Ahmed Atique U, Schmidt Rebecca L, Park Cheol Hong, Reed Nanette R, Hesse Shayla E, Thomas Charles F, Molina Julian R, Deschamps Claude, Yang Ping, Aubry Marie C, Tang Amy H
Departments of Surgery and Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
J Natl Cancer Inst. 2008 Nov 19;100(22):1606-29. doi: 10.1093/jnci/djn365. Epub 2008 Nov 11.
Hyperactivated epidermal growth factor receptor (EGFR) and/or RAS signaling drives cellular transformation and tumorigenesis in human lung cancers, but agents that block activated EGFR and RAS signaling have not yet been demonstrated to substantially extend patients' lives. The human homolog of Drosophila seven-in-absentia--SIAH-1 and SIAH-2--are ubiquitin E3 ligases and conserved downstream components of the RAS pathway that are required for mammalian RAS signal transduction. We examined whether inhibiting SIAH-2 function blocks lung cancer growth.
The antiproliferative and antitumorigenic effects of lentiviral expression of anti-SIAH-2 molecules (ie, a dominant-negative protease-deficient mutant of SIAH-2 [SIAH-2(PD)] and short hairpin RNA [shRNA]-mediated gene knockdown against SIAH-2) were assayed in normal human lung epithelial BEAS-2B cells and in human lung cancer BZR, A549, H727, and UMC11 cells by measuring cell proliferation rates, by assessing MAPK and other activated downstream components of the RAS pathway by immunoblotting, assessing apoptosis by terminal deoxynucleotidyltransferase-mediated UTP end-labeling (TUNEL) assay, quantifying anchorage-independent cell growth in soft agar, and assessing A549 cell-derived tumor growth in athymic nude mice (groups of 10 mice, with two injections of 1 x 10(6) cells each at the dorsal left and right scapular areas). All statistical tests were two-sided.
SIAH-2 deficiency in human lung cancer cell lines reduced MAPK signaling and statistically significantly inhibited cell proliferation compared with those in SIAH-proficient cells (P < .001) and increased apoptosis (TUNEL-positive A549 cells 3 days after lentivirus infection: SIAH-2(PD) vs control, 30.1% vs 0.0%, difference = 30.1%, 95% confidence interval [CI] = 23.1% to 37.0%, P < .001; SIAH-2-shRNA#6 vs control shRNA, 27.9% vs 0.0%, difference = 27.9%, 95% CI = 23.1% to 32.6%, P < .001). SIAH-2 deficiency also reduced anchorage-independent growth of A549 cells in soft agar (mean number of colonies: SIAH-2(PD) vs control, 124.7 vs 57.3, difference = 67.3, 95% CI = 49.4 to 85.3, P < .001; shRNA-SIAH-2#6 vs shRNA control: 27.0 vs 119.7, difference = 92.7, 95% CI = 69.8 to 115.5, P < .001), and blocked the growth of A549 cell-derived tumors in nude mice (mean tumor volume on day 36 after A549 cell injection: SIAH-2(PD) infected vs uninfected, 191.0 vs 558.5 mm(3), difference = 367.5 mm(3), 95% CI = 237.6 to 497.4 mm(3), P < .001; SIAH-2(PD) infected vs control infected, 191.0 vs 418.3 mm(3), difference = 227.5 mm(3), 95% CI = 87.4 to 367.1 mm(3), P = .003; mean resected tumor weight: SIAH-2(PD) infected vs uninfected, 0.12 vs 0.48 g, difference = 0.36 g, 95% CI = 0.23 to 0.50 g, P < .001; SIAH-2(PD) infected vs control infected, 0.12 vs 0.29 g, difference = 0.17 g, 95% CI = 0.04 to 0.31 g, P = .016).
SIAH-2 may be a viable target for novel anti-RAS and anticancer agents aimed at inhibiting EGFR and/or RAS-mediated tumorigenesis.
表皮生长因子受体（EGFR）和/或RAS信号过度激活驱动人类肺癌的细胞转化和肿瘤发生，但阻断激活的EGFR和RAS信号的药物尚未被证明能显著延长患者寿命。果蝇七缺失蛋白的人类同源物——SIAH-1和SIAH-2——是泛素E3连接酶，是哺乳动物RAS信号转导所需的RAS途径的保守下游成分。我们研究了抑制SIAH-2功能是否能阻止肺癌生长。
通过测量细胞增殖率，用免疫印迹法评估MAPK和RAS途径的其他激活下游成分，用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）法评估细胞凋亡，在软琼脂中定量非锚定依赖性细胞生长，以及评估无胸腺裸鼠中A549细胞衍生肿瘤的生长（每组10只小鼠，在左、右肩胛背部区域各注射两次1×10⁶个细胞），检测慢病毒表达抗SIAH-2分子（即SIAH-2的显性负性蛋白酶缺陷突变体[SIAH-2(PD)]和短发夹RNA[shRNA]介导的针对SIAH-2的基因敲低）在正常人类肺上皮BEAS-2B细胞和人类肺癌BZR、A549、H727和UMC11细胞中的抗增殖和抗肿瘤作用。所有统计检验均为双侧检验。
与SIAH功能正常的细胞相比，人肺癌细胞系中SIAH-2缺乏降低了MAPK信号传导，统计学上显著抑制了细胞增殖（P <.001），并增加了细胞凋亡（慢病毒感染后3天TUNEL阳性的A549细胞：SIAH-2(PD)与对照相比，30.1%对0.0%，差异 = 30.1%，95%置信区间[CI] = 23.1%至37.0%，P <.001；SIAH-2-shRNA#6与对照shRNA相比；27.9%对0.0%，差异 = 27.9%，95% CI = 23.1%至32.6%，P <.001）。SIAH-2缺乏还降低了A549细胞在软琼脂中的非锚定依赖性生长（集落平均数：SIAH-2(PD)与对照相比，124.7对57.3，差异 = 67.3，95% CI = 49.4至85.3，P <.001；shRNA-SIAH-2#6与shRNA对照相比：27.0对119.7，差异 = 92.7，95% CI = 69.8至115.5，P <.001），并阻断了裸鼠中A549细胞衍生肿瘤的生长（A549细胞注射后第36天的平均肿瘤体积：SIAH-2(PD)感染组与未感染组相比，191.0对558.5 mm³，差异 = 367.5 mm³，95% CI = 237.6至497.4 mm³，P <.001；SIAH-2(PD)感染组与对照感染组相比，191.0对418.3 mm³，差异 = 227.5 mm³，95% CI = 87.4至367.1 mm³，P =.003；平均切除肿瘤重量：SIAH-2(PD)感染组与未感染组相比；0.12对0.48 g，差异 = 0.36 g，95% CI = 0.23至0.50 g，P <.001；SIAH-2(PD)感染组与对照感染组相比，0.12对0.29 g，差异 = 0.17 g；95% CI = 0.04至0.31 g，P =.016）。
SIAH-2可能是新型抗RAS和抗癌药物的一个可行靶点，旨在抑制EGFR和/或RAS介导的肿瘤发生。
