肿瘤防治研究  2022, Vol. 49 Issue (11): 1119-1125
本刊由国家卫生和计划生育委员会主管,湖北省卫生厅、中国抗癌协会、湖北省肿瘤医院主办。
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文章信息

降脂药对肝癌细胞增殖、侵袭及中性粒细胞外诱捕网形成的影响
Effects of Cholesterol-lowering Agents on Proliferation, Invasion and Neutrophil Extracellular Trap Formation in Liver Cancer Cells
肿瘤防治研究, 2022, 49(11): 1119-1125
Cancer Research on Prevention and Treatment, 2022, 49(11): 1119-1125
http://www.zlfzyj.com/CN/10.3971/j.issn.1000-8578.2022.22.0280
收稿日期: 2022-03-21
修回日期: 2022-07-18
降脂药对肝癌细胞增殖、侵袭及中性粒细胞外诱捕网形成的影响
汤琦琦1,2 ,    李艳2 ,    孙国伟3 ,    梁蓓蓓2 ,    赵健1,2     
1. 201203 上海,上海中医药大学研究生院;
2. 201318 上海,上海健康医学院 上海市分子影像重点实验室;
3. 100191 北京,北京卫戍区第29离职干部休养所门诊部
摘要: 目的 探讨降脂药对肝癌细胞增殖、干性特征、迁移、侵袭和中性粒细胞外诱捕网(NETs)形成的作用。方法 下调ASPP2或HMGCR基因建立胆固醇合成增加或减弱的小鼠肝癌细胞Hepa1-6,分别用辛伐他汀和黄连素两种降脂药处理。CCK-8和平板克隆实验检测肝癌细胞增殖能力;低吸附板成球和qRT-PCR分析细胞干性特征和相关基因表达;划痕和Transwell实验分析细胞迁移、侵袭能力。免疫荧光染色分析NETs形成。结果 降脂药可显著抑制Hepa1-6细胞贴壁和成球生长及干性基因表达(P < 0.001);显著抑制Hepa1-6细胞迁移和侵袭能力(P < 0.001);显著抑制中性粒细胞诱导的Hepa1-6细胞侵袭和NETs的生成(P < 0.001)。结论 降脂药通过抑制肝癌细胞的干性特征和NETs形成,抑制肝癌细胞增殖和转移,是治疗肝癌转移的一种潜在方式。
关键词: 肝癌    胆固醇合成    增殖    侵袭    干性特征    中性粒细胞外诱捕网    
Effects of Cholesterol-lowering Agents on Proliferation, Invasion and Neutrophil Extracellular Trap Formation in Liver Cancer Cells
TANG Qiqi1,2 , LI Yan2 , SUN Guowei3 , LIANG Beibei2 , ZHAO Jian1,2     
1. Graduate School, Shanghai University of Traditional Medicine, Shanghai 201203, China;
2. Shanghai Key Laboratory of Molecular Imaging, Shanghai University of Medicine and Health Sciences, Shanghai 201318, China;
3. Out-patient Department, Beijing Garrison District 29th Retired Cadres Rest Home, Beijing 100191, China
Abstract: Objective To investigate the effects of cholesterol-lowering agents on the proliferation, stemness characters, migration, invasion, and neutrophil extracellular traps formation (NETs) formation in liver cancer cells. Methods ASPP2 or HMGCR gene was knocked down in mouse liver cancer cell Hepa1-6 to establish cells with high or low cholesterol, respectively. Simvastatin and berberine were used to reduce cholesterol synthesis. CCK-8 and plate cloning assays were conducted to detect the proliferation ability of liver cancer cells. Sphere formation assay and qRT-PCR were used to analyze the stemness character and expression of related genes. Wound-healing assay and Transwell assay were used to analyze the ability of cell migration and invasion. Immunofluorescence staining was carried out to analyze the effect of lipid-lowering agent on NETs formation. Results Cholesterol-lowering agents significantly inhibited the proliferation and stemness-related gene expression of Hepa1-6 cells (P < 0.001), significantly inhibited the migration and invasion of Hepa1-6 cells (P < 0.001), and significantly inhibited the neutrophil-induced invasion and formation of NETs (P < 0.001). Conclusion Cholesterol-lowering agents suppress the proliferation and invasion via inhibiting the stemness characters and NETs formation in liver cancer cells. It is a potential strategy for the treatment of liver cancer metastasis.
Key words: Liver cancer    Cholesterol biosynthesis    Proliferation    Invasion    Stemness character    Neutrophil extracellular traps    
0 引言

