皮非他林-μ使mTOR-活化的癌症对索拉非尼治疗敏感-动脉网

Pifithrin-μ sensitizes mTOR-activated liver cancer to sorafenib treatment

Nature 等信源发布 2025-01-26 10:13



可切换为仅中文







Abstract

摘要

TSC2, a suppressor of mTOR, is inactivated in up to 20% of HBV-associated liver cancer. This subtype of liver cancer is associated with aggressive behavior and early recurrence after hepatectomy. Being the first targeted regimen for advanced liver cancer, sorafenib has limited efficacy in HBV-positive patients.

TSC2是mTOR的抑制剂，在高达20%的HBV相关肝癌中失活。这种亚型肝癌与肝切除术后的侵袭行为和早期复发有关。索拉非尼是第一个针对晚期肝癌的靶向治疗方案，对HBV阳性患者的疗效有限。

In this study, we observed that mTOR-activated cells, due to the loss of either TSC2 or PTEN, were insensitive to the treatment of sorafenib. Mechanistically, HSP70 enhanced the interaction between active mTOR-potentiated CREB1 and CREBBP to boost the transcription of the antioxidant response regulator SESN3.

在这项研究中，我们观察到由于TSC2或PTEN的丢失，mTOR激活的细胞对索拉非尼的治疗不敏感。从机制上讲，HSP70增强了活性mTOR增强的CREB1和CREBBP之间的相互作用，以促进抗氧化反应调节剂SESN3的转录。

In return, elevated SESN3 enhanced cellular antioxidant capacity and rendered cells resistant to sorafenib. Pifithrin-μ, an HSP70 inhibitor, synergized with sorafenib in the induction of ferroptosis in mTOR-activated liver cancer cells and suppression of TSC2-deficient hepatocarcinogenesis. Our findings highlight the pivotal role of the mTOR-CREB1-SESN3 axis in sorafenib resistance of liver cancer and pave the way for combining pifithrin-μ and sorafenib for the treatment of mTOR-activated liver cancer..

作为回报，升高的SESN3增强了细胞的抗氧化能力，并使细胞对索拉非尼具有抗性。HSP70抑制剂Pifithrin-μ与索拉非尼协同诱导mTOR激活的肝癌细胞中的铁浓化和抑制TSC2缺陷的肝癌发生。我们的研究结果突出了mTOR-CREB1-SESN3轴在肝癌索拉非尼耐药中的关键作用，并为pifithrin和索拉非尼联合治疗mTOR激活的肝癌铺平了道路。。

Introduction

简介

Primary liver cancer is the sixth most common cancer and the third most common cause of cancer-related deaths globally [

原发性肝癌是全球第六大常见癌症，也是与癌症相关的死亡的第三大常见原因[

]. The number of new cases of hepatic cancer is projected to increase by >55% between 2020 and 2040 [

]。2020年至2040年间，新发肝癌病例预计将增加55%以上[

]. As persistent infection with hepatitis B virus (HBV) is a major risk factor, nearly half of the liver cancer cases diagnosed globally occur in China [

]。由于持续感染乙型肝炎病毒（HBV）是一个主要的危险因素，因此全球确诊的肝癌病例中近一半发生在中国[

]. Hepatic cancer is the fourth most common cancer and the second leading cause of cancer deaths in China [

]。肝癌是中国第四大常见癌症，也是导致癌症死亡的第二大原因[

]. Hepatocellular carcinoma (HCC) accounts for up to 90% of liver cancer [

]。肝细胞癌（HCC）占肝癌的90%[

]. The majority of HCC cases are diagnosed at advanced stages, resulting in limited treatment efficacy and unfavorable prognoses [

]。大多数HCC病例被诊断为晚期，导致治疗效果有限和预后不良[

]. Therefore, there is a compelling need to establish better treatment strategies for advanced hepatic cancer.

]。因此，迫切需要为晚期肝癌建立更好的治疗策略。

Sorafenib, a multi-target tyrosine kinase inhibitor, is the first targeted drug for the treatment of advanced HCC [

索拉非尼是一种多靶点酪氨酸激酶抑制剂，是第一种治疗晚期肝癌的靶向药物[

]. Although sorafenib has been widely used in clinics for stabilizing HCC progression, different etiological factors may contribute to various response rates of cells to sorafenib [

]。[

]. While 40% to 60% of HCC patients may benefit from sorafenib initially, this population develops drug resistance within 6 months of treatment [

]。虽然40%至60%的HCC患者最初可能受益于索拉非尼，但该人群在治疗后6个月内出现耐药性[

]. As drug resistance contributes to the limited survival benefits of sorafenib, strategies to overcome both primary and acquired resistance are urgently needed [

]。由于耐药性导致索拉非尼的生存益处有限，因此迫切需要克服原发性和获得性耐药性的策略[

]. The documented mechanisms of sorafenib resistance include activation of the hypoxia-induced pathway, epithelial-mesenchymal transition, reduced levels of reactive oxygen species (ROS) and others [

]。索拉非尼耐药的机制包括缺氧诱导途径的激活、上皮-间质转化、活性氧（ROS）水平的降低等[

]. Under normal conditions, cells maintain a balance between ROS generation and scavenging through oxidation and antioxidation processes. Numerous studies have shown that sorafenib induces ferroptosis by promoting the generation of ROS and iron accumulation [

]。在正常条件下，细胞通过氧化和抗氧化过程维持ROS产生和清除之间的平衡。大量研究表明，索拉非尼通过促进活性氧的产生和铁的积累来诱导铁浓化[

]. Therefore, strategies aimed at reducing the antioxidant capacity of tumor cells hold the potential for reversing sorafenib resistance.

]。因此，旨在降低肿瘤细胞抗氧化能力的策略具有逆转索拉非尼耐药性的潜力。

Mechanistic target of rapamycin (mTOR), a serine/threonine kinase, regulates various cellular processes, including cell metabolism, growth, proliferation, and survival. Loss of tumor suppressor phosphatase and tensin homolog (PTEN) or tuberous sclerosis complex 2 (TSC2) activates mTOR signaling pathway and is associated with various cancers [.

雷帕霉素（mTOR）是一种丝氨酸/苏氨酸激酶，其机制靶标可调节各种细胞过程，包括细胞代谢，生长，增殖和存活。肿瘤抑制性磷酸酶和张力蛋白同源物（PTEN）或结节性硬化症复合物2（TSC2）的缺失激活了mTOR信号通路，并与各种癌症有关[。

]. Multiple mTOR signaling pathway components were recurrently mutated in HBV-associated HCC. Up to 59% of HCC patients exhibit mTOR activation, which is linked to early recurrence and poor prognosis [

]。。高达59%的HCC患者表现出mTOR激活，这与早期复发和预后不良有关[

]. PTEN is mutated or silenced in approximately half of primary hepatoma patients [

]。大约一半的原发性肝癌患者PTEN突变或沉默[

]. About 6.3% to 20% of Asian HCC are associated with loss of TSC2 function [

]。大约6.3%至20%的亚洲HCC与TSC2功能丧失有关[

]. Array-based pathway profiling revealed mTOR activation in sorafenib-resistant HCC cells [

]。基于阵列的通路分析显示索拉非尼耐药HCC细胞中mTOR的激活[

]. Lack of efficacy for Sorafenib has been reported in HBV-positive patients [

]。据报道，HBV阳性患者对索拉非尼缺乏疗效[

]. Therefore, mTOR-activated cells might be resistant to sorafenib. Although mTOR inhibitors are effective in the treatment of benign tumor with mTOR activation [

]。因此，mTOR激活的细胞可能对索拉非尼具有抗性。虽然mTOR抑制剂在mTOR激活的良性肿瘤治疗中是有效的[

], their efficacy is limited in malignant tumor [

]，它们在恶性肿瘤中的疗效有限[

], largely due to their cytostatic nature. There is no solid evidence suggesting a true benefit of mTOR inhibitors in liver cancer treatment [

]，主要是由于它们的细胞抑制性质。没有确凿的证据表明mTOR抑制剂在肝癌治疗中有真正的益处[

]. Moreover, clinical trials of combining sorafenib and mTOR inhibitor everolimus did not improve overall survival of HCC patients [

]。此外，索拉非尼和mTOR抑制剂依维莫司联合应用的临床试验并没有提高肝癌患者的总生存率[

In this study, we found that mTOR-activated cells were resistant to sorafenib treatment. Antioxidant protein sestrin 3 (SESN3) was a novel effector of mTOR-cAMP responsive element-binding protein 1 (CREB1) signaling cascade. mTOR activation-mediated sorafenib resistance resulted from the accumulation of SESN3 and consequent enhancement of antioxidant capacity.

在这项研究中，我们发现mTOR激活的细胞对索拉非尼治疗具有抗性。抗氧化蛋白sestrin 3（SESN3）是mTOR-cAMP反应元件结合蛋白1（CREB1）信号级联的新型效应物。mTOR激活介导的索拉非尼耐药性是由SESN3的积累和抗氧化能力的增强引起的。

Pifithrin-μ, also known as 2-phenylethyenesulfonamide (PES), is a heat shock protein 70 (HSP70) inhibitor that disrupted the interaction between CREB1 and CREB-binding protein (CREBBP), ultimately suppressed SESN3 to enhance the anti-tumor effects of sorafenib. A combination of pifithrin-μ and sorafenib may thus represent a novel therapeutic approach to overcome sorafenib resistance in mTOR-activated liver cancer..

Pifithrin-μ，也称为2-苯基乙炔磺酰胺（PES），是一种热休克蛋白70（HSP70）抑制剂，可破坏CREB1和CREB结合蛋白（CREBBP）之间的相互作用，最终抑制SESN3以增强索拉非尼的抗肿瘤作用。因此，pifithrin-μ和索拉非尼的组合可能代表了克服mTOR激活的肝癌中索拉非尼耐药性的新型治疗方法。。

Materials and methods

材料和方法

Reagents

试剂

Sorafenib (#HY-10201) and SGC-CBP30 (#HY-15826) were purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA). Deferoxamine mesylate (DFO, #D9533) was obtained from Sigma-Aldrich (St. Louise, MO, USA). Ferrostatin-1 (Fer-1, #S7243), N-acetylcysteine (NAC, #S1623), necrostatin-1 (Nec-1, #S8037), chloroquine (CQ, #S6999) and pifithrin-μ (PES, #S2930) were purchased from Selleck Chemicals (Houston, TX, USA)..

索拉非尼（#HY-10201）和SGC-CBP30（#HY-15826）购自MedChemExpress（MCE，Monmouth Junction，NJ，USA）。甲磺酸去铁胺（DFO，#D9533）获自Sigma-Aldrich（美国密苏里州圣路易斯）。。。

Cell lines and culture

细胞系和培养

Wild-type (WT) mouse embryonic fibroblasts (MEFs),

野生型（WT）小鼠胚胎成纤维细胞（MEF），

Tsc2

Tsc2型

−/−

MEFs,

MEFs，

Pten

Pten公司

−/−

MEFs, 293FT, HCCLM3, HepG2, MHCC97H, SNU886, and SNU398 cell lines have been previously described [

先前已经描述了MEF，293FT，HCCLM3，HepG2，MHCC97H，SNU886和SNU398细胞系[

]. The Human Li7 cell line was generously provided by Huang Lin (Dalian Medical University, Dalian, China). These cell lines were cultured in DMEM (#11995-065, Gibco, Grand Island, NY, USA) or RPMI 1640 (#72400047, Gibco) supplemented with 10% FBS (#10100147 C, Gibco) and 1% penicillin-streptomycin (#15140163, Life Technologies, Carlsbad, CA, USA) under 5% CO.

]。人Li7细胞系由黄林（大连医科大学，大连，中国）慷慨提供。将这些细胞系在补充有10%FBS（10100147，Gibco）和1%青霉素-链霉素（15140163，Life Technologies，Carlsbad，CA，USA）的DMEM（11995-065，Gibco，Grand Island，NY，USA）或RPMI 1640（72400047，Gibco）中在5%CO下培养。

at 37 °C.

37°C。

Cell viability

细胞活力

MEFs and liver cancer cells were seeded at a density of 3 × 10

MEF和肝癌细胞以3×10的密度接种

cells per well in 96-well plates. After treatment with various compounds for a specific time, 10 μL of CCK8 solution (#40203ES60, Yeasen, Shanghai, China) were added to each well and incubated for 2 h. The absorbance at 450 nm was detected using a Thermo Multiskan MK3 Microplate reader (Thermo Fisher Scientific, Waltham, MA, USA)..

96孔板中每孔的细胞数。在用各种化合物处理特定时间后，将10μlCCK8溶液（40203ES60，Yeasen，Shanghai，China）加入每个孔中并孵育2小时。使用Thermo Multiskan MK3酶标仪（Thermo Fisher Scientific，Waltham，MA，USA）检测450 nm处的吸光度。。

Intracellular ROS and lipid ROS measurement

The levels of intracellular ROS and lipid peroxidation were quantified using DCFH-DA (#S0033M, Beyotime, Shanghai, China) and C11-BODIPY (#D3861, Thermo Fisher Scientific), respectively, following the manufacturer’s instructions. In brief, cells were incubated with probes for 20 min at 37 °C in a light-shielded environment.

按照制造商的说明，分别使用DCFH-DA（#S0033M，Beyotime，Shanghai，China）和C11-BODIPY（#D3861，Thermo Fisher Scientific）定量细胞内ROS和脂质过氧化的水平。简而言之，将细胞与探针在遮光环境中于37℃温育20分钟。

Subsequently, cells were collected and washed with PBS, and the fluorescence intensity was measured using a CytoFlex flow cytometer (Beckman-Coulter, Fullerton, CA, USA). Data analysis was performed using FlowJo version 10 software..

。使用FlowJo版本10软件进行数据分析。。

Plasmid constructions and lentiviral infection

质粒构建和慢病毒感染

pcDNA3-Flag-mTOR was generously provided by Jie Chen (plasmid #26603, Addgene, Cambridge, MA, USA) [

pcDNA3-Flag-mTOR由Jie Chen慷慨提供（质粒#26603，Addgene，Cambridge，MA，USA）[

]. Open reading frame human CREB1 and SESN3 cDNAs were subcloned into pcDNA3 plasmids, respectively. Short hairpin RNAs (shRNAs) targeting human mTOR, CREB1, and SESN3 were synthesized and subsequently cloned into pLKO.1 lentiviral vector, respectively. Virus particles produced in 293FT cells were harvested, filtered, and then infected into cells supplemented with polybrene.

]。将开放阅读框人CREB1和SESN3 cDNA分别亚克隆到pcDNA3质粒中。合成靶向人mTOR，CREB1和SESN3的短发夹RNA（shRNA），随后分别克隆到pLKO.1慢病毒载体中。收获293FT细胞中产生的病毒颗粒，过滤，然后感染到补充有凝聚胺的细胞中。

Cells were transfected with plasmids, shRNAs and HSP70 siRNAs using Lipofectamine 2000 (#11668019, Invitrogen, Carlsbad, CA, USA). The corresponding sequences for shRNAs/siRNAs are provided below:.

