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Molecular Cytogenetics of Solid Tumors

实体瘤的分子细胞遗传学

基本信息

批准号：
7965205
负责人：
NICOLAE POPESCU
金额：
$ 112.87万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/7965205
关键词：
Achievement Actins Adenovirus Vector Adult Alleles Anchorage-Independent Growth Biological Biological Models Cancer Prognosis Catalytic Domain Cell Line Cell Proliferation Cells Chromosomes Chromosomes, Human, Pair 3 Collaborations Colon Carcinoma Complementary DNA Complex Cytoskeleton DLC1 gene DNA DNA Sequence Rearrangement Data Development Diagnosis Down-Regulation Epigenetic Process Exhibits Exons Exposure to Focal Adhesions Frequencies GTPase-Activating Proteins Gene Mutation Gene Targeting Generations Genes Genetic Genomics Goals Growth Hematologic Neoplasms Hepatocarcinogenesis Histone Deacetylation Human Hypermethylation In Vitro Individual Knock-out Knockout Mice Laboratories Lesion Link Liver Malignant Epithelial Cell Malignant Neoplasms Malignant neoplasm of liver Maps Mediating Mediator of activation protein Methylation Molecular Molecular Biology Molecular Cytogenetics Molecular Target Monoclonal gammopathy of uncertain significance Multiple Myeloma Mus Neoplasm Metastasis Neoplasms Neoplastic Processes Nude Mice Oligonucleotide Microarrays Oncogenes Pathway interactions Primary carcinoma of the liver cells Process Proteins Reading Frames Recurrence Regulation Research Resistance Role SH3 Domains Secondary to Side Signal Pathway Signal Transduction Site Site Visit Solid Neoplasm Spectral Karyotyping Staging Structure Systems Analysis TNF gene Therapeutic Intervention Tissue-Specific Gene Expression Tissues Transgenic Animals Transgenic Mice Transgenic Organisms Translations Tumor Necrosis Factor Activation Tumor Suppressor Genes Tumorigenicity Yeasts bone c-myc Genes cancer cell cancer therapy carcinogenesis cell growth cell motility chemical carcinogen chromosome 15 gain chromosome 4 loss comparative comparative genomic hybridization embryonic stem cell established cell line gene function human DLC1 protein in vitro activity in vivo insight interstitial mRNA Expression microdeletion migration molecular marker mouse model mutant neoplastic cell novel novel marker overexpression polypeptide premature programs promoter ras GTPase-Activating Proteins receptor recombinase restoration rho rho GTP-Binding Proteins rho GTPase-activating protein therapeutic target tumor tumorigenesis yeast two hybrid system

项目摘要

In the past year novel and highly recurrent genomic alterations harboring critical cancer-related genes have been identified in human and mouse model for liver cancer and significant progress has been made in the understanding of the Deleted in Liver Cancer 1, (DLC1), a bona fide tumor suppressor gene function and its potential use for therapeutic interventions. During the neoplastic process certain tumor cells acquire resistance to the antiproliferative signaling of TGF&#946;. We examined a human hepatocellular carcinoma (HCC) cell line sensitive to TGF&#946;1 (Hep3B-TS) and its derivative (Hep3B-TR) rendered resistant to TGF&#946;1 by stepwise exposure to the agent by spectral karyotyping (SKY) and array comparative genomic hybridization (aCGH) analysis. We discovered that the acquisition of TGF&#946; resistance was caused by an interstitial microdeletion encompassing the site of TGF&#946; receptor II (TGF&#946;RII) gene, which occurred in the only apparently normal chromosome 3. Comparative differential gene expression analysis of two cell lines using an oligonucleotide microarray of 21397 genes revealed that 197 and 110 genes were up- and down-regulated, respectively in Hep3BTR. Among these, 6 genes were downstream targets of tumor necrosis factor (TNF) gene, suggesting that loss of TGF&#946;RII triggered the activation of TNF network, shown to be regulated by TGF&#946;1 pathway. Functionally, loss of the TGF&#946;RII in cells resistant to TGF&#946;1 significantly enhanced cell migration and anchorage independent growth in vitro and in vivo tumorigenicity compared with parental sensitive cells. Mouse models for HCC provide an experimental ground to dissect the genetic and biological complexities of human liver cancer and enhance our ability to gain insights into the relevance of candidate cancer genes. We used mouse SKY and aCGH to examine several cell lines derived from HCC developed in c-Myc transgenic animals, and four cell lines established from the tumors induced in nude mice by s.c. inoculation of the original HCC cell lines. All cell lines exhibited gain and loss of material from several chromosomes, and also the recurrent rearrangements whose breakpoints clustered in the regions previously identified as important for the early stages of c-Myc induced hepatocarcinogenesis. The results underscore the importance of recurrent genomic reorganizations, loss of chromosomes 4, 9 and 14, and gain of chromosomes 15 and 19 in mouse liver neoplasia. The identification of those five genomic imbalances represent an unique example, as identical imbalances have been found in HCC developed in other transgenic mouse models, in spontaneous mouse HCC and derivative cell lines, as well as in preneoplastic liver lesions induced with chemical carcinogens. These results demonstrate selective, non-random genomic changes involving chromosomal regions homologous to those implicated in human HCC. DLC1, that encodes a Rho GTPase-activating protein, is an authentic tumor suppressor gene in several common cancers. Loss of DLC-1 expression represents a frequent mechanism for activation of Rho GTPases, key mediators of human oncogenesis. The predominant mechanisms of silencing or down regulation of DLC-1 in solid tumors and hematological malignancies are promoter hypermethylation and/or histone deacetylation. We screened 44 multiple myeloma (MM) cell lines for abnormal DLC1 promoter methylation and mRNA expression and found that the majority of them exhibited various degree of promoter methylation that