A novel therapy for HER2 positive hormone refractory breast cancer

描述（由申请人提供）：该申请涉及广泛的挑战领域（15）转化科学，以及特定的挑战主题，15- ca -102：了解激素难治性癌症的治疗靶向机制。乳腺癌中激素耐药性的发展是一个重要的临床问题。ErbB2/3异源二聚体的表达增强导致乳腺癌细胞能够绕过正常的内分泌反应。然而，在临床上使用ErbB2靶向药物如曲妥珠单抗治疗激素耐药疾病的效果令人失望。因此，迫切需要降低ErbB2/3异源二聚体活性的新策略来治疗激素难治性乳腺癌。EBP1是我们实验室通过与ErbB3结合分离的一种蛋白，由于其下调ErbB2蛋白水平的能力，它是ErbB2/3信号转导的负调节因子。我们实验室的临床前研究表明，EBP1的外视表达增加了乳腺癌细胞对抗雌激素他莫昔芬的敏感性。此外，shRNA导致EBP1蛋白耗竭或PAK1（一种与乳腺癌进展和他莫昔芬耐药有关的激酶）磷酸化导致EBP1失活，导致激素难耐表型的发展。这些数据表明，EBP1通过降低ErbB2蛋白水平，可以减少导致激素抵抗的ErbB2/3受体信号。我们提出基于EBP1的治疗可能有效逆转激素抵抗。我们正在进行的体外和体内数据表明，了解EBP1的作用机制将阐明ErbB2的调控，从而为激素难治性乳腺癌的治疗提供新的途径。本研究的目的是了解EBP1降低ErbB2蛋白水平的机制，以及EBP1的缺失或失活如何导致激素抗性。此外，我们假设PAK1定向治疗可能通过恢复EBP1功能和降低ErbB2水平的能力在一定程度上逆转激素抵抗。我们期待这些研究将为ErbB2+、激素抵抗性乳腺癌的治疗带来新的疗法。在Specific Aim 1中，我们将确定EBP1抑制ErbB2水平的机制，最终目标是开发基于EBP1的疗法来下调ErbB2的表达。由于几个独立的研究小组已经证明EBP1既是DNA结合蛋白又是RNA结合蛋白，我们将研究EBP1对ErbB2转录和转录后事件的影响。因此，我们将a)使用启动子报告子和ChIP检测来确定EBP1诱导的ErbB2转录变化；b)测量EBP1诱导的ErbB2 mRNA稳定性变化。我们将扩展EBP1结合ErbB2 mRNA的研究，确定EBP1与RNA结合的关键区域，以及EBP1磷酸化对这种结合和ErbB2 mRNA稳定性的影响。ErbB2 mRNA结合EBP1的区域也将被确定，并评估该区域在维持ErbB2 mRNA稳定性方面的功能。在Specific Aim 2中，我们将确定如何克服PAK1失活的EBP1功能来治疗激素难治性癌症。在Specific Aim 2a中，我们将证明PAK1使EBP1失能，并有助于对他莫昔芬的耐药性。首先，我们将评估PAK1消除EBP1下调ErbB2 mRNA和蛋白水平的能力。我们还将确定不能被PAK1磷酸化的EBP1是否可以挽救PAK1诱导的他莫昔芬耐药。在特异性Aim 2b中，我们将测试PAK1抑制剂通过恢复EBP1活性来改善激素难耐表型的能力。PAK1抑制剂对激素耐药细胞株EBP1磷酸化、他莫昔芬敏感性和ErbB2水平的影响将在体外进行测试。此外，基于联合靶向ErbB2受体的方法可能有效的假设，我们将在体外测试PAK1和ErbB2抑制剂联合逆转激素抗性的能力。最后，我们将通过在动物模型中证明EBP1对他莫昔芬敏感性的重要性来证实体外结果。这些研究将为PAK1抑制剂在耐他莫昔芬乳腺癌细胞中的体内试验奠定基础。我们认为EBP1可以成为治疗ErbB2+、激素难治性乳腺癌的一类新的治疗剂的基础。药物可以基于EBP1的结构成分，也可以基于能够重新激活EBP1功能的PAK1抑制剂。基于EBP1的治疗，结合目前可用的ErbB2靶向药物，可能为逆转激素耐药性的治疗提供新的途径。

DESCRIPTION (provided by applicant): This application addresses the broad Challenge Area (15) Translational Science, and specific Challenge Topic, 15-CA-102: Understanding mechanisms of hormone refractory cancers for therapeutic targeting. The development of hormone resistance in breast cancer is a significant clinical problem. The enhanced expression of the ErbB2/3 heterodimer leads to the ability of breast cancer cells to bypass normal endocrine responsiveness. However, the use of ErbB2 targeted- agents such as Trastuzumab for hormone resistant disease in the clinic has been disappointing. Thus, new strategies that reduce the activity of the ErbB2/3 heterodimer are urgently needed for treatment of hormone refractory breast cancer. EBP1, a protein isolated in our laboratory by its binding to ErbB3, is a negative regulator of ErbB2/3 signal transduction due to its ability to downregulate ErbB2 protein levels. Preclinical studies from our laboratory demonstrate that ecoptic expression of EBP1 increases sensitivity of breast cancer cells to the antiestrogen tamoxifen. Further, depletion of EBP1 protein by shRNA or inactivation of EBP1 by phosphorylation by PAK1, a kinase implicated in both breast cancer progression and tamoxifen resistance, results in development of the hormone refractory phenotyope. These data suggest that EBP1, by decreasing ErbB2 protein levels, can diminish ErbB2/3 receptor signaling that leads hormone resistance. We propose that EBP1 based therapies may be effective in reversing hormone resistance. Our ongoing in vitro and in vivo data indicate that an understanding of EBP1's mechanism of action will illuminate ErbB2 regulation and thus offer a new avenue for treatment of hormone refractory breast cancer. The aim of this proposal is to understand the mechanism by which EBP1 decreases ErbB2 protein levels and how deletion or inactivation of EBP1 contributes to hormone resistance. In