Molecular Cytogenetics of Solid Tumors

实体瘤的分子细胞遗传学

• 批准号: 6762671
• 负责人: NICOLAE POPESCU
• 金额: --
• 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/6762671
• 关键词: animal genetic material tag; cancer risk; chemical structure function; chromosome aberrations; cytogenetics; family genetics; fluorescent in situ hybridization; gene targeting; genetic mapping; genetic markers; genetic screening; genetic susceptibility; genetically modified animals; human genetic material tag; human tissue; karyotype; laboratory mouse; molecular oncology; natural gene amplification; neoplasm /cancer; neoplasm /cancer genetics; neoplastic process; nucleic acid structure; oncoproteins; patient oriented research

The major objective of our research is to identify recurrent chromosomal alterations and to isolate new genes that are relevant to neoplastic development and may serve as targets for therapy. Our program combines molecular cytogenetics and molecular biology to study chromosome and gene alterations in solid tumors and hematological malignancies as well as in transformed cells. In the past year progress has been made in molecular genetics and cytogenetics of liver cancer, breast, and cervical carcinoma, in Burkitt's lymphoma (BL), as well as in liver tumors in transgenic mice. This resulted in the identification of new recurrent genomic alterations and isolation of new genes relevant to the initiation and progression of neoplasia and possibly useful markers for prognosis and diagnosis of the disease. Deletion of the short arm of chromosome 8 is one of the most common alterations in liver tumors as well as in several other cancers. DLC-1 is a gene located at a critical region of deletions on chromosome 8p. Several lines of evidence indicate that DLC-1 acts as a tumor suppressor gene in more than one form of cancer. The mouse DLC-1 gene was isolated, and the exon/intron organization was characterized. An intragenic polymorphic microsatellite marker was identified that was useful for linkage mapping and LOH analysis. Fragments of the gene were cloned into a targeting vector that was successfully used to disrupt the gene by homologous recombination in embryonic stem (ES) cells to generate DLC-1 deficient mice. From the 11q13 region commonly amplified in several cancers, a fragment having homology with EMS1 oncogene was isolated and subsequently found to be amplified in some primary human hepatocellular carcinoma (HCC) and overexpressed in HCC cell lines in the absence of gene amplification. The EMS1 gene encodes cortactin, a cortical actin-associated protein that is a substrate for Src kinase and is involved in cytoskeleton organization. Alterations of the EMS1 gene that lead to overexpression of cortactin may be associated with tumor development of HCC. EMS1 amplification and overexpression is indicative of an unfavorable prognosis in several cancers and may have similar prognostic implications in liver cancer. Among the newly isolated genes is mouse GTF2IRD1 that was identified near the breakpoint of recurrent balanced chromosome translocation t (5; 6) in HCC that developed in MYC transgenic mice. GTF2IRD1 encodes a transcription factor that interacts with the retinoblastoma protein; and the 5 end of the gene is deleted and its expression greatly reduced in mice homozygous for the translocation. The balanced translocation t (5; 6) occurs at the earliest stage in tumor development and may play a critical role in the initiation of neoplastic transformation. In addition, the mouse GTF2IRD1 gene is one of the genes hemizygously deleted in Williams-Beuren syndrome. Since the transgenic mice line represent the first "knock out" of one of the genes in the Williams-Beuren syndrome deletion, they may aid in understanding the pathogenesis of this disorder. Recurrent gain of chromosome 19 and double minute chromosomes (DM) derived from chromosome 19 has been identified in HCC developed in MYC transgenic mice. DM are cytological manifestations of high level DNA amplification. At the site of DNA amplification the MYCS and MXI1 genes are located, both known to interfere with MYC gene activity, and also the mouse RelA gene, which promote cell survival by inhibiting apoptosis, allowing MYC to drive proliferation of transformed cells. Further characterization of the PCR probes from DM showed that one of these genes, MYCS, a member of the intronless MYC was incorrectly assigned on chromosome 19. We provided the correct assignment MYCS on chromosome X. In the past, we localized the sites of breakage within the most active fragile site in human genome, FRA3B encompassing FHIT, a tumor suppressor gene implicated in a large number of cancers. A significantly