Wang Rong, Qi Bing, Dong Yi Wei, Cai Qian Qian, Deng Nian Hui, Chen Qi, Li Chao, Jin Yu Tong, Wu Xing Zhong
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, PR. China.
Key Laboratory of Glycoconjugate Research, Ministry of Public Health, Shanghai, PR. China.
Oncotarget. 2016 Jun 14;7(24):36563-36576. doi: 10.18632/oncotarget.9095.
Integrin αVβ3 is a malignant driver of anchorage-independence and tumor angiogenesis, but its dysregulation in hepatocellular carcinoma (HCC) remains unclear. In this study, we observed that sulfatide significantly promoted integrin αV(ITGAV) expression and wound closure in HCC. We also noted that elevated sulfatide profoundly stimulated integrin αVβ3 clustering and signaling. In the cells with integrin αVβ3 clustering induced by sulfatide, integrin β3 subunit was phosphorylated. Simultaneously, focal adhesion kinase (FAK), Src and paxillin were also phosphorylated. Treatment with FAK inhibitor resulted in robust suppression of FAK-Y397 and Src-Y416 phosphorylation stimulated by sulfatide, but not suppression of integrin β3 phosphorylation. Src inhibitors repressed Src-Y416 and FAK Y861 and Y925 phosphorylation, but not FAK-Y397 and integrin β3 phosphorylation. After mutation of integrin β3 (Y773F and Y785F), FAK or Src phosphorylation failed to be stimulated by sulfatide. Moreover, β3 Y773 and Y785 phosphorylation was suppressed by insulin-like growth factor receptor knockdown even in cells stimulated by sulfatide. In assays of immunoprecipitation and immunostaining with integrin αV or β3 antibody, labeled sulfatide was found in the complex and co-localized with integrin αVβ3. Taken together, this study demonstrated that elevated sulfatide bound to integrin αVβ3 and induced clustering and phosphorylation of αVβ3 instead of matrix ligand binding, triggering outside-in signaling.
整合素αVβ3是锚定非依赖性和肿瘤血管生成的恶性驱动因子，但其在肝细胞癌（HCC）中的失调仍不清楚。在本研究中，我们观察到硫苷显著促进HCC中整合素αV（ITGAV）的表达和伤口闭合。我们还注意到硫苷水平升高会强烈刺激整合素αVβ3的聚集和信号传导。在由硫苷诱导整合素αVβ3聚集的细胞中，整合素β3亚基发生磷酸化。同时，粘着斑激酶（FAK）、Src和桩蛋白也发生磷酸化。用FAK抑制剂处理可强烈抑制硫苷刺激的FAK-Y397和Src-Y416磷酸化，但不能抑制整合素β3磷酸化。Src抑制剂可抑制Src-Y416以及FAK Y861和Y925磷酸化，但不能抑制FAK-Y397和整合素β3磷酸化。整合素β3（Y773F和Y785F）突变后，硫苷无法刺激FAK或Src磷酸化。此外，即使在硫苷刺激的细胞中，胰岛素样生长因子受体敲低也会抑制β3 Y773和Y785磷酸化。在用整合素αV或β3抗体进行的免疫沉淀和免疫染色试验中，发现标记的硫苷存在于复合物中，并与整合素αVβ3共定位。综上所述，本研究表明，升高的硫苷与整合素αVβ3结合，诱导αVβ3的聚集和磷酸化，而不是与基质配体结合，从而触发由外向内的信号传导。