correlated with downregulation or silencing of DLC1 expression. Recently, others provided conclusive evidence that an asymptomatic monoclonal gammopathy of unknown significance (MGUS) preceded the diagnosis of MM in all cases and emphasized that novel markers are necessary to predict progression to MM in individuals with MGUS. Thus, high frequency of DLC1 methylation and downregulation may provide such a useful molecular marker for MGUS and MM. We also demonstrated that treatment of MM cell lines lacking DLC1 expression due to full promoter hypermethylation with demethylating and acetylating agents significantly augmented the expression of DLC1 and inhibited cell proliferation. In addition and most importantly, when cells exhibiting complete promoter methylation and absence of DLC1 expression were transduced by an adenoviral vector containing DLC1 cDNA, resulted in restoration of DLC1 expression. Reexpression of DLC1 inhibited myeloma cell invasion and migration, reduced RhoA activity and resulted in reorganization of actin cytoskeleton. These results provide the first evidence for antiproliferative effect of DLC1 in a hematological cancer and implicate RhoA pathway in suppression of MM migration and invasion. DLC1-mediated suppression of cell migration and invasion is of significant importance as the two processes are instrumental in the myeloma cell movement within the bone morrow and metastasis to secondary sites. Given the myeloma cells sensitivity to reactivation of DLC-1 function, the potential for molecular targeted therapy of DLC-1 mediated pathways as well as epigenetic therapies hold prospects. A yeast two-hybrid screen first identified 14 proteins that interact of DLC1 protein and several interactions were confirmed in human cells. Identification of such interacting partners could enhance our understanding of the mechanisms that regulate the function of DLC1. We demonstrated DLC1 protein interaction with p120Ras-GAP protein. Endogenous DLC1 forms a stable complex with endogenous Ras-GAP, and the two proteins co-localize in focal adhesions. The interaction was mapped to the Rho-GAP catalytic domain of DLC1 and the SH3 domain of Ras-GAP. This interaction induced a dramatic reduction of DLC1Rho-GAP activity in vitro. Moreover, overexpression of Ras-GAP, in colon carcinoma cells that harbored mutant Ras and thus were resistant to the negative regulation of Ras by Ras-GAP, increased the level of endogenous active Rho in a DLC1-dependent manner and antagonized the growth suppressive effects of DLC1. Our data showing that Ras-GAP can promote the growth of cells containing mutant Ras add to a growing body of evidence suggesting that Ras-GAP might represent a valid therapeutic target. Another significant achievement was the generation of mice with a conditional knockout allele of the DLC1 gene by using gene targeting in ES cells to introduce loxP sites on either side of exon 4. Expression of Cre recombinase deleted the loxP-flanked DNA segment containing exon 