CARP-1: A Potential Therapeutic Agent for Breast Cancer

描述（由申请人提供）： 乳腺癌的分子复杂性和治疗相关的副作用通常限制了许多治疗的有效性，阻碍了针对特定分子靶标的新药物的开发，同时最大限度地减少脱靶效应。我们的目标是开发新的，更安全，有效的人类乳腺癌（HBC）的治疗方法，利用细胞凋亡调节蛋白CCAR 1/CARP-1的功能。CARP-1的功能是抑制细胞生长以及化疗（阿霉素、依托泊苷或易瑞沙）依赖性抑制信号传导。CARP-1表达与HBC肿瘤分级呈负相关。酵母双杂交（Y2 H）筛选揭示CARP- 1与E3泛素连接酶亚基后期促进复合物（APC）-2蛋白结合。在绘制CARP-1和APC-2的相互作用表位之后，我们开发了荧光偏振测定（FPA）并筛选了化学文库以鉴定CARP-1：APC-2结合的小分子抑制剂（SMI）。被称为CARP-1功能模拟物（CFM）-4的主要SMI在体外和体内抑制HBC生长。CFM-4还减弱他莫昔芬（TAM）或阿霉素（ADR）抗性HBC细胞的生长，但不抑制非致瘤性和永生化MCF-10A细胞的生长。CFM-4与CARP-1结合，导致CARP- 1水平增加，激活促凋亡p38丝裂原活化蛋白激酶（MAPK）、半胱天冬酶-9和3以及凋亡。CFM-4诱导有丝分裂细胞周期蛋白B1和Cdc 20蛋白的丢失，可能是通过半胱天冬酶依赖性但泛素蛋白酶体途径（UPP）非依赖性机制。假设：CARP-1是一种核周磷酸化蛋白，具有调节HBC细胞生长和凋亡的功能，是ADR信号传导的关键调节因子。我们进一步假设CFM-4刺激CARP-1水平将为治疗乳腺癌和其他癌症提供一种新的机制。目的：我们的长期目标是阐明CARP-1依赖性HBC生长抑制的机制，并利用这些知识开发靶向HBC的药物。这一目标的基本原理是基于肿瘤抑制因子p53的缺失促进侵袭性HBCs的发展以及对ADR的耐药性仍然是一个问题的事实。由于CARP-1通过ADR调节HBC生长抑制并且是p53的共激活剂，因此结合并升高CARP-1、激活促凋亡p38 MAPK和半胱天冬酶并抑制Cdc 20和细胞周期蛋白B1水平的CFM具有靶向HBC的潜力，包括具有突变型p53以及对ADR或TAM具有抗性的那些HBC。我们将通过追求以下目标来检验我们的假设。(1)研究p38激活的分子机制，以及在CFM-4或ADR存在下CARP-1与p38结合调节HBC生长的程度。(2)阐明CARP-1依赖性激活半胱天冬酶-9和-3的机制，并确定在CFM-4存在下激活的半胱天冬酶靶向细胞周期蛋白B1的程度。(3)在HBC小鼠异种移植模型中进行药代动力学（PK）分析并证明CFM的抗肿瘤活性。对退伍军人医疗保健的潜在影响：这项调查将促进制定战略，以预防/打击乳腺癌，这将有利于妇女在退伍军人事务部的劳动力，有助于退伍军人医疗保健和进一步的退伍军人事务部的使命。

DESCRIPTION (provided by applicant): The molecular complexity of breast cancers and therapy-associated side effects often limit effectiveness of many therapies, warranting the development of new agents for specific molecular targets while minimizing the off-target effects. Our goal is to develop novel, safer, and effective human breast cancer (HBC) therapies by exploiting functions of an apoptosis regulatory protein CCAR1/CARP-1. CARP-1 functions to transduce cell growth as well as chemotherapy (adriamycin, etoposide, or Iressa)-dependent inhibitory signaling. CARP-1 expression correlates inversely with HBC tumor grades. A yeast-two-hybrid (Y2H) screen revealed that CARP- 1 binds with the E3 ubiquitin ligase subunit anaphase promoting complex (APC)-2 protein. Following mapping of the interacting epitopes of CARP-1 and APC-2, we developed a fluorescence polarization assay (FPA) and screened a chemical library to identify small molecule inhibitors (SMIs) of CARP-1:APC-2 binding. The lead SMI termed CARP-1 Functional Mimetic (CFM)-4, inhibits HBC growth in vitro and in vivo. CFM-4 also attenuates growth of tamoxifen (TAM) or adriamycin (ADR)-resistant HBC cells but does not suppress growth of the non-tumorigenic and immortalized MCF-10A cells. CFM-4 binds with CARP-1, leads to increased CARP- 1 levels, activates pro-apoptotic p38 mitogen-activated protein kinase (MAPK), caspases-9 and 3, and apoptosis. CFM-4 induces loss of mitotic cyclin B1 and Cdc20 proteins, possibly through caspase-dependent but ubiquitin proteasome pathway (UPP)-independent mechanism(s). Hypothesis: CARP-1, a peri-nuclear phospho-protein, functions to regulate HBC cell growth and apoptosis, and is a key regulator of ADR signaling. We further hypothesize that CFM-4 stimulation of CARP-1 levels will provide a novel mechanism for treating breast and other cancers. Objectives: Our long-term objective is to elucidate mechanisms of CARP-1-dependent HBC growth inhibition, and to exploit this knowledge to develop agents for targeting of HBC. The rationale for this objective is based on the facts that loss of tumor suppressor p53 promotes development of aggressive HBCs and that resistance to ADR continues to be a problem. Since CARP-1 regulates HBC growth inhibition by ADR and is a co- activator of p53, the CFMs that bind and elevate CARP-1, activate pro-apoptotic p38 MAPK and caspases, and suppress Cdc20 and cyclin B1 levels have potential to target HBCs including those that have mutant p53 as well as have resistance to ADR or TAM. We will test our hypothesis by pursuing the following objectives. (1) To investigate molecular mechanism(s) of p38 activation, and the extent to which CARP-1 binding with p38 regulates HBC growth in the presence of CFM-4 or ADR. (2) To elucidate mechanism(s) of CARP-1-dependent activation of caspases-9 and -3, and determine the extent to which activated caspases target Cyclin B1 in the presence of CFM-4. (3) To conduct pharmacokinetic (PK) profiling and to demonstrate the anti-tumor activities of CFM(s) in mouse xenograft models of HBC. Potential Impact on Veterans Health Care: This investigation will facilitate development of strategies to prevent/combat breast cancer that will benefit women in the VA workforce, contribute to Veterans Health Care and further the mission of the VA.