Molecular Cytogenetics of Solid Tumors

在过去的一年中，在人类和小鼠肝癌模型中发现了包含关键癌症相关基因的新型和高度复发的基因组改变，并且在理解肝癌1中缺失（dcl1），一种真正的肿瘤抑制基因功能及其用于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抗增殖信号的抗性。我们检测了一种对tgf敏感的人肝细胞癌（HCC）细胞系；1 （Hep3B-TS）及其衍生物（Hep3B-TR）对TGF&amp；#946；通过光谱核型分析（SKY）和阵列比较基因组杂交（aCGH）分析，逐步暴露于该试剂。我们发现收购TGF&amp；#946；耐药是由包含TGF&amp；#946位点的间质性微缺失引起的；受体II （TGF&#946；RII）基因，发生在唯一明显正常的3号染色体上。利用21397个基因的寡核苷酸芯片对两种细胞系的差异基因表达进行比较分析，发现Hep3BTR中分别有197个和110个基因被上调和下调。其中，6个基因是肿瘤坏死因子（TNF）基因的下游靶点，提示TGF&amp；#946；RII触发TNF网络的激活，被证明是由TGF&amp；#946；1通路。功能上，TGF&#946；RII在对tgf耐药的细胞中；1与亲代敏感细胞相比，显著增强了细胞迁移和锚定独立生长的体外和体内致瘤性。小鼠肝癌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检测了在c-Myc转基因动物中产生的几种HCC细胞系，以及通过sc接种原HCC细胞系在裸鼠中诱导的肿瘤中建立的四种细胞系。所有细胞系都表现出来自若干染色体的物质的增加和损失，以及复发性重排，其断点聚集在先前确定的对早期c-Myc诱导的肝癌发生重要的区域。这些结果强调了复发性基因组重组，染色体4、9和14的丢失以及染色体15和19的获得在小鼠肝脏肿瘤中的重要性。这五种基因组失衡的鉴定是一个独特的例子，因为在其他转基因小鼠模型中发展的HCC，在自发性小鼠HCC和衍生细胞系中，以及在化学致癌物诱导的肿瘤前肝脏病变中都发现了相同的失衡。这些结果表明，选择性的、非随机的基因组变化涉及与人类HCC相关的染色体区域。编码Rho gtpase激活蛋白的DLC1在几种常见癌症中是一种真正的肿瘤抑制基因。DLC-1表达缺失是激活Rho GTPases的常见机制，Rho GTPases是人类肿瘤发生的关键介质。在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超甲基化和/或组蛋白去乙酰化。我们对44株多发性骨髓瘤（MM）细胞系进行了DLC1启动子甲基化和mRNA表达异常筛选，发现其中大多数细胞系都表现出不同程度的启动子甲基化，这与DLC1表达下调或沉默相关。最近，其他人提供了确凿的证据，证明在所有病例中，未知意义的无症状单克隆γ病（MGUS）先于MM的诊断，并强调需要新的标记物来预测MGUS患者向MM的进展。因此，高频率的DLC1甲基化和下调可能为MGUS和MM提供了这样一个有用的分子标记。我们还证明，用去甲基化和乙酰化剂处理由于启动子完全超甲基化而缺乏DLC1表达的MM细胞系，可以显著增强DLC1的表达并抑制细胞增殖。此外，最重要的是，当含有DLC1 cDNA的腺病毒载体转导具有完全启动子甲基化和缺乏DLC1表达的细胞时，会导致DLC1表达的恢复。dcl1的重表达抑制骨髓瘤细胞的侵袭和迁移，降低RhoA活性，导致肌动蛋白细胞骨架重组。这些结果为dcl1在血液学癌症中的抗增殖作用提供了第一个证据，并暗示RhoA途径抑制MM的迁移和侵袭。dlc1介导的细胞迁移和侵袭的抑制是非常重要的，因为这两个过程在骨髓瘤细胞在骨内的运动和向继发部位的转移中起着重要的作用。鉴于骨髓瘤细胞对DLC-1功能再激活的敏感性，DLC-1介导途径的分子靶向治疗以及表观遗传治疗的潜力具有前景。酵母双杂交筛选首次鉴定出14种与DLC1蛋白相互作用的蛋白，并在人类细胞中证实了几种相互作用。识别这些相互作用的伙伴可以增强我们对调节dcl1功能的机制的理解。我们证实DLC1蛋白与p120Ras-GAP蛋白相互作用。内源性DLC1与内源性Ras-GAP形成稳定的复合物，两种蛋白在局灶黏附中共定位。相互作用映射到dcl1的Rho-GAP催化结构域和Ras-GAP的SH3结构域。这种相互作用在体外诱导DLC1Rho-GAP活性的显著降低。此外，在携带突变Ras的结肠癌细胞中，Ras- gap对Ras的负调控具有抗性，其过表达以DLC1依赖的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可能是一个有效的治疗靶点。另一个重要的成就是，通过在胚胎干细胞中使用基因靶向，在4号外显子两侧引入loxP位点，产生了具有dcl1基因条件敲除等位基因的小鼠。Cre重组酶的表达删除了含有外显子4的loxp侧DNA片段，导致阅读框移位，导致DLC1多肽的过早翻译终止。flxed等位基因（Dlc1fl/fl）的纯合子小鼠是活的和可育的，并且可以在各种组织特异性和可诱导启动子的控制下与表达Cre重组酶的转基因菌株杂交。这种条件基因敲除小鼠是与dr。Snorri Thorgeirsson和Douglas Lowy将为分析dcl1缺乏在正常发育和成人组织以及癌症和其他病理条件下的影响提供一个有价值的模型系统。

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 TGFβ. We examined a human hepatocellular carcinoma (HCC) cell line sensitive to TGFβ1 (Hep3B-TS) and its derivative (Hep3B-TR) rendered resistant to TGFβ1 by stepwise exposure to the agent by spectral karyotyping (SKY) and array comparative genomic hybridization (aCGH) analysis. We discovered that the acquisition of TGFβ resistance was caused by an interstitial microdeletion encompassing the site of TGFβ receptor II (TGFβRII) 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 TGFβRII triggered the activation of TNF network, shown to be regulated by TGFβ1 pathway. Functionally, loss of the TGFβRII in cells resistant to TGFβ1 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.