SIPA1调控乳腺癌及其微环境细胞氧化还原状态促进癌细胞逃逸的分子机制研究

Cancer cells escape is the main cause of death in patients with tumor metastasis. As one of the primary candidate underlying the metastasis efficiency modifier locus 1, SIPA-1 has been found to be capable of promoting breast cancer cell invasion and metastasis. It is well known that regulation of cellular redox status plays important role during breast cancer metastasis. In this project, we are going to detect the cellular redox status and SIPA1 expression in breast cancer cells, to clarify the correlation of cellular redox status and SIPA1 expression level with the metastatic status of breast cancer. We will also study the functional feather and ATP production in mitochondria of breast cancer cells, incorporating the candidate genes selected from chip-seq data, to identify the key molecules and signaling pathways to participate the regulation on redox status of breast cancer cell by SIPA1. Moreover, we are going to treat vascular endothelial cells and macrophage cells with exosomes secreted from breast cancer cells, to clarify the molecules and pathways that play crucial roles during the process that SIPA1 promotes certain genes expression in breast cancer cells, to affect the composition of exosomes from breast cancer cells and regulate the cellular redox status. Finally, we will discover the molecular mechanism of SIPA1 on regulating the breast cancer cell escape process and invasion into vascular endothelial cells to approach metastasis. Furthermore, we are going to discover the new targets to regulate the breast cancer metastasis, and to provide theoretical and technic supporting for breast cancer diagnose and therapy development.

癌细胞逃逸是癌症转移导致患者死亡的主要原因之一。信号诱导增殖相关蛋白SIPA1能促进乳腺癌细胞侵袭转移，且氧化还原状态的调控在乳腺癌细胞转移进程中起重要作用。本项目拟检测乳腺癌细胞Redox值和SIPA1表达水平，明晰氧化还原状态和SIPA1表达水平与乳腺癌转移程度的相关性；检测乳腺癌细胞中线粒体功能特征以及ATP量，结合Chip测序分析候选基因，确定SIPA1直接调控乳腺癌细胞氧化还原状态的关键分子及信号通路；进一步用乳腺癌细胞外泌体处理肿瘤微环境相关的血管内皮细胞和巨噬细胞，检测细胞氧化还原状态、血管内皮细胞通透性和巨噬细胞转型，明晰SIPA1通过调控乳腺癌细胞基因表达、改变癌细胞外泌体组分、调节氧化还原状态的关键分子和信号通路，揭示SIPA1调控乳腺癌细胞逃逸、透过血管内皮细胞实现转移的调控机制。本项目将发现调控乳腺癌细胞转移新靶点，为转移性乳腺癌检测和治疗提供技术支撑和理论依据。

本项目确定了SIPA1调控乳腺癌细胞有氧糖代谢的靶基因及其相关信号通路分子，明确癌细胞分泌微囊泡，募集巨噬细胞至肿瘤微环境，进而促进乳腺癌细胞侵袭转移机制；明确了SIPA1分子是一个新的转录因子，通过其特定DNA结合域靶向一系列下游基因，对调控癌细胞转移起关键作用。具体结果如下：（1）通过检测乳腺癌细胞Redox值和SIPA1表达，明晰了癌细胞氧化还原状态和SIPA1表达水平与乳腺癌转移程度的相关性；通过分析转录组数据和细胞实验，发现SIPA1蛋白正调节与有氧糖酵解途径相关基因HK2和LDHA表达以及LDH活性，增强癌细胞有氧糖酵解；通过检测线粒体功能特征以及ATP量，发现SIPA1抑制细胞总ATP生成，且调节ATP来源由线粒体向有氧糖酵解转变；通过上调PDK1抑制线粒体活性。Chip-seq数据分析及细胞和小鼠实验，明晰SIPA1蛋白正调节HIF-2a介导低氧应答过程和有氧糖酵解代谢，靶向调控细胞氧化还原状态关键基因EPAS1启动子，增强EPAS1基因转录活性和关键信号通路分子HK1和PDK2的表达，以及下游糖酵解、线粒体抑制基因PDK1以及MMP9表达，促进SIPA1/HIF-2α介导的有氧糖酵解，进而抑制乳腺癌生长和转移。（2）发现富含TSP1的外泌体组分能显著促进肿瘤细胞穿过血管内皮细胞，显著降低血管内皮细胞胞间连接分子VE-cadherin和ZO-1表达，减弱其成管能力，通过破坏血管内皮胞间完整性，促进癌细胞转移。（3）解析了SIPA通过直接靶向乳腺癌细胞MYH9的启动子结合并上调MYH9转录活性，通过分泌含MYH9分子的外泌体至肿瘤微环境，募集巨噬细胞，进而促进乳腺癌细胞逃逸和转移。（4）确定了纤黏蛋白诱导SIPA1通过与importinβ1相互作用入核，SIPA1蛋白通过其C端Ser764-Ala864结构域与具有TGAGTCAB基序的DNA序列直接结合，确定了一系列SIPA1的靶基因，为进一步阐明SIPA1蛋白功能奠定了基础。本项目解析了SIPA1调控乳腺癌细胞转移新机制，明确了一个新转录因子，为转移性乳腺癌检测和治疗提供了理论依据和技术支撑。