Cloning and functional analysis of tumor suppressor gene

抑癌基因的克隆及功能分析

• 批准号: 6950492
• 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/6950492
• 关键词: Von Hippel Lindau syndrome; bioinformatics; biotechnology; carcinogenesis; cytogenetics; differential display technique; functional /structural genomics; genetic mapping; genetically modified animals; human genetic material tag; human tissue; immunofluorescence technique; laboratory mouse; lung neoplasms; molecular cloning; molecular oncology; neoplasm /cancer genetics; northern blottings; nucleic acid sequence; oncogenes; transfection; tumor suppressor genes; yeast two hybrid system

INTRODUCTION The centrality of oncogenes and tumor suppressor genes (TSG) in causation and development of human cancer provides the means to predict, detect, early diagnose and treat/cure human malignancies. The products of cancer causing genes are components of signal transduction circuits that control embryonic development, cellular proliferation and/or apoptotic death. Identification of these signaling pathways allows identifying molecular markers that herald the onset of steps in cancer development and predict clinical outcome or response to treatment. Cancer-causing genes are usually discovered in the inherited family cancer syndromes or through genetic mapping in tumor tissues or cell lines. The development of tumors from incipient malignant cells to metastases is driven by Darwinian expansion of clonal cell populations involving additional mutated cancer genes. Very recent discoveries in stem cell research are revolutionizing the field of cancer biology with tremendous implications for treatment strategies. It is becoming increasingly clear that a minute population of tumor stem cells exists within the tumor tissue. Therefore of paramount importance is the need to discover the lung cancer stem cells that constitute a small population of cells with self-renewal capacity essential for both tumor maintenance and spread. Fundamentally, these immortal tumor cells finally determine the response to treatment and tumor recurrence. Evidence from lung embryology and the morphology of combined lung tumors point to a common airway epithelial stem/progenitor cell of origin for most if not all epithelial lung tumors. Our research program began in 1987 and culminated in the identification in 1993 of the VHL TSG located at 3p25. In 1993-1999 we investigated the sequence structure of the VHL gene and identified VHL target genes. In parallel we intensified research on mapping and molecular cloning of TSGs located on 3p21.3 involved in the origin and /or development of major forms of lung cancer and carcinomas of the breast, ovary, cervix, testes, head and neck, prostate, and colon. In 2000-2003 we investigated: (1) the methylation code of the VHL locus itself and the function of VHL target genes (carbonic anhydrases, CA9 and CA12, and the transcription regulator STRA13); (2) continued deletion mapping in 3p21.3 by real time PCR in tumor tissues and cell lines; and (3) completed the isolation and initial characterization of candidate cancer-causing genes from both 3p21.3 regions (centromeric, LUCA, and telomeric, AP20). These 3p21.3 regions should be considered contiguous cancer gene regions harboring clusters of TSG. We then focused our research on functional analysis (finding interacting proteins, analyzing null mutants in mice, and bioinformatics annotations) of some of the strong candidate genes. VHL TSG (3p25) Using the VHL locus as a model system, we investigated the mechanisms of aberrant DNA methylation in cancer. We