Investigating the Role of Cyr61 in Breast Cancer

描述（由申请人提供）：我们已经发现Cyr 61在人乳腺癌中过表达，并且其在非转化乳腺细胞中的强制表达导致裸鼠中稳健的肿瘤形成。我们推测，乳腺癌细胞产生和分泌的高水平Cyr 61，结合到整合素受体，以自分泌和旁分泌的方式刺激生长。具体目标1：乳腺癌中Cyr 61的表达与临床参数相关。将对乳腺癌组织阵列进行Cyr 61染色，并将结果与各种临床信息相关联。我们假设Cyr 61通过与整合素结合激活β-连环蛋白/TCF和Pi 3 KIAKT/mTORI 4 E-BPI 1 SK 6通路。将对复制的乳腺癌阵列进行染色，以确定这些下游蛋白质是否被激活。具体目标2：确定Cyr 61刺激乳腺癌细胞生长的途径。乳腺细胞系基因工程具有高Cyr 61表达，将用于解开Cyr 61信号转导途径，采用各种技术：显性负表达载体，阻断化学品，siRNA，反义和纯化的Cyt 61。具体目标3：研究Cyr 61过表达对p53功能的影响，我们发现过表达Cyr 61的乳腺细胞具有无功能的p53。这一极其重要的发现的机制将被探索。具体目标4：确定Cyr 61的哪些结构域是增强乳腺癌细胞转化能力所必需的。Cyr 61具有明确定义的保守模块。Cyr缺失突变体将稳定地置于乳腺细胞中并检查表型变化。具体目标5：纯化Cyr 61，以便在乳腺细胞上进行测试并开发ELISA。Cyr 61将被纯化并用于检查其对转化和非转化乳腺细胞的影响。同时，我们还将建立第一个Cyr 61 ELISA，这可能具有临床和实验室实用性。具体目的6：确定乳腺组织中Cyr 61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Cyr 61的转基因小鼠，以研究乳腺癌的体内发展。具体目的7：分析乳腺癌细胞中Cyr 61基因沉默的结果。利用慢病毒和siRNA技术，Cyr 61的表达将在组成型过表达的乳腺癌细胞系中被抑制;将检查它们的表型变化。具体目标8：研究Cyr 61过表达在乳腺癌中的治疗意义。我们的初步观察，Cyr 61过表达的乳腺癌细胞是化疗耐药将继续进行。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Cyr 61促生长/抗凋亡途径。总之，我们提出的研究将提供独特的见解Cyr 61在乳腺癌中发挥的关键作用，他们可能会导致新的治疗方法。

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.