最新统计分析显示我国肝癌发病率位居第5位,死亡率高居第2位[1]。肝脏是胆固醇合成的主要器官,小鼠肝癌模型提示胆固醇合成增加有利于肝癌形成[2-3]。胆固醇是通过甲羟戊酸(mevalonate, MVA)通路的一系列酶促反应产生的,HMG-CoA还原酶(HMGCR)是其限速酶[4],可被他汀类药物阻断。甾醇调节元素结合蛋白转录因子2(SREBP2)是MVA通路的关键转录调节因子[5],我们之前的研究发现P53凋亡刺激蛋白(apoptosis stimulating proteins of P53, ASPP)家族成员ASPP2可以与SREBP2结合,负向调节肝癌细胞HMGCR表达和胆固醇合成[6]。本研究通过下调ASPP2建立胆固醇合成增强的小鼠肝癌细胞Hepa1-6,分析降脂药辛伐他汀(simvastatin, sim)和黄连素(berberine, BBR)对肝癌细胞增殖、侵袭和中性粒细胞外诱捕网(neutrophil extracellular traps, NETs)形成的影响,以期为肝癌的临床治疗提供更多的实验依据。

1 材料与方法 1.1 主要试剂

C57/L小鼠中产生的BW7756小鼠肝癌的衍生株Hepa1-6由海军军医大学国际肿瘤研究所提供;小鼠乳腺癌细胞株4T1由中科院上海生物科技研究所提供,小鼠乳腺癌细胞株D2F2由海军军医大学国际肿瘤研究所提供;DMEM-F12培养基购自美国Hyclone公司。DMEM培养基、胎牛血清均购自美国Gibco公司。靶向小鼠ASPP2和HMGCR基因的shRNA慢病毒购自上海汉恒生物科技上海有限公司;辛伐他汀、ASPP2抗体购自美国Sigma公司。黄连素购自美国MCE公司。H3cit抗体购自美国Cell Signaling Technology公司;MPO抗体购自美国R&D公司。辛伐他汀用DMSO配置成10 mmol/L的储备液,使用时稀释至2 μmol/L工作液。黄连素用DMSO配置成10 mmol/L的储备液,使用时稀释成10 μmol/L的工作液。

1.2 细胞培养

Hepa1-6和D2F2细胞使用不完全高糖DMEM培养基培养,4T1细胞使用不完全1640培养基。培养基中加入10%胎牛血清,1%青链霉素并将细胞置于37℃、5%CO2恒温培养箱培养。

1.3 细胞感染慢病毒(1/2体积感染法)

细胞生长状态良好的Hepa1-6细胞,以2×105个/孔接种于6孔板中,待细胞生长密度达到40%~50%时换液,加入1 ml新鲜培养基和适量慢病毒感染细胞,并加入2 μl Polybrene促进慢病毒透膜。4 h后,加入1 ml新鲜培养基。24 h后弃含有慢病毒的培养基,换新鲜培养基继续培养。72 h后收集细胞沉淀,提取RNA,qRT-PCR检测ASPP2和HMGCR基因水平,评价shRNA干扰效率。后续培养时定期加入10 μg/ml嘌呤霉素以构建稳定转染细胞株,即可得shNon、shASPP2和shHMGCR组。

1.4 实时荧光定量PCR(qRT-PCR)

慢病毒稳定转染的Hepa1-6细胞,给予10 μmol/L黄连素或2 μmol/L辛伐他汀处理24 h,提取细胞总RNA,转录合成为cDNA,实时荧光定量PCR扩增。引物序列如下:ASPP2正义:5′-AGATAGTGATGGATGGACGCC-3′,反义:5′-CTGGGTCATCTTTTCACGGT-3′;HMGCR正义:5′-CGATCCTTCCTTATTGGCGG-3′,反义:5′-CGGATCTCAATGGAGGCCAA-3′;CD133正义:5′-CTGGACCGGAGAGGGATG-3′,反义:5′-AGCCATAGTTGTTGGCCTGA-3′;EPCAM正义:5′-ATCGCTGTCATTGTGGTGGT-3′,反义:5′-ACCCATCTCCTTTATCTCAGCC-3′;OCT-4正义:5′-CAGTGGGGCGGTTTTGAGT-3′,反义:5′-GGCTGAACACCTTTCCAAAGAGA-3′;CD44正义5′-CACCTTGGCCACCACTCCTAAT-3′,反义:5′-GATGGTTGTTGTGGGCCGAA-3′。

