Cloning and functional analysis of tumor suppressor gene

抑癌基因的克隆及功能分析

• 批准号: 6762015
• 负责人: MICHAEL LERMAN
• 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/6762015
• 关键词: Von Hippel Lindau syndrome; bioinformatics; carcinogenesis; cytogenetics; differential display technique; genetic mapping; genetically modified animals; human genetic material tag; human tissue; laboratory mouse; lung neoplasms; molecular cloning; molecular oncology; neoplasm /cancer genetics; nucleic acid sequence; transfection; tumor suppressor genes

VHL TSG (3p25) To analyze the function(s) of the VHL gene and its carcinogenic pathway(s) we obtained the entire genomic sequence of the gene including the promoter, introns, and flanks, and constructed a set of VHL minigenes (wild type and mutant) and a complete intronless VHL gene driven by the VHL promoter. We then set out to discover target genes controlled by pVHL. The differential display technology was employed to discover these genes using the UMRC6 and 786-0 cells stably transfected with wt and mutant VHL minigenes. To date (September 2002) six down regulated genes were identified, namely, NOTCH2 and STRA13, that specify cell fate determination and may have oncogenic potential, two transmembrane type carbonic anhydrases, CA9 and CA12, and two new unknown genes. The CA9 and CA12 genes are overexpressed in many tumor types due to hypoxia causing the loss of functional pVHL. The CAIX/XII enzymes could sense the intracellular pH and control the acidity (extracellular pH) of the miliew surrounding the cancer cells and thus create a microenvironment conducive to tumor growth and spread. They also play fundamental roles in normal physiology such as production of eye humor, brain and kidney functions etc. The STRA13 a bHLH transcription factor is negatively controlled by the VHL gene and hypoxia (a condition prevalent in cancer tissues) in many human tumors. We have now shown that STRA13 is a powerful regulator of the transcriptional factor STAT3 that is activated in many cancers. Because the STAT3 protein is fundamentally involved in regulating cancer cell growth and survival it would be beneficial to disrupt its function in cancer cells to block tumor progression. Analysis of the methylation of the VHL promoter in renal carcinoma cells carrying a methylated VHL endogene by monochromosome gene transfer, cell fusion, and VHL gene transfections showed that the meth+ phenotype is dominant in the UOK 21 cells probably resulting from changes in cis-acting elements of the VHL locus.We then created a mouse transgenic model expressing the human genomic VHL locus and demonstrated that human VHL methylation pattern was reproduced during mouse development and was very similar to that of the mouse VHL gene. This model would allow studying the local methylation protection mechanisms in the VHL locus and the effect of chromosomal context on de novo methylation of various elements of the VHL locus, such as repetitive sequences and the VHL CpG promoter. The future work will be focused (i) on the role of carbonic anhydrases (CAs) in the regulation of tumor pH and its impact on cancer growth, (ii) discovery of new specific inhibitors of these enzymes to treat cancer, (iii) on development of CAIX/XII cDNA based vaccines to treat cancer, (iv) on the design of inhibitors that could disrupt the STRA13-STAT3 signals to directly target STRA13 and/or STAT3 to improve treatment of cancer; and (v) on the nature of the cis-acting elements in the VHL locus involved in de novo aberrant methylation. The 3p21.3 TSG We used overlapping and nested homozygous deletions, contig building, genomic sequencing, physical, and transcript mapping to further define a ~630-kb lung cancer homozygous deletion region harboring one or more