使用Lipofectamine 2000（#11668019，Invitrogen，Carlsbad，CA，USA）用质粒，shRNA和HSP70 siRNA转染细胞。shRNA/siRNA的相应序列如下：。

sh-

mTOR

-1:5’-CCGCTAGTAGGGAGGTTTATT-3′

-1:5’-ccg标记-3’

sh-

mTOR

-2:5’-CCTGGCAACAATAGGAGAATT-3′

-2:5′-CCTGGCAACAATAGGAATT-3′

sh-

CREB1

-1:5’-GCTCGATAAATCTAACAGTTA-3′

-1:5’-总平面图

sh-

CREB1

-2:5’-GCAAACATTAACCATGACCAA-3′

2:5′-GCAAACATTAACCATGACCAA-3′

sh-

SESN3

-1:5’-GCTGAACTTCTTTATGCTCTT-3′

-1： 5'-GCTGAACTTCTTATGCTCTT-3'

sh-

SESN3

-2:5’-CAGTTCTCTAGTGTCAAAGTT-3′

-2： 5'-CAGTTCTCTAGTGTCAAAGTT-3'

si-

si公司-

HSP70

热休克蛋白70

: 5’-GCCTTTCCAAGATTGCTGT-3′

： 5′-GCCTTTCCAAGATTGCTGT-3′

Immunoblotting

免疫印迹

Cellular proteins were extracted using SDS sample buffer supplemented with a Protease and Phosphatase Inhibitor Cocktail (New Cell & Molecular Biotech, Suzhou, China). The samples were denatured, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto nitrocellulose membranes.

使用补充有蛋白酶和磷酸酶抑制剂混合物的SDS样品缓冲液（New Cell＆Molecular Biotech，Suzhou，China）提取细胞蛋白。将样品变性，通过SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离，然后转移到硝酸纤维素膜上。

After blocking with milk, the membranes were incubated with primary antibodies as follows: β-actin (#sc-47778, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), TSC2 (#4308, Cell Signaling Technologies [CST], Danvers, MA, USA), mTOR (#2983, CST), 4EBP1 (#9644, CST), P-4EBP1 (#2855, CST), P70S6K (#2708, CST), P-P70S6K (#9205, CST), PTEN (#9559, CST), P-AKT (#4060, CST), AKT (#4691, CST), PDK1(#3062, CST), CREB1 (#9197, CST), CREBBP (#7389, CST), HSP70 (#M20033, Abmart, Shanghai, China), SESN3 (#11431-2-AP, Proteintech, Rosemont, IL, USA), TFRC (#ab214039, Abcam, Cambridge, UK) and Ferritin (#T55648, Abmart).

Subsequently, the membranes were probed with IRDye secondary antibodies (#32210/#68071, LI-COR Biosciences, Lincoln, NE, USA) and imaged using LI-COR Odyssey CLX..

。。

Coimmunoprecipitation (Co-IP) assay

共免疫沉淀（Co-IP）测定

Protein extracts were incubated with antibodies-coupled magnetic beads (#HY-K0202, MCE) to pull down CREB1 (#9197, CST), CREBBP (#7389, CST), or HSP70 (#M20033, Abmart) at 4 °C overnight. IgG (#32935, CST) served as a negative control for antibodies. The beads were washed with Co-IP buffer three times and then boiled in SDS loading buffer at 98°C for 10 minutes.

将蛋白质提取物与抗体偶联的磁珠（#HY-K0202，MCE）一起温育，以在4℃下将CREB1（#9197，CST），CREBBP（#7389，CST）或HSP70（#M20033，Abmart）下拉过夜。IgG（#32935，CST）作为抗体的阴性对照。将珠子用Co-IP缓冲液洗涤三次，然后在98℃的SDS上样缓冲液中煮沸10分钟。

Subsequently, the proteins were subjected to SDS-PAGE analysis..

随后，对蛋白质进行SDS-PAGE分析。。

Luciferase reporter assay

荧光素酶报告基因测定

For luciferase reporter assays, cells (5 × 10

对于荧光素酶报告基因测定，细胞（5×10

cells per well) were seeded in a 24-well culture plate and incubated overnight. Renilla construct and reporter constructs cloned in pGL3-basic vector (#E1751, Promega Corporation, Madison, WI, USA) were co-transfected with either CREB1 overexpression plasmid or corresponding negative controls, using Lipofectamine 2000.

将每孔细胞接种在24孔培养板中并孵育过夜。使用Lipofectamine 2000将克隆到pGL3-basic载体（#E1751，Promega Corporation，Madison，WI，USA）中的Renilla构建体和报告构建体与CREB1过表达质粒或相应的阴性对照共转染。

After 48 h, luciferase activity was quantified using Dual Luciferase Reporter Gene Assay Kit (#11402ES, Yeasen) and normalized to Renilla luciferase activity according to the manufacturer’s instructions. The primers for human SESN3 gene were as follows: Response element 1 (RE1), 5’-ATCCTGGTACGCTGGAGACC-3’ and 5’-CTTGCATCGCCTACTGGCAA-3’; Response element 1 (RE2), 5’-GGAGACCTGGCTCCCCTAC-3’ and 5’-GCCCTGCTCAGAAAGGAAGG-3’..

48小时后，使用双荧光素酶报告基因测定试剂盒（11402ES，Yeasen）定量荧光素酶活性，并根据制造商的说明将其标准化为海肾荧光素酶活性。人SESN3基因的引物如下：响应元件1（RE1），5'-ATCCTGGTACGCTGGAGACC-3'和5'-CTTGCATCGCCTACTGGCAA-3'；响应元素1（RE2），5'-GGAGACCTGGCTCCCCTAC-3'和5'-GCCCTGCTCAGAAGGAAGG-3'。。

Chromatin-immunoprecipitation (ChIP) assay

The ChIP assay was performed using an Enzymatic Chromatin IP Kit (#9005, CST). Chromatin extracts containing DNA fragments were isolated through immunoprecipitation using CREB1 antibody (#9197, CST), subjected to qPCR using the previously described primers, and DNA enrichment was calculated relative to the total input chromatin: 2% × 2.

使用酶染色质IP试剂盒（#9005，CST）进行ChIP测定。使用CREB1抗体（#9197，CST）通过免疫沉淀分离含有DNA片段的染色质提取物，使用前述引物进行qPCR，并相对于总输入染色质计算DNA富集：2%××2。

(C[T] [2%Input] Sample − C[T] IP Sample)

（C[T][2%输入]样本-C[T]IP样本）

Measurement of total antioxidant capacity, NADPH/NADP+ ratio and GSH level

总抗氧化能力、NADPH/NADP+比值和GSH水平的测定

The total antioxidant activity was determined using a rapid 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) assay (#S0121, Beyotime) following the manufacturer’s protocol. ABTS is oxidized to green ABTS•

按照制造商的方案，使用快速3-乙基苯并噻唑啉-6-磺酸（ABTS）测定法（#S0121，Beyotime）测定总抗氧化活性。ABTS被氧化成绿色ABTS•

in the presence of suitable oxidants. The antioxidant molecules in cell lysates inhibited ABTS radical scavenging activity which was measured by recording the absorbance at 414 nm. The total antioxidant capacity of the samples was determined from a standard curve and reported as Trolox concentration.

在合适的氧化剂存在下。细胞裂解物中的抗氧化分子抑制ABTS自由基清除活性，这是通过记录414nm处的吸光度来测量的。从标准曲线确定样品的总抗氧化能力，并报告为Trolox浓度。

The NADP + /NADPH Assay Kit with WST-8 (#S0179, Beyotime) and the Total Glutathione Assay Kit (#E2015, Pplygen, Beijing, China) were used to assess NADPH/NADP+ ratio and total GSH level, respectively, in accordance with the manufacturers’ instructions. Cell lysates were evaluated by measuring the absorbance at 450 nm or 412 nm.

使用具有WST-8（#S0179，Beyotime）和总谷胱甘肽测定试剂盒（#E2015，Pplygen，Beijing，China）的NADP+/NADPH测定试剂盒分别根据制造商的说明评估NADPH/NADP+比率和总GSH水平。通过测量450 nm或412 nm处的吸光度来评估细胞裂解物。

All results were normalized based on protein content..

所有结果均基于蛋白质含量进行标准化。。

RNA extractions and real-time quantitative PCR

Total RNA was extracted from cells using TRIZOL reagent (Invitrogen). The RNA was reverse transcribed into cDNA using the Hifair® II 1st Strand cDNA Synthesis Kit (#11121ES60, Yeasen). qRT-PCR analysis was performed to quantify gene expression using SYBR qPCR Mix (#abs60086, Absin Biosciences, Shanghai, China).

使用TRIZOL试剂（Invitrogen）从细胞中提取总RNA。使用Hifair®II第一链cDNA合成试剂盒（#11121ES60，Yeasen）将RNA逆转录成cDNA。。

The sequences of the PCR primers used are as follows:.

所用PCR引物的序列如下：。

Human

人类

β-actin

β-阿克汀

Forward 5’-AGAGGGAAATCGTGCGTGAC-3’

Human

人类

β-actin

β-阿克汀

Reverse 5’-CAATAGTGATGACCTGGCCGT-3’;

反向5'-CAATAGTGATGACCTGGCCGT-3'；

Human

人类

CREB1

Forward 5’-GACCACTGATGGACAGCAGATC-3’

前进 5’-GACCACTGATGGACAGCAGATC-3’

Human

人类

CREB1

Reverse 5’-GAGGATGCCATAACAACTCCAGG-3’;

反向5'-GAGGATGCCATAACAACTCCAGG-3'；

Human

人类

SESN3

Forward 5’-GACAGTGACCTGCTATCCTGAG-3’

正向5'-GACAGTGACCTGCTATCCTGAG-3'

Human

人类

SESN3

Reverse 5’-CCGAGTTATGGCACGAAGAGCA-3’;

反向5'-CCGAGTTATGGCACGAAGAGCA-3'；

Human

人类

BCL2

BCL2级

Forward 5’-ATCGCCCTGTGGATGACTGAGT-3’

前进5'-ATCGCCCTGTGGAGTAGAGAGAGT-3'

Human

人类

BCL2

BCL2级

Reverse 5’-GCCAGGAGAAATCAAACAGAGGC-3’;

反向5'-GCCAGGAAATCAAACAGAGGC-3'；

Human

人类

BDNF

脑源性神经营养因子

Forward 5’-CATCCGAGGACAAGGTGGCTTG-3’

前进5'-CATCGAGACAGGTGTG-3'

Human

人类

BDNF

脑源性神经营养因子

Reverse 5’-GCCGAACTTTCTGGTCCTCATC-3’;

反向5'-GCCGAACTTTCTGGTCCTCATC-3'；

Human

人类

ATF3

Forward 5’-CGCTGGAATCAGTCACTGTCAG-3’

正向5'-CGCTGGAATCAGTCATGTCAG-3'

Human

人类

ATF3

Reverse 5’-CTTGTTTCGGCACTTTGCAGCTG-3’;

反向5'-CTTGTTTCGGCACTTTGCAGCTG-3'；

Human

人类

PDK1

Forward 5’-CATGTCACGCTGGGTAATGAGG-3’

Human

人类

PDK1

Reverse 5’-CTCAACACGAGGTCTTGGTGCA-3’;

反向5'-CTCAACAGAGGTCTTGGTGCA-3'；

Mouse

鼠标

β-actin

β-阿克汀

Forward 5’-CATTGCTGACAGGATGCAGAAGG-3’

前进 5’-CATTGCTGACAGGATGCAGAAGG-3’

Mouse

鼠标

β-actin

β-阿克汀

Reverse 5’-TGCTGGAAGGTGGACAGTGAGG-3’;

反向5'-TGCTGGAAGGTGGACAGTGAGG-3'；

Mouse

鼠标

Sesn3

Forward 5’-GCGCATGTATGACAGCTACTGG-3’

Mouse

鼠标

Sesn3

Reverse 5’-TCAGATGCCGAGTTATGGCTCG-3’;

反向5'-TCAGATGCCGAGTTATGGCTCG3'；反向5'-TCAGATGCCGCGCGCGCGCGTCG-3'；反向5'-TCAGATGCCGCGCGCGCGCGCGTCG-3'；反向5'-TCAGATGCCG；

Mouse

鼠标

Pdk1

Forward 5’-CCACTGAGGAAGATCGACAGAC-3’

前进5'-CCACTGAGAAGATCGACAGAC-3'

Mouse

鼠标

Pdk1

Reverse 5’-AGAGGCGTGATATGGGCAATCC-3’;

反向5'-agagggcgattgattggcaatcc-3'；

Mouse

鼠标

Gpx7

Forward 5’-CGACTTCAAGGCGGTCAACATC-3’

前进 5’-CGACTTCAAGGCGGTCAACATC-3’

Mouse

鼠标

Gpx7

Reverse 5’-AAGGCTCGGTAGTTCTGGTCTG-3’;

反向5'-AAGGCTCGGTAGTTCTGGTCTG-3'；

Mouse

鼠标

Ak4

Ak4型

Forward 5’-GAAGCAGTTGCTGCCAGGCTAA-3’

前进 5’-GAAGCAGTTGCTGCCAGGCTAA-3’

Mouse

鼠标

Ak4

Ak4型

Reverse 5’-GCCAGATTCTGTTAGTCTCCGTC-3’

反向5'-GCCAGATTCTGTTAGTCTCCGTC-3'

Fluorescence Imaging

荧光成像

For co-localization, cells were fixed with 4% paraformaldehyde (PFA, #G1101, Servicebio, Wuhan, China) for 20 min, washed three times with PBS, and then treated with blocking buffer (5% BSA, 0.2% Triton X-100 in PBS) for 1 h. After incubation with CREB1 (#9197, CST), CREB1 (#9104, CST), HSP70 (#MA9192S, Abmart) or CREBBP (#TP51023, Abmart) at 4 °C overnight, coverslips were washed with PBS and stained with Alexa-488/Cy3 fluorogenic secondary antibodies (#GB22403/#GB21401, Servicebio) for 2 h at room temperature.

为了共定位，将细胞用4%多聚甲醛（PFA，#G1101，Servicebio，武汉，中国）固定20 分钟，用PBS洗涤3次，然后用封闭缓冲液（5%BSA，0.2%Triton X-100的PBS溶液）处理1次 h、与CREB1（#9197，CST），CREB1（#9104，CST），HSP70（#MA9192S，Abmart）或CREBBP（#TP51023，Abmart）在4孵育后 °C过夜，盖玻片用PBS洗涤，并在室温下用Alexa-488/Cy3荧光二抗（#GB22403/#GB21401，Servicebio）染色2小时。

For intracellular iron content analysis, cells were fixed in 4% PFA after pretreatment with Phen Green.

