Department of Molecular Pharmacology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Clin Cancer Res. 2011 Apr 1;17(7):1796-806. doi: 10.1158/1078-0432.CCR-10-3216. Epub 2011 Feb 25.
We previously reported that nelfinavir (NFV) induces G(1) cell-cycle block and apoptosis selectively in liposarcoma cell lines due to increased SREBP-1 (sterol regulatory element binding protein-1) expression in the absence of increased transcription. We postulate that NFV interferes with regulated intramembrane proteolysis of SREBP-1 and ATF6 (activating transcription factor 6).
Time-lapse, confocal microscopic studies show that NFV inhibits the nuclear translocation of full-length SREBP-1-EGFP and ATF6-EGFP fusion proteins. siRNA-mediated knockdown of site-1 protease (S1P) and/or site-2 protease (S2P) leads to inhibition of SREBP-1 intracellular trafficking to the nucleus and reduces liposarcoma cell proliferation. Treatment of LiSa-2 liposarcoma cells with 3,4-dichloroisocoumarin, a serine protease inhibitor of S1P, did not affect SREBP-1 processing. In contrast, 1,10-phenanthroline, an S2P-specific inhibitor, reproduces the molecular and biological phenotypes observed in NFV-treated cells, which implicates S2P as a target of NFV. In vivo evaluation of NFV in a murine liposarcoma xenograft model leads to inhibition of tumor growth without significant toxicity.
NFV-induced upregulation of SREBP-1 and ATF6 results from inhibition of S2P, which together with S1P mediates regulated intramembrane proteolysis from their precursor to their transcriptionally active forms. The resulting endoplasmic reticulum (ER) stress and concurrent inhibition of the unfolded protein response induce caspase-mediated apoptosis.
These results provide new insight into the mechanism of NFV-mediated induction of ER stress and cell death in liposarcomas and are the first to report targeting S2P for cancer therapy.
我们之前报道过，奈非那韦（NFV）在不存在转录增加的情况下，通过增加 SREBP-1（固醇调节元件结合蛋白-1）的表达，选择性地诱导脂肪肉瘤细胞系中的 G1 细胞周期阻滞和细胞凋亡。我们假设 NFV 干扰 SREBP-1 和 ATF6（激活转录因子 6）的调节性跨膜蛋白水解。
延时、共聚焦显微镜研究表明，NFV 抑制全长 SREBP-1-EGFP 和 ATF6-EGFP 融合蛋白的核转位。通过 siRNA 介导的位点 1 蛋白酶（S1P）和/或位点 2 蛋白酶（S2P）的敲低，导致 SREBP-1 向细胞核内的细胞内转运受到抑制，并减少脂肪肉瘤细胞增殖。用 3,4-二氯异香豆素（S1P 的丝氨酸蛋白酶抑制剂）处理 LiSa-2 脂肪肉瘤细胞不会影响 SREBP-1 的加工。相比之下，1,10-菲咯啉（一种 S2P 特异性抑制剂）再现了 NFV 处理细胞中观察到的分子和生物学表型，这表明 S2P 是 NFV 的靶标。在脂肪肉瘤异种移植模型中体内评价 NFV 导致肿瘤生长抑制而无明显毒性。
NFV 诱导的 SREBP-1 和 ATF6 的上调是由于 S2P 的抑制，S2P 与 S1P 一起介导从其前体到其转录活性形式的调节性跨膜蛋白水解。由此产生的内质网（ER）应激和同时抑制未折叠蛋白反应诱导 caspase 介导的细胞凋亡。
这些结果为 NFV 介导的 ER 应激和脂肪肉瘤细胞死亡的机制提供了新的见解，并且是首次报道针对 S2P 进行癌症治疗。
