PTEN TUMOR SUPPRESSOR AND CELL CYCLE REGULATION

肿瘤抑制功能的阐明为肿瘤的发生机制提供了许多有价值的见解。我们之前已经确定了一种新的磷酸酶TEP1，它与几种细胞骨架蛋白（如张力蛋白）具有大量同源性。编码TEP1的基因被定位到染色体10q23上，这个位点在人类癌症中经常被删除。TEP1与候选肿瘤抑制因子PTEN或MMAC1相同，通过定位克隆方法分离得到。PTEN/MMAC1/TEP1在许多原发性人类癌症中缺失或突变，包括胶质母细胞瘤、子宫内膜瘤、乳腺癌和前列腺癌，以及一些癌症易感综合征，如考登病。该应用的广泛、长期目标是阐明细胞信号通路与细胞周期进展和细胞存活的调控过程相关的分子机制。特别地，本提案的重点是研究肿瘤抑制蛋白PTEN调节细胞周期进程的机制以及该肿瘤抑制蛋白的失活如何促进人类癌症的发生。我们最近的研究强烈表明，在体内，PTEN作为一种特定的磷酸酶作用于磷脂酰肌醇3,4,5-三磷酸（PIP3）。我们通过同源重组获得了Pten基因缺失的小鼠胚胎干细胞（Pten-/-细胞）。我们已经证明Pten-/-细胞含有PIP3水平升高和Akt/PKB激活增加，Akt/PKB是pi3激酶途径的下游信号分子。因此，Pten-/-细胞表现出增强的细胞增殖和细胞存活。我们进一步证明PTEN缺失导致细胞周期进展到s期，PTEN影响细胞周期的一个关键靶点是p27KIP1，一种CDK抑制剂。我们拟阐明PTEN调控p27水平和细胞周期进程的分子机制。我们的具体目标是：1)确定PTEN通路如何调节p27水平；2)分析参与p27调控的PI 3激酶下游因子；3)建立生化和分子检测方法，鉴定控制p27的PTEN响应因子；4)利用结构和功能分析探讨PTEN活性的调控。在这个应用程序中提出的研究计划应该为PTEN肿瘤抑制功能的分子机制提供新的见解。这些知识也应该有助于为设计诊断和治疗人类癌症的新策略提供分子基础。

Elucidation of tumor suppressor function provides much valuable insights into the mechanism of tumorigenesis. We have previously identified a novel phosphatase, TEP1, that shares substantial homology to several cytoskeletal proteins such as tensin. The gene encoding TEP1 is mapped to chromosome 10q23, a locus frequently deleted in human cancers. TEP1 is identical to the candidate tumor suppressor PTEN or MMAC1, which was isolated by positional cloning method. PTEN/MMAC1/TEP1 is deleted or mutated in many primary human cancers including glioblastma, endometric tumor, breast and prostate cancers and in several cancer predisposition syndromes such as Cowden disease. The broad, long term objective of this application is to elucidate the molecular mechanisms by which the cellular signaling pathways are linked to the regulatory processes for cell cycle progression and cell survival. In particular, this proposal is focused on investigating the mechanism by which the tumor suppressor protein PTEN functions to regulate cell cycle progression and how the inactivation of this tumor suppressor contributes to the genesis of human cancer. Our recent studies strongly suggest that in vivo, PTEN acts as a specific phosphatase towards phosphatidylinositol 3,4,5-trisphosphate (PIP3). We have generated the mouse embryonic stem cells in which the Pten gene was deleted by homologous recombination (Pten-/- cells). We have shown that Pten-/- cells contained elevated level of PIP3 and increased activation of Akt/PKB, a downstream signaling molecule in the PI 3-kinase pathway. Consequently, Pten-/- cells exhibited enhanced cell proliferation and cell survival. We further demonstrated that PTEN deletion caused advanced cell cycle progression into S-phase and a critical target for PTEN effect on cell cycle is p27KIP1, a CDK inhibitor. We propose to elucidate the molecular mechanism by which PTEN regulates p27 level and cell cycle progression. Our specific aims are: 1) To determine how the PTEN pathway regulates p27 level; 2) To analyze the PI 3-kinase downstream factors involved in regulation of p27; 3) To establish biochemical and molecular assays to identify the PTEN responsive factors that control p27; 4) To investigate the regulation of PTEN activity using structure and function analysis. The proposed research program in this application should provide novel insights into the molecular mechanisms that underlie the PTEN tumor suppressor function. Such knowledge should also help to provide a molecular basis for designing novel strategies for the diagnosis and treatment of human cancer.