higher frequency of loss of heterozygosity at the FHIT locus was observed for breast cancers with deleterious mutations of BRCA2. We examined a series of breast tumors with BRCA1 mutations and found that loss of FHIT expression was significantly more frequent in the BRCA1 cancers compared to sporadic breast tumors, suggesting that the BRCA1 pathway is also important in protecting the FRA3B/FHIT locus from damage. To further shed light on the association between repair and the FRA3B/FHIT locus, the incidence of apidicholin (apc)-induced gaps and breaks was examined in tumor cell lines and lymphoblastoid cell lines completely lacking mismatch repair as well as mouse repair deficient cell lines. The incidence of chromosomal lesions induced by apc was significantly greater in all repair-deficient cells than in control cells implicating proteins involved in mismatch repair and double strand break repair in maintenance of the integrity of fragile sites. Characteristic for BL are reciprocal translocations involving the MYC locus at fragile site 8C to any of the three immunoglobulin genes, resulting in MYC gene deregulation. In a comprehensive combined molecular cytogenetic study, we demonstrated two more complex t(8;14;18) and (7;8;14) translocations in addition to the reciprocal translocation t(8;14).These novel rearrangements in BL resulted in transposition of MYC sequences in a new genomic configuration. We also also observed partial duplication of the long arm of chromosome 1q23, the second most common alteration in BL and known to be associated with aggressive tumors and poor prognosis. While translocations involving the MYC gene are important in the initial stages of BL, duplication of 1q may be critical during progression and the acquisition of an invasive tumor phenotype. This region might harbor gene(s) associated with tumor cell invasiveness and serve as a prognostic marker in BL. The specificity of viral integration is fundamental in determining the biological significance of this phenomenon to various pathological conditions including cancer. Human papilloma viruses (HPV) are closely associated with cervical cancer. HPV16 integration in the genome of a rapidly progressive, lethal cervical cancer in a 39-year-old woman was localized on chromosome 14. HPV integration resulted in the disruption of the TNFAIP2 cytokine/retinoic acid-inducible gene, thus contributing to the rapid progression of cervical cancer. Acquisition of drug resistance is a major problem in cancer therapy. In a human colon carcinoma cell line rendered resistant to ecteinascidine a critical cytogenetic abnormality detected by SKY that led to the demonstration of novel mechanism of action for the drug. Thus, for the first time a specific cytogenetic abnormality was directly linked to the acquisition of drug resistance. The elucidation of the requirements for conversion of normal cells into neoplastic cells is critical in cancer research. Our cytogenetic analysis of neoplastically transformed human by the introduction of mutant alleles of a small number of genes, demonstrated that malignant transformation of normal human mammary cells required nonrandom chromosome changes involving the MYC gene, which is commonly altered in spontaneous breast cancer. In contrast the acquisition of tumorigenicity of kidney epithelial cells can occur in the absence of widespread genetic instability and aneuploidy. Introduction of a limited set of genes to induce a specific phenotype could be applied to other disorders and the resulting phenotype may be manifested by specific chromosome changes. The advances could lead to an improved early detection. A number of new cancer-related human, mouse, and rat genes have been mapped. These localizations will permit linkage analysis and facilitate both the identification of loci associated with genetic diseases of unknown etiology and examination of their involvement in cancer cell chromosome rearrangements. Among the mouse genes are: Sca-1, an antigen commonly used for purification of murine pluripotent hematopoietic cells, a member of the Ly-6 family and pancreatic phospholipase A2 which plays an important role in pancreatic and extrapancreatic tissue and UGRP, uteroglobin related protein which is down-regulated in lung and might be involved in inflammation. We also mapped the rat RMT-1 gene whose expression is correlated with mammary carcinogenesis. Among the human genes localized are: mitochondrial topoisomerase 1 gene; the claudin 18 gene which plays an important role in the structure and function of tight junctions in lung and stomach; and UGRP1, encoding uteroglobin protein 1, and is associated with an increased risk of asthma.

