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

摘要： 大约15-20%的乳腺癌具有三阴性表型（ER和PR表达阴性，缺乏ErbB 2过表达），这与侵袭性癌症和有限的治疗选择相关。我们已经开发了一种用于确定特定激酶在乳腺癌细胞的肿瘤发生和转移中的作用的体内筛选。三阴性和三阳性乳腺癌细胞系用于shRNA介导的特异性激酶敲低。将遗传改变的细胞系（除了特异性shRNA基因敲低之外还表达荧光素酶）注射到雌性SCID小鼠的乳房脂肪垫中。肿瘤生长和血管化是 使用微探头超声系统在8周时间内纵向监测。通过生物发光成像监测转移。该测定提供了用于分析控制乳腺肿瘤生长、血管形成和转移的蛋白质的体内筛选。MAP 3 K是调节MAP的第一层激酶， 激酶信号通路，其导致MAPK、ERK 1/2、p38、JNK和ERK的活化。MAPSKs控制对调节细胞周期、细胞因子和蛋白酶表达以及细胞凋亡重要的基因的表达。在9种MAP 3 K的筛选中，使用MDA-MB-231鉴定MEKK 2为转移的关键调节剂 在体内肿瘤发生测定中，对BT474（三阴性基底）和BT474（三阳性管腔）乳腺腺癌细胞进行比较。MEKK 2是一种MAP 3 K，通过激活MKK 7和MEKS调节JNK和ERK 5通路的激活。我们已经表明MEKK 2表达是EGFR（ErbBI）和EGFR（ErbBI）所必需的。 ErbB 2/Neu分别在MDA-MB-231和BT474细胞中激活ERKS。我们的假设是MAP 3 K MEKK 2作为细胞信号网络中的关键信号节点，响应ErbB和可能的其他酪氨酸激酶刺激肿瘤生长和转移。该提案的目标是从遗传学上确定MEKK 2在三阴性乳腺癌肿瘤生长和转移中的作用，并开发MEKK 2小分子抑制剂。具体目标1涉及使用体内异种移植物测定来定义MEKK 2-MEK 5-ERK 5信号传导在三阴性乳腺癌细胞的肿瘤发生和转移中的作用。在特定 目的2，我们建议阐明MEKK 2被ErbB 1/2激活的机制，使用MEKK 2突变体在生物化学和基于细胞的激活试验中。在具体目标3中，将开发小分子生物化学筛选以鉴定特异性抑制MEKK 2激酶活性的化合物。MEKK 2抑制剂将是 在基于细胞的测定中测试MAPK途径特异性，并分析针对激酶组的特异性。在未来的研究中，这些MEKK 2抑制剂将在乳腺癌的基因工程小鼠模型（GEMM）中测试抗肿瘤功效。

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.