对于细胞内铁含量分析，用Phen Green预处理后，将细胞固定在4%PFA中。

TM公司

SK (PGSK) fluorescence probe (#P14313, Thermo Fisher Scientific) and washed with PBS three times. Images were captured using a stimulated emission depletion (STED) microscope (Leica SP8 STED, Wetzlar, Germany).

。使用受激发射耗尽（STED）显微镜（Leica SP8 STED，Wetzlar，Germany）捕获图像。

Animal study

动物研究

All animal experiments were approved by the Animal Research Committee, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College. For subcutaneous xenograft study, 2 × 10

所有动物实验均经中国医学科学院基础医学研究所动物研究委员会和北京协和医学院批准。对于皮下异种移植研究，2×10

HCCLM3 cells transfected with vector or mTOR plasmid, or 3 × 10

用载体或mTOR质粒或3×10转染的HCCLM3细胞

SNU886 cells, were subcutaneously injected into the posterior flanks of BALB/c nude mice (female; aged 6 weeks; procured from HFK Bio-Technology, Beijing, China). Once tumor volume reached around 100 mm

将SNU886细胞皮下注射到BALB/c裸鼠（雌性；6周龄；购自HFK Bio Technology，Beijing，China）的后侧。

, mice were randomized into 4 groups (n = 6/group): vehicle, sorafenib (20 mg/kg, i.g.), pifithrin-μ (10 mg/kg, i.p.) and a combination of sorafenib (20 mg/kg, i.g.) and pifithrin-μ (10 mg/kg, i.p.) every other day. Body weights and tumor volumes were measured every 3 days. Tumor volumes were calculated using formula: .

，将小鼠随机分为4组（n=6/组）：载体，索拉非尼（20mg/kg，i.g.），匹非他林-μ（10mg/kg，i.p.）和索拉非尼（20mg/kg，i.g.）和匹非他林-μ（10mg/kg，i.p.）的组合每隔一天。每3天测量体重和肿瘤体积。使用公式计算肿瘤体积：。

五

= (length × width

（长度×宽度）

) × 0.5. For spontaneous liver cancer study,

)对于自发性肝癌研究，

Tsc2

Tsc2型

flox/flox

(stock no. 027458) and

（库存号027458）和

Alb-Cre

Alb-Cre公司

(stock no. 003574) mice were obtained from Jackson Laboratory (Bar Harbor, Maine, USA). 8-month-old

。8个月大

Tsc2

Tsc2型

flox/flox

;

Alb

Alb公司

Cre

Cre公司

mice (a.k.a.

小鼠（又称小鼠）。

Tsc2

Tsc2型

−/−

) were randomly divided into 4 groups (

)随机分为4组(

= 8/group): vehicle, sorafenib (20 mg/kg, i.g.), pifithrin-μ (10 mg/kg, i.p.), and combined sorafenib (20 mg/kg, i.g.) and pifithrin-μ (10 mg/kg, i.p.) every other day for 2 months. Body weights were monitored every 5 days.

。每5天监测一次体重。

Database analysis

数据库分析

Microarray dataset GSE21755 was obtained from Gene Expression Omnibus database (GEO,

微阵列数据集GSE21755是从Gene Expression Omnibus database（GEO，

http://www.ncbi.nlm.nih.gov/geo

) [

]. Differentially expressed gene (DEG) analysis was performed using GEO2R online software. Gene ontology (GO) enrichment analysis was conducted with DAVID functional annotation clustering tool (

]。使用GEO2R在线软件进行差异表达基因（DEG）分析。使用DAVID功能注释聚类工具进行基因本体论（GO）富集分析(

https://david.ncifcrf.gov/

). Genecards database (

)。Genecards数据库(

https://www.genecards.org/

) was used to acquire genes involved in protection against oxidative stress with the potential to regulate cellular sorafenib resistance. We obtained SESN3 promoter sequence, which is 2 kb upstream of transcription start site (TSS) from UCSC genome browser (

)用于获得参与保护免受氧化应激的基因，并具有调节细胞索拉非尼耐药性的潜力。我们从UCSC基因组浏览器获得了SESN3启动子序列，该序列位于转录起始位点（TSS）上游2 kb(

https://genome.ucsc.edu/

) using GRCh38/hg38 assembly. JASPAR Transcription Factor Binding Site database (

)使用GRCh38/hg38组件。JASPAR转录因子结合位点数据库(

https://jaspar.genereg.net/

) was used to predict potential transcription factor of SESN3, generate sequence logo of potential transcription factor and analyze potential response elements on SESN3 promoter sequence. STRING database (

)用于预测SESN3的潜在转录因子，生成潜在转录因子的序列徽标，并分析SESN3启动子序列上的潜在响应元件。字符串数据库(

https://cn.string-db.org/

) was used to conduct protein-protein interaction (PPI) network analysis.

)用于进行蛋白质-蛋白质相互作用（PPI）网络分析。

Statistical analysis

统计分析

Data were presented as mean ± SD. All data were repeated three times, and statistical analyses were performed using a two-tailed

数据以平均SD表示。所有数据重复三次，并使用两尾进行统计分析

test or one-way analysis of variance in GraphPad Prism version 8 (n.s., not statistically significant. *

GraphPad Prism版本8（n.s.，无统计学意义）中的测试或单向方差分析*

< 0.05, **

< 0.05， **

< 0.01, ***

< 0.001).

（0.001）

Results

结果

mTOR activation confers cell resistance to sorafenib

mTOR激活赋予细胞对索拉非尼的耐药性

Loss of tumor suppressor TSC2 or PTEN causes constitutive mTOR activation. To investigate sorafenib sensitivity of mTOR-activated cells, we first treated WT and

肿瘤抑制因子TSC2或PTEN的缺失会导致组成型mTOR激活。为了研究mTOR激活细胞对索拉非尼的敏感性，我们首先处理了WT和

Tsc2

Tsc2型

−/−

MEFs with sorafenib.

MEFs与索拉非尼。

Tsc2

Tsc2型

−/−

MEFs were less sensitive to sorafenib (Fig.

MEF对索拉非尼不太敏感（图）。

1A, B

1A、B

). Compared with WT MEFs,

)。与WT MEF相比，

Pten

Pten公司

−/−

MEFs also exhibited insensitivity to sorafenib (Fig.

MEF对索拉非尼也不敏感（图）。

S1A, B

S1A、B

). We then assessed the impact of mTOR on sorafenib resistance in human liver cancer cells by using 3 TSC2-deficient cell lines (SNU886, SNU398 and Li7) and 3 TSC2 WT cell lines (HCCLM3, HepG2 and MHCC97H) (Fig.

)。然后，我们通过使用3种TSC2缺陷细胞系（SNU886，SNU398和Li7）和3种TSC2 WT细胞系（HCCLM3，HepG2和MHCC97H）评估了mTOR对人肝癌细胞索拉非尼耐药性的影响（图）。

1C级

). mTOR-activated cells were less sensitive to sorafenib than WT cells (Fig.

)。mTOR激活的细胞对索拉非尼的敏感性低于WT细胞（图）。

1维

). Moreover, overexpressing mTOR conferred resistance to HCCLM3 and HepG2 cells (Fig.

)。此外，过表达mTOR赋予HCCLM3和HepG2细胞抗性（图）。

1E, F

1E、F

). In contrast, knockdown of mTOR impaired sorafenib resistance of SNU886 and SNU398 cells (Fig.

)。相反，mTOR的敲低损害了SNU886和SNU398细胞对索拉非尼的耐药性（图）。

1G, H

1克，高

). Furthermore, sorafenib blocked tumorigenesis of HCCLM3 cells more effectively than that of HCCLM3/mTOR cells, manifesting as reduced tumor volumes and tumor weights, with minimal effects on body weights of nude mice (Fig.

)。此外，索拉非尼比HCCLM3/mTOR细胞更有效地阻断HCCLM3细胞的肿瘤发生，表现为肿瘤体积和肿瘤重量减少，对裸鼠体重的影响最小（图）。

1I–L

). Taken together, mTOR confers cell resistance to sorafenib.

)。总之，mTOR赋予细胞对索拉非尼的抗性。

Fig. 1: mTOR activation confers cell resistance to sorafenib.

图1:mTOR激活赋予细胞对索拉非尼的抗性。

Immunoblotting of

免疫印迹

Tsc2

Tsc2型

−/−

MEFs.

MEFs。

B类

Viability of

生存能力

Tsc2

Tsc2型

−/−

MEFs treated with different concentrations of sorafenib for 24 h,

用不同浓度的索拉非尼处理MEF 24小时，

= 3.

= 3。

C级

Immunoblotting of human liver cancer cell lines.

人肝癌细胞系的免疫印迹。

Viability of liver cancer cells treated with sorafenib for 24 h,

索拉非尼治疗肝癌细胞24小时的活力，

= 3.

= 3。

–

小时

CCK8 analysis of HCCLM3 (

HCCLM3的CCK8分析(

) or HepG2 (

)或HepG2(

F级

) cells transfected with vector or mTOR plasmid, SNU886 (

)用载体或mTOR质粒SNU886转染的细胞(

克

) or SNU398 (

或SNU398（SNU398）

小时

) cells transfected with control or mTOR shRNA, followed by treatment with sorafenib for 24 h,

)用对照或mTOR shRNA转染细胞，然后用索拉非尼处理24小时，

= 3.

= 3。

我

–

Nude mice were xenografted with HCCLM3 cells transfected with vector or mTOR plasmid. Once tumor volume reached ~100 mm

用载体或mTOR质粒转染的HCCLM3细胞异种移植裸鼠。一旦肿瘤体积达到〜100毫米

, mice were treated with vehicle or sorafenib (20 mg/kg, i.g.) every other day (

，每隔一天用媒介物或索拉非尼（20 mg/kg，i.g.）治疗小鼠(

= 6 per group). Measurements of body weights (

每组6个）。体重测量(

我

) and tumor volumes (

)和肿瘤体积(

) of nude mice every 3 days. Representative images of tumors (

)裸鼠每3天一次。肿瘤的代表性图像(

) and tumor weights (

)和肿瘤重量(

) were plotted at the end of treatment. Data are displayed as mean ± SD (error bars). *

)在治疗结束时绘制。数据显示为平均值±SD（误差线）*

< 0.05, **

< 0.05， **

< 0.01, ***

< 0.01， ***

< 0.001. Sora: sorafenib.

< 0.001.姐姐：索拉非尼。

Full size image

全尺寸图像

mTOR attenuates sorafenib-mediated ROS accumulation and oxidative stress in liver cancer cells

mTOR减弱索拉非尼介导的肝癌细胞中ROS的积累和氧化应激

Sorafenib wields therapeutic effects mainly through the accumulation of lipid ROS and induction of ferroptosis [

索拉非尼主要通过脂质ROS的积累和铁浓化的诱导发挥治疗作用[

]. Consistently, the effect of sorafenib on HCCLM3 cells was partially reversed by Fer-1 (ferroptosis inhibitor), NAC (ROS scavenger) or DFO (iron chelator), but not Nec-1 (necrosis inhibitor) or CQ (autophagy inhibitor) (Fig.

]。。

2A级

). Sorafenib increased intracellular ROS and lipid peroxidation in HCCLM3 cells (Fig.

)。索拉非尼增加HCCLM3细胞内ROS和脂质过氧化（图）。

2B, C

2B，C

). Furthermore, sorafenib raised intracellular Fe

)。此外，索拉非尼提高了细胞内铁

level, as shown by the quenching of PGSK fluorescence (Fig.

水平，如PGSK荧光的猝灭所示（图）。

二维

, Fig.

，图。

S2A

S2A公司

). As tumor cells often exhibit increased metabolic antioxidant capacity through Warburg effect to promote proliferation [

)。由于肿瘤细胞通常通过Warburg效应表现出增强的代谢抗氧化能力，从而促进增殖[

], we speculated that activation of mTOR facilitates sorafenib resistance through increasing cellular antioxidant capacity of liver cancer cells. Indeed, sorafenib induction of ROS was compromised in HCCLM3/mTOR cells but not HCCLM3 cells (Fig.

]，我们推测mTOR的激活通过增加肝癌细胞的细胞抗氧化能力来促进索拉非尼的耐药性。事实上，索拉非尼诱导ROS在HCCLM3/mTOR细胞中受损，但在HCCLM3细胞中没有受损（图）。

2E级

). Furthermore, total antioxidant capacity, GSH level and NADPH/NADP+ ratio were increased in HCCLM3/mTOR cells as compared to controls (Fig.

)。此外，与对照组相比，HCCLM3/mTOR细胞的总抗氧化能力，GSH水平和NADPH/NADP+比值增加（图）。

2楼

). Similar findings were also observed in HepG2/mTOR cells (Fig.

)。在HepG2/mTOR细胞中也观察到类似的发现（图）。

S2B, C

S2B、C

). By contrast, sorafenib increased intracellular ROS level in SNU886/sh

)。相比之下，索拉非尼增加了SNU886/sh中的细胞内ROS水平

mTOR

cells (Fig.

细胞（图）。

2克

) and SNU398/sh

)和SNU398/sh

mTOR

cells (Fig.

细胞（图）。

S2D

S2D公司

). Additionally, knockdown of mTOR reduced total antioxidant capacity, GSH level and NADPH/NADP+ ratio in SNU886 cells (Fig.

)。此外，mTOR的敲低降低了SNU886细胞的总抗氧化能力，GSH水平和NADPH/NADP+比值（图）。

2小时

) and SNU398 cells (Fig.

)和SNU398细胞（图）。

S2E

S2E系列

). These results suggest that mTOR improves the antioxidant ability of liver cancer cells and attenuates sorafenib-induced ferroptosis.

)。这些结果表明，mTOR可提高肝癌细胞的抗氧化能力，并减轻索拉非尼诱导的铁浓化。

Fig. 2: mTOR attenuates sorafenib-mediated ROS accumulation and oxidative stress in liver cancer cells.

图2:mTOR减弱索拉非尼介导的肝癌细胞中ROS积累和氧化应激。

Viability of HCCLM3 cells treated with sorafenib (10 μM) in the presence or absence of specific inhibitors for 24 h,

在存在或不存在特异性抑制剂的情况下，用索拉非尼（10m）处理HCCLM3细胞24小时的活力，

= 3. Ferrostatin-1 (Fer-1, 10 μM), N-acetylcysteine (NAC, 1 mM), deferoxamine (DFO, 10 μM), necrostatin-1 (Nec-1, 20 μM) and chloroquine (CQ, 10 μM).

