Investigating the Role of Cyr61 in Breast Cancer

研究 Cyr61 在乳腺癌中的作用

• 批准号: 7092204
• 负责人: Harold Phillip Koeffler
• 金额: $ 28.75万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-26 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7092204
• 关键词: SDS polyacrylamide gel electrophoresis; biological signal transduction; breast neoplasms; cell growth regulation; cell line; cytoskeletal proteins; enzyme linked immunosorbent assay; gene expression; gene induction /repression; genetically modified animals; human tissue; immunocytochemistry; integrins; laboratory mouse; neoplastic growth; p53 gene /protein; posttranslational modifications; protein structure function; receptor binding; small interfering RNA; western blottings

DESCRIPTION (provided by applicant): We have found that Cyr61 is overexpressed in human breast cancers, and its forced expression in non-transformed breast cells results in robust tumor formation in nude mice. We hypothesize that high levels of Cyr61 produced and secreted by breast cancer cells, bind to integrin receptors to stimulate growth in an autocrine and paracrine fashion. Specific Aim 1: Correlate expression of Cyr61 in breast cancers with clinical parameters. Breast cancer tissue arrays will be stained for Cyr61, and results will be correlated with a variety of clinical information. We hypothesize that Cyr61 by binding to integrins activates the beta-catenin/TCFand Pi3KIAKT/mTORI4E-BPIlSK6 pathways. Replicate breast cancer arrays will be stained to determine if these downstream proteins are activated. Specific Aim 2: Determine pathways by which Cyr61 stimulates growth of breast cancer cells. Breast cell lines genetically engineered to have high Cyr61 expression, will be used to unravel Cyr61 signaling pathways employing a variety of techniques: dominant negative expression vectors, blocking chemicals, siRNA, anti-sense and purified Cyt61. Specific Aim 3: Investigate the effects of overexpression of Cyr61 on p53 function, we discovered that breast cells that overexpress Cyr61, have a non-functional p53. The mechanism of this extremely important finding will be explored. Specific Aim 4: Determine which domains of Cyr61 are necessary for enhancing the transforming ability of breast cancer cells. Cyr61 has well defined, conserved modules. Cyr deletional mutants will be stably placed into breast cells and examined for phenotypic changes. Specific Aim 5: Purify Cyr61 in order to test on breast cells and to develop an ELISA. Cyr61 will be purified and used to examine its effect on transformed and non-transformed breast cells. Also, we will make the first Cyr61 ELISA, which may have clinical and laboratory utility. Specific Aim 6: Determine the effects, in vivo of selective overexpression of Cyr61 in breast tissue. Transgenic mice that selectively overexpress wild type and deletionally mutant Cyr61 will be created to study the in vivo development of breast cancer. Specific Aim 7: Analyze the results of gene silencing of Cyr61 in breast cancer cells. Taking advantage of lentiviral and siRNA technologies, expression of Cyr61 will be inhibited in breast cancer lines that constitutively overexpress it; their phenotypic changes will be examined. Specific Aim 8: Study the therapeutic implications of overexpression of Cyr61 in breast cancers. Our preliminary observations that Cyr61 overexpressing breast cancer cells are chemo-resistant will be pursued. Also, to explore therapeutic possibilities, we will try to block the Cyr61 pro-growth/anti-apoptotic pathways in breast cancers using a variety of approaches. Taken together, our proposed studies will provide unique insights into the pivotal role that Cyr61 plays in breast cancer ant they may lead to novel therapeutic approaches.

描述（申请人提供）：我们发现Cyr61在人类乳腺癌中过度表达，并且其在非转化乳腺细胞中的强制表达导致裸鼠中强烈的肿瘤形成。我们假设乳腺癌细胞产生和分泌的高水平 Cyr61 与整合素受体结合，以自分泌和旁分泌方式刺激生长。具体目标 1：将乳腺癌中 Cyr61 的表达与临床参数相关联。乳腺癌组织阵列将进行 Cyr61 染色，结果将与各种临床信息相关联。我们假设 Cyr61 通过与整合素结合激活 β-连环蛋白/TCF 和 Pi3KIAKT/mTORI4E-BPI1SK6 途径。复制的乳腺癌阵列将被染色以确定这些下游蛋白质是否被激活。具体目标 2：确定 Cyr61 刺激乳腺癌细胞生长的途径。经过基因改造具有高 Cyr61 表达的乳腺细胞系将用于采用多种技术来解开 Cyr61 信号通路：显性失活表达载体、封闭化学物质、siRNA、反义和纯化 Cyt61。具体目标3：研究Cyr61过度表达对p53功能的影响，我们发现过度表达Cyr61的乳腺细胞具有无功能的p53。我们将探讨这一极其重要的发现的机制。具体目标4：确定Cyr61的哪些结构域对于增强乳腺癌细胞的转化能力是必需的。 Cyr61 具有明确定义的保守模块。 Cyr 缺失突变体将被稳定地放入乳腺细胞中并检查表型变化。具体目标 5：纯化 Cyr61 以测试乳腺细胞并开发 ELISA。 Cyr61 将被纯化并用于检查其对转化和非转化乳腺细胞的影响。此外，我们将进行第一个 Cyr61 ELISA，它可能具有临床和实验室实用性。具体目标 6：确定乳腺组织中 Cyr61 选择性过度表达的体内影响。将创建选择性过表达野生型和缺失突变体 Cyr61 的转基因小鼠来研究乳腺癌的体内发展。具体目标7：分析乳腺癌细胞中Cyr61基因沉默的结果。利用慢病毒和siRNA技术，Cyr61的表达将在组成性过度表达的乳腺癌细胞系中受到抑制；将检查它们的表型变化。具体目标 8：研究 Cyr61 过度表达在乳腺癌中的治疗意义。我们将继续研究过表达 Cyr61 的乳腺癌细胞具有化疗耐药性的初步观察结果。此外，为了探索治疗的可能性，我们将尝试使用多种方法阻断乳腺癌中的 Cyr61 促生长/抗凋亡途径。总而言之，我们提出的研究将为 Cyr61 在乳腺癌中发挥的关键作用提供独特的见解，并可能带来新的治疗方法。