addition, we hypothesize that PAK1 directed therapies may reverse hormone resistance in part via their ability to restore EBP1 function and reduce ErbB2 levels. We anticipate that these studies will lead to new therapies for treatment of ErbB2+, hormone resistant breast cancer. In Specific Aim 1, we will determine the mechanism of EBP1's attenuation of ErbB2 levels with the ultimate goal of developing EBP1 based therapies to downregulate ErbB2 expression. As several independent groups have demonstrated that EBP1 is both a DNA and RNA binding protein, we will examine effects on EBP1 on ErbB2 transcription and post-transcriptional events. We will therefore a) determine EBP1 induced changes in ErbB2 transcription using both promoter reporter and ChIP assays b) measure EBP1-induced changes in ErbB2 mRNA stability. We will extend studies indicating EBP1 binds ErbB2 mRNA and determine the region of EBP1 critical for RNA binding and the effects of phosphorylation of EBP1 on such binding and ErbB2 mRNA stability. The region of ErbB2 mRNA that binds EBP1 will also be determined and the function of this region in maintaining ErbB2 mRNA stability assessed. In Specific Aim 2, we will determine how to overcome PAK1 inactivation of EBP1 function for treatment of hormone refractory cancer. In Specific Aim 2a, we will demonstrate that PAK1 disables EBP1 and contributes to tamoxifen resistance. First, the ability of PAK1 to abrogate the EBP1 downregulation of ErbB2 mRNA and protein levels will be assessed. We will also determine if an EBP1 that cannot be phosphorylated by PAK1 can rescue PAK1 induced tamoxifen resistance. In Specific Aim 2b, we will test the ability of PAK1 inhibitors to ameliorate the hormone refractory phenotype via restoration of EBP1 activity. The effect of PAK1 inhibitors on EBP1 phosphorylation, tamoxifen sensitivity and ErbB2 levels in hormone resistant cell lines will be tested in vitro. In addition, the ability of combinations of PAK1 and ErbB2 inhibitors to reverse hormone resistance will be tested in vitro based on the hypothesis that a combined approach to target the ErbB2 receptor may be efficacious. Finally, we will confirm in vitro results by demonstrating the importance of EBP1 for tamoxifen sensitivity in animal models. These studies will then form the basis for in vivo trials of PAK1 inhibitors in tamoxifen resistant breast cancer cells. We suggest that EBP1 can form the basis of a new class of therapeutic agents for treatment of ErbB2+, hormone refractory breast cancer. Drugs can be based either on the structural components of EBP1 or on PAK1 inhibitors that will reactivate EBP1 function. EBP1 based therapies, in conjunction with currently available ErbB2 targeted drugs, may offer a new avenue for the development of treatments to reverse hormone resistance. PUBLIC HEALTH RELEVANCE: Despite numerous advances in therapies for breast cancer in recent years, recurrence and mortality remain high. Deregulation of the ErbB receptor family, a group of enzymes which stimulates cell growth, leads to resistance to chemotherapy and hormone treatment. We have cloned and characterized EBP1, an ErbB binding protein that decreases levels and activity of ErbB receptors, thus restoring sensitivity to hormone treatments. The current study is designed to determine how EBP1 regulates ErbB expression and hormone sensitivity. Our studies would establish EBP1 as a new therapeutic agent in breast cancer to restore hormone sensitivity in cells which overexpress ErbB receptors.