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与治疗相关的副作用往往限制了许多治疗方法的有效性，因此有必要针对特定的分子靶点开发新的药物，同时将靶外效应降至最低。我们的目标是通过开发凋亡调节蛋白CCAR1/CARP-1的功能来开发新的、更安全和有效的人类乳腺癌(HBC)治疗方法。CARP-1的功能是转导细胞生长和化疗(阿霉素、依托泊苷或易瑞沙)依赖的抑制信号。CARP-1的表达与HBC肿瘤分级呈负相关。酵母双杂交(Y2H)筛选表明，CARP-1与E3泛素连接酶亚基后期促进复合体(APC)-2蛋白结合。在绘制了CARP-1和APC-2相互作用表位的图谱后，我们建立了荧光偏振分析(FPA)，并筛选了一个化学文库来鉴定CARP-1：APC-2结合的小分子抑制物(SMI)。前导SMI被称为CARP-1功能模拟物(CFM)-4，在体外和体内抑制HBC的生长。CFM-4还抑制耐他莫昔芬()或阿霉素(ADR)的HBc细胞的生长，但不抑制非致瘤和永生化的MCF-10A细胞的生长。CFM-4与CARP-1结合，导致CALP-1水平升高，激活促凋亡的p38丝裂原活化蛋白激酶(MAPK)、caspase-9和caspase-3，以及细胞凋亡。CFM-4诱导有丝分裂周期蛋白B1和CDC20蛋白的丢失，可能是通过caspase依赖但泛素蛋白酶体途径不依赖的机制(S)。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信号转导的关键调节因子。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依赖于CARP-1的HBC生长抑制的机制，并利用这一知识开发靶向HBC的药物。这一目标的基本原理是基于这样一个事实，即肿瘤抑制基因p53的缺失促进了侵袭性HBCs的发展，而且对ADR的耐药性仍然是一个问题。由于CARP-1调节ADR对HBc生长的抑制，并且是P53的共同激活剂，因此结合和上调CARP-1，激活促凋亡的p38MAPK和caspase，抑制CDC20和Cyclin B1水平的CFM有可能靶向HBCs，包括那些突变的P53和对ADR或具有耐药性的HBCs。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(S)，以及在CFM-4或ADR存在下，CARP-1与p38结合在多大程度上调节HBC的生长。(2)阐明CALP-1依赖激活caspase-9和caspase-3的机制(S)，并确定在CFM-4存在下，激活的caspase靶向Cyclin B1的程度。(3)在小鼠异种肝细胞移植模型上进行药代动力学(PK)分析，并验证S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预防/抗击乳腺癌的战略，使退伍军人劳动力中的女性受益，为退伍军人医疗保健做出贡献，并促进退伍军人管理局的使命。

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.