TMEM7表达的丧失，并表明IFN-α对TMEM7表达的修饰可能在HCC的一部分中具有潜在的治疗相关性。