我们研究的主要目标是识别反复发生的染色体改变并分离与肿瘤发展相关并可作为治疗靶点的新基因。我们的项目结合了分子细胞遗传学和分子生物学来研究实体瘤、血液恶性肿瘤以及转化细胞中的染色体和基因改变。 去年，肝癌、乳腺癌、宫颈癌、伯基特淋巴瘤（BL）以及转基因小鼠肝脏肿瘤的分子遗传学和细胞遗传学取得了进展。这导致了新的复发性基因组改变的鉴定和与肿瘤的起始和进展相关的新基因的分离以及对于疾病的预后和诊断可能有用的标记。 8 号染色体短臂的缺失是肝肿瘤以及其他几种癌症中最常见的改变之一。 DLC-1是位于染色体8p缺失关键区域的基因。多项证据表明 DLC-1 在多种癌症中充当肿瘤抑制基因。分离小鼠 DLC-1 基因，并表征外显子/内含子组织。鉴定出可用于连锁作图和 LOH 分析的基因内多态性微卫星标记。该基因的片段被克隆到靶向载体中，该载体成功地用于通过胚胎干 (ES) 细胞中的同源重组破坏该基因，从而产生 DLC-1 缺陷小鼠。 从几种癌症中普遍扩增的11q13区域中，分离出与EMS1癌基因具有同源性的片段，随后发现该片段在一些原发性人肝细胞癌（HCC）中扩增，并且在没有基因扩增的情况下在HCC细胞系中过度表达。 EMS1 基因编码 cortactin，一种皮质肌动蛋白相关蛋白，是 Src 激酶的底物，参与细胞骨架组织。导致 Cortactin 过度表达的 EMS1 基因的改变可能与 HCC 的肿瘤发展有关。 EMS1 扩增和过度表达表明多种癌症的预后不良，并且可能对肝癌具有类似的预后影响。 新分离的基因之一是小鼠 GTF2IRD1，它是在 MYC 转基因小鼠中发生的 HCC 中反复平衡染色体易位 t (5; 6) 的断点附近发现的。 GTF2IRD1 编码与视网膜母细胞瘤蛋白相互作用的转录因子；并且该基因的5末端被删除，其在易位纯合小鼠中的表达大大降低。平衡易位t（5；6）发生在肿瘤发展的最早阶段，并且可能在肿瘤转化的启动中发挥关键作用。此外，小鼠GTF2IRD1基因是Williams-Beuren综合征中半合子缺失的基因之一。由于转基因小鼠品系代表了威廉姆斯-博伊伦综合征缺失中一个基因的首次“敲除”，因此它们可能有助于了解这种疾病的发病机制。 在 MYC 转基因小鼠的 HCC 中发现了 19 号染色体和源自 19 号染色体的双小染色体 (DM) 的反复获得。 DM 是高水平 DNA 扩增的细胞学表现。 DNA 扩增位点上有 MYCS 和 MXI1 基因（已知这两种基因都会干扰 MYC 基因活性），还有小鼠 RelA 基因，该基因通过抑制细胞凋亡来促进细胞存活，从而使 MYC 能够驱动转化细胞的增殖。来自 DM 的 PCR 探针的进一步表征表明，这些基因之一 MYCS（无内含子 MYC 的成员）被错误地分配在 19 号染色体上。我们在 X 染色体上提供了正确的 MYCS 分配。 过去，我们将断裂位点定位在人类基因组中最活跃的脆弱位点，即包含 FHIT 的 FRA3B，FHIT 是一种与大量癌症有关的肿瘤抑制基因。对于具有 BRCA2 有害突变的乳腺癌，观察到 FHIT 基因座杂合性丢失的频率显着更高。