3.Ferrostatin-1（Fer-1,10μM），N-乙酰半胱氨酸（NAC，1mM），去铁胺（DFO，10μM），necrostatin-1（Nec-1,20μM）和氯喹（CQ，10μM）。

B类

–

HCCLM3 cells were treated with sorafenib (10 μM) for 24 h,

用索拉非尼（10m）处理HCCLM3细胞24小时，

= 3. The intracellular ROS (

细胞内活性氧(

B类

) and lipid peroxidation (

)和脂质过氧化(

C级

) were measured with flow cytometry. Cytosolic Fe

)。胞质铁

levels were assayed by PGSK probe, fluorescence intensity was observed under the fluorescence microscope, scale bar=50 μm (

通过PGSK探针测定水平，在荧光显微镜下观察荧光强度，比例尺=50μm(

F级

HCCLM3 cells were transfected with vector or mTOR plasmid and treated with sorafenib (10 μM) for 24 h,

用载体或mTOR质粒转染HCCLM3细胞，并用索拉非尼（10m）处理24小时，

= 3. The intracellular ROS were measured with flow cytometry (

流式细胞仪检测细胞内活性氧(

). Relative total antioxidant capacity, GSH level, and NADPH/NADP+ ratio were measured (

)。测定相对总抗氧化能力、谷胱甘肽水平和NADPH/NADP+比值(

F级

克

小时

SNU886 cells were transfected with control or mTOR shRNA and treated with sorafenib (10 μM) for 24 h,

用对照或mTOR shRNA转染SNU886细胞，并用索拉非尼（10m）处理24小时，

= 3. The intracellular ROS were measured with flow cytometry (

流式细胞仪检测细胞内活性氧(

克

). Relative total antioxidant capacity, GSH level, and NADPH/NADP+ ratio were measured (

)。测定相对总抗氧化能力、谷胱甘肽水平和NADPH/NADP+比值(

小时

). Data are displayed as mean ± SD (error bars). **

)。数据显示为平均值±SD（误差线）**

< 0.01, ***

< 0.01， ***

< 0.001. Sora: sorafenib.

< 0.001。索拉尼布（Sorafenib）

Full size image

全尺寸图像

mTOR-enhanced SESN3 promotes sorafenib resistance

mTOR增强的SESN3促进索拉非尼耐药

To identify the potential key factor(s) responsible for sorafenib resistance in mTOR-activated cells, we analyzed differentially expressed genes (DEGs) between

为了确定mTOR激活细胞中索拉非尼耐药的潜在关键因素，我们分析了两者之间的差异表达基因（DEGs）

Tsc2

Tsc2型

−/−

MEFs and WT MEFs in GEO database (GSE21755) (Fig.

GEO数据库（GSE21755）中的MEF和WT MEF（图）。

3A级

). The upregulated genes but not the downregulated genes were enriched in the pathways relevant to oxidoreductase activity, aldehyde dehydrogenase activity, and cellular responses to hypoxia (Fig.

)。上调的基因而不是下调的基因富含与氧化还原酶活性，醛脱氢酶活性和细胞对缺氧反应相关的途径（图）。

S3A, B

S3A、B

). We then overlapped the upregulated genes in

)。然后，我们将上调的基因重叠在

Tsc2

Tsc2型

−/−

MEFs with the genes associated with oxidative stress protection from the Genecards database and identified four potential candidates responsible for sorafenib resistance:

来自Genecards数据库的具有与氧化应激保护相关基因的MEF，并确定了导致索拉非尼耐药的四个潜在候选者：

Pdk1, Gpx7, Sesn3

Pdk1、Gpx7、Sesn3

, and

，以及

Ak4

Ak4型

(Fig.

（图。

3B级

). qRT-PCR confirmed overexpression of

)。qRT-PCR证实过表达

Sesn3

and

和

Pdk1

在里面

Tsc2

Tsc2型

−/−

MEFs (Fig.

MEF（图）。

S3C

S3C公司

). Additionally, SESN3 but not PDK1 was increased at mRNA and protein levels in both HCCLM3/mTOR cells and HepG2/mTOR cells (Figs.

)。此外，在HCCLM3/mTOR细胞和HepG2/mTOR细胞中，SESN3而非PDK1在mRNA和蛋白质水平均增加（图）。

and

和

S3D, E

S3D，E

). In contrast, silencing mTOR decreased mRNA and protein of SESN3 in both SNU886 and SNU398 cells (Fig.

)。相反，沉默mTOR降低了SNU886和SNU398细胞中SESN3的mRNA和蛋白（图）。

). Next, we sought to determine whether SESN3 was involved in the regulation of sorafenib resistance in mTOR-activated liver cancer cells. We overexpressed SESN3 in HCCLM3 cells and silenced SESN3 in SNU886 cells (Fig.

)。接下来，我们试图确定SESN3是否参与mTOR激活的肝癌细胞中索拉非尼耐药性的调节。我们在HCCLM3细胞中过表达SESN3，在SNU886细胞中沉默SESN3（图）。

3E, F

3E，F

). Overexpressed SESN3 increased sorafenib resistance of HCCLM3 cells, as indicated by reduction of intracellular ROS and enhancement of cell viability (Fig.

)。过表达的SESN3增加了HCCLM3细胞对索拉非尼的耐药性，如细胞内ROS的减少和细胞活力的增强所示（图）。

3G, H

3G，小时

). Conversely, silencing SESN3 enhanced sorafenib sensitivity by increasing intracellular ROS and decreasing the viability of SNU886 cells (Fig.

)。相反，沉默SESN3通过增加细胞内ROS和降低SNU886细胞的活力来增强索拉非尼的敏感性（图）。

3I, J

3I，J

). To confirm sorafenib resistance of mTOR-activated liver cancer cells relying on high expression of SESN3, we overexpressed SESN3 in SNU886/sh

)。为了证实依赖于SESN3高表达的mTOR激活的肝癌细胞对索拉非尼的耐药性，我们在SNU886/sh中过表达了SESN3

mTOR

cells (Fig.

细胞（图）。

3公里

). Overexpression of SESN3 reversed the impairment of sorafenib resistance caused by mTOR silencing, as evidenced by enhanced cell viability (Fig.

)。SESN3的过度表达逆转了由mTOR沉默引起的索拉非尼耐药性的损害，如细胞活力增强所证明的（图）。

3升

). SESN3 is thus positively regulated by mTOR and is critical for mTOR-mediated sorafenib resistance by reducing oxidative stress.

)。因此，SESN3受mTOR正调控，通过减少氧化应激对mTOR介导的索拉非尼耐药至关重要。

Fig. 3: mTOR-enhanced SESN3 promotes sorafenib resistance.

图3:mTOR增强的SESN3促进索拉非尼耐药。

Volcano plot exhibits the differentially expressed genes (DEGs) of

火山图显示了

Tsc2

Tsc2型

−/−

MEFs vs WT MEFs from GSE21755 dataset in GEO database. The thresholds were set as false discovery rate (FDR) < 0.01 and fold change >1.5.

GEO数据库中GSE21755数据集中的MEF与WT MEF。阈值设置为错误发现率（FDR）<0.01，倍数变化>1.5。

B类

Schematic delineation of flows and results of screening for potential candidates.

潜在候选人筛选流程和结果的示意图。

C级

mRNA and protein levels of SESN3 in HCCLM3 or HepG2 cells (

HCCLM3或HepG2细胞中SESN3的mRNA和蛋白水平(

C级

) transfected with vector or mTOR plasmid, and SNU886 or SNU398 cells (

)用载体或mTOR质粒以及SNU886或SNU398细胞转染(

) transfected with control or mTOR shRNA.

)用对照或mTOR shRNA转染。

F级

Immunoblotting of HCCLM3 cells (

HCCLM3细胞的免疫印迹(

) transfected with vector or SESN3 plasmid, and SNU886 cells (

)用载体或SESN3质粒转染SNU886细胞(

F级

) transfected with control or SESN3 shRNA.

)用对照或SESN3 shRNA转染。

克

小时

HCCLM3 cells were transfected with vector or SESN3 plasmid,

用载体或SESN3质粒转染HCCLM3细胞，

= 3.The intracellular ROS of cells treated with sorafenib (10 μM) for 24 h (

3.索拉非尼（10m）处理细胞24小时后细胞内活性氧（ROS）的变化(

克

). Viability of cells treated with different concentrations of sorafenib for 24 h (

)。用不同浓度的索拉非尼处理24小时的细胞活力(

小时

我

SNU886 cells were transfected with control or SESN3 shRNA,

用对照或SESN3 shRNA转染SNU886细胞，

= 3. The intracellular ROS of cells treated with sorafenib (10 μM) for 24 h (

用索拉非尼（10μM）处理24小时的细胞内ROS(

我

). Viability of cells treated with different concentrations of sorafenib for 24 h (

)。用不同浓度的索拉非尼处理24小时的细胞活力(

SNU886/sh

mTOR

cells were transfected with vector or SESN3 plasmid. Immunoblotting of cells (

用载体或SESN3质粒转染细胞。细胞免疫印迹(

). Viability of cells treated with different concentrations of sorafenib for 24 h,

)。，

= 3 (

（3）

). Data are displayed as mean ± SD (error bars).

)。数据显示为平均值±SD（误差线）。

< 0.01,

***

< 0.001. Sora: sorafenib.

< 0.001。索拉尼布（Sorafenib）

Full size image

全尺寸图像

CREB1 stimulates SESN3 transcription

CREB1刺激SESN3转录

To determine whether mTOR activation of SESN3 expression is at the transcriptional level, we obtained SESN3 promoter sequence using UCSC genome browser and identified potential transcription factors in JASPAR database (Fig.

为了确定SESN3表达的mTOR激活是否在转录水平，我们使用UCSC基因组浏览器获得了SESN3启动子序列，并在JASPAR数据库中鉴定了潜在的转录因子（图）。

S4A, B

S4A，B

). Among them, CREB1 is a transcription factor that is positively regulated by mTOR in multiple biological processes [

)。其中，CREB1是一种转录因子，在多种生物过程中受mTOR正调控[

]. CREB1 was enriched in both HCCLM3/mTOR and HepG2/mTOR cells (Fig.

]。CREB1富含HCCLM3/mTOR和HepG2/mTOR细胞（图）。

4A级

). Conversely, silencing mTOR reduced CREB1 expression in SNU886 and SNU398 cells (Fig.

)。相反，沉默mTOR降低了SNU886和SNU398细胞中CREB1的表达（图）。

4B级

). Furthermore, CREB1 overexpression increased SESN3 mRNA and protein levels in HCCLM3 and HepG2 cells (Fig.

)。此外，CREB1过表达增加了HCCLM3和HepG2细胞中SESN3 mRNA和蛋白水平（图）。

4C, D

4C，D

) while knockdown of CREB1 reduced SESN3 expression in SNU886 and SNU398 cells (Fig.

)而CREB1的敲低降低了SNU886和SNU398细胞中SESN3的表达（图）。

4E, F

4E，F

). Moreover, overexpressing CREB1 reversed the impairment of sorafenib resistance caused by mTOR silencing in SNU886 cells, as evidenced by enhanced cell viability (Fig.

)。此外，过表达CREB1逆转了SNU886细胞中mTOR沉默引起的索拉非尼耐药性的损害，如细胞活力增强所证明的（图）。

4G, H

4G，小时

). These findings suggest that CREB1 activation of SESN3 expression is critical for sorafenib resistance mediated by active mTOR.

)。。

Fig. 4: CREB1 stimulates SESN3 transcription.

图4:CREB1刺激SESN3转录。

B类

Immunoblotting of HCCLM3 or HepG2 cells (

HCCLM3或HepG2细胞的免疫印迹(

) transfected with vector or mTOR plasmid, and SNU886 or SNU398 cells (

)用载体或mTOR质粒以及SNU886或SNU398细胞转染(

B类

) transfected with control or mTOR shRNA.

)用对照或mTOR shRNA转染。

C级

mRNA (

mRNA(

C级

) and protein (

)和蛋白质(

) levels of SESN3 in HCCLM3 and HepG2 cells transfected with vector or CREB1 plasmid.

)用载体或CREB1质粒转染的HCCLM3和HepG2细胞中SESN3的水平。

F级

mRNA (

mRNA(

) and protein (

)和蛋白质(

F级

) levels of SESN3 in SNU886 and SNU398 cells transfected with control or CREB1 shRNA.

)用对照或CREB1 shRNA转染的SNU886和SNU398细胞中SESN3的水平。

克

小时

SNU886/sh

mTOR

cells were transfected with vector or CREB1 plasmid. Immunoblotting of cells (

用载体或CREB1质粒转染细胞。细胞免疫印迹(

克

). Viability of cells treated with different concentrations of sorafenib for 24 h,

)。，

= 3 (

（3）

小时

我

Sequence logo of CREB1 binding motif generated from JASPAR database.

从JASPAR数据库生成的CREB1结合基序的序列徽标。

Schematic representation of human SESN3 genomic structure. Shown are two potential CREB1 response elements (RE1 and RE2) and the corresponding mutant response elements (RE1 mut, RE2 mut).

人类SESN3基因组结构的示意图。显示了两个潜在的CREB1响应元件（RE1和RE2）和相应的突变响应元件（RE1 mut，RE2 mut）。

Relative luciferase activity detected after transfection of luciferase reporter constructs containing RE1, RE2, RE1 mut, or RE2 mut into HCCLM3 cells. Renilla vector was used as a transfection internal control,

将含有RE1，RE2，RE1-mut或RE2-mut的荧光素酶报告基因构建体转染到HCCLM3细胞后，检测到相对荧光素酶活性。Renilla载体用作转染内部对照，

= 5.

= 5。

Interaction between CREB1 and promoter region of SESN3 analyzed in SNU886 cells by CHIP assay. Three PCR probe sets were designed, namely RE1, RE2, and NC-RE, a negative control probe that is 5 kb upstream of the transcription start site of SESN3,

通过CHIP分析在SNU886细胞中分析CREB1与SESN3启动子区域之间的相互作用。设计了三种PCR探针组，即RE1，RE2和NC-RE，这是一种阴性对照探针，位于SESN3转录起始位点上游5 kb，

= 3. Data are displayed as mean ± SD (error bars).

数据显示为平均值±SD（误差线）。

< 0.01,

< 0.01，

***

< 0.001. Sora: sorafenib. TSS: transcription start site.

<0.001。索拉：索拉非尼。TSS：转录开始网站。

Full size image

全尺寸图像

To find out whether SESN3 is a transcriptional target of CREB1, JASPAR database was used to search for potential CREB1 response elements on the promoter region of SESN3 (Fig.

为了确定SESN3是否是CREB1的转录靶标，使用JASPAR数据库搜索SESN3启动子区域上潜在的CREB1反应元件（图）。

4I号

). Two putative CREB1 response elements (RE1, RE2) were identified with the highest predicted scores. Subsequently, we cloned sequences of wild-type response elements (RE1, RE2) and corresponding mutant response elements (RE1 mut, RE2 mut), respectively, into firefly luciferase reporter plasmids (Fig.