1.5 细胞总胆固醇含量检测

慢病毒稳定转染的Hepa1-6细胞经黄连素或辛伐他汀处理24 h后,收集细胞沉淀。加入适量提取液,冰浴超声破碎细胞。离心取上清液,依据试剂盒(北京索莱宝科技有限公司)说明书加入检测试剂后用酶标仪于500 nm处进行检测。

1.6 CCK-8细胞存活检测

将细胞以1×104个/孔的密度接种到96孔板中,并在37℃培养箱中培养。用10 μmol/L黄连素或2 μmol/L辛伐他汀或黄连素和辛伐他汀联合给药处理细胞。依据试剂盒说明书,CCK-8(碧云天生物科技有限公司)在治疗24和48 h后测量相对细胞活力。测量每个样品在450 nm处的吸光度(OD)值,并将细胞活力计算为药物处理细胞和未处理细胞之间OD值的比率,以shNon组细胞为对照组。

1.7 平板克隆形成实验

消化Hepa1-6细胞以2×103个/孔接种于六孔板,加入含20%FBS的培养基,shASPP2细胞给予黄连素或辛伐他汀处理。10天后,弃培养基用4%多聚甲醛固定,1%结晶紫染色。水洗,拍照,计数集落形成情况。

1.8 低吸附板成球实验

消化Hepa1-6细胞以2×103个/孔加至低吸附板(24孔板)中,加入培养基(以50 ml为例,基础培养基DMEM-F12中加入胰岛素4 μg/ml;0.4%BSA;B27添加剂1 ml;表皮生长因子EGF 20 ng/ml;成纤维细胞生长因子FGF 20 ng/ml)。shASPP2细胞中加入黄连素或辛伐他汀。每天观察细胞状态,6天后光学显微镜拍照、计数,记为一次成球。随后用PBS吹打细胞至单细胞悬液,离心后加入新鲜培养基继续培养,15天后拍照、计数,记为二次成球。

1.9 细胞划痕实验

消化Hepa1-6细胞,以5×105个/孔均匀铺于六孔板中,待细胞密度长至90%时,使用10 μl枪头进行垂直划痕处理。PBS清洗至无漂浮细胞后在显微镜下于0 h、24 h和48 h拍照,分析细胞伤口愈合率。

1.10 Transwell侵袭实验

消化Hepa1-6细胞,计数为1×106个/毫升。每孔添加100 μl细胞悬液至铺有50 μl稀释基质胶的Transwell小室中。下室孔板中添加含有20% FBS的新鲜培养基或2.5×105个经佛波醇12-肉豆蔻酸酯13-乙酸(phorbol 12-myristate 13-acetate, PMA)刺激不含FBS的中性粒细胞悬液。24 h后取出Transwell小室,置于4%多聚甲醛中固定,后用0.1%结晶紫染色。水洗后,用棉签小心擦去膜上未穿孔细胞,风干后置于显微镜随机选取五个视野拍照,并计数。

1.11 共培养体系诱导NETs形成

从小鼠股骨和胫骨中分离出中性粒细胞,用不含FBS的培养基重悬并计数为每毫升5×105个细胞,添加500 μl到包被多聚赖氨酸的载玻片上用20 nm PMA刺激30 min后,加入或不加DNase 1(0.32 u/ml, shASPP2+DNase1组)。将Hepa1-6细胞添加至铺有基质胶的Transwell小室中,含中性粒细胞的载玻片置于下室孔上。22~24 h后,取出下室载玻片,免疫荧光染色检测髓过氧化物酶(myeloperoxidase, MPO)和瓜氨酸化组蛋白H3(citrullinated histone-3, H3Cit),NETs的生成情况使用以下公式计算,NET形成百分比=(NET形成中性粒细胞/中性粒细胞)×100%。荧光值通过Image J软件获得。