我们检查了一系列具有 BRCA1 突变的乳腺肿瘤，发现与散发性乳腺肿瘤相比，BRCA1 癌症中 FHIT 表达缺失的频率明显更高，这表明 BRCA1 通路在保护 FRA3B/FHIT 位点免受损害方面也很重要。为了进一步阐明修复与 FRA3B/FHIT 基因座之间的关联，在完全缺乏错配修复的肿瘤细胞系和淋巴母细胞系以及小鼠修复缺陷细胞系中检查了阿匹胆碱 (apichololin, apc) 诱导的间隙和断裂的发生率。在所有修复缺陷细胞中，由 apc 诱导的染色体损伤的发生率显着高于对照细胞，这表明蛋白质参与错配修复和双链断裂修复，以维持脆弱位点的完整性。 BL 的特征是易位，涉及脆弱位点 8C 的 MYC 基因座到三个免疫球蛋白基因中的任何一个，导致 MYC 基因失调。在一项全面的组合分子细胞遗传学研究中，我们证明了除了相互易位 t(8;14) 之外，还有两种更复杂的 t(8;14;18) 和 (7;8;14) 易位。BL 中的这些新重排导致 MYC 序列转座在新的基因组配置中。我们还观察到染色体 1q23 长臂的部分重复，这是 BL 中第二常见的改变，已知与侵袭性肿瘤和不良预后相关。虽然涉及 MYC 基因的易位在 BL 的初始阶段很重要，但 1q 的重复在进展和侵袭性肿瘤表型的获得过程中可能至关重要。该区域可能含有与肿瘤细胞侵袭性相关的基因，并可作为 BL 的预后标记。 病毒整合的特异性对于确定这种现象对包括癌症在内的各种病理状况的生物学意义至关重要。人乳头瘤病毒（HPV）与宫颈癌密切相关。一名 39 岁女性的快速进展、致命性宫颈癌基因组中的 HPV16 整合位于 14 号染色体上。HPV 整合导致 TNFAIP2 细胞因子/视黄酸诱导基因的破坏，从而导致宫颈癌的快速进展。 耐药性的获得是癌症治疗中的一个主要问题。 SKY 检测到对海鞘素产生耐药性的人类结肠癌细胞系中出现了严重的细胞遗传学异常，从而证明了该药物的新作用机制。因此，第一次特定的细胞遗传学异常与耐药性的获得直接相关。 阐明正常细胞转化为肿瘤细胞的要求对于癌症研究至关重要。我们通过引入少量基因的突变等位基因对肿瘤转化的人类进行细胞遗传学分析，证明正常人类乳腺细胞的恶性转化需要涉及 MYC 基因的非随机染色体变化，而 MYC 基因在自发性乳腺癌中通常发生改变。相反，肾上皮细胞获得致瘤性可以在不存在广泛的遗传不稳定性和非整倍性的情况下发生。引入一组有限的基因来诱导特定的表型可以应用于其他疾病，并且所产生的表型可以通过特定的染色体变化来体现。这些进步可能会改善早期检测。 许多新的与癌症相关的人类、小鼠和大鼠基因已经被绘制出来。这些定位将允许进行连锁分析，并有助于识别与未知病因的遗传性疾病相关的基因座，并检查它们与癌细胞染色体重排的关系。小鼠基因包括：Sca-1，一种常用于纯化小鼠多能造血细胞的抗原，Ly-6家族的成员；胰腺磷脂酶A2，在胰腺和胰腺外组织中发挥重要作用；以及UGRP，子宫珠蛋白相关蛋白，在肺部下调，可能与炎症有关。我们还绘制了大鼠 RMT-1 基因图谱，该基因的表达与乳腺癌发生相关。定位的人类基因包括：线粒体拓扑异构酶 1 基因； Claudin 18 基因在肺和胃紧密连接的结构和功能中发挥重要作用； UGRP1，编码子宫珠蛋白1，与哮喘风险增加相关。