Vimentin Phospho-Malleability is Critical for Maintaining Stemness and Metastatic Properties

项目摘要 转移仍然是乳腺癌治疗的主要障碍。转移的一个关键因素是 上皮向间充质转化(EMT)计划。在EMT过程中，癌细胞获得间质，迁移， 侵袭性和干细胞特性。肿瘤干细胞(CSCs)在肿瘤的转移过程中起着关键作用。 复发和化疗耐药。迫切需要CSC靶向疗法来对抗复发 并防止肿瘤转移。利用CSC漏洞的首要策略是确定基本因素 支持茎的表型。最近的研究表明，EMT过程不会移动细胞 在“上皮”和“间充质”两种状态之间。相反，它是一个连续体，细胞可以维持 同时具有上皮和间充质特征(杂合的E/M)，并具有高度的细胞可塑性和干性。 在EMT过程中，中间丝的变化，特别是间充质相关的上调 细丝蛋白Vimentin，已有文献记载。然而，波形蛋白在EMT和干性中的作用以及 波形蛋白和细胞角蛋白(CK)在高干性杂交E/M细胞中共表达的后果 为人所知。此外，Vimentin的功能还通过动态磷酸化和去磷酸化来调节 在关键部位，也称为“波形蛋白磷化延展性”。初步数据显示，抑制磷- 波形蛋白丝氨酸-56(S56)的延展性抑制三阴性乳房的茎形成并诱导多核形成 肿瘤(TNBC)细胞。它不影响不表达波形蛋白的上皮表型癌细胞， 或者成纤维细胞，它们只表达波形蛋白。在各种磷酸化位点中，磷酸化延展性 单独使用S56可以诱导多核，抑制茎的形成。因此，我们假设Vimentin S56 磷酸化延展性对于维持表达波形蛋白的癌细胞的干性至关重要， 而且它也是肿瘤转移的驱动力。干扰波形蛋白S56的磷酸化将导致选择性 消除肿瘤干细胞，抑制肿瘤转移，提高化疗敏感性。我们提出以下建议 目的是验证这一假设：(1)表征Vimentin S56磷酸化延展性如何影响EMT和茎 单元格属性。(2)确定磷酸化可延展性波形蛋白和CK之间的相互作用如何影响小麦的茎 具有可塑性的杂交E/M细胞。(3)确定Vimentin S56磷酸化延展性对 转移。通过使用混合E/M细胞系的创新实验，我们将识别信号通路和 波形蛋白S56磷酸化延展性背景下的波形蛋白相互作用蛋白。使用患者的TNBC样本， 同基因肿瘤异种移植，以及人患者来源的异种移植，我们将展示其生物学特性 波形蛋白S56磷酸化延展性对体外干细胞形成和体内转移的重要性。了解 Vimentin S56磷酸化延展性的重要性及其受损的影响将有助于开发新的 EMT和CSC靶向治疗易转移的TNBCs。

Project Summary Metastasis remains a major obstacle in the treatment of breast cancer. A key contributor to metastasis is the epithelial-to-mesenchymal transition (EMT) program. During EMT, cancer cells gain mesenchymal, migratory, invasive, and stem cell properties. Cancer stem cells (CSCs) play crucial roles in metastatic progression, cancer recurrence, and chemoresistance. There is an urgent need for CSC-targeting therapies to combat recurrence and prevent metastasis. The premiere strategy to exploit the vulnerabilities of CSCs is to identify essential factors that support the stemness phenotype. Recent research has shown that the EMT process does not move cells between the binary “epithelial” and “mesenchymal” states. Instead, it is a continuum, and the cells can maintain both epithelial and mesenchymal characteristics (hybrid E/M) and possess high cellular plasticity and stemness. During EMT, changes in intermediate filaments, particularly upregulation of the mesenchymal-associated filament protein vimentin, have been documented. However, the role of vimentin in EMT and stemness and the consequence of co-expression of vimentin and cytokeratin (CK) in hybrid E/M cells with high stemness are not known. Besides, the function of vimentin is regulated through dynamic phosphorylation and dephosphorylation on critical sites, also known as “vimentin phospho-malleability. Preliminary data show that inhibiting phospho- malleability of vimentin serine-56 (S56) inhibits stemness and induces multinucleation in triple-negative breast cancer (TNBC) cells. It does not affect cancer cells with epithelial phenotypes, which do not express vimentin, or fibroblasts, which only express vimentin. Among the various phosphorylation sites, the phospho-malleability of S56 alone can induce multinucleation and inhibit stemness. Therefore, we hypothesize that vimentin S56 phospho-malleability is critical for maintaining the stemness of carcinoma cells expressing vimentin, and it is a driver of metastasis. Interfering with vimentin S56 phosphorylation will result in selective elimination of CSCs, inhibition of metastasis, and increased chemo-sensitivity. We propose the following aims to test this hypothesis: (1) Characterize how vimentin S56 phospho-malleability influences EMT and stem cell properties. (2) Define how the interaction between phospho-malleable vimentin and CK affects stemness in hybrid E/M cells with plasticity. (3) Determine the importance of vimentin S56 phospho-malleability on metastasis. Through innovative experiments using hybrid E/M cell lines, we will identify signaling pathways and vimentin-interacting proteins in the context of vimentin S56 phospho-malleability. Using patient TNBC samples, syngeneic tumor xenografts, and human patient-derived xenografts, we will demonstrate the biological importance of vimentin S56 phospho-malleability for stemness in vitro and metastasis in vivo. Understanding the importance of vimentin S56 phospho-malleability and the impact of impairing this will aid in developing novel EMT and CSC-targeting therapies to treat metastasis-prone TNBCs.