Mawji Imtiaz A, Simpson Craig D, Hurren Rose, Gronda Marcela, Williams Moyo A, Filmus Jorge, Jonkman James, Da Costa Ralph S, Wilson Brian C, Thomas Michael P, Reed John C, Glinsky Gennadi V, Schimmer Aaron D
Ontario Cancer Institute, Princess Margaret Hospital, 610 University Ave, Toronto, ON, Canada, M5G 2M9.
J Natl Cancer Inst. 2007 May 16;99(10):811-22. doi: 10.1093/jnci/djk182.
Normal 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.
We 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.
PPC-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 microM, 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.
FLIP may be a suppressor of anoikis and therefore a possible target for antimetastatic therapeutic strategies.
正常上皮细胞在失去与细胞外基质的锚定后会发生失巢凋亡，即通过启动半胱天冬酶激活的死亡受体途径而凋亡。然而，具有转移潜能的恶性上皮细胞能够抵抗失巢凋亡，并能以不依赖锚定的方式存活。我们推测，c-Fas相关死亡结构域样白介素-1转换酶样抑制蛋白（FLIP），一种死亡受体信号的内源性抑制剂，可能会抑制失巢凋亡。
我们使用3-（4,5-二甲基噻唑-2-基）-5-（3-羧甲氧基苯基）-2-（4-磺基苯基）-2H-四唑内盐和膜联蛋白V染色试验，评估在贴壁和悬浮条件下培养的PPC-1前列腺癌细胞的活力和凋亡情况。使用流式细胞术检测死亡受体Fas的表达和半胱天冬酶8的激活情况。通过逆转录-聚合酶链反应测量Fas配体的表达。通过免疫印迹法测量FLIP蛋白的表达。使用FLIP的小分子抑制剂（包括死亡受体敏化剂5809354）和针对FLIP的小干扰（si）RNA来评估FLIP抑制对前列腺癌细胞体外和体内失巢凋亡的影响。所有统计检验均为双侧检验。
尽管Fas（0小时与8小时相比，平均相对表达百分比=100%对135%，差异=35%，95%置信区间[CI]=10%至61%；P=0.02）和Fas L（0小时与24小时相比，平均相对表达百分比=100%对208%，差异=108%，95%CI=18%至197%；P=0.02）的表达增加，但在悬浮培养的PPC-1细胞仍能抵抗失巢凋亡。通过siRNA敲低FLIP表达或用5809354处理可使前列腺癌细胞对失巢凋亡敏感（10 nM时对照siRNA与FLIP siRNA相比，平均相对活力百分比=95%对51%，差异=44%，95%CI=34%至54%；P<0.001；20 microM时对照与5809354相比，平均相对活力百分比=96%对52%，差异=44%，95%CI=13%至75%；P=0.015）。FLIP表达的抑制特异性地激活了悬浮而非贴壁条件下生长的PPC-1细胞中的半胱天冬酶8，并降低了体内循环PPC-1细胞的转移潜能。
FLIP可能是失巢凋亡的抑制因子，因此可能是抗转移治疗策略的一个潜在靶点。
