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）在人类癌症的发生和发展中发挥着核心作用，为预测、检测、早期诊断和治疗/治愈人类恶性肿瘤提供了手段。致癌基因的产物是控制胚胎发育、细胞增殖和/或细胞凋亡的信号转导电路的组成部分。识别这些信号通路可以识别预示癌症发展步骤开始并预测临床结果或治疗反应的分子标记。致癌基因通常是在遗传性家族癌症综合征中或通过肿瘤组织或细胞系的基因图谱发现的。 肿瘤从初期恶性细胞发展到转移是由涉及额外突变癌症基因的克隆细胞群的达尔文扩张驱动的。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，肿瘤组织内存在极少量的肿瘤干细胞。因此，最重要的是需要发现肺癌干细胞，这些细胞构成了一小群具有自我更新能力的细胞，这对肿瘤的维持和扩散至关重要。从根本上说，这些不朽的肿瘤细胞最终决定了对治疗的反应和肿瘤复发。来自肺胚胎学和合并肺肿瘤形态学的证据表明，大多数（如果不是全部）上皮性肺肿瘤具有共同的气道上皮干/祖细胞起源。 我们的研究计划始于 1987 年，最终于 1993 年识别出位于 3p25 的 VHL TSG。 1993-1999年我们研究了VHL基因的序列结构并鉴定了VHL靶基因。与此同时，我们加强了对位于 3p21.3 的 TSG 的定位和分子克隆的研究，这些 TSG 涉及主要形式的肺癌和乳腺癌、卵巢癌、子宫颈癌、睾丸癌、头颈癌、前列腺癌和结肠癌的起源和/或发展。 2000-2003年我们研究了：（1）VHL位点本身的甲基化密码和VHL靶基因（碳酸酐酶、CA9和CA12以及转录调节因子STRA13）的功能； (2)通过实时PCR在肿瘤组织和细胞系中继续进行3p21.3缺失定位； (3) 完成了 3p21.3 两个区域（着丝粒、LUCA 和端粒、AP20）候选致癌基因的分离和初步表征。这些 3p21.3 区域应被视为含有 TSG 簇的连续癌症基因区域。 然后，我们将研究重点放在一些强候选基因的功能分析（寻找相互作用的蛋白质、分析小鼠的无效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密码（CpG 和 H3/H4 组蛋白密码）来了解它们是否含有与个体差异相关的变异，这些变异可能决定散发性癌症的风险。我们假设，在正常衰老过程中也表现出 TSG 基因座表观遗传密码的个体差异，这可能是 VHL 基因引起的常见癌症的遗传易感性的基础。我们对人和小鼠 VHL 位点（分别为 17 和 6.7 kb）进行了详细且连续的“亚硫酸氢盐测序”图谱，以揭示除 H3/H4 组蛋白甲基化图谱之外的 CpG 代码。在表达 VHL 的细胞中，只有启动子 CpG 岛受到保护且不受甲基化，而其余基因座则严重甲基化。这种甲基化模式在转基因小鼠的发育过程中从头重建并维持在人类基因组VHL转基因上，但在培养的转染畸胎瘤ES细胞中则不然。然而，体细胞在体细胞杂交体融合后以及人-小鼠单染色体 3 转移后能够保留预先存在的甲基化模式。我们现在将重点关注 VHL 基因座中保护性顺式元件的鉴定以及 VHL 表观遗传密码变异的研究。 我们发现 CA 9 /CA12 基因在许多肿瘤类型中被特异性诱导和过度表达，应被视为癌症生长的分子标记和新的治疗靶点。这些酶可以控制受损的肿瘤微环境，并应被视为开发新治疗方式（基于 cDNA 的疫苗和新的 CA 抑制剂）的分子靶点。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 转录因子受到 VHL 基因的负向控制，并由缺氧（癌症组织中普遍存在的情况）诱导。我们通过 Northern 分析和免疫染色确定该基因在许多人类肿瘤中表达水平非常高，但在周围和匹配的正常组织中却没有。我们发现 STRA13 结合并调节强大的转录调节因子 STAT3 的活性，STAT3 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，因此有必要通过实验研究 Stra13 在 mESC 中的表达。 3p21.3 TSG 我们使用酵母双杂交系统、肿瘤细胞中转基因的受控表达以及生物信息学来发现常驻假定 TSG 的功能。 我们发现 RASSF1A 基因是一个涉及多种肿瘤的多 TSG，包括肺癌、乳腺癌、前列腺癌、肾癌、头颈癌、宫颈癌等。我们假设 RASSF1 基因及其旁系同源物在大约 70% 的人类癌症中失活。 HYAL2 蛋白被鉴定为绵羊肺癌逆转录病毒 JSRV 的 GPI 锚定受体，并且证明了 HYAL2 蛋白失活的隔离机制。 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 HYAL2 蛋白。 HYAL2 的缺失（由假定的病毒或突变失活介导）会导致 RON 受体酪氨酸激酶及其下游信号通路（Akt 和 MAPK）的配体独立激活。 我们也一直在研究 RON 在 SCLC 中的参与。我们发现，在 SCLC 中，RON 的启动子因过度甲基化而沉默，导致假定的内部启动子同时激活。源自该内部启动子的新转录物主要编码受体的细胞质部分，该部分被组成型激活并且可能充当原癌基因。因此，RON 正在成为肺癌的潜在致癌因素和治疗干预的目标。 我们发现 PL6 蛋白结合并调节位于内质网中的两个相似的孕酮受体 (MAPR) 的功能，我们使用免疫荧光染色将 PL6 定位于高尔基体，并将其重命名为 Prego（孕酮受体相关高尔基体蛋白）。 我们将 HYA22 基因鉴定为一种蛋白磷酸酶，可调节 RB TSG 的磷酸化状态，从而负向控制细胞周期，并将其重命名为 RBSP。 我们将 NPRL2/G21 鉴定为潜在的新型错配修复基因和顺铂的靶标。 当前/未来的计划重点是： (一)VHL基因和VHL靶基因的功能分析，具体为：(1)鉴定保护VHL CpG启动子的顺式元件和反式元件。 (2)进一步研究碳的表达和作用