Role of TGFBI gene in cell proliferation and tumor progression

描述（由申请人提供）：TGFBI由TGF-b1诱导，是一种肌束蛋白样蛋白，已被证明是多种人体组织中细胞外基质的组成部分。利用体外转化系统，申请人和他的共同研究者先前已经表明，TGFBI表达的缺失与辐射/石棉纤维处理的人支气管上皮细胞的致瘤表型有因果关系，这一研究进一步证实了TGFBI在人肺癌细胞中的重组表达完全消除了其致瘤性。TGFBI基因在多种人类肿瘤细胞系和三分之一的原发性人肺癌中均有显著下调。此外，TGFBI启动子CpG岛的高甲基化与基因沉默有关。这些发现提示TGFBI基因在人类肿瘤进展中的重要作用。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-/-小鼠自发性肿瘤易感增加，TGFBI-/- mef细胞增殖增强，CREB和cyclin D1异常活化。这就提出了以下问题：TGFBI-/-小鼠自发性肿瘤的发生率是否明显高于野生型和杂合型小鼠？在TGFBI基因破坏小鼠中，细胞周期蛋白D1上调在细胞增殖和自发性肿瘤发展中的潜在作用是什么？tgfbi调控的cyclin D1表达由哪条信号通路负责？为了解决这些问题，提出了一系列3个具体目标来解决3个可测试的假设。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）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编码一种含有整合素结合位点并促进细胞粘附和扩散的分泌蛋白。我们之前的研究表明，TGFBI在正常人体组织中普遍表达，而在多种人类肿瘤细胞系以及三分之一的原发性人肺肿瘤样本中，其表达要么减少，要么消失。在缺乏内源性TGFBI蛋白的人癌细胞中恢复TGFBI的表达可显著抑制其致瘤性。我们进一步证明，启动子超甲基化与TGFBI启动子的沉默有关，这是人类癌症中肿瘤抑制基因失活的机制之一。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，我们成功地培育了tgfbi缺陷小鼠。基于该模型的初步数据显示，TGFBI表达缺失导致小鼠自发肿瘤发展的趋势增加，TGFBI缺失的细胞增殖增强，CREB和cyclin D1异常活化。这就提出了以下问题：TGFBI-/-小鼠自发性肿瘤的发生率是否明显高于野生型和杂合小鼠？在TGFBI-/-小鼠中，细胞周期蛋白D1上调在细胞增殖增加和自发肿瘤发展增强中的潜在作用是什么？tgfbi调控的cyclin D1表达由哪条信号通路负责？为了解决这些问题，提出了一系列3个具体目标来解决3个可测试的假设。假设1:TGFBI的破坏可能有助于致瘤性进展。特异性Aim 1A-C将解决这一假设，并使用TGFBI-/-、杂合子和野生型小鼠来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tgfbi调节的细胞增殖和肿瘤进展中起关键作用。Aim 2A将探讨TGFBI沉默在野生型mef中是否能激活cyclin D1，诱导异常细胞增殖和细胞转化。在特异性Aim 2B中，将通过沉默cyclin D1表达来检测cyclin D1上调在TGFBI-/-细胞中的重要作用。细胞增殖和转化表型的抑制将在细胞周期蛋白d1沉默的TGFBI-/-细胞中定义。Aim 2C将通过体内小鼠模型进一步阐明cyclin D1上调的重要性。假设3:TGFBI作为一种分泌蛋白，可能通过整合素相关信号通路调控下游靶点。在Aim 3A中，将定义CREB激活在cyclin D1上调中的因果作用。Aim 3B将确定CREB和cyclin D1的异常激活是否由于PKA（蛋白激酶a）活性失调。在Aim 3C中，将鉴定参与TGFBI-整合素相互作用、调控CREB和cyclin D1激活的整合素受体和TGFBI的功能域。在Aim 3D中，TGFBI-/-细胞中发现的异常信号将在TGFBI-/-小鼠产生的自发肿瘤组织中进行检测，以确定其在肿瘤进展过程中的意义。本研究将为TGFBI具有抗肿瘤功能的假说提供体内证据。由于TGFBI在原发性人类肿瘤中经常下调，因此该应用产生的数据可能会对我们目前对TGFBI缺失在人类癌症发展中的功能作用的理解产生重大影响。

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.