Molecular Cytogenetics of Solid Tumors

实体瘤的分子细胞遗传学

• 批准号: 6950933
• 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/6950933
• 关键词: acute myelogenous leukemia; animal genetic material tag; breast neoplasms; cancer risk; chromosome aberrations; cytogenetics; fluorescent in situ hybridization; gene targeting; genetic mapping; genetic markers; genetic screening; genetic susceptibility; genetically modified animals; hepatocellular carcinoma; human genetic material tag; laboratory mouse; molecular oncology; natural gene amplification; neoplasm /cancer genetics; neoplastic process; nucleic acid structure; oncoproteins; prostate neoplasms; renal cell carcinoma; tumor suppressor genes

Our research program combines molecular cytogenetics and molecular biology approaches to study the genetic alterations in solid tumors, hematological malignancies and in transformed cells. The major objective of this project is to identify recurrent chromosomal alterations and to isolate new genes relevant both to neoplastic development, and as potential targets for therapy. In the past year,progress has been made in molecular genetics and cytogenetics of several solid tumors and hematological malignancies, as well as of tumors developed in transgenic mice. This resulted in the identification of new recurrent genomic alterations, and isolation of new genes, relevant to both initiation and progression of neoplasia and as possible useful markers in prognosis and diagnosis of the disease. Novel genomic regions of recurrent DNA amplification and deletions, as well as chromosome translocations in cancer cells were identified by fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH) and spectral karyotyping (SKY). Deletion of the short arm of chromosome 8 is one of the most common alterations in several human cancers including hepatocellular carcinoma (HCC). From this critical region of deletion and loss of heterozygosity, long suspected to harbor tumor suppressor genes, we identified and isolated the DLC-1 gene, a regulator of the Rho family small GTPases that control actin cytoskeleton organization, membrane trafficking, gene expression, cell proliferation, malignant transformation, metastasis. In the past year, compelling evidence was provided that DLC-1 acts as a tumor suppressor gene. Restoration of DLC-1 expression in cell lines derived from metastatic breast adenocarcinomas lacking endogenous gene expression caused significant growth inhibition and prevented the development of tumors in athymic nude mice. In two isogenic breast cancer cell lines with diametrically opposite metastatic capabilities, DLC-1 was found to be down-regulated in the metastatic relative to the non-metastatic cells. Similarly, in a different model, DLC-1 was down-regulated in breast cancer cell populations highly metastatic to bone and not in populations weakly metastatic to adrenal gland.The ability of DLC-1 to prevent the growth of cells derived from metastatic tumors shows that this gene has tumor suppressive activity in breast cancer and may be involved in the process of metastasis. Also, the findings suggest a potential clinical application in the treatment of aggressive metastatic breast cancer or prevention of metastasis. Downregulation or inactivation of the DLC-1 gene during the tumor development occurs either due to the copy-number deletion at the genomic level, or to aberrant promoter methylation at the transcriptional level, and in certain cancers may be associated with