细胞表面热休克蛋白90在人前列腺癌细胞中的表达及其对侵袭能力的影响

FAK/c-Src signaling pathway mediates the expression of cell surface HSP90 in cultured human prostate cancer cells and its association with their invasive capability

目的 观察细胞表面热休克蛋白90（HsPgo）在高侵袭性人前列腺癌细胞PC3中的表达,及其对细胞侵袭能力的影响,探讨其可能的作用机制.方法 采用免疫荧光染色和膜蛋白生物素标记法,检测HSPgO在PC3细胞表面的表达;采用Boyden小室侵袭实验观察细胞侵袭能力的改变;采用Western blot和免疫共沉淀法,检测局部黏着斑激酶（FAK）、c-Src蛋白磷酸化水平及其两者相互作用的变化;采用RNA干扰技术下调FAK蛋白表达,应用抑制剂PP2抑制Src激酶活性,观察细胞FAK/Src信号通路及其侵袭能力的改变.结果 PC3细胞表面表达HSP00.HSPg0特异性抗体可显著降低PC3细胞体外侵袭能力,且伴有FAK tyr 397、576、577、925及c-Src tyr 416磷酸化水平的显著下降,以及FAK与c-Src蛋白相互结合的明显减弱.经小分子干扰RNA（siRNA）有效干扰FAK表达,或采用PP2抑制Src激酶活性,均可显著抑制PC3细胞的侵袭性,但同时联合应用抗HSPg0抗体,未有协同作用.小室侵袭实验结果显示,PC3细胞经抗HSPg0抗体作用后,穿膜细胞数为（39.57 4±7.54）个,而对照组和lgG对照组分别为（105.70±12.16）个和（110.60 4±11.61）个,差异均有统计学意义（P〈0.001）.结论 细胞表面HSP90可经FAK/c-Src信号通路促进体外培养前列腺癌细胞的侵袭,提示其可能是潜在的对肿瘤转移实施防治的一个靶点.

Objective To investigate the expression of heat shock protein 90 （HSP90） on the cell surface of highly invasive human prostate cancer cells PC3 and its possible molecular mechanisms of its effect on cell invasion through analyzing FAK/Src signaling pathway. Methods The expression of cell surface HSP90 on PC3 cells was studied by immunofluorescence staining and surface biotinylation assay respectively. A specific HSP90 antibody was used to inhibit the cell surface HSP90. In vitro cell invasion was assessed by modified Boyden chambers. Phosphorylated FAK on tyr 397, 576, 577 and 925, and phosphorylated c-Src on tyr 416 were examined by Western blot assay. The association between FAK and c-Src was analyzed by immunoprecipitation. The effects of FAK knockdown by siRNA or Src kinases inhibitor PP2, with or without anti-HSP90 antibody, on PC3 cell invasion were also evaluated. Results A pool of HSP90 was detected on the cell surface of PC3 cells. A specific HSP90 antibody significantly retarded tumor cell invasion. Concomitant with this finding, targeting cell surface HSP90 significantly inhibited the phosphorylations of FAK and c-Src, and also the interactions between FAK and c-Src. FAK knockdown or PP2 dramatically suppressed cell invasion, however, anti-HSP90 antibody didn＇t further inhibit cell invasion. Conclusions Cell surface HSP90 promotes human prostate cancer cell invasion through a FAK/c-Src signaling, with may be a novel therapeutic target against metastatic tumors.