Signal Transduction Events and the Regulation of Cell Gr

该项目旨在通过研究生长、存活和转移调节信号转导事件来确定抗癌药物开发的分子靶点，从而开发一种新的癌症治疗方法。我们的工作通过临床前开发研究核心分为基础研究和转化研究，这是我们建立的转化药物开发机构。我们目前的工作集中在(1)组蛋白脱乙酰酶作为抗肿瘤药物开发的靶点，(2)β-连环蛋白调节造血细胞的分子机制和确定β-连环蛋白作为血液系统恶性肿瘤的靶标(3)开发新的药效学分析方法，包括抗血管生成治疗的分析方法。(1)我们对抑制激素耐药的前列腺癌细胞生长的信号转导通路的基础研究使我们确定组蛋白脱乙酰酶是该肿瘤的关键靶点。在本财政年度，我们已经完成了一种新的药效学方法的开发，用于评估体内HDAC抑制剂的活性。NCI已经为我们的工作申请了专利，它是唯一能够分析像手指棒一样少的血液中的HDAC抑制剂活性的设备，并且可以通过同时检测7个参数来观察联合治疗的药效学反应。我们已经在3项临床试验中使用了这项技术，其中两项已经撰写发表。我们已经与布罗德研究所的Jay Bradner博士和Stuart Schreiber博士建立了合作关系，利用我们的技术开发新的HDAC抑制剂。(2)在研究洛伐他汀的抗癌作用时，我们发现，对洛伐他汀促凋亡活性敏感性的一个关键决定因素是β-连环蛋白的完整性。洛伐他汀是作为我们研究的直接翻译而在NCI进行的I期临床试验。这让我们研究了β-连环蛋白在细胞凋亡中的作用。我们使用恶性血液病作为我们的模型，发现β-连环蛋白在这些细胞中发挥着出人意料的重要作用。我们的数据表明，β-连环蛋白调节白血病细胞的存活、增殖和黏附特性。这些数据表明，β-连环蛋白是治疗血液系统恶性肿瘤的抗癌药物开发的新靶点。我们还在研究β-连环蛋白在成熟外周淋巴细胞中的作用，我们发现β-连环蛋白在外周T细胞激活中起关键作用。我们的数据表明，T细胞激活需要大量的β-连环蛋白信号，而未能适当下调β-连环蛋白信号会促进转化。此外，我们还描述了人外周血淋巴细胞中β-连环蛋白的翻译后调节途径。这些数据表明，β-连环蛋白是由与β-TrCP1相关的泛素连接酶复合体控制的，而通过TCR连接来稳定β-连环蛋白是通过钙流动、钙调神经磷酸酶激活和β-连环蛋白丝氨酸/苏氨酸去磷酸化来实现的，从而阻止了β-连环蛋白对蛋白酶体的靶向。这会导致胞质和核内β-连环蛋白的积累，并激活β-连环蛋白靶基因。此外，我们发现IL-2是一个β-连环蛋白反应基因，并已开始定位IL-2启动子上的反应部位。我们以前发表过，β-连环蛋白促进成人T细胞白血病(ATL)细胞系HUT102的增殖。我们现在已经研究了β-连环蛋白在ATL中的信号转导机制，发现β-连环蛋白是由HTLV-1 Tax诱导的，并与Tax在转录激活中协同作用。在ATL细胞系HUT102和Jurkat T-ALL细胞中，Tax的表达增加了由β-连环蛋白启动子驱动的荧光素酶报告基因的活性。实时荧光定量RT-PCR证实，税收增加了β-连环蛋白的表达。

This project is designed to develop a new approach to cancer treatment through the study of growth, survival, and metastasis regulatory signal transduction events that identify molecular targets for anticancer drug development. Our work is divided into basic research and translational research through the Preclinical Development Research Core, a translational drug development facility that we have established. Our work is currently focused on (1) histone deacetylase as a target for anticancer drug development, and (2) the molecular mechanisms of hematopoietic cell regulation by beta-catenin and the identification of beta-catenin as a target in hematologic malignancies (3) development of novel pharmacodynamic assays, including assays for antiangiogenic therapy. (1) Our basic research on signal transduction pathways that can inhibit the growth of hormone-refractory prostate cancer cells led us to the identification of histone deacetylase as a critical target in this neoplasm. During this fiscal year we have finished development of a novel pharmacodynamic assay for assessment of HDAC inhibitor activity in vivo. The NCI has applied for a patent for our work, which is uniquely capable of analyzing HDAC inhibitor activity in as little blood as in a finger-stick, and can look at combination therapy pharmacodynamic responses by examining 7 parameters simultaneously. We have used this technology in 3 clinical trials, two of which have been written for publication. We have established a collaboration with Drs. Jay Bradner and Stuart Schreiber of the Broad Institute to use our technology to develop new HDAC inhibitors. (2) While studying the anticancer action of lovastatin, a drug that was brought to Phase I clinical trial at the NCI as a direct translation of our research, we found that a critical determinant of sensitivity to the proapoptotic activity of lovastatin was the integrity of beta-catenin protein. This led us to examine the role of beta-catenin in apoptosis. We used hematologic malignancies as our model and found that beta-catenin plays an unexpectedly vital role in these cells. Our data demonstrated that beta-catenin regulates leukemia cell survival, proliferation, and adhesive properties. These data identified beta-catenin as a novel target for anticancer drug development in hematologic malignancies. We are also studying the role of beta-catenin in mature peripheral lymphocytes, where we found that beta-catenin is critical in peripheral T-cell activation. Our data suggest that a burst of beta-catenin signaling is required for T-cell activation, and that failure to appropriately down-regulate beta-catenin signaling promotes transformation. Furthermore we delineated the pathway for posttranslational regulation of beta-catenin in human PBL. These data demonstrate that beta-catenin is controlled by the beta-TrCP1-associated ubiquitin ligase complex and that stabilization of beta-catenin with TCR ligation is achieved by calcium flux, calcineurin activation, and beta-catenin serine/threonine dephosphorylation, which blocks targeting of beta-catenin to the proteasome. This leads to accumulation of cytoplasmic and nuclear beta-catenin and activation of beta-catenin target genes. Furthermore we found that IL-2 is a beta-catenin-responsive gene and have begun mapping the responsive sites on the IL-2 promoter.We had published previously that beta-catenin promotes proliferation of the adult T-cell leukemia (ATL) cell line HUT102. We have now investigated the mechanism of beta-catenin signaling in ATL and discovered that beta-catenin is induced by HTLV-1 Tax and cooperates with Tax in transcriptional activation. In the ATL cell line HUT102 and Jurkat T-ALL cells, Tax expression increased activity of a luciferase reporter driven by the beta-catenin promoter. Real-time RT-PCR confirmed that Tax increased beta-catenin expression.