)。确定了两个推定的CREB1反应元件（RE1，RE2），其预测得分最高。随后，我们将野生型反应元件（RE1，RE2）和相应的突变反应元件（RE1 mut，RE2 mut）的序列分别克隆到萤火虫荧光素酶报告质粒中（图）。

4J型

). Only RE1 but not RE2, RE1 mut, or RE2 mut induced luciferase expression in response to CREB1 overexpression in HCCLM3 cells (Fig.

)。只有RE1而不是RE2，RE1 mut或RE2 mut诱导荧光素酶表达以响应HCCLM3细胞中CREB1的过表达（图）。

). To check the potential direct interaction between CREB1 and SESN3 promoter, we performed a ChIP assay in SNU886 cells and observed that CREB1 was preferentially enriched in the RE1 genomic region (Fig.

)。为了检查CREB1和SESN3启动子之间潜在的直接相互作用，我们在SNU886细胞中进行了ChIP分析，观察到CREB1优先富集在RE1基因组区域（图）。

4升

). Therefore, CREB1 transcriptionally stimulates

)。

SESN3

expression by directly binding to

SESN3

promoter.

发起人。

HSP70 facilitates the interaction between CREB1 and CREBBP

HSP70促进CREB1和CREBBP之间的相互作用

It has been documented that CREBBP, as a coactivator, participates in the transcriptional regulation of CREB1 [

已有文献表明，CREBBP作为共激活因子参与CREB1的转录调控[

]. To investigate the role of CREBBP in mTOR regulation of CREB1, we first assessed CREBBP expression in SNU886 and SNU398 cells with or without mTOR depletion. mTOR did not affect CREBBP expression (Fig.

]。为了研究CREBBP在CREB1的mTOR调节中的作用，我们首先评估了有或没有mTOR消耗的SNU886和SNU398细胞中CREBBP的表达。mTOR不影响CREBBP表达（图）。

5A级

). Furthermore, SGC-CBP30, a CREBBP inhibitor, reduced the mRNA levels of SESN3 and other CREB1 target genes, including

)。此外，CREBBP抑制剂SGC-CBP30降低了SESN3和其他CREB1靶基因的mRNA水平，包括

ATF3, BCL2 and BDNF

ATF3，BCL2和BDNF

(Fig.

（图。

5B条

). CREBBP maintained CREB1 transcriptional activity without affecting CREB1 expression (Fig.

)。CREBBP维持CREB1转录活性而不影响CREB1表达（图）。

5摄氏度

). To confirm the direct interaction between CREB1 and CREBBP, we performed immunofluorescent staining of SNU886 and SNU398 cells. CREB1 and CREBBP were colocalized in nuclei (Fig.

)。为了证实CREB1和CREBBP之间的直接相互作用，我们对SNU886和SNU398细胞进行了免疫荧光染色。CREB1和CREBBP在细胞核中共定位（图）。

5天

). The Co-IP experiment showed that CREBBP interacted with CREB1 in SNU886 and SNU398 cells (Fig.

)。Co-IP实验表明，CREBBP与SNU886和SNU398细胞中的CREB1相互作用（图）。

5E, F

5E，F

). These results indicate that CREBBP probably binds to CREB1 and facilitates CREB1 transcriptional regulation in mTOR-activated cells.

)。这些结果表明，CREBBP可能与CREB1结合，并促进mTOR激活细胞中CREB1的转录调控。

Fig. 5: HSP70 facilitates the physical interaction between CREB1 and CREBBP.

图5:HSP70促进CREB1和CREBBP之间的物理相互作用。

Immunoblotting of SNU886 or SNU398 cells transfected with control or mTOR shRNA.

用对照或mTOR shRNA转染的SNU886或SNU398细胞的免疫印迹。

B类

C级

SNU886 cells were treated with SGC-CBP30 (5 μM) for 24 h. mRNA levels of CREB1 target genes,

用SGC-CBP30（5μM）处理SNU886细胞24小时。CREB1靶基因的mRNA水平，

= 3 (

（3）

B类

). Immunoblotting of cells (

)。细胞免疫印迹(

C级

Confocal images illustrating the co-localization of CREB1 and CREBBP in SNU886 and SNU398 cells. Scale bar = 10 μm.

共聚焦图像显示CREB1和CREBBP在SNU886和SNU398细胞中的共定位。比例尺=10μm。

F级

Co-IP demonstrating the interaction of CREB1 and CREBBP in SNU886 (

Co-IP证明了SNU886中CREB1和CREBBP的相互作用(

) and SNU398 (

（SNU398）

F级

) cells.

)细胞。

克

小时

Confocal images showing the interaction of HSP70 with CREB1 (

共聚焦图像显示HSP70与CREB1的相互作用(

克

) or CREBBP (

（或CREBBP）

小时

) in SNU886 and SNU398 cells. Scale bar = 10 μm.

)在SNU886和SNU398细胞中。比例尺=10μm。

我

Co-IP experiments showing the interaction of HSP70, CREB1, and CREBBP in SNU886 (

Co-IP实验显示SNU886中HSP70，CREB1和CREBBP的相互作用(

我

) and SNU398 (

（SNU398）

) cells.

)细胞。

Co-IP analysis for CREB1 interaction with CREBBP after silencing HSP70 in SNU886 cells. Data are displayed as mean ± SD (error bars). n.s., not statistically significant,

在SNU886细胞中沉默HSP70后，CREB1与CREBBP相互作用的Co-IP分析。数据显示为平均值±SD（误差线）。n、在美国，没有统计学意义，

< 0.01,

< 0.01，

***

< 0.001.

< 0.001。

Full size image

全尺寸图像

Even though SGC-CBP30 hinders the transcriptional activity of CREB1 and has the potential to alleviate sorafenib resistance in mTOR-activated liver cancer, SGC-CBP30 is metabolized too quickly in vivo and might not be suitable for clinical application [

[

]. Therefore, we sought to identify other bioactive molecules with disruptive effects on CREB1 transcription. HSP70 is a ubiquitous chaperone and plays crucial roles in biological processes such as protein folding and assembly of protein complexes [

]。因此，我们试图鉴定对CREB1转录具有破坏作用的其他生物活性分子。HSP70是一种无处不在的伴侣蛋白，在蛋白质折叠和蛋白质复合物组装等生物过程中起着至关重要的作用[

]. Supported by prediction from PPI network in STRING database, we speculated that HSP70 might participate in the assembly of CREB1/CREBBP complexes to influence CREB1-mediated transcription (Fig.

]。在STRING数据库中PPI网络的预测支持下，我们推测HSP70可能参与CREB1/CREBBP复合物的组装，从而影响CREB1介导的转录（图）。

S5级

). We thus investigated the subcellular location of HSP70 and its relationship with CREB1 and CREBBP in SNU886 and SNU398 cells. HSP70 was colocalized with both CREB1 and CREBBP in nuclei (Fig.

)。因此，我们研究了HSP70的亚细胞定位及其与SNU886和SNU398细胞中CREB1和CREBBP的关系。。

5G, H

5G，小时

). Co-IP experiments further confirmed the interaction of HSP70 with CREB1/CREBBP complexes (Fig.

)。Co-IP实验进一步证实了HSP70与CREB1/CREBBP复合物的相互作用（图）。

5I, J

5I，J

). Next, we silenced HSP70 in SNU886 cells to test whether HSP70 is necessary for the formation of CREB1/CREBBP complexes. Depleted HSP70 reduced co-precipitation of CREB1 and CREBBP in SNU886 cells (Fig.

)。接下来，我们沉默了SNU886细胞中的HSP70，以测试HSP70是否是形成CREB1/CREBBP复合物所必需的。。

5公里

). Transcriptional activity of CREB1 thus relies on the formation of CREB1/CREBBP complexes. HSP70 is crucial in maintaining the interaction between CREB1 and CREBBP.

)。因此，CREB1的转录活性依赖于CREB1/CREBBP复合物的形成。HSP70对于维持CREB1和CREBBP之间的相互作用至关重要。

HSP70 inhibitor pifithrin-μ augments sorafenib-induced ferroptosis in mTOR-activated liver cancer cells

HSP70抑制剂pifithrin-μ增强索拉非尼诱导的mTOR激活的肝癌细胞铁浓化

To test whether HSP70 contributed to sorafenib resistance of mTOR-activated cells, we treated SNU886 cells with HSP70 inhibitor pifithrin-μ (PES). Pifithrin-μ disrupted the interaction between CREB1 and CREBBP (Fig.

为了测试HSP70是否有助于mTOR激活细胞对索拉非尼的耐药性，我们用HSP70抑制剂pifithrin-μ（PES）处理了SNU886细胞。Pifithrin-μ破坏了CREB1和CREBBP之间的相互作用（图）。

) and reduced the mRNA levels of CREB1 target genes (Fig.

)并降低CREB1靶基因的mRNA水平（图）。

). Furthermore, pifithrin-μ suppressed SESN3 expression by disrupting the recruitment of CREB1 to genomic region of SESN3-RE1, but not NC-RE, a negative control probe which is 5 kb upstream of transcription start site of SESN3 (Fig.

)。此外，pifithrinμ通过破坏CREB1向SESN3-RE1基因组区域的募集来抑制SESN3表达，而不是NC-RE，这是一种在SESN3转录起始位点上游5kb的阴性对照探针（图）。

6C, D

6C，D

). Moreover, pifithrin-μ potentiated the cell death caused by sorafenib in SNU886 cells (Fig.

)。此外，pifithrin-μ增强了索拉非尼在SNU886细胞中引起的细胞死亡（图）。

6E型

). On the other hand, SESN3 overexpression canceled the lethality caused by combination of pifithrin-μ and sorafenib (Figs.

)。另一方面，SESN3过表达抵消了由pifithrin-μ和索拉非尼联合引起的致死率（图）。

6F级

and

和

S6A

S6A系列

). To ascertain that pifithrin-μ and sorafenib-induced ferroptosis in mTOR-activated cells, we examined biomarkers of ferroptosis in SNU886 cells treated with pifithrin-μ and sorafenib. Compared to sorafenib alone, combined pifithrin-μ and sorafenib increased intracellular Fe

)。为了确定pifithrin-μ和索拉非尼在mTOR激活的细胞中诱导铁浓化，我们检测了用pifithrin-μ和索拉非尼处理的SNU886细胞中铁浓化的生物标志物。与单独使用索拉非尼相比，联合使用pifithrin和索拉非尼可增加细胞内铁

level illustrated as quenching of PGSK probe (Fig.

水平显示为PGSK探针的淬火（图）。

6克

, Fig.

，图。

S6B

S6B系列

). Moreover, pifithrin-μ potentiated sorafenib-induced generation of intracellular ROS and lipid peroxidation in SNU886 cells (Fig.

)。此外，pifithrinμ增强了索拉非尼诱导的SNU886细胞内ROS和脂质过氧化的产生（图）。

6H, I

6小时，我

). These in vitro findings prompted us to test the efficacy of this combinatory strategy in vivo. We injected SNU886 cells subcutaneously into nude mice. Combined pifithrin-μ and sorafenib treatment achieved greater inhibition of tumor growth than single drug application (Fig.

)。这些体外发现促使我们在体内测试这种组合策略的功效。我们将SNU886细胞皮下注射到裸鼠体内。。

6J–L

). There were no significant differences on body weights of nude mice with various combination of the two drugs (Fig.

)。两种药物的不同组合对裸鼠体重没有显着差异（图）。

S6C

S6C系列

). Taken together, pifithrin-μ suppresses CREB1/CREBBP complex formation, CREB1-mediated transcription, and sorafenib resistance of mTOR-activated liver cancer cells.

)。总之，pifithrin-μ抑制mTOR激活的肝癌细胞的CREB1/CREBBP复合物形成，CREB1介导的转录和索拉非尼抗性。

Fig. 6: HSP70 inhibitor Pifithrin-μ augments sorafenib-induced ferroptosis in mTOR-activated liver cancer cells.

图6:HSP70抑制剂Pifithrin-μ增强了索拉非尼诱导的mTOR激活的肝癌细胞中的铁浓化。

–

SNU886 cells were treated with pifithrin-μ (5 μM) for 24 h. Co-IP analysis demonstrates the interaction between CREB1, CREBBP, and HSP70 (

用pifithrinμ（5μM）处理SNU886细胞24小时。Co-IP分析证明了CREB1，CREBBP和HSP70之间的相互作用(

). mRNA levels of CREB1 target genes,

)。CREB1靶基因的mRNA水平，

= 3 (

（3）

B类

). Immunoblotting of cells (

)。细胞免疫印迹(

C级

). ChIP assay testing the recruitment of CREB1 to SESN3 genomic region RE1,

)。ChIP分析测试CREB1向SESN3基因组区域RE1的募集，

= 3 (

（3）

–

我

Concentration of pifithrin-μ and sorafenib were 5 μM and 10 μM, respectively. The treatment time was 24 h,

pifithrin-μ和索拉非尼的浓度分别为5μM和10μM。治疗时间为24小时，

= 3. Viability of SNU886 (

=3.SNU886的可行性(

) and SNU886 (

（冷落886）

F级

) cells transfected with vector or SESN3 plasmid. Representative confocal images using fluorescent PGSK dye, scale bar = 50 μm (

)用载体或SESN3质粒转染的细胞。使用荧光PGSK染料的代表性共焦图像，比例尺=50μm(

克

), relative ROS level (

)，相对活性氧水平(

小时

), and lipid peroxidation (

)和脂质过氧化(

我

) of SNU886 cells.

)SNU886细胞。

–

Nude mice were xenografted with SNU886 cells. Once tumor volume reached ~100 mm

裸鼠用SNU886细胞异种移植。一旦肿瘤体积达到〜100毫米

, mice were treated with vehicle, pifithrin-μ (10 mg/kg, i.p.), sorafenib (20 mg/kg, i.g.), or combined pifithrin-μ (10 mg/kg, i.p.) and sorafenib (20 mg/kg, i.g.) every other day (

，每隔一天用媒介物，匹非替林（10mg/kg，i.p.），索拉非尼（20mg/kg，i.g.）或联合匹非替林（10mg/kg，i.p.）和索拉非尼（20mg/kg，i.g.）治疗小鼠(

= 6 per group). Measurements of tumor volumes of nude mice every 3 days (

每组6个）。每3天测量裸鼠的肿瘤体积(

). Representative tumor images (

)。代表性的肿瘤图像(

) and tumor weights (

)和肿瘤重量(

) were recorded after sacrifice at the end of treatment. Data are displayed as mean ± SD (error bars). n.s., not statistically significant,

)在治疗结束时处死后记录。数据显示为平均值±SD（误差线）。n、在美国，没有统计学意义，

< 0.01,

< 0.01，

***

< 0.001. PES: pifithrin-μ, Sora: sorafenib.

