Critical role for Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein in anoikis resistance and distant tumor formation
Critical role for Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein in anoikis resistance and distant tumor formation
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DOI：
10.1093/jnci/djk182
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发表时间：
2007-05-16
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影响因子：
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通讯作者：
Schimmer, Aaron D.
中科院分区：
文献类型：
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作者：
Mawji, Imtiaz A.;Simpson, Craig D.;Schimmer, Aaron D.
背景：正常上皮细胞通过启动caspase激活的死亡受体途径，在失去对细胞外基质的锚定时发生凋亡。然而，具有转移潜能的恶性上皮细胞能够抵抗肿瘤，并且能够以不依赖于锚定的方式存活。我们假设c- fas相关的死亡结构域样白介素-1转换酶样抑制蛋白（FLIP），一种内源性的死亡受体信号抑制剂，可能抑制anoikis。方法采用3-（4,5-二甲基噻唑-2-基）-5-（3-羧基甲氧基苯基）-2-（4-磺胺苯基）- 2h -四氮唑内盐法和Annexin V染色法观察贴壁和悬浮培养的PPC-1前列腺癌细胞的生存能力和凋亡情况。流式细胞术检测死亡受体Fas的表达和caspase 8的活化。用逆转录-聚合酶链反应测定Fas配体的表达。免疫印迹法检测FLIP蛋白表达。利用FLIP的小分子抑制剂（包括死亡受体敏化剂5809354）和靶向FLIP的小干扰RNA (si)，在体外和体内评估FLIP抑制对前列腺癌细胞的影响。所有统计检验均为双侧检验。结果悬浮培养的sppc -1细胞，尽管Fas（0小时比8小时，平均相对表达百分比= 100%比135%，差异= 35%，95%可信区间[CI] = 10% ~ 61%， P= 0.02）和Fas L（0小时比24小时，平均相对表达百分比= 100%比208%，差异= 108%,95% CI = 18% ~ 197%， P= 0.02）的表达增加，但仍能抵抗anoikis。siRNA或5809354使前列腺癌细胞对anoikis敏感（对照siRNA与FLIP siRNA在10 nM，平均相对存活率= 95%对51%，差异= 44%,95% CI = 34%至54%，P< 0.001；对照与5809354在20 μM，平均相对存活率= 96%对52%，差异= 44%,95% CI = 13%至75%，P= 0.015）。抑制FLIP表达特异性激活悬浮而非贴壁条件下生长的PPC-1细胞中的caspase 8，并降低体内循环PPC-1细胞的转移潜力。结论flip可能是anoikis的抑制因子，因此可能是抗转移治疗策略的靶点。
BackgroundNormal epithelial cells undergo anoikis, or apoptosis on loss of anchorage to the extracellular matrix, by initiating the death receptor pathway of caspase activation. However, malignant epithelial cells with metastatic potential resist anoikis and can survive in an anchorage-independent fashion. We hypothesized that c-Fas–associated death domain–like interleukin-1–converting enzyme–like inhibitory protein (FLIP), an endogenous inhibitor of death receptor signaling, may suppress anoikis.MethodsWe assessed viability and apoptosis of PPC-1 prostate cancer cells cultured in adherent and suspension conditions using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt and Annexin V staining assays. Expression of the death receptor Fas and activation of caspase 8 were measured using flow cytometry. Expression of Fas ligand was measured by reverse transcription–polymerase chain reaction. FLIP protein expression was measured by immunoblotting. Small-molecule inhibitors of FLIP (including the death receptor sensitizer 5809354) and small-interfering (si) RNA directed against FLIP were used to assess the effects of FLIP inhibition on anoikis of prostate cancer cells in vitro and in vivo. All statistical tests were two-sided.ResultsPPC-1 cells cultured in suspension resisted anoikis, despite increased expression of Fas (0 versus 8 hours, mean relative percent expression = 100% versus 135%, difference = 35%, 95% confidence interval [CI] = 10% to 61%;P= .02) and Fas L (0 versus 24 hours, mean relative percent expression = 100% versus 208%, difference = 108%, 95% CI = 18% to 197%;P= .02). Knockdown of FLIP expression by siRNA or treatment with 5809354 sensitized prostate cancer cells to anoikis (control siRNA versus FLIP siRNA at 10 nM, mean relative percent viability = 95% versus 51%, difference = 44%, 95% CI = 34% to 54%;P<.001; control versus 5809354 at 20 μM, mean relative percent viability = 96% versus 52%, difference = 44%, 95% CI = 13% to 75%;P= .015). Inhibition of FLIP expression specifically activated caspase 8 in PPC-1 cells grown in suspension but not adherent conditions and decreased the metastatic potential of circulating PPC-1 cells in vivo.ConclusionsFLIP may be a suppressor of anoikis and therefore a possible target for antimetastatic therapeutic strategies.