剪接因子U2AF2通过介导CD44s/v亚型转化调控黑色素瘤转移中力学信号转导分子机制

Under blood flow conditions, the molecular mechanisms of mechanotransduction regulating melanoma hematogenous metastasis remain elusive. The aberrantly high levels of variant isoforms of CD44 (CD44v) on tumor are closely related to tumor metastatic potentials. The applicant’s previous studies funded by National Natural Science Foundation for Youth found: 1) Highly metastatic melanoma cell lines express aberrantly high levels of CD44v8-v10; 2) Splicing factor U2AF2 promotes CD44s/v isoform switching on melanoma cells. These findings imply that U2AF2 guides alternative splicing of CD44v exons, thereby enhancing the adhesion of melanoma to endothelium, transmitting mechano-signals and promoting tumor migration. Therefore, in the current proposal, we aim to conduct in vitro and in vivo investigation: 1) to determine CD44v exon splicing mechanism mediated by U2AF2 and the effect of U2AF2-mediated CD44 alternative splicing on melanoma adhesion under shear conditions; 2) to study the spatial-temporal dynamics of signaling initiated by CD44v-selectin bond tension to promote tumor migration with living cell imaging; 3) to determine the effect of tumor U2AF2-mediated CD44s/v switching on melanoma adhesion-induced endothelial adherens junction breakdown in flow by immunofluorescence and traction force microscopy; 4) to elucidate the effect of CD44s/v switching on tumor adhesion to blood vessel wall, vessel permeability and experimental lung metastasis in nude mouse models. The results from this proposal will shed light on the molecular mechanisms underlying CD44v-mediated mechanotransduction in tumor and enrich the theory related to tumor metastasis.

血液流场环境中黑色素瘤血道转移的力学信号机制尚不清楚。癌细胞表面变异型CD44v高表达与肿瘤侵袭密切相关。申请人承担的青年基金项目发现：1）侵袭性黑色素瘤异常表达CD44v8-v10；2）剪接因子U2AF2促进CD44s/v亚型转化，提示U2AF2指导CD44v8-v10选择性剪接，增强黑色素瘤与内皮细胞粘附，传递力学信号，促进肿瘤迁移。拟开展体内外研究：1）考察U2AF2对CD44v剪接的调控方式及CD44选择性剪接对黑色素瘤细胞流场下粘附能力的影响；2）活细胞成像揭示CD44v-选择素键张力激活肿瘤迁移信号的时空动态规律；3）免疫荧光及牵引力显微镜研究U2AF2介导的CD44s/v转化参与流场下肿瘤粘附调控内皮细胞粘合连接打开的机制；4）裸鼠模型阐明CD44s/v转化对肿瘤细胞血管壁粘附、血管通透性及肺转移的影响。该研究可望解释CD44v调控的肿瘤力学信号转导机制、丰富肿瘤转移理论。

血液流场环境中黑色素瘤黏附对细胞的表观调控及力学信号转导的机制并不清楚。项目研究了CD44v介导的黑色素瘤与内皮细胞的黏附机制。采用CRISPR敲除，流式细胞技术，免疫荧光及活体成像等技术，发现了细胞骨架及分子马达蛋白，FAK, Src, PKC, MLC, CaMKII, RacGAP1等参与CD44v介导的黑色素瘤细胞对内皮细胞的黏附及迁移。利用准3-D 显微镜检测Rho 家族小G 蛋白激酶时序性活化，发现研究RacGAP1、FAK、Src及PKC的干预等影响黑色素瘤细胞在内皮细胞表面的迁移。通过spinning-disk confocal microscopy, 发现敲除或药物抑制btk或FAK显著降低肿瘤细胞对内皮细胞细胞的稳定黏附及肿瘤穿越内皮细胞的能力。利用RNA-seq高通量检测了原代病人细胞黏附及干预后差异表达的转录组，发现黏附能引起表观基因，SIRT6, JMJD6及HDAC9的表达上升,CD44v的敲除能降低这些基因的表达。通路富集分析发现：细胞骨架通路及细胞迁移通路基因富集。并且我们发现JMJD6参与U2AF2对CD44v的选择性剪辑。JMJD6及SIRT6的抑制能有效降低肿瘤细胞增殖及转移。该项目的完成为进一步研究力学信号转导与表观调控的关系及寻找抑制血道转移的分子靶标奠定了基础。