< 0.001.PES：吡菲林-μ，索拉非尼。

Full size image

全尺寸图像

Pifithrin-μ potentiates sorafenib sensitivity of mTOR-activated primary liver tumor

Pifithrin-μ增强mTOR激活的原发性肝肿瘤对索拉非尼的敏感性

To test the effect of pifithrin-μ and sorafenib on mTOR-activated liver cancer development, we bred

为了测试pifithrin和索拉非尼对mTOR激活的肝癌发展的影响，我们繁殖了

Tsc2

Tsc2型

-（笑声）

floxed

floxed 的

mice with

小鼠

Alb-Cre

Alb-Cre公司

transgenic mice to generate

转基因小鼠产生

Tsc2

Tsc2型

flox/flox

;

Alb

Alb公司

cre

cre公司

mice (a.k.a.

小鼠（又称小鼠）。

Tsc2

Tsc2型

−/−

) (Fig.

)（图。

S7A, B

S7A，B

). At 8 months of age, macroscopic liver tumors were observed in

)。在8个月大时，在

Tsc2

Tsc2型

−/−

mice but not in WT mice (Fig.

小鼠，但不是WT小鼠（图）。

7A个

Tsc2

Tsc2型

−/−

mice had disordered liver tissue structures, manifested by the lack of clear hepatic lobules. Positive Heppar1 and negative CK19 stainings indicated that the tumors were HCC (Fig.

小鼠肝脏组织结构紊乱，表现为缺乏清晰的肝小叶。阳性Heppar1和阴性CK19染色表明肿瘤为HCC（图）。

7B条

). We treated

)。我们治疗过

Tsc2

Tsc2型

−/−

mice with pifithrin-μ and sorafenib for two months (Fig.

用pifithrin和索拉非尼治疗小鼠两个月（图）。

7摄氏度

). This combination regimen exerted synergistic suppression on tumor numbers, liver-to-body weight ratios, serum ALT and AST levels as compared to sorafenib or pifithrin-μ alone (Figs.

)。与单独使用索拉非尼或匹非他林相比，该联合方案对肿瘤数量，肝体重比，血清ALT和AST水平具有协同抑制作用（图）。

7D–G

and

和

S7D–E

), without significant impact on body weights of mice (Fig.

)，对小鼠体重没有显着影响（图）。

S7C

S7C系列

). In addition, combined treatment reduced SESN3 expression in mouse liver tumors, accompanied by Fe

)。此外，联合治疗可降低小鼠肝肿瘤中SESN3的表达，并伴有铁

accumulation (Fig.

积累（图）。

7小时

). These data suggest that combined treatment of pifithrin-μ and sorafenib inhibits liver tumor advancement of

)。这些数据表明，pifithrin-μ和索拉非尼的联合治疗可抑制肝肿瘤的进展

Tsc2

Tsc2型

−/−

mice.

老鼠。

Fig. 7: Pifithrin-μ potentiates sorafenib sensitivity of mTOR-activated primary liver tumor.

图7：Pifithrin-μ增强了mTOR激活的原发性肝肿瘤对索拉非尼的敏感性。

B类

Tumor formation of 8-month-old mice. Representative liver images (

8个月大小鼠的肿瘤形成。代表性肝脏图像(

), liver tissue staining of H&E, Heppar1, and CK19, scale bar = 100 μm (

)，H＆E，Heppar1和CK19的肝组织染色，比例尺=100μm(

B类

C级

–

小时

Liver tumors in

肝脏肿瘤

Tsc2

Tsc2型

−/−

mice at 8 months. Mice were treated with vehicle, pifithrin-μ (10 mg/kg, i.p.), sorafenib (20 mg/kg, i.g.), or combined pifithrin-μ (10 mg/kg, i.p.) and sorafenib (20 mg/kg, i.g.) every other day (

小鼠在8个月时。每隔一天用媒介物，匹非替林（10mg/kg，i.p.），索拉非尼（20mg/kg，i.g.）或联合匹非替林（10mg/kg，i.p.）和索拉非尼（20mg/kg，i.g.）治疗小鼠(

= 8 per group) for 2 months. Schematic depiction of treatment schedules (

每组8个），持续2个月。治疗时间表示意图(

C级

), representative liver images (

)，代表性的肝脏图像(

), liver tissue staining of H&E and Ki67, scale bar = 100 μm (

)，H＆E和Ki67的肝组织染色，比例尺=100μm(

), serum ALT levels (

)，血清ALT水平(

F级

), serum AST levels (

)，血清AST水平(

克

), and immunoblotting (

)，以及免疫印迹(

小时

). Data are displayed as mean ± SD (error bars).

)。数据显示为平均值±SD（误差线）。

< 0.01,

< 0.01，

***

< 0.001. PES pifithrin-μ, Sora sorafenib.

< 0.001.PES吡菲林-μ，索拉非尼姐妹。

Full size image

全尺寸图像

Discussion

讨论

As a multi-target tyrosine kinase inhibitor, sorafenib has been used in the clinic for stabilizing HCC progression by inducing oxidative stress. However, different etiological factors of HCC may contribute to various responses of cells to sorafenib [

作为一种多靶点酪氨酸激酶抑制剂，索拉非尼已在临床上用于通过诱导氧化应激来稳定HCC进展。然而，肝癌的不同病因可能导致细胞对索拉非尼的不同反应[

]. In this study, we observed that mTOR-activated cells were resistant to sorafenib treatment. Mechanistically, HSP70 potentiated CREB1 transcription to participate in SESN3-mediated antioxidant capacity in mTOR-activated cells. HSP70 inhibitor pifithrin-μ enhanced the efficacy of sorafenib to repress mTOR-activated tumorigenesis (Fig.

]。在这项研究中，我们观察到mTOR激活的细胞对索拉非尼治疗具有抗性。从机制上讲，HSP70增强CREB1转录，参与mTOR激活细胞中SESN3介导的抗氧化能力。HSP70抑制剂pifithrin-μ增强了索拉非尼抑制mTOR激活的肿瘤发生的功效（图）。

Fig. 8

图8

Illustration of pifithrin-μ enhancing sorafenib efficacy by targeting mTOR/CREB1/SESN3 axis in liver cancer cells.

通过靶向肝癌细胞中的mTOR/CREB1/SESN3轴来说明pifithrin-μ增强索拉非尼功效。

Full size image

全尺寸图像

mTOR signaling pathway is activated in a subtype of HCC [

[

]. Notably, TSC1 and TSC2 emerge as the most frequently mutated genes linked to activated mTOR signaling in HCC tumor samples. We found that mTOR-activated liver cancer cells were resistant to sorafenib-induced ferroptosis. Targeting the mTOR signaling pathway is thus expected to be an important strategy to reverse sorafenib resistance for liver cancer.

]。值得注意的是，TSC1和TSC2是HCC肿瘤样品中与活化的mTOR信号传导相关的最常见突变基因。我们发现mTOR激活的肝癌细胞对索拉非尼诱导的ferroptosis具有抗性。因此，靶向mTOR信号传导途径有望成为逆转索拉非尼对肝癌耐药性的重要策略。

However, a combination of sorafenib and everolimus did not prolong the survival of HCC patients [.

然而，索拉非尼和依维莫司的联合治疗并没有延长HCC患者的生存期[。

]. Therefore, exploring the mechanism of mTOR resistance to sorafenib is crucial for improving targeted therapy for liver cancer.

]。因此，探索mTOR对索拉非尼耐药的机制对于改善肝癌的靶向治疗至关重要。

Heightened ROS levels are believed to hinder tumor growth. ROS interacts with polyunsaturated fatty acids in lipid membranes, resulting in lipid ROS formation and triggering ferroptosis. Targeting ROS thus represents a promising therapeutic strategy. However, tumors possess an inherent antioxidant capacity that enables them to combat oxidative stress and augment their resistance to drugs.

ROS水平升高被认为会阻碍肿瘤生长。ROS与脂质膜中的多不饱和脂肪酸相互作用，导致脂质ROS形成并引发铁浓化。因此，靶向ROS代表了一种有前途的治疗策略。然而，肿瘤具有固有的抗氧化能力，使其能够抵抗氧化应激并增强其对药物的抵抗力。

In HCC, specific antioxidant enzymatic genes are overexpressed, contributing to sorafenib resistance [.

在HCC中，特定的抗氧化酶基因过表达，导致索拉非尼耐药[。

]. We observed that mTOR activation results in sorafenib resistance in liver cancer cells by enhancing cellular antioxidant capacity. These findings are corroborated by studies indicating a mechanistic connection between mTOR and redox balance [

]。我们观察到mTOR激活通过增强细胞抗氧化能力导致肝癌细胞对索拉非尼的耐药性。这些发现得到了研究的证实，这些研究表明mTOR和氧化还原平衡之间存在机械联系[

It is well established that SESN3 encodes antioxidant modulators of peroxiredoxins, participating in the maintenance of redox homeostasis [

众所周知，SESN3编码过氧化物酶的抗氧化调节剂，参与维持氧化还原稳态[

]. We demonstrated that SESN3 was a target gene of mTOR and played a crucial role in conferring active mTOR cells resistance to sorafenib. CREB1, a transcription factor governing genes linked to both cell survival and apoptosis, undergoes regulation by numerous protein kinases and phosphatases [

]。我们证明SESN3是mTOR的靶基因，并且在赋予活性mTOR细胞对索拉非尼的抗性方面起着至关重要的作用。CREB1是一种控制与细胞存活和凋亡相关的基因的转录因子，受到许多蛋白激酶和磷酸酶的调节[

]. Previously, we identified that activation of mTOR induced CREB1 phosphorylation and its subsequent accumulation [

]。以前，我们发现mTOR的激活诱导CREB1磷酸化及其随后的积累[

]. Here, CREB1 enhanced SESN3 expression by binding to SESN3 promoter and subsequently boosted the antioxidant capacity of liver cancer cells.

]。在这里，CREB1通过与SESN3启动子结合来增强SESN3表达，并随后增强肝癌细胞的抗氧化能力。

CREB1 operates as a cAMP-regulated transcription factor that stimulates target gene expression, partly through interaction with coactivator paralogs such as CREBBP. We observed that HSP70 facilitated the binding of CREB1 and CREBBP. The chaperone role of HSP70 involves binding to folded proteins and inducing conformational changes that impact PPIs [.

CREB1作为cAMP调节的转录因子起作用，部分通过与共激活因子旁系同源物（例如CREBBP）的相互作用来刺激靶基因表达。我们观察到HSP70促进了CREB1和CREBBP的结合。HSP70的伴侣作用涉及与折叠蛋白结合并诱导影响PPI的构象变化[。

]. Additionally, HSP70 has the potential to serve as a sensitive marker for distinguishing early HCC from precancerous lesions or noncancerous liver conditions [

]。此外，HSP70有可能作为区分早期HCC与癌前病变或非癌性肝病的敏感标志物[

]. Pifithrin-μ functions by interacting with the substrate-binding domain of the HSP70 carboxyl-terminal and disrupting the association between HSP70 and its co-chaperones. This inhibitor has exhibited cytotoxic effects on various types of tumor cells including acute leukemia, bladder cancer cells, and prostate cancer cells, with little toxicity towards normal cells [.

]。Pifithrin-μ通过与HSP70羧基末端的底物结合结构域相互作用并破坏HSP70与其伴侣之间的缔合而起作用。该抑制剂对包括急性白血病，膀胱癌细胞和前列腺癌细胞在内的各种类型的肿瘤细胞表现出细胞毒性作用，对正常细胞的毒性很小[。

]. However, its efficacy in the treatment of liver cancer has not been tested yet. In our study, pifithrin-μ inhibited HSP70 to reduce the transcriptional activity of CREB1 and the expression of SESN3, consequently suppressing liver cancer cell proliferation and tumorigenesis.

]。然而，其在肝癌治疗中的疗效尚未得到测试。在我们的研究中，pifithrin-μ抑制HSP70以降低CREB1的转录活性和SESN3的表达，从而抑制肝癌细胞增殖和肿瘤发生。

Current combination therapies, such as radiotherapy, cytotoxic chemotherapy, and molecular targeted therapy, may overcome sorafenib resistance and improve the effectiveness of sorafenib [

目前的联合治疗，如放疗、细胞毒性化疗和分子靶向治疗，可以克服索拉非尼的耐药性，提高索拉非尼的疗效[

]. Our study reveals that pifithrin-μ synergizes with sorafenib to suppress the proliferation and tumorigenesis of mTOR-activated cells. This innovative combination therapy presents a compelling strategy to enhance therapeutic efficacy of sorafenib.

]。我们的研究表明，pifithrin-μ与索拉非尼协同抑制mTOR激活细胞的增殖和肿瘤发生。这种创新的联合疗法为提高索拉非尼的治疗效果提供了一种令人信服的策略。

In conclusion, activated mTOR confers cells resistance to sorafenib treatment by increasing cellular antioxidant capacity through CREB1/CREBBP/HSP70-induced SESN3 expression. Pifithrin-μ in conjunction with sorafenib exerts the therapeutic potential in the treatment of TSC2-deficient human liver cancer cell-derived xenograft tumors and spontaneous mouse liver cancer.

总之，活化的mTOR通过CREB1/CREBBP/HSP70诱导的SESN3表达增加细胞抗氧化能力，从而赋予细胞对索拉非尼治疗的抗性。Pifithrin-μ与索拉非尼联合在治疗TSC2缺陷型人肝癌细胞衍生的异种移植肿瘤和自发性小鼠肝癌方面发挥了治疗潜力。

Targeting mTOR-CREB1-SESN3 axis may offer a promising therapeutic strategy to alleviate sorafenib resistance of TSC2 deficiency-associated mTOR-activated liver cancer..

靶向mTOR-CREB1-SESN3轴可能为缓解TSC2缺陷相关mTOR激活的肝癌对索拉非尼的耐药性提供了一种有前途的治疗策略。。

Data availability

数据可用性

The data analyzed in this study are included in both the published article and the supplemental data files.

本研究中分析的数据包含在已发表的文章和补充数据文件中。

References

参考文献

Singal AG, Kanwal F, Llovet JM. Global trends in hepatocellular carcinoma epidemiology: implications for screening, prevention and therapy. Nat Rev Clin Oncol. 2023;20:864–84.

Singal AG，Kanwal F，Llovet JM。肝细胞癌流行病学的全球趋势：对筛查，预防和治疗的影响。Nat Rev Clin Oncol。2023年；20： 864年至84年。

Article

文章

PubMed

Google Scholar

谷歌学者

Tan EY, Danpanichkul P, Yong JN, Yu Z, Tan DJH, Lim WH, et al. Liver cancer in 2021: global burden of disease study. J Hepatol. 2024;S0168-8278:02652-7.

