Cloning and functional analysis of tumor suppressor gene

背景/概述：癌基因和肿瘤抑制基因（TSG）在人类癌症的发生和发展中起着核心作用，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。这些致癌基因的产物是控制胚胎发育、细胞增殖和/或凋亡的信号转导回路的组成部分。了解这些信号通路可以识别预示癌症发展开始的分子标记，并预测对治疗的反应和临床结果。肿瘤从早期恶性细胞发展到转移是由涉及额外突变癌基因的克隆细胞群的达尔文式扩张所驱动的。每一个肿瘤组织都含有一小部分具有自我更新能力的肿瘤干细胞，这些干细胞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的复发。我们的研究计划始于1987年，最终于1993年确定了位于3p25的VHL TSG。1993-2003年，我们研究了VHL基因的序列结构，并对VHL靶基因进行了鉴定和分析。与此同时，我们加强了位于3p21.3的TSG的定位和分子克隆研究，这些TSG参与了肺癌和其他常见癌症的主要形式的起源和/或发展。2003-2004年，我们研究了VHL基因座本身的甲基化编码和VHL靶基因（碳酸酐酶CA9和CA12、转录调节因子STRA13和其他VHL中继基因STAT1和TIEG1）的功能；(2)利用real - time PCR在肿瘤组织和细胞系中持续定位3p21.3缺失位点；(3)完成了3p21.3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区域应被认为是含有TSG簇的连续癌症基因区域。然后，我们将研究重点放在了一些强候选基因的功能分析上（发现相互作用蛋白，分析小鼠中的零突变体，以及生物信息学注释）。VHL TSG （3p25）

Background/outline: Oncogenes and tumor suppressor genes (TSG) play a central role in causation and development of human cancer and provide the means to predict, detect, early diagnose and treat/cure human malignancies. The products of these cancer-causing genes are components of signal transduction circuits that control embryonic development, cellular proliferation and/or apoptotic death. Knowledge of these signaling pathways allows to identify molecular markers that herald the onset of cancer development and to predict response to treatment and clinical outcome. The development of tumors from incipient malignant cells to metastases is driven by Darwinian expansion of clonal cell populations involving additional mutated cancer genes. Every tumor tissue contains a minute population of tumor stem 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. Our research program began in 1987 and culminated in the identification in 1993 of the VHL TSG located at 3p25. In 1993-2003 we investigated the sequence structure of the VHL gene and identified and analyzed VHL target genes. In parallel we intensified research on mapping and molecular cloning of TSG located on 3p21.3 involved in the origin and /or development of major forms of lung cancer and other common carcinomas. In 2003-2004 we investigated: (1) the methylation code of the VHL locus itself and the function of VHL target genes (carbonic anhydrases, CA9 and CA12, the transcription regulator STRA13 and other VHL relay genes, STAT1 and TIEG1); (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/familial differences that may determine risk of sporadic VHL type cancers. 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. Somatic cells were also able to preserve the pre-existing methylation pattern after fusion in somatic cell hybrids and after human-to-mouse single chromosome 3 transfer. We found that CTCF a ubiquitous chromatin DNA binding protein has binding sites in human and mouse VHL CpG promoter islands and may play an important role in protecting against aberrant silencing of the gene in kidney cancer. We will now focus on the identification of other protecting cis elements and binding proteins in the VHL locus and on studies of the VHL epigenetic code variations.We discovered that CA 9 /CA12 genes are specifically induced and over-expressed in many tumor types. These enzymes may control the acidic tumor microenvironment and should be considered molecular targets for development of new treatment modalities. Using purified CAIX/XII enzymes we tested novel and classical (clinically used for glaucoma treatment) aromatic sulfonamide inhibitors that may have potent anti-tumor activity. We identified among them several compounds that showed nanomolar inhibition specific for each enzyme. We aim now to initiate clinical trials with the FDA approved compounds. Our computer modeling studies of the enzyme-inhibitor interactions showed the affinity for each carbonic anhydrase correlates with how well the inhibitor fills the spatial volume of the inhibitor-binding pocket. This specificity is promising for the design of even more potent agents with reduced toxicity.The STRA13 transcription factor is negatively controlled by the VHL gene and induced by hypoxia, a condition prevalent in cancer tissues. We discovered that STRA13 binds to and modulates the activity of the powerful regulator of transcription STAT3 that is fundamentally involved in regulating cancer cell growth and survival and embryonic and probably cancer stem cell self-renewal. Importantly STRA13 is also involved in regulating acquired immunity. Our characterization of the STRA13 protein network is broadening the spectrum of potential therapeutic targets and expands our knowledge of the VHL carcinogenic pathway(s). Hypoxia and transforming growth factor -beta1 (TGF-beta 1) play important roles in regulation of the immune system, cell adhesion, migration, and tissue remodeling. Using TGF-beta1/BMP cDNA arrays and RNA isolated from CCRCC cell lines with different VHL status we outlined a set of targets common for pVHL and TGF-beta1 pathways. We associated one of these targets, the hypoxia-inducible Kruppel-like zinc finger transcriptional factor TIEG1, with transcriptional regulation of pVHL/hypoxia targets CA12, TGFBi, PAI-1, and others. We also showed that transcriptional modulation of another novel pVHL target, STAT1, is mediated via STRA13, which was previously implicated in both pVHL/HIF and TGF-beta1 pathways. These results provide novel insights on transcriptional cooperation between the pVHL/HIF, TGF-beta1, and JAK/STAT pathways and emphasize a new mechanism that employs relay genes to amplify and diversify the original primary hypoxia signal. In addition, these findings will provide a rapid and accurate identification of drug targets and tumor markers with true potential. The 3p21.3 TSG Functional studies on the 3p21.3 tumor suppressor genes: We used the yeast two-hybrid system, controlled expression of trangenes in tumor cells, targeted inactivation in mice (mouse knockout models), 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 and 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 hypermethylation 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 drive cell proliferation. Thus RON is emerging as a potential oncogenic factor in lung cancer and a target for therapeutic intervention.