Data_Sheet_2_ERK5 Is Required for Tumor Growth and Maintenance Through Regulation of the Extracellular Matrix in Triple Negative Breast Cancer.DOCX

Conventional mitogen-activated protein kinase (MAPK) family members regulate diverse cellular processes involved in tumor initiation and progression, yet the role of ERK5 in cancer biology is not fully understood. Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. ERK5 signaling contributes to drug resistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT). More recently a role for ERK5 in regulation of the extracellular matrix (ECM) has been proposed, and here we investigated the necessity of ERK5 in TNBC tumor formation. Depletion of ERK5 expression using the CRISPR/Cas9 system in MDA-MB-231 and Hs-578T cells resulted in loss of mesenchymal features, as observed through gene expression profile and cell morphology, and suppressed TNBC cell migration. In vivo xenograft experiments revealed ERK5 knockout disrupted tumor growth kinetics, which was restored using high concentration Matrigel™ and ERK5-ko reduced expression of the angiogenesis marker CD31. These findings implicated a role for ERK5 in the extracellular matrix (ECM) and matrix integrity. RNA-sequencing analyses demonstrated downregulation of matrix-associated genes, integrins, and pro-angiogenic factors in ERK5-ko cells. Tissue decellularization combined with cryo-SEM and interrogation of biomechanical properties revealed that ERK5-ko resulted in loss of key ECM fiber alignment and mechanosensing capabilities in breast cancer xenografts compared to parental wild-type cells. In this study, we identified a novel role for ERK5 in tumor growth kinetics through modulation of the ECM and angiogenesis axis in breast cancer.

经典丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）家族成员可调控肿瘤发生与进展相关的多种细胞进程，但ERK5在癌症生物学中的作用尚未完全阐明。三阴性乳腺癌（triple-negative breast cancer, TNBC）因肿瘤侵袭性强且缺乏靶向治疗手段，成为临床诊疗的难题。ERK5信号通路可通过多种不同机制，包括激活上皮间质转化（epithelial-to-mesenchymal transition, EMT），参与耐药性产生与肿瘤转移进展。近年来有研究提出ERK5可调控细胞外基质（extracellular matrix, ECM），本研究旨在探究ERK5在三阴性乳腺癌肿瘤形成中的必要性。采用CRISPR/Cas9系统敲除MDA-MB-231与Hs-578T细胞中的ERK5基因后，经基因表达谱与细胞形态学观察发现，细胞间质表型丢失，且三阴性乳腺癌细胞的迁移能力受到抑制。体内异种移植实验结果显示，ERK5敲除会破坏肿瘤生长动力学，而高浓度Matrigel™可逆转这一现象；同时ERK5敲除会降低血管生成标志物CD31的表达水平。上述研究结果表明ERK5参与调控细胞外基质（ECM）及其结构完整性。RNA测序分析结果显示，在ERK5敲除细胞中，基质相关基因、整合素以及促血管生成因子的表达均出现下调。组织脱细胞联合冷冻扫描电镜（cryo-SEM）以及生物力学特性检测结果显示，与亲本野生型细胞相比，ERK5敲除的乳腺癌异种移植瘤中关键ECM纤维排列及机械感应能力均出现丧失。本研究明确了ERK5可通过调控细胞外基质与血管生成轴，影响乳腺癌的肿瘤生长动力学，揭示了其在癌症生物学中的全新功能。