Tan EY，Danpanichkul P，Yong JN，Yu Z，Tan DJH，Lim WH等。2021年肝癌：全球疾病负担研究。J Hepatol。2024年；S0168-8278:02652-7。

Rumgay H, Arnold M, Ferlay J, Lesi O, Cabasag CJ, Vignat J, et al. Global burden of primary liver cancer in 2020 and predictions to 2040. J Hepatol. 2022;77:1598–606.

Rumgay H，Arnold M，Ferlay J，Lesi O，Cabasag CJ，Vignat J等。2020年原发性肝癌的全球负担和2040年的预测。J Hepatol。2022年；77:1598年至606年。

Article

文章

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Li C, He WQ. Global prediction of primary liver cancer incidences and mortality in 2040. J Hepatol. 2023;78:e144–e146.

李C，何WQ。2040年原发性肝癌发病率和死亡率的全球预测。J Hepatol。2023年；78:e144–e146。

Article

文章

PubMed

Google Scholar

谷歌学者

Li Q, Cao M, Lei L, Yang F, Li H, Yan X, et al. Burden of liver cancer: from epidemiology to prevention. Chin J Cancer Res. 2022;34:554–66.

李青，曹敏，雷丽，杨芳，李红，严X，等。肝癌的负担：从流行病学到预防。Chin J Cancer Res.2022；34:554-66。

Article

文章

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Han B, Zheng R, Zeng H, Wang S, Sun K, Chen R, et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent. 2024;4:47–53.

韩B，郑R，曾H，王S，孙K，陈R等。中国癌症发病率和死亡率，2022年。J Natl癌症中心。2024年；4： 47-53岁。

Article

文章

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Rumgay H, Ferlay J, de Martel C, Georges D, Ibrahim AS, Zheng R, et al. Global, regional and national burden of primary liver cancer by subtype. Eur J Cancer. 2022;161:108–18.

Rumgay H，Ferlay J，de Martel C，Georges D，Ibrahim AS，Zheng R等。按亚型划分的原发性肝癌的全球，地区和国家负担。Eur J癌症。2022年；161:108-18。

Article

文章

PubMed

Google Scholar

谷歌学者

Yang C, Zhang H, Zhang L, Zhu AX, Bernards R, Qin W, et al. Evolving therapeutic landscape of advanced hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol. 2023;20:203–22.

杨C，张H，张L，朱AX，伯纳德R，秦W，等。进展期肝细胞癌的治疗前景。Nat Rev胃肠肝病。2023年；20： 203年至22年。

Article

文章

PubMed

Google Scholar

谷歌学者

Zheng S, Chan SW, Liu F, Liu J, Chow PKH, Toh HC, et al. Hepatocellular carcinoma: current drug therapeutic status, advances and challenges. Cancers (Basel). 2024;16:1582.

郑S，Chan SW，Liu F，Liu J，Chow PKH，Toh HC等。肝细胞癌：目前的药物治疗现状，进展和挑战。癌症（巴塞尔）。2024年；16： 1582年。

Article

文章

PubMed

Google Scholar

谷歌学者

Jackson R, Psarelli EE, Berhane S, Khan H, Johnson P. Impact of viral status on survival in patients receiving sorafenib for advanced hepatocellular cancer: a meta-analysis of randomized phase III trials. J Clin Oncol. 2017;35:622–8.

Jackson R，Psarelli EE，Berhane S，Khan H，Johnson P.病毒状态对接受索拉非尼治疗晚期肝细胞癌患者生存的影响：随机III期试验的荟萃分析。J临床肿瘤学。2017年；35:622-8。

Article

文章

PubMed

Google Scholar

谷歌学者

Huang A, Yang XR, Chung WY, Dennison AR, Zhou J. Targeted therapy for hepatocellular carcinoma. Signal Transduct Target Ther. 2020;5:146.

。信号传输目标Ther。2020年；5： 146页。

Article

文章

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Tang W, Chen Z, Zhang W, Cheng Y, Zhang B, Wu F, et al. The mechanisms of sorafenib resistance in hepatocellular carcinoma: theoretical basis and therapeutic aspects. Signal Transduct Target Ther. 2020;5:87.

Tang W，Chen Z，Zhang W，Cheng Y，Zhang B，Wu F，等。肝细胞癌索拉非尼耐药机制：理论基础和治疗方面。信号传输目标Ther。2020年；5： 87页。

Article

文章

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Fornari F, Giovannini C, Piscaglia F, Gramantieri L. Elucidating the molecular basis of sorafenib resistance in HCC: current findings and future directions. J Hepatocell Carcinoma. 2021;8:741–57.

Fornari F，Giovannini C，Piscaglia F，Gramantieri L.阐明HCC中索拉非尼耐药的分子基础：当前发现和未来方向。J肝细胞癌。2021年；8： 741年至57年。

Article

文章

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Chen J, Duda DG. Overcoming sorafenib treatment-resistance in hepatocellular carcinoma: a future perspective at a time of rapidly changing treatment paradigms. EBioMedicine. 2020;52:102644.

陈J，杜达DG。克服肝细胞癌中索拉非尼治疗耐药性：在治疗模式迅速变化的时代的未来前景。EBioMedicine。2020年；52:102644。

Article

文章

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Xia S, Pan Y, Liang Y, Xu J, Cai X. The microenvironmental and metabolic aspects of sorafenib resistance in hepatocellular carcinoma. EBioMedicine. 2020;51:102610.

夏S，潘Y，梁Y，徐J，蔡X.肝细胞癌索拉非尼耐药的微环境和代谢方面。EBioMedicine。2020年；51:102610。

Article

文章

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Mendez-Blanco C, Fondevila F, Garcia-Palomo A, Gonzalez-Gallego J, Mauriz JL. Sorafenib resistance in hepatocarcinoma: role of hypoxia-inducible factors. Exp Mol Med. 2018;50:1–9.

门德斯-布兰科C，丰德维拉F，加西亚-帕洛莫A，冈萨雷斯-加列戈J，莫里斯JL。索拉非尼在肝癌中的耐药性：缺氧诱导因子的作用。Exp Mol Med。2018；50:1-9。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Xu J, Ji L, Ruan Y, Wan Z, Lin Z, Xia S, et al. UBQLN1 mediates sorafenib resistance through regulating mitochondrial biogenesis and ROS homeostasis by targeting PGC1beta in hepatocellular carcinoma. Signal Transduct Target Ther. 2021;6:190.

Xu J，Ji L，Ruan Y，Wan Z，Lin Z，Xia S等。UBQLN1通过靶向肝细胞癌中的PGC1beta来调节线粒体生物发生和ROS稳态，从而介导索拉非尼耐药。信号传输目标Ther。2021年；6： 190页。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Guo L, Hu C, Yao M, Han G. Mechanism of sorafenib resistance associated with ferroptosis in HCC. Front Pharm. 2023;14:1207496.

郭L，胡C，姚M，韩G.索拉非尼耐药与HCC铁浓化相关的机制。前药学2023；14： 1207496页。

Article

文章

CAS

中科院

Google Scholar

谷歌学者

Li Q, Chen K, Zhang T, Jiang D, Chen L, Jiang J, et al. Understanding sorafenib-induced ferroptosis and resistance mechanisms: Implications for cancer therapy. Eur J Pharm. 2023;955:175913.

。Eur J Pharm。2023年；955:175913年。

Article

文章

CAS

中科院

Google Scholar

谷歌学者

Kwiatkowski DJ, Zhang H, Bandura JL, Heiberger KM, Glogauer M, el-Hashemite N, et al. A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1 null cells. Hum Mol Genet. 2002;11:525–34.

Kwiatkowski DJ，Zhang H，Bandura JL，Heiberger KM，Glogauer M，el Hashimite N等。TSC1的小鼠模型揭示了肝血管瘤的性别依赖性致死性，以及TSC1无效细胞中p70S6激酶活性的上调。哼，摩尔基因。2002年；11： 525-34岁。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Inoki K, Li Y, Zhu T, Wu J, Guan KL. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol. 2002;4:648–57.

Inoki K，Li Y，Zhu T，Wu J，Guan KL。TSC2被Akt磷酸化和抑制，并抑制mTOR信号传导。Nat细胞生物学。2002年；4： 648-57岁。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Potter CJ, Pedraza LG, Xu T. Akt regulates growth by directly phosphorylating Tsc2. Nat Cell Biol. 2002;4:658–65.

Potter CJ，Pedraza LG，Xu T.Akt通过直接磷酸化Tsc2来调节生长。Nat细胞生物学。2002年；4： 658-65岁。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Gao X, Zhang Y, Arrazola P, Hino O, Kobayashi T, Yeung RS, et al. Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling. Nat Cell Biol. 2002;4:699–704.

Gao X，Zhang Y，Arrazola P，Hino O，Kobayashi T，Yeung RS等。Tsc肿瘤抑制蛋白拮抗氨基酸TOR信号传导。Nat细胞生物学。2002年；4： 699-704年。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell. 2002;10:151–62.

Manning BD，Tee AR，Logsdon MN，Blenis J，Cantley LC。鉴定结节性硬化症复合物-2肿瘤抑制基因产物结节蛋白作为磷酸肌醇3-激酶/akt途径的靶标。摩尔细胞。2002年；10： 151年至62年。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Mossmann D, Park S, Hall MN. mTOR signalling and cellular metabolism are mutual determinants in cancer. Nat Rev Cancer. 2018;18:744–57.

Mossmann D，Park S，Hall MN。mTOR信号传导和细胞代谢是癌症的相互决定因素。Nat Rev癌症。2018年；18： 744年至57年。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Bhat M, Sonenberg N, Gores GJ. The mTOR pathway in hepatic malignancies. Hepatology. 2013;58:810–8.

Bhat M，Sonenberg N，Gores GJ。肝脏恶性肿瘤中的mTOR途径。肝病学。2013年；58:810-8。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Zhou L, Huang Y, Li J, Wang Z. The mTOR pathway is associated with the poor prognosis of human hepatocellular carcinoma. Med Oncol. 2010;27:255–61.

Zhou L，Huang Y，Li J，Wang Z.mTOR通路与人肝细胞癌预后不良有关。医学Oncol。2010年；27:255-61。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Matter MS, Decaens T, Andersen JB, Thorgeirsson SS. Targeting the mTOR pathway in hepatocellular carcinoma: current state and future trends. J Hepatol. 2014;60:855–65.

Matter MS，Decans T，Andersen JB，Thorgeirsson SS。靶向肝细胞癌中的mTOR途径：现状和未来趋势。J Hepatol。2014年；60:855-65。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Hu TH, Huang CC, Lin PR, Chang HW, Ger LP, Lin YW, et al. Expression and prognostic role of tumor suppressor gene PTEN/MMAC1/TEP1 in hepatocellular carcinoma. Cancer. 2003;97:1929–40.

Hu TH，Huang CC，Lin PR，Chang HW，Ger LP，Lin YW，等。抑癌基因PTEN/MMAC1/TEP1在肝细胞癌中的表达和预后作用。癌症。2003年；97:1929年至40年。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Ho DWH, Chan LK, Chiu YT, Xu IMJ, Poon RTP, Cheung TT, et al. TSC1/2 mutations define a molecular subset of HCC with aggressive behaviour and treatment implication. Gut. 2017;66:1496–506.

。肠道。2017年；66:1496年至506年。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Huynh H, Hao HX, Chan SL, Chen D, Ong R, Soo KC, et al. Loss of tuberous sclerosis complex 2 (TSC2) is frequent in hepatocellular carcinoma and predicts response to mTORC1 inhibitor everolimus. Mol Cancer Ther. 2015;14:1224–35.

Huynh H，Hao HX，Chan SL，Chen D，Ong R，Soo KC等。结节性硬化症复合物2（TSC2）的丢失在肝细胞癌中很常见，并预测对mTORC1抑制剂依维莫司的反应。摩尔癌症治疗。2015年；14： 1224-35年。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Song K, He F, Xin Y, Guan G, Huo J, Zhu Q, et al. TSC2 mutations were associated with the early recurrence of patients with HCC underwent hepatectomy. Pharmgenomics Pers Med. 2021;14:269–78.

Song K，He F，Xin Y，Guan G，Huo J，Zhu Q等。TSC2突变与接受肝切除术的HCC患者的早期复发有关。Pharmgenomics Pers Med。2021；14： 269年至78年。

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Chen L, Zhang C, Xue R, Liu M, Bai J, Bao J, et al. Deep whole-genome analysis of 494 hepatocellular carcinomas. Nature. 2024;627:586–93.

陈丽，张超，薛若，刘敏，白洁，鲍洁，等。494例肝细胞癌的全基因组深度分析。自然。2024年；627:586-93。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Masuda M, Chen WY, Miyanaga A, Nakamura Y, Kawasaki K, Sakuma T, et al. Alternative mammalian target of rapamycin (mTOR) signal activation in sorafenib-resistant hepatocellular carcinoma cells revealed by array-based pathway profiling. Mol Cell Proteom. 2014;13:1429–38.

Masuda M，Chen WY，Miyanaga A，Nakamura Y，Kawasaki K，Sakuma T等。通过基于阵列的途径分析揭示索拉非尼耐药肝细胞癌细胞中雷帕霉素（mTOR）信号激活的替代哺乳动物靶标。摩尔细胞蛋白质组学。2014年；13： 1429年至1428年。

Article

文章

CAS

中科院

Google Scholar

谷歌学者

Bissler JJ, McCormack FX, Young LR, Elwing JM, Chuck G, Leonard JM, et al. Sirolimus for angiomyolipoma in tuberous sclerosis complex or lymphangioleiomyomatosis. N. Engl J Med. 2008;358:140–51.

Bissler JJ，McCormack FX，Young LR，Elwing JM，Chuck G，Leonard JM等。西罗莫司治疗结节性硬化症或淋巴管平滑肌瘤病中的血管平滑肌脂肪瘤。N、英国医学杂志2008；358:140-51。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

McCormack FX, Inoue Y, Moss J, Singer LG, Strange C, Nakata K, et al. Efficacy and safety of sirolimus in lymphangioleiomyomatosis. N. Engl J Med. 2011;364:1595–606.

McCormack FX，Inoue Y，Moss J，Singer LG，Strange C，Nakata K等。西罗莫司治疗淋巴管平滑肌瘤病的疗效和安全性。N、英国医学杂志2011；364:1595年至606年。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Wood KC, Gutkind JS. Challenges and emerging opportunities for targeting mTOR in cancer. Cancer Res. 2022;82:3884–7.

伍德KC，古特金JS。针对癌症中mTOR的挑战和新兴机遇。癌症研究2022；82:3884-7。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Adib E, Klonowska K, Giannikou K, Do KT, Pruitt-Thompson S, Bhushan K, et al. Phase II clinical trial of everolimus in a pan-cancer cohort of patients with mTOR pathway alterations. Clin Cancer Res. 2021;27:3845–53.