1.12 免疫荧光实验

载玻片上的细胞经4%多聚甲醛固定,0.5% Trionx-100透化,1% BSA封闭1 h后,孵育ASPP2(1:100)抗体、H3cit(1:400)或MPO(10 μg/ml)过夜(4℃)。第二天孵育同种属二抗室温孵育2 h。PBS清洗后用含有DAPI的封片剂封片。风干后置于共聚焦显微镜拍照。

以上每个独立实验重复三次以上。

1.13 统计学方法

所有实验采用GraphPad Prism 7软件分析,数据采用单因素方差分析方法,P < 0.05为差异有统计学意义。

2 结果 2.1 ASPP2表达水平和胆固醇水平检测

qRT-PCR结果显示,与小鼠乳腺癌细胞4T1和D2F2相比,小鼠肝癌细胞Hepa1-6细胞中有较高的HMGCR基因表达和较低的ASPP2基因表达。Hepa1-6细胞中总胆固醇水平较高。免疫荧光结果显示ASPP2蛋白在Hepa1-6细胞的胞内和核内都有表达,且核内表达较高,见图 1A~C

*: P < 0.05; **: P < 0.01; ***: P < 0.001; BBR: berberine; sim: simvastatin. A: differences in mRNA expression of ASPP2 and HMGCR among different mouse tumor cells; B: localization of ASPP2 in different mouse tumor cells. Scale bar: 25 μm; C: cells were collected and used for cholesterol content assays; D: Hepa1-6 cells were infected with lentivirus expressing shRNA targeting mouse ASPP2 or HMGCR gene, and scramble shRNA (shNon) was used as control. Drugs were added to LV-shASPP2-infected cells. After 24h, cells were collected and used to detect ASPP2 and HMGCR mRNA levels and cholesterol content. 图 1 ASPP2表达水平和胆固醇水平检测 Figure 1 ASPP2 expression level and cholesterol level detection

为了建立胆固醇合成增强和减弱的肝癌细胞,用shRNA分别干扰ASPP2和HMGCR表达。qRT-PCR结果显示,LV-shASPP2的抑制效率大于70%,LV-shHMGCR的抑制效率大于80%,且shASPP2肝癌细胞表现出了旺盛的胆固醇生物合成(P=0.0075)和HMGCR基因表达增加(P=0.0496)。辛伐他汀和黄连素可以抑制这种由于下调ASPP2引起的胆固醇合成增加(P=0.0009)和HMGCR增强(P=0.01)。shHMGCR的Hepa1-6细胞胆固醇合成被抑制(P=0.0081),见图 1D

2.2 降脂药抑制肝癌细胞增殖

降脂药处理24 h后,CCK-8细胞存活检测结果显示,shASPP2的Hepa1-6细胞存活率比对照细胞(shNon)高(64.8±7.401)%,而shHMGCR肝癌细胞存活率较对照细胞低(24.12±1.157)%。给予黄连素或辛伐他汀后可以显著抑制shASPP2肝癌细胞的增殖(P=0.005,P=0.002)。黄连素和辛伐他汀连用后对肿瘤细胞增殖的抑制作用更显著(P=0.0002),见图 2A。降脂药处理48 h后,观察到相似的结果。与对照组相比,shASPP2组肿瘤细胞的克隆数显著增多(P=0.0008),给予黄连素或辛伐他汀显著抑制克隆形成(P=0.0006,P=0.0005),而敲低HMGCR基因表达后克隆形成显著减少(P=0.0001),见图 2B

*: P < 0.05; **: P < 0.01; ***: P < 0.001; NC: negative control. A: *, **, ***: compared with NC group. Cell viability of Hepa1-6 cells treated with simvastatin or berberine or their combination for 24 and 48 h; B: Hepa1-6 cells were cultured in medium containing 20% FBS for 12–15 days. Colony number and representative images were obtained. 图 2 降脂药抑制小鼠肝癌细胞增殖 Figure 2 Cholesterol-lowering agents inhibited proliferation of liver cancer cells
2.3 降脂药抑制肝癌细胞干性特征

shASPP2肝癌细胞表现出旺盛的胆固醇生物合成,促进了Hepa1-6细胞在低吸附板中的连续成球能力,细胞球体大且数量多,干性相关标志物CD44、CD133、EpCAM和转录因子OCT4的表达显著增加(P=0.0270)。给予黄连素、辛伐他汀显著抑制ASPP2下调引起的肝癌细胞成球生长(P=0.0218)和干性基因表达(P=0.0187)。抑制HMGCR基因表达后,肝癌细胞细胞干性特征减弱,见图 3

