CARP-1 Functional Mimetics: A Novel Class of Small Molecule Inhibitors of Medulloblastoma Cell Growth

Medulloblastomas (MBs) constitute an aggressive class of intracranial pediatric tumors. Current multimodality treatments for MBs include surgery, ionizing radiation, and chemotherapy. Toxic side effects of therapies coupled with high incidence of recurrence and the metastatic spread warrant development of more effective, less toxic therapies for this disease. CARP-1/CCAR1 is a peri-nuclear phospho-protein that is a co-activator of the cell cycle regulatory anaphase promoting complex/cyclosome (APC/C) E3 ligase. CARP-1 functional mimetics (CFMs) are a novel class of small molecule compounds that interfere with CARP-1 binding with APC/C subunit APC-2, and suppress growth of a variety of cancer cells in part by promoting apoptosis. Here we investigated MB growth inhibitory potential of the CFMs and found that CFM-4 inhibits growth of MB cells in part by inducing CARP-1 expression, promoting PARP cleavage, activating pro-apoptotic stress-activated protein kinases (SAPK) p38 and JNK, and apoptosis. Gene-array-based analysis of the CFM-4-treated Daoy MB cells indicated down-regulation of a number of key cell growth and metastasis-promoting genes including cell motility regulating small GTP binding protein p21Rac1, and extracellular matrix metallopeptidase (MMP)-10. Moreover, CFM-4 treatment stimulated expression of a number of molecules such as neurotrophin (NTF)3, and NF-κB signaling inhibitors ABIN1 and 2 proteins. Overexpression of NTF3 resulted in reduced MB cell viability while knock-down of NTF3 interfered with CFM-4-dependent loss of viability. CFMs also attenuated biological properties of the MB cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Together our data support anti-MB properties of CFM-4, and provide a proof-of-concept basis for further development of CFMs as potential anti-cancer agents for MBs.髓母细胞瘤（Medulloblastomas, MBs）是一类侵袭性颅内儿童恶性肿瘤。当前针对MBs的多模态治疗方案包括手术、电离辐射及化疗。但治疗带来的毒性不良反应、较高的复发率以及肿瘤转移扩散问题，亟待开发更为高效且低毒的MBs治疗策略。CARP-1/CCAR1是一种核周磷酸化蛋白，作为细胞周期调控的后期促进复合物/周期体（anaphase promoting complex/cyclosome, APC/C）E3泛素连接酶的共激活因子发挥作用。CARP-1功能模拟物（CARP-1 functional mimetics, CFMs）为一类新型小分子化合物，可通过干扰CARP-1与APC/C亚基APC-2的结合，在一定程度上通过诱导细胞凋亡抑制多种癌细胞的增殖。本研究探究了CFMs对MB细胞的生长抑制潜力，结果显示CFM-4可通过诱导CARP-1表达、促进PARP剪切、激活促凋亡应激激活蛋白激酶（stress-activated protein kinases, SAPK）p38与JNK通路以及诱导细胞凋亡，在一定程度上抑制MB细胞的生长。对经CFM-4处理的Daoy MB细胞开展基于基因芯片的分析后发现，多个关键的细胞生长与转移促进基因表达下调，包括调控细胞运动的小GTP结合蛋白p21Rac1以及细胞外基质金属肽酶（matrix metallopeptidase, MMP）-10。此外，CFM-4处理可上调多种分子的表达水平，例如神经营养因子（neurotrophin, NTF）3，以及NF-κB信号通路抑制剂ABIN1与ABIN2蛋白。过表达NTF3可降低MB细胞的活力，而敲低NTF3则会干扰CFM-4介导的细胞活力丧失。CFMs还可通过阻断MB细胞的迁移能力、悬浮集落形成能力以及穿透基质包被膜的侵袭能力，削弱其恶性生物学特性。综上，本研究数据证实了CFM-4的抗髓母细胞瘤活性，为将CFMs开发为治疗MBs的潜在抗癌药物提供了概念验证依据。