Adib E，Klonowska K，Giannikou K，Do KT，Pruitt-Thompson S，Bhushan K等。依维莫司在mTOR途径改变的泛癌患者队列中的II期临床试验。Clin Cancer Res.2021；27:3845-53。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Ferrin G, Guerrero M, Amado V, Rodriguez-Peralvarez M, De la Mata M. Activation of mTOR signaling pathway in hepatocellular carcinoma. Int J Mol Sci. 2020;21:1266.

Ferrin G，Guerrero M，Amado V，Rodriguez-Peralvarez M，De la Mata M.肝细胞癌中mTOR信号通路的激活。国际分子科学杂志。2020年；21:1266年。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Zhu AX, Kudo M, Assenat E, Cattan S, Kang YK, Lim HY, et al. Effect of everolimus on survival in advanced hepatocellular carcinoma after failure of sorafenib: the EVOLVE-1 randomized clinical trial. JAMA. 2014;312:57–67.

Zhu AX，Kudo M，Assenat E，Cattan S，Kang YK，Lim HY等。依维莫司对索拉非尼失败后晚期肝细胞癌生存的影响：EVOLVE-1随机临床试验。杰玛。2014年；312:57-67。

Article

文章

PubMed

Google Scholar

谷歌学者

Koeberle D, Dufour JF, Demeter G, Li Q, Ribi K, Samaras P, et al. Sorafenib with or without everolimus in patients with advanced hepatocellular carcinoma (HCC): a randomized multicenter, multinational phase II trial (SAKK 77/08 and SASL 29). Ann Oncol. 2016;27:856–61.

Koeberle D，Dufour JF，Demeter G，Li Q，Ribi K，Samaras P等。索拉非尼联合或不联合依维莫司治疗晚期肝细胞癌（HCC）：一项随机多中心，多国II期临床试验（SAKK 77/08和SASL 29）。安·昂科尔。2016年；27:856-61。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Liu F, Gai X, Wu Y, Zhang B, Wu X, Cheng R, et al. Oncogenic beta-catenin stimulation of AKT2-CAD-mediated pyrimidine synthesis is targetable vulnerability in liver cancer. Proc Natl Acad Sci USA. 2022;119:e2202157119.

Liu F，Gai X，Wu Y，Zhang B，Wu X，Cheng R等。致癌β-连环蛋白刺激AKT2-CAD介导的嘧啶合成是肝癌的靶向易感性。美国国家科学院院刊2022；119:e2202157119。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Liu F, Wu Y, Zhang B, Yang S, Shang K, Li J, et al. Oncogenic beta-catenin-driven liver cancer is susceptible to methotrexate-mediated disruption of nucleotide synthesis. Chin Med J (Engl) 2023;137:181–9.

Liu F，Wu Y，Zhang B，Yang S，Shang K，Li J等。致癌β-连环蛋白驱动的肝癌易受甲氨蝶呤介导的核苷酸合成破坏的影响。Chin Med J（英语）2023；137:181-9。

Li C, Chen H, Lan Z, He S, Chen R, Wang F, et al. mTOR-dependent upregulation of xCT blocks melanin synthesis and promotes tumorigenesis. Cell Death Differ. 2019;26:2015–28.

Li C，Chen H，Lan Z，He S，Chen R，Wang F等。依赖mTOR的xCT上调阻断黑色素合成并促进肿瘤发生。细胞死亡不同。2019年；26:2015-28。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Vilella-Bach M, Nuzzi P, Fang Y, Chen J. The FKBP12-rapamycin-binding domain is required for FKBP12-rapamycin-associated protein kinase activity and G1 progression. J Biol Chem. 1999;274:4266–72.

Viella Bach M，Nuzzi P，Fang Y，Chen J.FKBP12-雷帕霉素结合结构域是FKBP12-雷帕霉素相关蛋白激酶活性和G1进展所必需的。生物化学杂志。1999年；274:4266-72。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Duvel K, Yecies JL, Menon S, Raman P, Lipovsky AI, Souza AL, et al. Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol Cell. 2010;39:171–83.

Duvel K，Yecies JL，Menon S，Raman P，Lipovsky AI，Souza等。mTOR复合物1下游代谢基因调控网络的激活。摩尔细胞。2010年；39:171-83。

Article

文章

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Chen X, Kang R, Kroemer G, Tang D. Broadening horizons: the role of ferroptosis in cancer. Nat Rev Clin Oncol. 2021;18:280–96.

Chen X，Kang R，Kroemer G，Tang D.拓宽视野：ferroptosis在癌症中的作用。Nat Rev Clin Oncol。2021年；18： 280–96。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Hayes JD, Dinkova-Kostova AT, Tew KD. Oxidative stress in cancer. Cancer Cell. 2020;38:167–97.

Hayes JD，Dinkova Kostova AT，Tew KD。癌症中的氧化应激。癌细胞。2020年；38:167-97。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Wang Y, Hu Z, Liu Z, Chen R, Peng H, Guo J, et al. MTOR inhibition attenuates DNA damage and apoptosis through autophagy-mediated suppression of CREB1. Autophagy. 2013;9:2069–86.

Wang Y，Hu Z，Liu Z，Chen R，Peng H，Guo J，et al。MTOR抑制通过自噬介导的CREB1抑制来减轻DNA损伤和凋亡。自噬。2013年；9： 2069-86年。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Ohayon S, Yitzhaky A, Hertzberg L. Gene expression meta-analysis reveals the up-regulation of CREB1 and CREBBP in Brodmann Area 10 of patients with schizophrenia. Psychiatry Res. 2020;292:113311.

Ohayon S，Yitzhaky A，Hertzberg L.基因表达荟萃分析揭示了精神分裂症患者Brodmann 10区CREB1和CREBBP的上调。精神病学Res.2020；292:113311。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Perez-Salvia M, Esteller M. Bromodomain inhibitors and cancer therapy: from structures to applications. Epigenetics. 2017;12:323–39.

Perez-Salvia M，Esteller M.溴结构域抑制剂和癌症治疗：从结构到应用。表观遗传学。2017年；12： 323年至39年。

Article

文章

PubMed

Google Scholar

谷歌学者

Rosenzweig R, Nillegoda NB, Mayer MP, Bukau B. The Hsp70 chaperone network. Nat Rev Mol Cell Biol. 2019;20:665–80.

Rosenzweig R，Nillegoda NB，Mayer MP，Bukau B.Hsp70伴侣网络。Nat Rev Mol Cell Biol。2019年；20：。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Villanueva A, Chiang DY, Newell P, Peix J, Thung S, Alsinet C, et al. Pivotal role of mTOR signaling in hepatocellular carcinoma. Gastroenterology. 2008;135:1972–83.

Villanueva A，Chiang DY，Newell P，Peix J，Thung S，Alsinet C等。mTOR信号在肝细胞癌中的关键作用。胃肠病学。2008年；135:1972年至83年。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Lee D, Xu IM, Chiu DK, Leibold J, Tse AP, Bao MH, et al. Induction of oxidative stress through inhibition of thioredoxin reductase 1 is an effective therapeutic approach for hepatocellular carcinoma. Hepatology. 2019;69:1768–86.

Lee D，Xu IM，Chiu DK，Leibold J，Tse AP，Bao MH等。通过抑制硫氧还蛋白还原酶1诱导氧化应激是治疗肝细胞癌的有效方法。肝病学。2019年；69:1768-86年。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Zhao Y, Hu X, Liu Y, Dong S, Wen Z, He W, et al. ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway. Mol Cancer. 2017;16:79.

赵Y，胡X，刘Y，董S，温Z，何W，等。代谢应激下的ROS信号传导：AMPK和AKT途径之间的串扰。。2017年；16： 79页。

Article

文章

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Kopnin PB, Agapova LS, Kopnin BP, Chumakov PM. Repression of sestrin family genes contributes to oncogenic Ras-induced reactive oxygen species up-regulation and genetic instability. Cancer Res. 2007;67:4671–8.

Kopnin PB，Agapova LS，Kopnin BP，Chumakov PM。抑制sestrin家族基因有助于致癌Ras诱导的活性氧上调和遗传不稳定性。癌症研究2007；67:4671-8。

Article

文章

CAS

中科院

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Steven A, Friedrich M, Jank P, Heimer N, Budczies J, Denkert C, et al. What turns CREB on? And off? And why does it matter? Cell Mol Life Sci. 2020;77:4049–67.

史蒂文A，弗里德里希M，詹克P，海默N，布奇斯J，丹科特C等。是什么让CREB开启了？然后离开？为什么这很重要？细胞分子生命科学。2020年；77:4049-67。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Kampinga HH, Craig EA. The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat Rev Mol Cell Biol. 2010;11:579–92.

坎平加HH，克雷格EA。HSP70伴侣机制：J蛋白作为功能特异性的驱动因素。Nat Rev Mol细胞生物学。2010年；11： 579年至92年。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Chuma M, Sakamoto M, Yamazaki K, Ohta T, Ohki M, Asaka M, et al. Expression profiling in multistage hepatocarcinogenesis: identification of HSP70 as a molecular marker of early hepatocellular carcinoma. Hepatology. 2003;37:198–207.

Chuma M，Sakamoto M，Yamazaki K，Ohta T，Ohki M，Asaka M等。多阶段肝癌发生中的表达谱：HSP70作为早期肝细胞癌分子标志物的鉴定。肝病学。2003年；37:198-207。

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Leu JI, Pimkina J, Frank A, Murphy ME, George DL. A small molecule inhibitor of inducible heat shock protein 70. Mol Cell. 2009;36:15–27.

。诱导型热休克蛋白70的小分子抑制剂。摩尔细胞。2009年；36:15-27。

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Download references

下载参考资料

Funding

资金

This study was supported by the Haihe Laboratory of Cell Ecosystem Innovation Fund (22HHXBSS00012), the National Natural Science Foundation of China (81730078), and the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2021-I2M-1-018).

这项研究得到了海河细胞生态系统创新基金实验室（22HHXBSS00012），国家自然科学基金（81730078）和中国医学科学院医学科学创新基金（2021-I2M-1-018）的支持。

Author information

作者信息

Authors and Affiliations

作者和隶属关系

Department of Organ Transplantation and Hepatobiliary Surgery, Key Laboratory of Organ Transplantation of Liaoning Province, The First Hospital of China Medical University, Shenyang, China

中国医科大学第一医院辽宁省器官移植重点实验室器官移植与肝胆外科，沈阳

Jiarui Lv & Hongbing Zhang

吕家瑞和张红兵

Department of Physiology, State Key Laboratory of Common Mechanism Research for Major Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Basic Medical Sciences and School of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

中国医学科学院和北京协和医科大学基础医学研究所和基础医学院海河细胞生态系统实验室重大疾病共同机制研究国家重点实验室生理学系

Jiarui Lv, Yanan Wang, Cuiting Zheng, Xinyu Zhang, Linyan Wan, Fangming Liu & Hongbing Zhang

吕家瑞，王亚南，郑翠婷，张新宇，万林燕，刘方明和张红兵

Department of Plastic Surgery, The First Hospital of China Medical University, Shenyang, China

中国医科大学第一医院整形外科，沈阳

Jiacheng Lv

嘉成 Lv

Department of Radiology, State Key Laboratory of Complex, Severe and Rare Diseases, Chinese Academy of Medical Sciences, Peking Union Medical College and Peking Union Medical College Hospital, Beijing, China

中国医学科学院复杂、重症和罕见病国家重点实验室放射科，北京协和医学院和北京协和医院，北京

Xinyu Zhang

张兴宇

Department of Gastroenterology, Yichang Central People’s Hospital, The First College of Clinical Medical Science, China Three Gorges University, Yichang, China

中国三峡大学第一临床医学院宜昌市中心人民医院消化内科，宜昌

Linyan Wan

万林燕

Department of Breast Oncology, Key Laboratory of Carcinogenesis and Translational Research, Peking University Cancer Hospital and Institute, Beijing, China

北京大学肿瘤医院和研究所癌症发生与转化研究重点实验室乳腺肿瘤学系，北京

Jiayang Zhang

张嘉阳

Authors

作者

Jiarui Lv

吕嘉瑞

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Yanan Wang

王亚南

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Jiacheng Lv

嘉成 Lv

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Cuiting Zheng

郑翠婷（音）

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Xinyu Zhang

张兴宇

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Linyan Wan

万林燕

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Jiayang Zhang

张嘉阳

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Fangming Liu

方明灯

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Hongbing Zhang

Honbgingengang

View author publications

查看作者出版物

You can also search for this author in

您也可以在中搜索此作者

PubMed

Google Scholar

谷歌学者

Contributions

捐款

Hongbing Zhang supervised the project. Jiarui Lv conceived, designed, and performed experiments, analyzed the data, and wrote the manuscript. Yanan Wang supervised the experiments. Jiacheng Lv analyzed the database and designed the mechanism diagram. Cutting Zheng and Xinyu Zhang participated in the construction of a subcutaneous xenograft model.

张红兵监督了该项目。吕家瑞构思，设计并进行了实验，分析了数据并撰写了手稿。王亚南监督了实验。Jiacheng Lv分析了数据库并设计了机制图。Cutting Zheng和Xinyu Zhang参与了皮下异种移植模型的构建。

Linyan Wan participated in the generation of a spontaneous liver cancer model. Fangming Liu and Jiayang Zhang advised data analysis..

万林燕参与了自发性肝癌模型的建立。刘方明和张嘉阳建议进行数据分析。。

Corresponding author

通讯作者

Correspondence to

通信对象

Hongbing Zhang

Honbgingengang

Ethics declarations

道德宣言

Competing interests

相互竞争的利益

The authors declare no competing interests.

作者声明没有利益冲突。

Ethical approval

道德认可

All the animal protocols were approved by the Animal Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and Peking Union Medical College (ACUC-A01-2022-023) and abided by the regulations of the Beijing Administration Office of Laboratory Animal.

所有动物实验方案均经中国医学科学院基础医学研究所动物中心和北京协和医学院（ACUC-A01-2022-023）批准，并遵守北京实验动物管理办公室的规定。

Additional information

其他信息

Publisher’s note

出版商注释

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。

Edited by Giovanni Blandino

编辑：乔瓦尼·布兰迪诺

Supplementary information

补充信息

Supplementary materials

补充材料

Full and uncropped western blots

Rights and permissions

权限和权限

Open Access

开放存取

This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.

本文根据知识共享署名4.0国际许可证进行许可，该许可证允许以任何媒体或格式使用，共享，改编，分发和复制，只要您对原始作者和来源给予适当的信任，提供知识共享许可证的链接，并指出是否进行了更改。

The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this licence, visit .

要查看此许可证的副本，请访问。

http://creativecommons.org/licenses/by/4.0/

Reprints and permissions

重印和许可

About this article

关于本文

Cite this article

引用本文

Lv, J., Wang, Y., Lv, J.

Lv，J，Wang，Y，Lv，J

et al.