SPA-1蛋白调控乳腺癌细胞浸润和转移分子机制研究

Cancer metastasis is the main cause for the dearth of cancer patients. To unravel the mechanism of cancer metastasis is now becoming the focus of current cancer researches. SPA-1 (signal induced proliferation -associated protein-1) is a Rap1GAP which plays an important role in cell proliferation, adhesion, and migration. During a survey of breast cancer patients, we found SPA-1 protein is overexpressed in high grade breast tissues and migrated cancer cells both in breast tissue and lymph nodes. SPA-1 also promotes MDA-MB-231 cell invation and migration in vitro. These findings suggest that SPA-1 protein is probably a novel target molecule of cancer metastasis.This project aims to dissect the mechanism by which SPA-1 promotes breast cancer metastasis at multiple levels in the following studies: 1) Confirm that SPA-1 promotes breast cancer cell metastasis both in vitro and in vivo. 2) Dissect the mechanisms that how SPA-1regulates breast cancer cell morphological changes and signal transduction pathways to accelerate its invasion and migration; 3) Investigate the interaction between SPA-1 and integrins; 4) Investigate the interaction of SPA-1 regulated breast cancer cell with endothelial cells to understand how cancer cells set up a benefit environment to metastasis. Our ultimate goals are to create a novel biomarker for early detection of cancer metastasis and to develop effective therapeutic target for cancer treatment.

癌细胞转移是癌症患者死亡的主要原因, 深入阐明其机制具有重要理论与实践意义。小G蛋白Rap1特异性GAP（SPA-1）是乳腺癌转移效率修饰位点Mtes1中主要候选基因之一。我们前期研究发现：乳腺癌转移患者癌细胞SPA-1表达显著高于非转移患者，而SPA-1表达沉默的人乳腺癌细胞迁移能力明显下降；SPA-1可与integrin相互作用；可促进MMP9表达；诱导VEGF表达促进肿瘤血管新生。上述实验进展提示，SPA-1是参与癌细胞转移的关键分子。本项目拟采用细胞力学、双光子显微成像和高分辨光声成像等技术在细胞和整体动物水平阐述SPA-1与integrrin直接相互作用调控肿瘤细胞骨架重塑；揭示通过影响FAK磷酸化等信号通路促进蛋白酶表达与活性而促进肿瘤组织胞外基质降解以及诱导VEGF表达而促进肿瘤组织血管新生的精细分子机制，为基于SPA-1的癌症转移早期诊断和干预策略提供实验依据。

癌细胞转移是导致癌症病人死亡的主要因素，深入阐明其机制具有重要理论与实践意义。项目期间，我们的研究发现小G蛋白Rap1特异性GAP, SIPA-1 ,促进乳腺癌细胞浸润和转移。SIPA-1蛋白在乳腺癌转移患者的癌细胞中表达显著高于非转移患者乳腺癌细胞。核定位SIPA-1通过作用于Integrinβ1启动子启动Integrin蛋白的表达，从而激活Integrin介导的FAK/AKT信号通路，增加基质金属蛋白酶MMP9的表达，促进乳腺癌细胞粘附、迁移和浸润。我们进一步发现SIPA1通过乳腺癌细胞外泌体调节血管内皮细胞通透性和能量代谢。Sipa1 基因沉默后的MDA-MB-231/sh-Sipa1外泌体刺激组的成管数目显著高于MDA-MB-231外泌体体刺激组的成管数目，能显著促进血管内皮细胞伪足形成。更进一步，采用Redox Ratio技术，利用共聚焦显微镜检测HUVCE细胞内FAD和NADH自发荧光强度，计算FAD/(FAD+NADH) Redox比值。结果表明MDA-MB-231及MDA-MB-231/sh-Sipa1细胞外泌体刺激，增加HUVCE细胞FAD/(FAD+NADH) 比值，提高HUVCE细胞能量代谢水平。Sipa1基因下调后的乳腺癌外泌体刺激后，能量代谢进一步增强。本项目不仅将揭示细胞内SIPA-1蛋白调控癌细胞发生浸润和迁移的时空规律，而且通过研究乳腺癌外泌体对肿瘤微环境的影响，特别是对HUVEC细胞的通透性和能量代谢情况做出初步研究，为SIPA-1成为癌症转移早期诊断新标记物和治疗新靶点、新方案提供新的理论依据和技术支撑。