4, leading to a reading frame shift that should result in premature translation termination of the DLC1 polypeptide. Mice homozygous for the floxed allele (Dlc1fl/fl) are viable and fertile and can be crossed with transgenic strains that express Cre recombinase under the control of various tissue-specific and inducible promoters. This conditional knockout mouse that was generated in collaboration with Drs. Snorri Thorgeirsson and Douglas Lowy will provide a valuable model system for analyzing the effects of DLC1 deficiency in normal developing and adult tissues and in cancer and other pathological conditions.

去年，在人类和小鼠肝癌模型中发现了含有关键癌症相关基因的新型且高度复发的基因组改变，并且在了解肝癌删除基因 1 (DLC1)（一种真正的肿瘤抑制基因功能）及其治疗干预的潜在用途方面取得了重大进展。在肿瘤形成过程中，某些肿瘤细胞获得了对 TGF&#946 的抗增殖信号传导的抵抗力。我们检查了对 TGFβ1 (Hep3B-TS) 及其衍生物 (Hep3B-TR) 敏感的人肝细胞癌 (HCC) 细胞系，通过光谱核型分析 (SKY) 和阵列比较基因组杂交 (aCGH) 分析逐步暴露于该药物，从而对 TGFβ1 产生耐药性。我们发现收购 TGFβ抗性是由包含 TGF-β 位点的间质微缺失引起的。受体 II (TGFβRII) 基因，出现在唯一明显正常的 3 号染色体上。使用 21397 个基因的寡核苷酸微阵列对两个细胞系进行比较差异基因表达分析，结果显示在 Hep3BTR 中分别有 197 个和 110 个基因上调和下调。其中，6 个基因是肿瘤坏死因子 (TNF) 基因的下游靶标，表明 TGFβRII 的缺失触发了 TNF 网络的激活，该网络受到 TGFβ1 通路的调节。从功能上讲，与亲代敏感细胞相比，对 TGF-1 具有抗性的细胞中 TGF-RII 的缺失显着增强了体外和体内的细胞迁移和贴壁独立生长的致瘤性。 HCC 小鼠模型为剖析人类肝癌的遗传和生物学复杂性提供了实验基础，并增强了我们深入了解候选癌症基因相关性的能力。我们使用小鼠 SKY 和 aCGH 来检查在 c-Myc 转基因动物中开发的几种源自 HCC 的细胞系，以及从裸鼠皮下诱导肿瘤中建立的四种细胞系。接种原始 HCC 细胞系。所有细胞系都表现出多条染色体物质的获得和损失，以及反复发生的重排，其断点聚集在先前确定对 c-Myc 诱导的肝癌发生早期阶段重要的区域。结果强调了小鼠肝肿瘤中反复基因组重组、4、9和14号染色体丢失以及15和19号染色体获得的重要性。这五种基因组失衡的鉴定代表了一个独特的例子，因为在其他转基因小鼠模型中开发的 HCC、自发性小鼠 HCC 和衍生细胞系以及化学致癌剂诱导的肿瘤前肝脏病变中也发现了相同的失衡。这些结果表明选择性的、非随机的基因组变化涉及与人类肝癌相关的染色体区域同源。 DLC1 编码 Rho GTP 酶激活蛋白，是几种常见癌症中真正的肿瘤抑制基因。 DLC-1 表达的丧失代表了 Rho GTPases（人类肿瘤发生的关键介质）激活的常见机制。实体瘤和血液恶性肿瘤中 DLC-1 沉默或下调的主要机制是启动子高甲基化和/或组蛋白脱乙酰化。我们筛选了 44 个多发性骨髓瘤 (MM) 细胞系的异常 DLC1 启动子甲基化和 mRNA 表达，发现其中大多数表现出不同程度的启动子甲基化，与 DLC1 表达下调或沉默相关。最近，其他人提供了确凿的证据，表明在所有病例中诊断出 MM 之前均存在无症状意义不明的单克隆丙种球蛋白病 (MGUS)，并强调需要新的标志物来预测 MGUS 个体进展为 MM 的情况。因此，DLC1 甲基化和下调的高频率可能为 MGUS 和 MM 提供有用的分子标记。我们还证明，用去甲基化剂和乙酰化剂处理由于启动子完全甲基化而缺乏 DLC1 表达的 MM 细胞系，可显着增强 DLC1 的表达并抑制细胞增殖。此外，最重要的是，当显示出启动子完全甲基化且不存在 DLC1 表达的细胞被含有 DLC1 cDNA 的腺病毒载体转导时，会恢复 DLC1 表达。 DLC1 的重新表达抑制骨髓瘤细胞侵袭和迁移，降低 RhoA 活性并导致肌动蛋白细胞骨架重组。这些结果为 DLC1 在血液癌中的抗增殖作用提供了第一个证据，并暗示 RhoA 通路在抑制 MM 迁移和侵袭中的作用。 DLC1 介导的细胞迁移和侵袭抑制具有重要意义，因为这两个过程有助于骨髓瘤细胞在骨髓内移动和转移到继发部位。鉴于骨髓瘤细胞对 DLC-1 功能重新激活的敏感性，DLC-1 介导途径的分子靶向治疗以及表观遗传疗法的潜力具有前景。酵母双杂交筛选首先鉴定了 14 种与 DLC1 蛋白相互作用的蛋白质，并且在人类细胞中证实了几种相互作用。识别此类相互作用的伙伴可以增强我们对调节 DLC1 功能的机制的理解。我们证明了 DLC1 蛋白与 p120Ras-GAP 蛋白的相互作用。内源性 DLC1 与内源性 Ras-GAP 形成稳定的复合物，并且这两种蛋白在粘着斑中共定位。该相互作用被映射到 DLC1 的 Rho-GAP 催化结构域和 Ras-GAP 的 SH3 结构域。这种相互作用导致体外 DLC1Rho-GAP 活性显着降低。此外，在携带Ras突变体的结肠癌细胞中，Ras-GAP的过表达可以抵抗Ras-GAP对Ras的负调节，以DLC1依赖性方式增加内源性活性Rho的水平，并拮抗DLC1的生长抑制作用。我们的数据显示 Ras-GAP 可以促进含有突变 Ras 的细胞的生长，这增加了越来越多的证据表明 Ras-GAP 可能代表一个有效的治疗靶点。另一个重大成就是通过在 ES 细胞中使用基因靶向在外显子 4 的两侧引入 loxP 位点，产生了具有 DLC1 基因条件性敲除等位基因的小鼠。Cre 重组酶的表达删除了包含外显子 4 的 loxP 侧翼 DNA 片段，导致阅读框移位，从而导致 DLC1 多肽的过早翻译终止。 floxed 等位基因 (Dlc1fl/fl) 纯合的小鼠具有存活力和生育力，可以与在各种组织特异性和诱导型启动子控制下表达 Cre 重组酶的转基因品系杂交。这种条件性基因敲除小鼠是与博士合作产生的。 Snorri Thorgeirsson 和 Douglas Lowy 将提供一个有价值的模型系统，用于分析 DLC1 缺陷对正常发育和成人组织以及癌症和其他病理状况的影响。