*: P < 0.05; **: P < 0.01; ***: P < 0.001. A: Hepa1-6 cells as indicated were cultured onto ultralow attachment plates. Number of sphere formation in primary (6 days) and secondary spheres (15 days) was calculated. Scale bar: 50 µm; B: qRT-PCR assay for CD133, CD44, EpCAM, and OCT-4 mRNA levels in treated Hepa1-6 cells. 图 3 降脂药抑制肝癌细胞干性特征 Figure 3 Cholesterol-lowering agents inhibited stemness of liver cancer cells
2.4 降脂药抑制肝癌细胞迁移和侵袭

通过下调ASPP2表达增强胆固醇生物合成使Hepa1-6细胞表现出更强的迁移(P=0.0207)和侵袭能力(P=0.0017),给予辛伐他汀和黄连素后显著抑制细胞的迁移和侵袭(P=0.0087)。然而,shHMGCR使Hepa1-6细胞迁移(P=0.0436)和侵袭(P=0.0216)能力下降,见图 4

*: P < 0.05; **: P < 0.01; ***: P < 0.001. A: wound healing assay and migration areas of treated Hepa1-6 cells. Scale bar: 100 μm; B: Hepa1-6 cells cultured with rehydrated matrigel in the upper chamber of the Transwells. 20% serum containing medium was added to the bottom wells. After culture for 22-26 h, invading cells were counted by a light microscope and analyzed by Image J software. Scale bar: 200 μm. 图 4 降脂药抑制肝癌细胞迁移和侵袭 Figure 4 Cholesterol-lowering agents inhibited liver cancer cells migration and invasion
2.5 降脂药抑制NETs生成

在与中性粒细胞的共培养系统中,shASPP2的Hepa1-6细胞组比对照组细胞表现出更多的侵袭性(P=0.0058),辛伐他汀和黄连素可以抑制这种侵袭(P=0.0044),见图 5A。DNase 1显著抑制了shASPP2细胞的侵袭能力(P=0.0027),这表明NETs形成参与了胆固醇合成增加诱导的肿瘤细胞侵袭。

*: P < 0.05; **: P < 0.01; ***: P < 0.001. A: Hepa1-6 cells cultured in the upper chamber of the Transwells. PMA-stimulated neutrophils in serum-free medium with or without DNase I were added to the bottom wells. After culture for 22-26 h, invading cells were counted by a light microscope; B: NET formation was analyzed by immunostaining of MPO (red) and H3cit (green) on neutrophil-cultured slides in different groups. Scale bar: 50 μm. 图 5 降脂药抑制NETs的生成 Figure 5 Cholesterol-lowering agents inhibited neutrophil extracellular traps formation

免疫荧光结果显示,NETs的生成在shASPP2组中显著升高(P=0.0182),而在shHMGCR组中减少,见图 5B。辛伐他汀和黄连素减弱了ASPP2下调诱导的NETs形成(P=0.0325),表明肿瘤细胞胆固醇合成增加可以促进NETs形成。

3 讨论

肿瘤细胞通常在新陈代谢方面具有特征性变化,异常的细胞代谢有助于细胞增殖和肿瘤进展[7]。细胞增殖是所有肿瘤的共同特征,胆固醇合成增加可以为肿瘤细胞提供原料,促进肿瘤发生和发展[8]。大规模基因筛查结果显示,多种MVA途径代谢酶对肿瘤细胞的存活至关重要[9-10]。此外,多个研究结果显示他汀类药物家族可以在体内和体外实验中抑制多种肿瘤的生长和促进细胞凋亡[11-12]。临床资料显示,高表达MVA通路基因的患者预后较差[3, 13],而他汀类药物可以降低肝癌患者的发病率和癌症相关的死亡率[14-15]。这些观察结果表明胆固醇合成不仅与肿瘤生长有关,还与肿瘤转移有关。靶向MVA通路和胆固醇代谢可能是肿瘤治疗的一个潜在治疗靶标。目前他汀类药物正在全球20多个临床机构开展治疗肝癌、乳腺癌等肿瘤的临床试验[5, 7]

