Investigating the Role of Cyr61 in Breast Cancer

描述（由申请人提供）：我们发现Cyr61在人类乳腺癌中过表达，其在非转化乳腺细胞中的强制表达导致裸鼠肿瘤的强烈形成。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，以自分泌和旁分泌的方式刺激生长。特异性目的1:Cyr61在乳腺癌中的表达与临床参数的相关性。乳腺癌组织阵列将进行Cyr61染色，其结果将与多种临床信息相关联。我们假设Cyr61通过结合整合素激活β -catenin/TCFand Pi3KIAKT/ mtor4e - bpilsk6通路。复制的乳腺癌阵列将被染色以确定这些下游蛋白是否被激活。特异性目标2：确定Cyr61刺激乳腺癌细胞生长的途径。通过基因工程使Cyr61高表达的乳腺细胞系，将使用多种技术来解开Cyr61信号通路：显性负表达载体、阻断化学物质、siRNA、反义和纯化Cyt61。特异性目的3：研究Cyr61过表达对p53功能的影响，我们发现过表达Cyr61的乳腺细胞具有非功能性p53。这一极其重要的发现的机制将被探索。特异性目的4：确定Cyr61的哪些结构域是增强乳腺癌细胞转化能力所必需的。Cyr61具有定义良好的保守模块。Cyr缺失突变体将被稳定地放置到乳腺细胞中并检查表型变化。特异性目的5：纯化Cyr61以检测乳腺细胞并开发ELISA。Cyr61将被纯化并用于检测其对转化和非转化乳腺细胞的影响。此外，我们将制作首个Cyr61 ELISA，可能具有临床和实验室用途。特异性目的6：确定Cyr61在乳腺组织中选择性过表达的体内效应。将建立选择性过表达野生型和缺失突变Cyr61的转基因小鼠来研究乳腺癌的体内发展。特异性目的7：分析Cyr61基因沉默在乳腺癌细胞中的结果。利用慢病毒和siRNA技术，Cyr61的表达将在组成性过表达的乳腺癌细胞系中被抑制；他们的表型变化将被检查。特异性目标8：研究Cyr61过表达在乳腺癌中的治疗意义。我们对Cyr61过表达的乳腺癌细胞具有耐药性的初步观察将继续进行。此外，为了探索治疗可能性，我们将尝试使用各种方法阻断Cyr61促生长/抗凋亡通路。综上所述，我们提出的研究将为Cyr61在乳腺癌中的关键作用提供独特的见解，并可能导致新的治疗方法。

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.