also aimed to use epigenetic codes (CpG and H3/H4 histone codes) to understand whether they harbor variations associated with inter-individual differences that may determine risk of sporadic cancers. We hypothesize that individual differences in epigenetic codes of TSG loci also manifested during normal aging may underlie the genetic predisposition to common cancers caused by the VHL gene. We produced detailed and contiguous "bisulfite-sequencing" profiles of the human and mouse VHL loci (17 and 6.7 kb respectively) to reveal CpG codes in addition to H3/H4 histone methylation profiles. In VHL-expressing cells, only the promoter CpG island is protected and free from methylation while the rest of the locus is heavily methylated. This methylation pattern was re-created de novo and maintained on the human genomic VHL transgene during development of transgenic mice, but not in transfected teratocarcinoma ES cells in culture. However, somatic cells were able to preserve the pre-existing methylation pattern after fusion in somatic cell hybrids and after human-to-mouse single chromosome 3 transfer. We will now focus on the identification of the protecting cis elements in the VHL locus and studies of the VHL epigenetic code variations. We discovered that CA 9 /CA12 genes are specifically induced and over-expressed in many tumor types and should be considered molecular markers of cancer growth and novel targets for treatment. These enzymes may control the compromised tumor microenvironment and should be considered molecular targets for development of new treatment modalities (cDNA based vaccines and new CA inhibitors). Using purified CAIX/XII enzymes we are testing novel CA inhibitors that may have potent anti-tumor activity (they also could be used to treat glaucoma). The STRA13 transcription factor is negatively controlled by the VHL gene and induced by hypoxia, a condition prevalent in cancer tissues. We established by Northern analysis and immunostaining very high expression levels of this gene in many human tumors but not in surrounding and matching normal tissues. We discovered that STRA13 binds to and modulates the activity of the powerful regulator of transcription STAT3 that is also activated in many cancers suggesting that STRA13 may be involved in regulating cancer cell growth and survival. Because the STAT3 transcriptional system is required for embryonal stem cell potency and self-renewal it would be necessary to investigate experimentally the expression of Stra13 in mESC. The 3p21.3 TSG We used the yeast two-hybrid system, controlled expression of trangenes in tumor cells, and bioinformatics to discover the function of the resident putative TSG. We identified the RASSF1A gene as a multiple TSG involved in many tumors, including lung, breast, prostate, kidney, head & neck, uterine cervix and others. We hypothesize that RASSF1 genes and their paralogs are inactivated in approximately 70% of human cancers. The HYAL2 protein was identified as a GPI-anchored receptor for the sheep lung cancer retrovirus, JSRV, and a sequestration mechanism inactivating HYAL2 protein was demonstrated. The env gene of JSRV was shown to transform human bronchial epithelial cells in vitro and sequester the HYAL2 protein. The absence of HYAL2 (mediated either by a putative virus or mutational inactivation) leads to ligand-independent activation of the RON receptor tyrosine kinase and its downstream signaling pathways (Akt and MAPK). We have been also studying the involvement of RON in SCLC. We discovered that in SCLC the promoter of RON is silenced by hypermathylation leading