Targeting the MEKK2-ERK5 Signaling Node in Triple Negative Breast Cancer

摘要： 大约15%-20%的乳腺癌具有三重阴性表型(ER和PR表达阴性，缺乏ErbB2过表达)，这与侵袭性癌症和有限的治疗选择有关。我们已经开发了一种体内筛查，以确定特定的激酶在乳腺癌细胞的肿瘤发生和转移中的作用。用三个阴性和三个阳性的乳腺癌细胞系进行shRNA介导的特异性激酶基因敲除。将基因改变的品系(除了特定shRNA基因敲除外还表达荧光素酶)注射到雌性SCID小鼠的乳房脂肪垫中。肿瘤的生长和血管生成 使用微探头超声系统在8周内进行纵向监测。转移是通过生物发光成像监测的。该分析为分析控制乳腺肿瘤生长、血管形成和转移的蛋白质提供了体内筛选。MAP3K是调节MAP的第一层激酶 导致MAPKs Erk1/2、p38、JNK和ERKs激活的信号通路。MAPSKs调控细胞周期、细胞因子和蛋白水解酶表达以及细胞凋亡等重要基因的表达。在9个MAP3K的筛选中，使用MDA-MB-231确定MEKK2是一个关键的转移调节因子 BT474(三重阳性)乳腺腺癌细胞体内成瘤实验。MEKK2是一种MAP3K，通过激活MKK7和MEKS来调节JNK和ERK5通路的激活。我们已经证明了MEKK2的表达对于EGFR(ErbBI)和 ERBB2/Neu分别在MDA-MB-231和BT474细胞中激活ERKS。我们的假设是，MAP3K MEKK2在细胞信号网络中作为一个关键的信号节点发挥作用，刺激肿瘤生长和转移，以响应ErbB和可能的其他酪氨酸激酶。本研究的目的是从基因上明确MEKK2在三阴性乳腺癌生长和转移中的作用，并开发一种MEKK2小分子抑制剂。具体目标1利用体内异种移植实验确定MEKK2-MEK5-ERK5信号在三阴性乳腺癌细胞发生和转移中的作用。具体而言 目的2、利用MEKK2突变体在生化和细胞激活实验中阐明ErbB1/2激活MEKK2的机制。在具体目标3中，将开发一种小分子生化筛查来鉴定特定抑制MEKK2激酶活性的化合物。MEKK2抑制剂将是 在基于细胞的分析中测试MAPK通路的特异性，并针对KYOME进行特异性分析。在未来的研究中，这些MEKK2抑制剂将在乳腺癌的基因工程小鼠模型(GEMM)中测试抗肿瘤效果。

Abstract: Approximately 15-20% of breast cancers have a triple negative phenotype (negative for ER and PR expression and lack ErbB2 overexpression) that correlates with aggressive cancer and limited treatment options. We have developed an in vivo screen for defining the role of specific kinases in tumorigenesis and metastasis of breast cancer cells. Triple negative and triple positive breast cancer lines were used for shRNA-mediated knock down of specific kinases. The genetically altered lines (expressing luciferase in addition to specific shRNA gene knockdown) are injected into the mammary fat pad of female SCID mice. Tumor growth and vascularization is monitored longitudinally over an 8 week period using a micro-probe ultrasound system. Metastasis is monitored by bioluminescence imaging. The assay provides an in vivo screen for analysis of proteins that control the growth, vascularization and metastasis of breast tumors. MAP3Ks are the first tier of kinases regulating the MAP kinase signaling pathways that lead to the activation of the MAPKs ERKl/2, p38, JNK and ERKS. MAPSKs control expression of genes important for regulating the cell cycle, cytokine and protease expression and apoptosis. In a screen of 9 MAP3Ks, MEKK2 was identified as a key regulator of metastasis using MDA-MB-231 (triple negative basal) and BT474 (triple positive luminal) breast adenocarcinoma cells in the in vivo tumorigenesis assay. MEKK2 is a MAP3K that regulates the activation of the JNK and ERK5 pathways via activation of MKK7 and MEKS. We have shown that MEKK2 expression is required for EGFR (ErbBI) and ErbB2/Neu activation of ERKS in MDA-MB-231 and BT474 cells, respectively. Our hypothesis is that the MAP3K MEKK2 functions as a critical signaling node within the cell signaling network stimulating tumor growth and metastasis in response to ErbB and possibly other tyrosine kinases. The goal of this proposal is to genetically define the role of MEKK2 in triple negative breast cancer tumor growth and metastasis and to develop a MEKK2 small molecule inhibitor. Specific aim 1 involves defining the role of MEKK2-MEK5-ERK5 signaling in tumorigenesis and metastasis of triple negative breast cancer cells using the in vivo xenograft assay. In specific aim 2, we propose to elucidate the mechanism by which MEKK2 gets activated by ErbB1/2 using MEKK2 mutants in biochemical and cell-based activation assays. In specific aim 3, a small molecule biochemical screen will be developed to identify compounds that specifically inhibit MEKK2 kinase activity. MEKK2 inhibitors will be tested for MAPK pathway specificity in cell-based assays and profiled for specificity against the kinome. In future studies, these MEKK2 inhibitors will be tested for anti-tumor efficacy in genetically engineered mouse models (GEMMs) of breast cancer.