cell dissemination and metastasis, the major cause of cancer death. A different degree of promoter hypermethylation was detected in all cell lines derived from of liver, breast, colon and prostate tumors with aberrant DLC-1 expression. The hypermethylation status was reversed by treatment with a demethylating agent. Altered DNA methylation is one of the most promising markers for early detection, prediction of cancer risk and prognosis of disease and the profile of DLC-1 methylation may serve as a useful marker. Point mutation is another common mechanism responsible for gene inactivation. Exonic missense mutations and intronic insertions/deletions of DLC-1 were found only in subset of HCC and other solid tumors. Meanhwile, several polymorphisms have been identified that could be used for determining whether DLC-1 is linked to cancer susceptibility loci. The mouse DLC-1 gene was also isolated, and the exon/intron organization was characterized. An intragenic polymorphic microsatellite marker was identified that was useful for linkage mapping and LOH analysis. To provide an animal model system for investigating the biological functions of DLC-1 in vivo, we successfully used homologous recombination in embryonic stem cells to generate mice with a disrupted DLC-1 gene. The phenotype of the mice was characterized and the data will be reported soon. Identification of two regions of high-level DNA amplification in HCC led us to examine alterations of two known genes, EMS1 and SMAD5. Over-representation of 11q13 chromosomal region was detected by CGH and a fragment of the EMS1 oncogene was isolated and subsequently found to be amplified in primary HCC and over expressed 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 the 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. The minimal region of DNA copy-number gains at 5q31 overlaps with the location of the common fragile site (FS), FRA5C, and the locus of the SMAD5 gene. Deletions and unbalanced translocations with breakpoints near the SMAD5 locus, recurrent formation of isochromosome 5q leading to selective loss of 5p and gain of 5q, and intrachromosomal amplification of SMAD5 gene at the fragile site FRA5C were detected in HCC, indicating that this locus is a frequent target in liver cancer. SMAD high-level amplification at FRA5C is one of the few examples of gene amplification at a common FS through breakage-fusion-bridge cycles. These results show that SMAD5 undergoes copy number gain, high level amplification and increased expression rather than loss of expression, suggesting SMAD5 does not function as a tumor suppressor gene in HCC. The consequences of SMAD5 overexpression on function of other genes, such bone morphogenetic protein genes might be important in hepatocarcinogenesis. A new constitutive translocation involving a FS was also characterized. Members of a family in which multifocal clear renal cell carcinoma (RCC) segregates with a balanced constitutional chromosome t (2; 3) translocation, YAC and BAC contigs encompassing the 2q and 3q breakpoints were constructed, and BACs crossing the breakpoints were partially sequenced. By FISH, the 3q breakpoint was placed at 3q13, possibly near the border with 3q21 and the 2q breakpoint was localized closely telomeric to fragile site FRA2G on chromosome 2.Based on the genomic map of the 2q breakpoint an EST was identified near the translocation, and the full length c DNA of novel gene, DIRC1 which is disrupted by the balanced translocation