tumor suppressor gene(s) (TSGs) on chromosome 3p21.3. This location was identified through somatic genetic mapping in cancers, cancer cell lines and pre-malignant lesions of the lung and breast including the discovery of several homozygous deletions. The combination of molecular manual methods and computational predictions permitted us to detect, isolate, characterize and annotate a set of 25 genes which likely constitute the complete set of protein-coding genes residing in this ~630-kb sequence. A subset of 19 of these genes were found within the deleted overlap region of ~370-kb. This region was further subdivided by a nesting 200-kb breast cancer homozygous deletion into two gene sets: 8 genes lying in the proximal ~120-kb segment and 11 genes lying in the distal ~250-kb segment. These 19 genes were analyzed extensively by computational methods and were tested by manual methods for loss of expression and mutations in lung cancers to identify candidate TSGs from within this group. Several genes showed loss-of-expression or reduced mRNA levels in non-small cell lung cancer (NSCLC) (CACNA2D2/ (a2d-2), SEMA3B (formerly SEMA(V),) BLU, RASSF1/A (formerly 123F2), and HYAL1) or small cell lung cancer (SCLC) (SEMA3B, BLU, RASSF1/A (formerly 123F2), and HYAL1) cell lines. We found six of the genes to have 2 or more amino acid sequence altering mutations including: BLU, NPRL2/Gene21, FUS1, HYAL1, FUS2, and SEMA3B. However, none of the 19 genes tested for mutation showed a frequent (>10%) mutation rate in lung cancer samples. This led us to exclude several of the genes in the region as classical tumor suppressors for sporadic lung cancer. On the other hand, we now also have to consider the newly recognized class of haploinsufficient TSGs, where the absence of only one wild-type allele facilitates tumorigenesis. Functional testing of the critical genes by gene transfer and gene disruption strategies is under way and will permit the identification of the putative lung cancer TSG(s), LUCA. To date (September, 2002) we identified the RASSF1/A gene as a multiple TSG involved in many tumors, including lung, breast, ,prostate, kidney, head & neck, uterine cervix and others. The HYAL2 gene was identified as a GPI-anchored receptor for the sheep lung cancer retrovirus, JSRV and a sequestration mechanism inactivating HYAL2 product was demonstrated. The Env gene of JSRV was shown to transform human bronchial epithelial cells in vitro and sequester the HYAL2 product which allow to study the signal transduction pathways leading to carcinogenesis in this system. We have now identified the oncogenic pathway negatively controlled by the HYAL2 product. The RON receptor tyrosine kinase is activated in human lung cancer. We have now shown that in SCLC the promoter of RON is hypermethylated resulting in silencing of the gene and simultaneous activation of an internal promoter; the shorter transcript originating from this internal promoter encodes only the cytoplasmic portion of the receptor that functions as an oncogene. A new FAS2 gene cDNA polymorphism was shown to be associated with NPC with predictive value in Asian populations. Current work is focused on (I) the detection and isolation of the putative human retrovirus that may cause a rapidly rising form of human lung cancer namely bronchioloalveolar adenocarcinoma (BAC), (ii) analysis of the oncogenic pathway activated by the truncated form of RON. The 3p12 TSG Cytogenetic deletions and LOH at human 3p12 are a consistent feature of lung cancer specimens and suggest the presence of a tumor suppressor gene(s) (TSG) at this location. Only one gene (DUTT1, Deleted in U Twenty Twenty) was so far cloned from the overlapping region deleted in several lung and breast cancer cell lines (U2020, NCI