Role of TGFBI gene in cell proliferation and tumor progression

TGFBI基因在细胞增殖和肿瘤进展中的作用

• 批准号: 7522949
• 负责人: Yongliang Zhao
• 金额: $ 32.8万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-18 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7522949
• 关键词: Acetone; Address; Adenocarcinoma Cell; Adhesions; Age; Anchorage-Independent Growth; Animals; Anthracenes; Antibodies; Apoptotic; Asbestos; Binding; Binding Sites; Biological Assay; Biological Models; Blocking Antibodies; Breast; Breeding; Bromodeoxyuridine; CCND1 gene; CREB1 gene; Cell Adhesion; Cell Culture System; Cell Cycle; Cell Line; Cell Proliferation; Cells; Cessation of life; Chemicals; Chinese Hamster; Count; CpG Islands; Culture Media; Cyclic AMP; Cyclic AMP Response Element; Cyclic AMP-Dependent Protein Kinases; Cyclic AMP-Responsive DNA-Binding Protein; Cyclin D1; Data; Databases; Development; Disruption; Dominant-Negative Mutation; Dose; Down-Regulation; Embryo; Enzyme Immunoassay; Epithelial Cells; Extracellular Matrix; Fiber; Fibroblasts; Frequencies; Gene Expression; Gene Silencing; Genotype; Goals; Growth; Heart; Hematopoietic; Heterozygote; Histology; Human; Human Development; Hypermethylation; In Vitro; Incidence; Individual; Integrin Binding; Integrins; Leukocytes; Link; Liver; Loss of Heterozygosity; Luciferases; Lung; Lung Neoplasms; Malignant Neoplasms; Malignant neoplasm of lung; Matrigel Invasion Assay; Measures; Mediating; Mesenchymal Cell Neoplasm; Modeling; Monitor; Mus; Mutate; Normal tissue morphology; Nude Mice; Null Lymphocytes; Numbers; Ovary; Phenotype; Phosphorylation; Phosphotransferases; Play; Predisposition; Principal Investigator; Process; Promoter Regions; Prostate; Protein Overexpression; Proteins; RGD (sequence); Radiation; Rate; Recombinants; Regulation; Relative (related person); Reporter; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Series; Signal Pathway; Signal Transduction; Skin Neoplasms; Small Interfering RNA; Small Intestines; Solid Neoplasm; Specific qualifier value; Spleen; Supplementation; System; TGFBI gene; Testing; Time; Transfection; Transforming Growth Factor-Beta Induced Protein IGH3; Transforming Growth Factors; Transgenic Organisms; Tumor Cell Line; Tumor Suppressor Genes; Tumor Suppressor Proteins; Tumor Tissue; Tumorigenicity; Up-Regulation; Week; Western Blotting; Wild Type Mouse; anthracene; base; c-myc Genes; cancer cell; cell growth; cell transformation; cell type; chromatin immunoprecipitation; day; dimethylbenzanthracene; human tissue; in vitro Model; in vivo; in vivo Model; inhibitor/antagonist; leukemia; lung Carcinoma; mouse model; mutant; neoplastic cell; peripheral blood; programs; promoter; receptor; reconstitution; tumor; tumor growth; tumor progression; tumorigenic; vector

DESCRIPTION (provided by applicant): TGFBI, induced by TGF-b1, is a fascilin-like protein and has been shown to be a component of extracellular matrix in a variety of human tissues. Using in vitro transformation systems, the applicant and his co-investigators have shown previously that loss of TGFBI expression is causally linked to tumorigenic phenotype in radiation/asbestos fibers-treated human bronchial epithelial cells, which is further substantiated by the studies that reconstituted expression of TGFBI in human lung cancer cells completely abrogate their tumorigenicity. Significant downregulation of TGFBI gene was found in various human tumor cell lines and in one-third of primary human lung carcinomas. Furthermore, hypermethylation of CpG island of TGFBI promoter correlates with the gene silencing. These findings suggest a significant role of TGFBI gene in human tumor progression. Using TGFBI-/- mouse model, the applicant and his co- investigators have provided the preliminary data showing an increased predisposition to spontaneous tumors in TGFBI-/- mice as well as an enhanced cell proliferation and aberrant activation of CREB and cyclin D1 in TGFBI-/- MEFs. This raises the following questions: Is increased frequency of spontaneous tumors in TGFBI-/- mice significantly higher than in wild type and heterozygous mice? What is the potential role of cyclin D1 upregulation in cell proliferation and spontaneous tumor