除了他汀药物作用于HMGCR抑制胆固醇合成,黄连素可以通过促进低密度脂蛋白受体(LDLR)水平抑制胆固醇合成[16],本研究选用辛伐他汀和黄连素作为降脂药,探讨降脂药对肝癌细胞增殖、迁移、侵袭和NETs形成的作用。本研究结果显示降脂药显著抑制肝癌细胞增殖及其干性特征。之前文献报道,MVA通路和胆固醇合成对于维持细胞的干性特征至为重要。p53突变可导致MVA通路激活,胆固醇合成增加,有利于乳腺癌细胞在3D培养中维持形态[17]。最近的研究结果提示胆固醇合成有利于低氧状态下肝癌细胞维持其干性特征[18]。本研究结果显示敲低ASPP2基因表达的肝癌细胞,一次成球和二次成球的球体大且数量多,给予降脂药后球体的生长被显著抑制。PCR结果显示肝干细胞标志物EpCAM[19]、CD133[20]和CD44[21]以及干性调节分子OCT-4的表达水平均被降脂药显著抑制。上述结果提示降脂药可以通过抑制肝癌细胞干性特征抑制细胞增殖。

胆固醇合成不仅参与调控细胞增殖,与肿瘤转移也密切相关。胆固醇可以促进中性粒细胞聚集和肿瘤转移[22]。最近的研究发现NETs通过激活“休眠”的肿瘤细胞和降解细胞外基质在肿瘤转移中发挥重要作用[23-24]。NETs形成过程为活化的中性粒细胞通过向细胞外释放去聚化的染色质,形成网状结构,它最初的作用是中性粒细胞诱捕和杀死细菌和真菌的一种免疫方式[25]。我们观察到ASPP2下调的肝癌细胞可以诱导中性粒细胞产生更强的NETs网状结构,这种NET网状结构可被降脂药和DNase 1抑制。上述结果提示肿瘤细胞胆固醇合成增加可以促进肿瘤侵袭能力,NETs形成参与了这一过程。HMGCR是胆固醇合成的关键酶,我们的研究结果提示靶向抑制肿瘤细胞HMGCR活性可望为抑制NETs形成和肿瘤转移提供新策略。

综上,胆固醇合成增加可以通过增强肝癌细胞的干性特征和诱导NETs形成,促进肝癌细胞增殖和转移。服用降脂药可望成为治疗肝癌转移的潜在治疗方式。

作者贡献:

汤琦琦:实验实施、数据处理及论文撰写

李艳:细胞培养及实验的实施

孙国伟:实验指导及数据处理

梁蓓蓓:实验指导及论文修改

赵健:课题的构思、实验设计及论文修改

参考文献
[1]
Zheng R, Zhang S, Zeng H, et al. Cancer incidence and mortality in China, 2016[J]. J Nat Cancer Center, 2022, 2(1): 1-9. DOI:10.1016/j.jncc.2022.02.002
[2]
Che L, Chi W, Qiao Y, et al. Cholesterol biosynthesis supports the growth of hepatocarcinoma lesions depleted of fatty acid synthase in mice and humans[J]. Gut, 2020, 69(1): 177-186. DOI:10.1136/gutjnl-2018-317581
[3]
Moon SH, Huang CH, Houlihan SL, et al. p53 represses the mevalonate pathway to mediate tumor suppression[J]. Cell, 2019, 176(3): 564-580. e519. DOI:10.1016/j.cell.2018.11.011
[4]
Simons K, Ikonen E. How cells handle cholesterol[J]. Science, 2000, 290(5497): 1721-1726. DOI:10.1126/science.290.5497.1721
[5]
Mullen PJ, Yu R, Longo J, et al. The interplay between cell signalling and the mevalonate pathway in cancer[J]. Nat Rev Cancer, 2016, 16(11): 718-731. DOI:10.1038/nrc.2016.76
[6]
Liang B, Chen R, Song S, et al. ASPP2 inhibits tumor growth by repressing the mevalonate pathway in hepatocellular carcinoma[J]. Cell Death Dis, 2019, 10(11): 830. DOI:10.1038/s41419-019-2054-7
[7]
Martinez-Outschoorn UE, Peiris-Pagés M, Pestell RG, et al. Cancer metabolism: a therapeutic perspective[J]. Nat Rev Clin Oncol, 2017, 14(1): 11-31. DOI:10.1038/nrclinonc.2016.60
[8]
Kuzu OF, Noory MA, Robertson GP. The Role of Cholesterol in Cancer[J]. Cancer Res, 2016, 76(8): 2063-2070. DOI:10.1158/0008-5472.CAN-15-2613
[9]
Hart T, Chandrashekhar M, Aregger M, et al. High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities[J]. Cell, 2015, 163(6): 1515-1526. DOI:10.1016/j.cell.2015.11.015
[10]
Wang T, Birsoy K, Hughes NW, et al. Identification and characterization of essential genes in the human genome[J]. Science, 2015, 350(6264): 1096-1101. DOI:10.1126/science.aac7041
[11]
Jiang W, Hu JW, He XR, et al. Statins: a repurposed drug to fight cancer[J]. J Exp Clin Cancer Res, 2021, 40(1): 241. DOI:10.1186/s13046-021-02041-2
[12]
Juarez D, Fruman DA. Targeting the Mevalonate Pathway in Cancer[J]. Trends Cancer, 2021, 7(6): 525-540. DOI:10.1016/j.trecan.2020.11.008
[13]
Liu M, Xia Y, Ding J, et al. Transcriptional Profiling Reveals a Common Metabolic Program in High-Risk Human Neuroblastoma and Mouse Neuroblastoma Sphere-Forming Cells[J]. Cell Rep, 2016, 17(2): 609-623. DOI:10.1016/j.celrep.2016.09.021
[14]
Thrift AP, Natarajan Y, Liu Y, et al. Statin Use After Diagnosis of Hepatocellular Carcinoma Is Associated With Decreased Mortality[J]. Clin Gastroenterol Hepatol, 2019, 17(10): 2117-2125. e3. DOI:10.1016/j.cgh.2018.12.046
[15]
Alipour TG, Trézéguet V, Merched A. Hepatocellular Carcinoma and Statins[J]. Biochemistry, 2020, 59(37): 3393-3400. DOI:10.1021/acs.biochem.0c00476
[16]
Kong W, Wei J, Abidi P, et al. Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins[J]. Nat Med, 2004, 10(12): 1344-1351. DOI:10.1038/nm1135
[17]
Freed-Pastor WA, Mizuno H, Zhao X, et al. Mutant p53 disrupts mammary tissue architecture via the mevalonate pathway[J]. Cell, 2012, 148(1-2): 244-258. DOI:10.1016/j.cell.2011.12.017
[18]
Husain A, Chiu YT, Sze KM, et al. Ephrin-A3/EphA2 axis regulates cellular metabolic plasticity to enhance cancer stemness in hypoxic hepatocellular carcinoma[J]. J Hepatol, 2022, 8278(22): 125-128.
[19]
Terris B, Cavard C, Perret C. EpCAM, a new marker for cancer stem cells in hepatocellular carcinoma[J]. J Hepatol, 2010, 52(2): 280-281. DOI:10.1016/j.jhep.2009.10.026
[20]
Kakinuma S, Ohta H, Kamiya A, et al. Analyses of cell surface molecules on hepatic stem/progenitor cells in mouse fetal liver[J]. J Hepatol, 2009, 51(1): 127-138. DOI:10.1016/j.jhep.2009.02.033
[21]
Kon J, Ooe H, Oshima H, et al. Expression of CD44 in rat hepatic progenitor cells[J]. J Hepatol, 2006, 45(1): 90-98. DOI:10.1016/j.jhep.2006.01.029
[22]
Coffelt SB, Kersten K, Doornebal CW, et al. IL-17-producing γδ T cells and neutrophils conspire to promote breast cancer metastasis[J]. Nature, 2015, 522(7556): 345-348. DOI:10.1038/nature14282
[23]
Park J, Wysocki RW, Amoozgar Z, et al. Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps[J]. Sci Transl Med, 2016, 8(361): 361ra138.
[24]
Albrengues J, Shields MA, Ng D, et al. Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice[J]. Science, 2018, 361(6409): eaao4227. DOI:10.1126/science.aao4227
[25]
Demkow U. Neutrophil Extracellular Traps (NETs) in Cancer Invasion, Evasion and Metastasis[J]. Cancers (Basel), 2021, 13(17): 4495. DOI:10.3390/cancers13174495