DLC1 缺失对内皮细胞接触生长抑制和血管肉瘤进展的影响。
Effects of DLC1 Deficiency on Endothelial Cell Contact Growth Inhibition and Angiosarcoma Progression.
机构信息
Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD.
Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan.
出版信息
J Natl Cancer Inst. 2018 Apr 1;110(4):390-399. doi: 10.1093/jnci/djx219.
BACKGROUND
Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene frequently deleted in cancer. However, DLC1 is not known to be deleted in angiosarcoma, an aggressive malignancy of endothelial cell derivation. Additionally, the physiologic functions of DLC1 protein in endothelial cells are poorly defined.
METHODS
We investigated the effects of shRNA-induced DLC1 depletion in endothelial cells. Cell growth was measured by 3H thymidine incorporation, IncuCyte imaging, and population doublings; cell death by cell cycle analysis; gene expression by Affimetrix arrays and quantitative polymerase chain reaction; NF-κB activity by reporter assays; and protein levels by immunoblotting and immunofluorescence staining. We tested Tanespimycin/17-AAG and Fasudil treatment in groups of nine to 10 mice bearing ISOS-1 angiosarcoma. All statistical tests were two-sided.
RESULTS
We discovered that DLC1 is a critical regulator of cell contact inhibition of proliferation in endothelial cells, promoting statistically significant (P < .001) cell death when cells are confluent (mean [SD] % viability: control DLC1 = 15.6 [19.3]; shDLC1 = 73.4 [13.1]). This prosurvival phenotype of DLC1-depleted confluent endothelial cells is attributable to a statistically significant and sustained increase of NF-κB activity (day 5, P = .001; day 8, P = .03) associated with increased tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) signaling. Consistently, we found that DLC1 is statistically significantly reduced (P < .001 in 5 of 6) and TNFAIP3/A20 is statistically significantly increased (P < .001 in 2 of 3 and P = 0.02 in 1 of 3) in human angiosarcoma compared with normal adjacent endothelium. Treatment with the NF-κB inhibitor Tanespimycin/17-AAG statistically significantly reduced angiosarcoma tumor growth in mice (treatment tumor weight vs control, 0.50 [0.19] g vs 0.91 [0.21] g, P = .001 experiment 1; 0.66 [0.26] g vs 1.10 [0.31] g, P = .01 experiment 2).
CONCLUSIONS
These results identify DLC1 as a previously unrecognized regulator of endothelial cell contact inhibition of proliferation that is depleted in angiosarcoma and support NF-κB targeting for the treatment of angiosarcoma where DLC1 is lost.
背景
肝癌缺失基因 1（DLC1）是一种在癌症中经常缺失的肿瘤抑制基因。然而，DLC1 在血管肉瘤中并不缺失，血管肉瘤是一种来源于内皮细胞的侵袭性恶性肿瘤。此外，DLC1 蛋白在内皮细胞中的生理功能也尚未明确。
方法
我们研究了短发夹 RNA 诱导的 DLC1 缺失对内皮细胞的影响。通过 3H 胸腺嘧啶掺入、Incucyte 成像和倍增时间测量细胞生长；通过细胞周期分析测量细胞死亡；通过 Affymetrix 微阵列和定量聚合酶链反应测量基因表达；通过报告基因测定测量 NF-κB 活性；通过免疫印迹和免疫荧光染色测量蛋白水平。我们在 9 至 10 只患有 ISOS-1 血管肉瘤的小鼠中测试了 Tanespimycin/17-AAG 和 Fasudil 的治疗效果。所有统计检验均为双侧检验。
结果
我们发现 DLC1 是内皮细胞接触性增殖抑制的关键调节因子，当细胞达到汇合状态时，可显著促进细胞死亡（对照 DLC1 存活率 [标准差]：15.6 [19.3]%；shDLC1：73.4 [13.1]%）。这种 DLC1 缺失的汇合内皮细胞的存活表型归因于 NF-κB 活性的持续显著增加（第 5 天，P =.001；第 8 天，P =.03），与肿瘤坏死因子-α诱导蛋白 3（TNFAIP3/A20）信号相关。一致地，我们发现与正常相邻内皮相比，DLC1 在 6 例中的 5 例（P <.001）显著降低，而 TNFAIP3/A20 在 3 例中的 2 例（P <.001）和 3 例中的 1 例（P = 0.02）显著增加（P <.001）。NF-κB 抑制剂 Tanespimycin/17-AAG 治疗可显著降低小鼠血管肉瘤肿瘤生长（治疗肿瘤重量与对照相比，0.50 [0.19] g 与 0.91 [0.21] g，P =.001 实验 1；0.66 [0.26] g 与 1.10 [0.31] g，P =.01 实验 2）。
结论
这些结果表明 DLC1 是内皮细胞接触性增殖抑制的一个以前未被识别的调节因子，在血管肉瘤中缺失，并支持针对 DLC1 缺失的血管肉瘤的 NF-κB 靶向治疗。
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