Investigating the Role of Cyr61 in Breast Cancer

描述(申请人提供)：我们发现Cyr61在人类乳腺癌中过表达，它在未转化的乳腺细胞中强制表达导致在裸鼠体内形成强大的肿瘤。我们推测，高水平的Cyr61由乳腺癌细胞产生和分泌，与整合素受体结合，以自分泌和旁分泌的方式刺激生长。具体目的1：Cyr61在乳腺癌中的表达与临床参数的关系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了β-catenin/TCFs和Pi3KIAKT/mTORI4E-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。具体目标2：确定Cyr61刺激乳腺癌细胞生长的途径。通过基因工程获得高表达Cyr61的乳腺细胞系，将利用多种技术来解开Cyr61信号通路：显性负表达载体、阻断化学物质、siRNA、反义和纯化的Cyt61。具体目的3：研究Cyr61过表达对P53功能的影响，我们发现过表达Cyr61的乳腺细胞有一个非功能的P53。这一极其重要的发现的机制将被探索。具体目标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、保守的模块。CyR缺失突变体将被稳定地放置到乳腺细胞中，并检查其表型变化。具体目的5：提纯Cyr61，用于乳腺细胞检测和建立一种酶联免疫吸附试验。Cyr61将被提纯，并用于检测其对转化和未转化的乳腺细胞的影响。此外，我们还将研制出第一个具有临床和实验室实用价值的Cyr61酶联免疫吸附试验。特异性目标6：在体内测定Cyr61在乳腺组织中选择性过表达的效果。将创建选择性过度表达野生型和缺失突变Cyr61的转基因小鼠，以研究乳腺癌的体内发展。特定目的7：分析Cyr61基因沉默乳腺癌细胞的结果。利用慢病毒和siRNA技术，Cyr61在结构性过度表达的乳腺癌株中的表达将被抑制；他们的表型变化将被检测。具体目标8：研究Cyr61在乳腺癌中过表达的治疗意义。我们将继续我们的初步观察，即过度表达Cyr61的乳腺癌细胞对化疗具有耐药性。此外，为了探索治疗的可能性，我们将尝试使用各种方法来阻断乳腺癌中的Cyr61促生长/抗凋亡通路。综上所述，我们提出的研究将为Cyr61在乳腺癌中发挥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.