to simultaneous activation of a putative internal promoter. The novel transcript originating from this internal promoter encodes mostly the cytoplasmic portion of the receptor that is constitutively activated and may function as a proto-oncogene. RON therefore is emerging as a potential oncogenic factor in lung cancer and a target for therapeutic intervention. We discovered that the PL6 protein binds to and modulates the function of two similar progesterone receptors (MAPRs) localized in the endoplasmic reticulum, We localized PL6 to the Golgi using immunofluorescence staining and renamed it Prego (Progesterone REceptor-associated GOlgi protein). We identified the HYA22 gene as a protein phosphatase that regulates the phosphorylation status of the RB TSG thereby negatively controlling the cell cycle and renamed it RBSP. We identified the NPRL2/G21 as a potential novel mismatch repair gene and target of cisplatine. Current/future plans are focused on: (I) Functional analysis of the VHL gene and VHL target genes, specifically: (1) to identify the cis elements and trans factors protecting the VHL CpG promoter. (2) further study the expression and role of carbon

介绍 癌基因和肿瘤抑制基因（TSG）在人类癌症的因果关系和发育中的中心性提供了预测，检测，早期诊断和治疗/治疗人类恶性肿瘤的手段。引起基因的癌症产物是控制胚胎发育，细胞增殖和/或凋亡死亡的信号转导电路的组成部分。这些信号传导途径的识别允许识别预示癌症发展步骤的分子标记，并预测临床结果或对治疗的反应。通常在遗传性家庭癌症综合征或通过肿瘤组织或细胞系中的遗传图中发现引起癌症的基因。 肿瘤从初期恶性细胞到转移的发展是由达尔文式的克隆细胞种群扩展驱动的，克隆细胞群涉及其他突变的癌症基因。干细胞研究的最新发现正在彻底改变癌症生物学领域，对治疗策略产生巨大影响。越来越清楚的是，肿瘤组织内存在一小撮肿瘤干细胞。因此，至关重要的是，需要发现构成少量细胞的肺癌干细胞，对肿瘤维持和扩散至关重要。从根本上讲，这些不朽的肿瘤细胞最终决定了对治疗和肿瘤复发的反应。肺胚胎学和联合肺部肿瘤的形态的证据表明，大多数（如果不是全部）上皮肺肿瘤的普通气道上皮茎/祖细胞。 我们的研究计划始于1987年，最终达到了1993年的VHL TSG的标识，位于3p25。在1993 - 1999年，我们研究了VHL基因的序列结构，并鉴定了VHL靶基因。同时，我们对位于3p21.3上的TSG的映射和分子克隆进行了加强研究，参与了主要形式和 /或开发主要形式的肺癌和乳腺癌，卵巢，子宫颈，遗迹，头部，头部和颈部，前列腺和结肠癌。在2000年至2003年，我们研究了：（1）VHL基因座本身的甲基化代码以及VHL靶基因的功能（碳酸酐酶，Ca9和Ca12，以及转录调节剂Stra13）； （2）通过肿瘤组织和细胞系中实时PCR在3P21.3中的持续删除映射； （3）完成了从3p21.3区域（Centromeric，Luca和Telomeric，AP20）的候选癌症基因的分离和初始表征。这些3p21.3区域应视为具有TSG簇的连续癌基因区域。 然后，我们将研究重点放在某些强候选基因的功能分析（发现相互作用的蛋白质，分析小鼠中的空突变体以及生物信息学注释）上。 VHL TSG（3P25） 使用VHL基因座作为模型系统，我们研究了癌症中异常DNA甲基化的机制。我们还旨在使用表观遗传代码（CPG和H3/H4组蛋白代码）来了解它们是否具有与个体间差异相关的变化，这可能决定了零星癌的风险。我们假设在正常衰老期间也体现的TSG基因座表观遗传代码的个体差异可能是由VHL基因引起的常见癌症的遗传易感性。我们产生了人和小鼠VHL基因座（分别为17和6.7 kb）的详细且连续的“ Bisulfite-sequer-sequer-sequering”曲线，以揭示除H3/H4组蛋白甲基化曲线外的CpG代码。在表达VHL的细胞中，只有启动子CpG岛受到保护并免受甲基化的影响，而其余的基因座则被大量甲基化。这种甲基化模式被重新创建，并在转基因小鼠的发育过程中维持在人类基因组VHL转基因上，但在培养中转染的畸形核ES ES细胞中不保留。然而，体细胞能够在体细胞杂交中和人向小鼠单染色体3转移后融合后保留现有的甲基化模式。现在，我们将专注于识别VHL基因座中的CIS元素以及对VHL表观遗传密码变化的研究。 我们发现Ca 9 /Ca12基因在许多肿瘤类型中被特异性诱导和表达过表达，应被视为癌症生长的分子标志物和治疗的新靶标。这些酶可以控制受损的肿瘤微环境，应视为开发新治疗方式的分子靶标（基于cDNA的疫苗和新的CA抑制剂）。使用纯化的CAIX/XII酶，我们正在测试可能具有有效抗肿瘤活性的新型CA抑制剂（它们也可用于治疗青光眼）。 Stra13转录因子由VHL基因负面控制，并由缺氧诱导，这是癌组织中普遍存在的病。我们通过北方分析和免疫染色在许多人类肿瘤中的非常高表达水平建立，但在周围和匹配正常组织中没有建立。我们发现Stra13与强大的转录STAT3调节剂的活性结合并调节，这在许多癌症中也被激活，这表明Stra13可能参与调节癌细胞的生长和存活。由于STAT3转录系统是胚胎干细胞效力和自我更新所必需的，因此有必要在MESC中实验研究Stra13的表达。 3P21.3 TSG 我们使用了酵母两杂交系统，肿瘤细胞中trangenes的表达以及生物信息学的控制来发现居民推定的TSG的功能。 我们将RASSF1A基因确定为许多参与许多肿瘤的TSG，包括肺，乳腺，前列腺，肾脏，头颈，子宫宫颈等。我们假设RASSF1基因及其旁系同源物在大约70％的人类癌症中被灭活。 透明蛋白被鉴定为绵羊肺癌逆转录病毒，JSRV的GPI锚定受体，并证明了一种失活透明蛋白的隔离机制。 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蛋白。透视感（由推定病毒或突变灭活介导的）导致RON受体酪氨酸激酶及其下游信号传导途径（AKT和MAPK）的配体非依赖性激活。 我们还一直在研究罗恩在SCLC中的参与。我们发现，在SCLC中，RON的启动子被过度甲基化沉默，从而导致推定的内部启动子同时激活。起源于该内部启动子的新型转录本主要编码受体激活的受体的细胞质部分，并可能充当原始癌基因。因此，罗恩（Ron）成为肺癌中潜在的致癌因子，也是治疗干预的靶标。 我们发现PL6蛋白与内质网中定位的两个相似的孕酮受体（MAPR）的功能结合并调节，我们使用免疫荧光染色将PL6局部在高尔基体中，并重命名为Prego（孕酮受体相关的高尔基蛋白）。 我们确定羟基基因是一种蛋白质磷酸酶，可调节RB TSG的磷酸化状态，从而负面控制细胞周期并将其更名为RBSP。 我们将NPRL2/G21确定为潜在的新型不匹配修复基因和顺铂的靶标。 当前/未来计划的重点是： （i）对VHL基因和VHL靶基因的功能分析，特别是：（1）确定保护VHL CpG启动子的顺式元素和反式因子。 （2）进一步研究碳的表达和作用