development in mice with disruption of TGFBI gene? And, which signaling pathway is responsible for TGFBI-regulated cyclin D1 expression? To address these issues, a series of 3 specific aims are proposed to address the 3 testable hypotheses. The proposed studies are likely to have a significant impact on our current understanding of functional role of TGFBI loss in cell proliferation and tumor progression. PROJECT NARRATIVE Interaction between integrin and extracellular matrix (ECM) plays a crucial role in the regulation of cell adhesion and proliferation, anti-apoptotic death and tumor progression. TGFBI encodes a secreted protein that contains an integrin-binding site and promotes cellular adhesion and spreading. We have shown previously that TGFBI is ubiquitously expressed in normal human tissues, whereas its expression was either decreased or lost in a variety of human tumor cell lines as well as in one-third of primary human lung tumor samples. Recovered expression of TGFBI in human cancer cells that lack endogenous TGFBI protein significantly suppresses their tumorigenicity. We further demonstrate that promoter hypermethylation, one of the mechanisms by which tumor suppressor genes are inactivated in human cancers, correlates with silencing of TGFBI Promoter. These data suggest a tumor suppressor function of TGFBI in vivo. To study this hypothesis, we have succeeded in generating TGFBI-deficient mice. Preliminary data based on this model showed that lack of TGFBI expression results in an increased tendency to spontaneous tumor development in mice, and an enhanced cell proliferation and aberrant activation of CREB and cyclin D1 in TGFBI-null cells. These raise the following questions: Is increased frequency of spontaneous tumors in TGFBI-/- mice significantly higher than in wild type and heterozygous mice? What is the potential role of cyclin D1 upregulation in an increased cell proliferation and an enhanced spontaneous tumor development in TGFBI-/- mice? And, which signaling pathway is responsible for TGFBI-regulated cyclin D1 expression? To address these issues, a series of 3 specific aims are proposed to address the 3 testable hypotheses. Hypothesis 1: Disruption of TGFBI may contribute to tumorigenic progression. Specific Aim 1A-C will address this hypothesis and use the TGFBI-/-, heterozygous and wild type mice to compare the difference in frequencies of both spontaneous tumor growth and DMBA-induced skin tumors. Hypothesis 2: The cell cycle regulator, cyclin D1, may be critical in mediating TGFBI-regulated cell proliferation and tumor progression. Aim 2A will address whether silence of TGFBI in wild type MEFs can activate cyclin D1 and induce aberrant cell proliferation and cell transformation. In Specific Aim 2B, essential role of cyclin D1 upregulation in TGFBI-/- cells will be examined by silence of cyclin D1 expression. Suppression of cell proliferation and transformation phenotypes will be defined in cyclin D1-silenced TGFBI-/- cells. And Aim 2C will further address the importance of cyclin D1 upregulation using an in vivo mouse model. Hypothesis 3: TGFBI, a secreted protein, may regulate downstream targets through an integrin-associated signaling pathway. In Aim 3A, causal role of CREB activation in cyclin D1 upregulation will be defined. Aim 3B will determine whether an aberrant activation of CREB and cyclin D1 are due to a dysregulated PKA (Protein kinase A) activity. In Aim 3C, integrin receptor(s) and functional domain of TGFBI that are involved in TGFBI- integrin interaction and in the regulation of CREB and cyclin D1 activation will be identified. And in Aim 3D, aberrant signaling found in TGFBI-/- cells will be examined in spontaneous tumor tissues arising from TGFBI-/- mice to determine their significance in process of tumor progression. The proposed studies will provide the in vivo evidence for supporting the hypothesis that TGFBI possesses anti-tumor function. Since TGFBI is frequently down-regulated in primary human tumors, the data generated from this application are likely to have a significant impact on our current understanding of functional role of TGFBI loss in development of human cancers.

