膜蛋白KCNN1通过ERLIN2/Cyclin B1通路促进乳腺癌增殖和转移的分子机制及临床意义

Membrane proteins are important in the development and target therapy of breast cancer. In previous study, we screened a novel gene, encoding the membrane protein KCNN1, which could promote breast cancer metastasis. We found that KCNN1 was overexpressed in breast cancer tissue and its expression correlated with the poor prognosis of breast cancer patients. Overexpression of KCNN1 enhanced the proliferation, migration and invasion abilities of breast cancer cell and promoted the growth of subcutaneous implanted tumor and lung metastasis. KCNN1 interacted with ERLIN2 and facilitated ERLIN2 phosphorylation. KCNN1 further promoted the ubiquitination and stability of Cyclin B1. However, the molecular mechanisms underlying how KCNN1 regulates ERLIN2/Cyclin B1 pathway is still unclear. In this study, we plan to use some modern molecular biology methods, such as CRISPR technology, LC-MS/MS and protein interaction assays. We aim to identify a protein kinase that can phosphorylate ERLIN2 and explore the mechanisms underlying how KCNN1 promotes the proliferation and metastasis of breast cancer through ERLIN2/Cyclin B1 pathway. We will also demonstrate the correlation between the expression of KCNN1, phosphorylated ERLIN2 and Cyclin B1 and expound their clinical significance. This study will provide the basis for looking for novel target protein and developing new drugs for curing breast cancer metastasis.

膜蛋白在乳腺癌发生发展及靶向治疗中具有重要作用。前期研究中，我们通过CRIPSRa技术筛选出促乳腺癌转移的新的膜蛋白基因KCNN1。研究发现：①KCNN1高表达于乳腺癌组织且与患者较差的预后相关；②过表达KCNN1增强乳腺癌细胞增殖、迁移和侵袭能力，促进裸鼠皮下成瘤及肺转移；③KCNN1与ERLIN2相互作用，促进ERLIN2的磷酸化及下游Cyclin B1的泛素化及稳定性，但KCNN1调控ERLIN2/Cyclin B1通路的具体机制仍不清楚。本研究拟利用CRISPR技术、质谱分析、相互作用研究等现代分子生物学方法，旨在：①鉴定能够磷酸化ERLIN2的激酶；②明确KCNN1通过ERLIN2/Cyclin B1信号通路促进乳腺癌增殖和转移的机理；③阐明KCNN1、磷酸化ERLIN2与Cyclin B1的表达相关性及临床意义。本研究将为寻找新的候选靶分子及研发新的肿瘤转移治疗药物提供依据。

乳腺癌转移是导致患者预后较差的主要原因。乳腺癌转移机制复杂，明确有效治疗靶点及作用机制，有助于为乳腺癌转移靶向治疗提供依据。项目前期研究利用细胞膜蛋白CRISPR/Cas9文库在乳腺癌肺转移模型中筛选出潜在的促转移基因KCNN1，初步发现KCNN1可能通过与ERLIN2相互作用影响Cyclin B1的K63泛素化和稳定性。本研究旨在进一步探索KCNN1在乳腺癌细胞和裸鼠成瘤模型中的生物学作用，明确KCNN1与ERLIN2相互作用的机制，探索KCNN1通过结合ERLIN2影响Cyclin B1泛素化和稳定性，介导乳腺癌细胞增殖转移的机制。本研究首先制备了KCNN1的单克隆抗体，研究发现过表达（敲低）KCNN1能够促进（抑制）乳腺癌细胞的增殖、迁移和侵袭；ERLIN2与KCNN1在细胞质中共定位，并通过其1-266aa氨基酸区域与KCNN1相互作用；KCNN1通过ERLIN2促进Cyclin B1的K63位点泛素化和稳定性；Cyclin B1作为KCNN1的下游分子介导了KCNN1对乳腺癌细胞增殖、迁移和侵袭作用。本研究为把 KCNN1 开发成一个新的抗乳腺癌治疗靶点奠定基础，为乳腺癌的靶向治疗提供新的方向。