H2198, HCC38). DUTT1 is the human ortholog of the fly gene ROBO that has homology to NCAM proteins. Extensive analyses of DUTT1 in lung cancer did not reveal any mutations, suggesting another gene(s) at this location could be associated with lung cancer initiation and/or development. We discovered in the overlapping critical region a new small (~230kb), nested homozygous deletion in the SCLC cell line GLC20. This deletion has been PCR-characterized using several polymorphic markers. P1 library screening produced three overlapping clones that cover the whole region and flanks. These clones were used to define by fiber-FISH the location and size of the deletion. Recently several BAC clones covering this region were sequenced by the MIT genome sequencing center providing a genomic tool to discover in silico the resident genes. Several genes represented by EST clusters were detected in the deletion and are being isolated. Subsequent mutation and functional studies will identify the potential 3p12 lung/breast cancer TSG.

VHL TSG（3P25） 为了分析VHL基因的功能及其致癌途径（S），我们获得了基因的整个基因组序列，包括启动子，内含子和侧面，并构建了一组VHL Minigenes（野生型和突变体）和一组无内在的无内在VHL基因驱动的VHL驱动器。然后，我们着手发现由PVHL控制的靶基因。使用差异显示技术使用WT和突变体VHL微型烯稳定转染的UMRC6和786-0细胞发现这些基因。迄今为止（2002年9月）鉴定了六个下调的基因，即Notch2和Stra13，这些基因指定了细胞命运的测定，并且可能具有致癌潜力，两个跨膜型碳纤维性赤霉酶Ca9和ca12，以及两个新的未知基因。由于缺氧导致功能性PVHL丧失，CA9和CA12基因在许多肿瘤类型中过表达。 CAIX/XII酶可以感知细胞内pH，并控制围绕癌细胞的Miliew的酸度（细胞外pH），从而产生有助于肿瘤生长和扩散的微环境。它们还在正常生理学中起着基本作用，例如眼睛幽默，脑和肾功能等。STRA13A BHLH转录因子在许多人类肿瘤中受VHL基因和缺氧（癌症组织中普遍存在的状况）负面控制。我们现在已经表明，Stra13是转录因子STAT3的强大调节剂，在许多癌症中被激活。由于STAT3蛋白从根本上参与调节癌细胞生长和存活，因此破坏其在癌细胞中的功能以阻断肿瘤进展是有益的。通过单色基因转移，细胞融合和VHL基因转染的单色体基因转移，细胞融合和VHL基因转染的分析在肾脏癌细胞中VHL启动子的甲基化分析分析METH+表型在UOK 21细胞中占主导地位。证明人VHL甲基化模式在小鼠发育过程中得到了复制，并且与小鼠VHL基因非常相似。该模型将允许研究VHL基因座中的局部甲基化保护机制，以及染色体环境对VHL基因座各种元素的从头甲基化的影响，例如重复序列和VHL CPG启动子。未来的工作将集中在（i）上（i）在碳pH的调节中的作用及其对癌症生长的影响，（ii）发现这些酶对癌症的新特定抑制剂（III）（iii）（iii）在开发CAIX/XII cDNA疫苗中对癌症的疫苗的开发，以治疗癌症，（IV），该疫苗对癌症的设计，（IIV）均可构成抑制作用的侵害，（IV）/可能会扰动抑制作用13 STAT3改善癌症治疗； （v）关于从头异常甲基化涉及的VHL基因座中顺式作用元件的性质。 3P21.3 TSG 我们使用重叠和嵌套的纯合缺失，重叠群建筑，基因组测序，物理和转录本映射，进一步定义了一个〜630-kb的肺癌纯合缺失区域，该区域内具有一个或多个肿瘤抑制基因（S）（TSG）3p21.3染色体上。通过癌细胞，癌细胞系和肺部和乳房的恶性病变中的体细胞遗传学图来鉴定该位置，包括发现几种纯合缺失。分子手动方法和计算预测的组合使我们能够检测，分离，表征和注释一组25个基因，这些基因可能构成了构成该〜630-KB序列中的一组完整的蛋白质编码基因。在〜370-kb的删除重叠区域内发现了其中19个基因的子集。该区域通过嵌套200-kb乳腺癌纯合缺失成两个基因组进一步细分：8个基因位于近端〜120-KB段中，而11个基因位于远端〜250-kb段中。通过计算方法对这19个基因进行了广泛的分析，并通过手动方法对肺癌中表达和突变的丧失进行测试，以鉴定该组内的候选TSG。 Several genes showed loss-of-expression or reduced mRNA levels in non-small cell lung cancer (NSCLC) (CACNA2D2/ (a2d-2), SEMA3B (formerly SEMA(V),) BLU, RASSF1/A (formerly 123F2), and HYAL1) or small cell lung cancer (SCLC) (SEMA3B, BLU, RASSF1/A (formerly 123F2), and透明1）细胞系。我们发现六个基因具有2个或更多的氨基酸序列改变突变，包括：BLU，NPRL2/Gene21，Fus1，hyal1，Fus2和Sema3b。然而，对突变测试的19个基因均未显示出肺癌样品中的频繁（> 10％）突变。这导致我们将该地区的几个基因排除在零星肺癌的经典肿瘤抑制因子中。另一方面，我们现在还必须考虑新认识的一类无弹性TSG，其中仅缺少一个野生型等位基因会促进肿瘤发生。通过基因转移和基因破坏策略对关键基因的功能测试正在进行中，并将允许鉴定推定的肺癌TSG LUCA。迄今为止（2002年9月），我们将RASSF1/A基因确定为参与许多肿瘤的多个TSG，包括肺，乳房，前列腺，前列腺，肾脏，头颈，子宫子宫颈等。透明基因被鉴定为绵羊肺癌逆转录病毒，JSRV的GPI锚定受体，并证明了隔离机制灭活透明产物。 JSRV的ENV基因被证明可以在体外转化人支气管上皮细胞，并隔离透明产物，该产物可以研究导致该系统癌变的信号转导途径。现在，我们已经确定了由透明产品负面控制的致癌途径。 RON受体酪氨酸激酶在人肺癌中被激活。我们现在已经表明，在SCLC中，RON的启动子是高甲基化的，导致基因沉默和同时激活内部启动子。源自该内部启动子的较短的转录本仅编码作为癌基因的受体的细胞质部分。新的FAS2基因cDNA多态性显示与亚洲人群中具有预测价值的NPC相关。当前的工作集中在（i）假定的人逆转录病毒的检测和分离上，该病毒可能引起人类肺癌的快速上升形式，即支支支支支支通电动型肺癌（BAC），（ii）分析由RON的截断形式激活的致癌途径。 3P12 TSG 人类3P12处的细胞遗传学缺失和LOH是肺癌标本的一致特征，建议在此位置存在肿瘤抑制基因（S）（TSG）。到目前为止，只有一个基因（DUTT1，在U二十二十）中被克隆了几个肺癌和乳腺癌细胞系（U2020，NCI H2198，HCC38）的重叠区域。 dutt1是与NCAM蛋白具有同源性的蝇基因机器人的人类直系同源物。 DUTT1在肺癌中的广泛分析没有发现任何突变，这表明该位置的另一个基因可能与肺癌的启动和/或发育有关。我们在重叠的临界区域中发现了一个新的小（〜230kb），嵌套的纯合缺失在SCLC细胞系GLC20中。该删除已使用多个多态标记物进行了PCR特征。 P1库筛选产生了三个覆盖整个区域和侧面的重叠克隆。这些克隆被用来通过纤维化删除的位置和大小来定义。最近，通过MIT基因组测序中心对几个覆盖该区域的BAC克隆进行了测序，该中心提供了一种基因组工具，可以在居民基因中发现。在缺失中检测到以EST簇为代表的几个基因，并正在分离。随后的突变和功能研究将确定潜在的3P12肺/乳腺癌TSG。