描述（由申请人提供）：由TGF-β 1诱导的TGFBI是一种肌成束蛋白样蛋白，已被证明是多种人体组织中细胞外基质的组分。使用体外转化系统，申请人和他的合作研究者先前已经表明，TGFBI表达的丧失与辐射/石棉纤维处理的人支气管上皮细胞中的致瘤性表型有因果关系，这通过人肺癌细胞中TGFBI的重建表达完全消除其致瘤性的研究进一步证实。TGFBI基因在多种人类肿瘤细胞系和三分之一的原发性肺癌中表达显著下调。此外，TGFBI启动子区CpG岛甲基化与基因沉默相关。这些发现表明TGFBI基因在人类肿瘤进展中起重要作用。使用TGFBI-/-小鼠模型，申请人及其合作研究者提供了初步数据，显示TGFBI-/-小鼠中自发性肿瘤的易感性增加，以及TGFBI-/-MEF中CREB和细胞周期蛋白D1的细胞增殖和异常激活增强。这就提出了以下问题：TGFBI-/-小鼠自发性肿瘤发生率的增加是否显著高于野生型和杂合子小鼠？在TGFBI基因破坏的小鼠中，细胞周期蛋白D1上调在细胞增殖和自发肿瘤发展中的潜在作用是什么？并且，哪个信号通路负责TGF β 1调节的细胞周期蛋白D1表达？为了解决这些问题，提出了一系列的3个具体目标，以解决3个可检验的假设。拟议的研究可能会对我们目前对TGFBI丢失在细胞增殖和肿瘤进展中的功能作用的理解产生重大影响。整合素与细胞外基质（ECM）之间的相互作用在调节细胞粘附和增殖、抗凋亡死亡和肿瘤进展中起着至关重要的作用。TGFBI编码含有整合素结合位点并促进细胞粘附和扩散的分泌蛋白。我们之前已经表明，TGFBI在正常人体组织中普遍表达，而其表达在多种人类肿瘤细胞系以及三分之一的原发性人类肺肿瘤样品中减少或丢失。缺乏内源性TGFBI蛋白的人癌细胞中TGFBI的过度表达显著抑制其致瘤性。我们进一步证明，启动子超甲基化，肿瘤抑制基因在人类癌症中失活的机制之一，与TGFBI启动子沉默相关。这些数据表明TGFBI在体内具有肿瘤抑制功能。为了研究这一假设，我们成功地产生了TGF β 1缺陷小鼠。基于该模型的初步数据显示，缺乏TGFBI表达导致小鼠中自发肿瘤发展的趋势增加，并且在TGFBI缺失的细胞中细胞增殖和CREB和细胞周期蛋白D1的异常激活增强。这就提出了以下问题：TGFBI-/-小鼠自发性肿瘤发生率的增加是否显著高于野生型和杂合子小鼠？在TGFBI-/-小鼠中，细胞周期蛋白D1上调在细胞增殖增加和自发性肿瘤发展中的潜在作用是什么？并且，哪个信号通路负责TGF β 1调节的细胞周期蛋白D1表达？为了解决这些问题，提出了一系列的3个具体目标，以解决3个可检验的假设。假设1：TGFBI的破坏可能有助于肿瘤发生进展。具体目标1A-C将解决这一假设，并使用TGFBI-/-、杂合和野生型小鼠比较自发肿瘤生长和DMBA诱导的皮肤肿瘤的频率差异。假设2：细胞周期调节因子cyclin D1可能在介导TGF β 1调节的细胞增殖和肿瘤进展中起关键作用。目的2A将研究野生型MEFs中TGFBI的沉默是否能激活细胞周期蛋白D1并诱导异常细胞增殖和细胞转化。在特异性目的2B中，将通过细胞周期蛋白D1表达的沉默来检查TGFBI-/-细胞中细胞周期蛋白D1上调的重要作用。将在细胞周期蛋白D1沉默的TGFBI-/-细胞中定义细胞增殖和转化表型的抑制。Aim 2C将使用体内小鼠模型进一步阐明细胞周期蛋白D1上调的重要性。假设3：TGFBI是一种分泌蛋白，可能通过整合素相关信号通路调节下游靶点。在目标3A中，CREB激活在细胞周期蛋白D1上调中的因果作用将被定义。目的3B将确定CREB和细胞周期蛋白D1的异常激活是否是由于PKA（蛋白激酶A）活性失调所致。在目标3C中，将鉴定参与TGFBI-整联蛋白相互作用和调节CREB和细胞周期蛋白D1活化的TGFBI的整联蛋白受体和功能结构域。在Aim 3D中，将在TGFBI-/-小鼠产生的自发肿瘤组织中检查TGFBI-/-细胞中发现的异常信号传导，以确定其在肿瘤进展过程中的意义。本研究为TGFBI具有抗肿瘤作用的假说提供了体内实验证据。由于TGFBI在原发性人类肿瘤中经常下调，因此从该应用中产生的数据可能对我们目前对TGFBI损失在人类癌症发展中的功能作用的理解产生重大影响。