PO.ET09.08 · 实验与分子治疗
新型 Ect2-Rac 抑制剂 CPV-337 在乳腺癌和胰腺癌中的应用
Novel Ect2-Rac inhibitor CPV-337 in breast and pancreatic cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
转移是癌症患者治疗失败和不良预后的主要原因；然而，针对转移的靶向疗法却很少。同源 Rho GTP 酶 Rac 和 Cdc42 由于在癌细胞增殖、活力、存活、迁移和侵袭中的核心作用，是转移性癌症中的可行靶点。在癌症中，Rac 和 Cdc42 通常不发生突变，而是被致癌性鸟嘌呤核苷酸交换因子（GEF）激活，后者将 GDP 交换为 GTP。我们开发了临床阶段化合物 MBQ-167（一种 Rac 和 Cdc42 抑制剂）作为转移抑制剂。CPV-337 是 MBQ-167 的衍生物，对 Rac（Rac1、2、3、Rac1b）具有特异性，在 HER2++ 乳腺癌中 IC50 约为 50nM（即比 MBQ-167 有效 2 倍），在胰腺癌中 IC50 <100nM。CPV-337 在转移性乳腺癌细胞中的 GI50 为 57nM，而不影响乳腺上皮细胞。在 HER2++ 乳腺癌实验性转移小鼠模型中，以 5mg/kg CPV-337 治疗使乳腺肿瘤生长和转移减少约 90%，与 10 mg/kg MBQ-167 的效果相当。本研究的目的是表征 CPV-337 作为抗癌药物的作用机制。我们发现 CPV-337 抑制 Rac 下游效应因子 PAK 和 Cofilin 的磷酸化，二者调控肌动蛋白细胞骨架结构（如片状伪足）。CPV-337 治疗后还观察到 Rac 下游效应因子 WAVE 蛋白（同样调控肌动蛋白细胞骨架）的显著减少。相应地，CPV-337 抑制 HER2++ 乳腺癌细胞的片状伪足形成、侵袭和迁移。为确定 CPV-337 抑制 Rac1 的机制，使用对活化 GEF 具有更高亲和力的 Rac1(G15A) 无核苷酸突变体进行了下拉实验。在所测试的 GEF 中，Ect2（上皮细胞转化因子 2）被确定为受 CPV-337 抑制的主要 Rac.GEF。尽管 Ect2 是一个调控胞质分裂和细胞周期进程的关键癌基因，但迄今尚无已知的 Rac-Ect2 特异性抑制剂。Ect2 在不同类型癌症中的过表达与不良预后和总生存期缩短相关。正在进行的研究使用 CRISPR-Cas9 Rac-1 敲除乳腺癌细胞评估 CPV-337 的抑制机制及其特异性。总之，CPV-337 是一种有前景的抗乳腺癌和胰腺癌药物，具有独特而特异的抑制机制。
查看英文原文 English abstract
Metastasis is the leading cause of treatment failure and poor prognosis in cancer patients; however, there are few metastasis targeted therapies. The homologous Rho GTPases Rac and Cdc42 are viable targets in metastatic cancer due to their central role in cancer cell proliferation, viability, survival, migration, and invasion. In cancer, Rac and Cdc42 are not generally mutated but activated by oncogenic guanine nucleotide exchange factors (GEFs), which exchange the GDP for a GTP. We developed the clinical-stage compound MBQ-167, a Rac and Cdc42 inhibitor, as a metastasis inhibitor. CPV-337 is an MBQ-167 derivative that is specific for Rac (Rac1,2, 3, Rac1b) with an IC50 of ~50nM (i.e., 2X more effective than MBQ-167) in HER2++ breast cancer and an IC50 of <100nM in pancreatic cancer. CPV-337 has a GI50 of 57nM in metastatic breast cancer cells without affecting mammary epithelial cells. In a mouse model of HER2++ breast cancer experimental metastasis, treatment with 5mg/kg CPV-337 resulted in ~90% reduction in mammary tumor growth and metastasis, to the same extent as 10 mg/kg MBQ-167. The objective herein was to characterize the mechanism by which CPV-337 acts as an anticancer agent. We show that CPV-337 inhibits the phosphorylation of Rac downstream effectors PAK and Cofilin, which regulate actin cytoskeletal structures, such as lamellipodia. A significant reduction of the Rac downstream effector WAVE protein that also regulates the actin cytoskeleton, was also observed following CPV-337 treatment. Accordingly, CPV-337 inhibits lamellipodia formation, invasion, and migration of HER2++ breast cancer cells. To identify the mechanism by which CPV-337 inhibits Rac1, pulldowns were conducted using the Rac1(G15A) nucleotide-free mutant, which has a higher affinity for activated GEFs. Of the GEFs tested, Ect2 (Epithelial Cell Transforming 2) was identified as the major Rac.GEF inhibited by CPV-337. To date there are no known Rac-Ect2 specific inhibitors, despite Ect2 being a critical oncogene that regulates cytokinesis and cell cycle progression. Overexpression of Ect2 in different types of cancer has been associated with poor prognosis and reduced overall survival. Ongoing studies are evaluating the inhibitory mechanism of CPV-337 and its specificity using CRISPR-Cas9 Rac-1 knockout breast cancer cells. In conclusion, CPV-337 is a promising anti breast and pancreatic cancer drug with a unique and specific mechanism of inhibition.
利益披露 Disclosure
J. Colon Gonzalez, None..
N. Grafals-Ruiz, None..
A. Torres-Sanchez, None..
C. Vlaar, None..
S. Dharmawardhane, None.