t (2; 3) was isolated. Low expression of this gene was detected in several tissues and a GFP- DIRC1 fusion protein was expressed in vitro. Lack of mutations and rare polymorphism in tumors and tumor cell lines obscure the role of DIRC1 gene in RCC. The genetic changes in tumors spontaneously developing in certain transgenic mice closely evoke those that occur during the progression of human cancers. The cytogenetic analyses of spontaneous HCC and acute promyelocytic leukemia (APL) in transgenic mice resulted in identification of novel genomic alterations and cloning of new genes. Our SKY analysis of HCC in MYC transgenic mice identified recurrent gains of chromosome 19 and double minute chromosomes (DM) derived from chromosome 19. DMs are cytological manifestations of high level DNA amplification. By screening a cDNA library of mouse chromosome 19, a new putative mouse ortholog of the human Rho GTPase activating protein 8, ARHGAP8 was cloned. The open reading frame encodes a peptide of 387 amino acids with high homology to human ARHGAP8 in its N-terminal

我们的研究项目结合了分子细胞遗传学和分子生物学方法来研究实体瘤、血液恶性肿瘤和转化细胞中的遗传改变。该项目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的新基因，并作为潜在的治疗靶点。在过去的一年里，几种实体瘤和血液恶性肿瘤以及转基因小鼠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，以及新基因的分离，这些基因与肿瘤的发生和进展相关，并且可能是疾病预后和诊断中有用的标记。 通过荧光原位杂交 (FISH)、比较基因组杂交 (CGH) 和光谱核型分析 (SKY) 鉴定了癌细胞中反复出现的 DNA 扩增和缺失以及染色体易位的新基因组区域。 8 号染色体短臂缺失是包括肝细胞癌 (HCC) 在内的多种人类癌症中最常见的改变之一。从这个长期被怀疑含有肿瘤抑制基因的缺失和杂合性丧失的关键区域，我们鉴定并分离了DLC-1基因，它是Rho家族小GTP酶的调节因子，控制肌动蛋白细胞骨架组织、膜运输、基因表达、细胞增殖、恶性转化、转移。去年，有令人信服的证据表明 DLC-1 具有抑癌基因的作用。在缺乏内源基因表达的转移性乳腺腺癌来源的细胞系中恢复 DLC-1 表达会导致显着的生长抑制并阻止无胸腺裸鼠中肿瘤的发展。在两种具有截然相反的转移能力的同基因乳腺癌细胞系中，DLC-1被发现在转移细胞中相对于非转移细胞下调。同样，在不同的模型中，DLC-1在高度骨转移的乳腺癌细胞群体中下调，而在肾上腺微弱转移的群体中则没有下调。DLC-1阻止源自转移性肿瘤的细胞生长的能力表明该基因在乳腺癌中具有肿瘤抑制活性，并且可能参与转移过程。此外，研究结果表明其在治疗侵袭性转移性乳腺癌或预防转移方面具有潜在的临床应用价值。 肿瘤发展过程中DLC-1基因的下调或失活要么是由于基因组水平的拷贝数缺失，要么是转录水平的异常启动子甲基化，并且在某些癌症中可能与细胞播散和转移有关，这是癌症死亡的主要原因。在源自具有异常 DLC-1 表达的肝脏、乳腺、结肠和前列腺肿瘤的所有细胞系中检测到不同程度的启动子高甲基化。通过去甲基化剂治疗可以逆转高甲基化状态。 DNA 甲基化改变是早期检测、预测癌症风险和疾病预后最有希望的标记之一，DLC-1 甲基化谱可能作为有用的标记。点突变是导致基因失活的另一种常见机制。 DLC-1 的外显子错义突变和内含子插入/缺失仅在 HCC 和其他实体瘤的子集中发现。同时，已鉴定出几种多态性，可用于确定 DLC-1 是否与癌症易感性位点相关。 还分离了小鼠 DLC-1 基因，并表征了外显子/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。为了提供研究 DLC-1 体内生物学功能的动物模型系统，我们成功地在胚胎干细胞中使用同源重组来产生 DLC-1 基因被破坏的小鼠。小鼠的表型已被表征，数据将很快报告。 HCC 中两个高水平 DNA 扩增区域的鉴定使我们检查了两个已知基因 EMS1 和 SMAD5 的改变。 CGH 检测到 11q13 染色体区域的过度表达，并分离出 EMS1 癌基因的片段，随后发现其在原发性 HCC 中扩增，并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 基因改变可能与 HCC 的发生有关。 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。 5q31 处 DNA 拷贝数增益的最小区域与共同脆弱位点 (FS)、FRA5C 的位置以及 SMAD5 基因的位点重叠。在 HCC 中检测到 SMAD5 位点附近断点的缺失和不平衡易位、同染色体 5q 的反复形成导致 5p 的选择性丢失和 5q 的增加，以及 SMAD5 基因在脆弱位点 FRA5C 处的染色体内扩增，表明该位点是肝癌的常见靶点。 FRA5C 上的 SMAD 高水平扩增是通过断裂-融合-桥循环在共同 FS 上进行基因扩增的少数例子之一。这些结果表明，SMAD5 经历了拷贝数增加、高水平扩增和表达增加而不是表达缺失，表明 SMAD5 在 HCC 中不起肿瘤抑制基因的作用。 SMAD5 过度表达对其他基因（例如骨形态发生蛋白基因）功能的影响可能在肝癌发生中很重要。 还描述了涉及 FS 的新的本构易位。多灶性透明肾细胞癌 (RCC) 家族成员因平衡染色体 t (2; 3) 易位而分离，构建了包含 2q 和 3q 断点的 YAC 和 BAC 重叠群，并对跨越断点的 BAC 进行了部分测序。通过FISH，3q断点位于3q13，可能靠近3q21的边界，2q断点位于端粒附近，靠近2号染色体上的脆弱位点FRA2G。根据2q断点的基因组图谱，在易位附近鉴定了EST，并分离了被平衡易位t（2；3）破坏的新基因DIRC1的全长c DNA。在多个组织中检测到该基因的低表达，并在体外表达了 GFP-DIRC1 融合蛋白。肿瘤和肿瘤细胞系中缺乏突变和罕见的多态性掩盖了 DIRC1 基因在 RCC 中的作用。 某些转基因小鼠中自发发生的肿瘤的遗传变化与人类癌症进展过程中发生的变化非常相似。对转基因小鼠中自发性肝癌和急性早幼粒细胞白血病（APL）的细胞遗传学分析导致​​了新基因组改变的鉴定和新基因的克隆。我们对 MYC 转基因小鼠的 HCC 进行 SKY 分析，发现 19 号染色体和源自 19 号染色体的双小染色体 (DM) 反复增加。DM 是高水平 DNA 扩增的细胞学表现。通过筛选小鼠 19 号染色体的 cDNA 文库，克隆了人类 Rho GTPase 激活蛋白 8 的新推定小鼠直系同源物 ARHGAP8。开放阅读框编码387个氨基酸的肽，其N端与人ARHGAP8高度同源