SLC7A11对HPV阳性宫颈癌患者免疫应答和生存的影响.pdf

2023,43(7)http:/J Clin Pathol Res 临床与病理杂志SLC7A11对HPV阳性宫颈癌患者免疫应答和生存的影响雷田雨1，王夫浩2，李晓慧2，黄睿3，邹文雪3，刘超2，胡钦勇11.武汉大学人民医院肿瘤中心，武汉 430060；2.山东第一医科大学附属肿瘤医院放疗科，济南 250117；3.武汉经济技术开发区(汉南区)人民医院内科，武汉 430060摘要 目的：宫颈癌是最常见的妇科肿瘤之一，其主要病因为高危型人乳头瘤病毒(human papillomavirus，HPV)的持续感染。HPV蛋白可以通过诱导-氧化和氧化应激促进细胞铁死亡，而铁死亡相关基因SLC7A11在其中的作用尚不清楚。本研究旨在探索人HPV阳性宫颈癌中SLC7A11的表达水平与抗肿瘤免疫及患者生存之间的关系。方法：纳入来自癌症基因组图谱数据库的169例HPV阳性宫颈癌患者，根据SLC7A11表达的水平，将患者分为SLC7A11低表达组(SLC7A11low)和SLC7A11高表达组(SLC7A11high)，分析SLC7A11表达水平对HPV阳性宫颈癌患者预后、肿瘤微环境中免疫细胞浸润的影响。结果：与SLC7A11low组相比，SLC7A11high组铁死亡抑制评分更高、免疫评分更低，差异均有统计学意义(均P0.05)。Kaplan-Meier生存分析发现SLC7A11high组相对于SLC7A11low组有更差的预后(P=0.025，HR=4.45，95%CI 1.9510.15)。差异基因富集分析也发现在SLC7A11high组中免疫相关基因及通路下调。与SLC7A11low组相比，SLC7A11high组有较低的免疫细胞浸润、较低的T细胞共刺激因子和T细胞共抑制因子的表达，提示SLC7A11high组可能存在较差的抗肿瘤免疫反应。进一步对免疫治疗相关标志物的分析提示SLC7A11的高表达与免疫检查点的表达水平低相关，且与免疫治疗效果差相关。结论：SLC7A11可能是影响宫颈癌肿瘤微环境免疫细胞浸润和宫颈癌患者预后的重要因素之一，其可能成为HPV相关宫颈癌潜在的治疗靶点。关键词 宫颈癌；SLC7A11；人乳头瘤病毒；铁死亡；免疫细胞浸润Impact of SLC7A11 on immune response and survival of patient with HPV-positive cervical cancerLEI Tianyu1,WANG Fuhao2,LI Xiaohui2,HUANG Rui3,ZOU Wenxue3,LIU Chao2,HU Qinyong11.Department of Oncology,Renmin Hospital of Wuhan University,Wuhan 430060;2.Department of Radiation Oncology,Shandong Cancer Hospital and Institute,Shandong First Medical University,Jinan 250117;3.Department of Internal Medicine,Peoples Hospital of Wuhan Economic and Technological Development Zone(Hannan District),Wuhan 430060,ChinaDOI：10.11817/j.issn.2095-6959.2023.220044收稿日期(Date of reception)：2023-02-15第一作者(First author)：雷田雨，Email:,ORCID:0009-0005-4240-8682通信作者(Corresponding author)：胡钦勇，Email:,ORCID:0000-0002-0764-3792基金项目(Foundation item)：国家重点研发计划(2020YFC2006000)；山东省自然科学基金(ZR2021QH006)。This work was supported by the National Key Research and Development Plan(2020YFC2006000)and Shandong Provincial Natural Science Foundation(ZR2021QH006),China.1323临床与病理杂志,2023,43(7)http:/ABSTRACT Objective:Cervical cancer is one of the most common gynecological tumors,and its main cause is the persistent infection of high-risk human papillomavirus(HPV).HPV proteins can promote cell ferroptosis by inducing-oxidation and oxidative stress,while the role of ferroptosis-related gene SLC7A11 in this process is still unclear.This study aims to explore the relationship between SLC7A11 expression,anti-tumor immunity,and survival of patient with HPV-related cervical cancer.Methods:Using 169 HPV-positive(HPV+)cervical cancer patients from the Cancer Genome Atlas(TCGA)database,this study divided them into 2 groups,SLC7A11low group and SLC7A11high group,based on the expression levels of SLC7A11 and analyzed the effect of SLC7A11 on the prognosis of HPV+cervical cancer and the immune cell infiltration in tumor microenvironment.Results:In HPV+cervical cancer,the ferroptosis suppressor score was higher and the immune score was lower in SLC7A11high group compared with SLC7A11low group(both P1 且 q0.05的基因作为差异基因。1.3 通路评分采用单样本基因富集分析(single sample gene set enrichment analysis，ssGSEA)法进行铁死亡抑制评分和免疫评分。1.4 基因集合富集分析采用 R 包 ClusterProfiler 对分子特征库“c5.all.v7.4.symbols.gmt”基因集进行基因集合富集分析(gene set enrichment analyses，GSEA)，从 而 评 估SLC7A11high组和 SLC7A11low组富集的经典通路16。错误发现率(false discovery rate，FDR)0.05被认为是显著富集的。1.5 基因集变异分析采用基因集变异分析(Gene Set Variation Analysis，GSVA)来源于分子特征库(MSigDB v7.5.1)的特征基因集，用GSVA的R包(版本1.40.1)评估每一个样本每一个基因集的通路活性。1.6 免疫浸润分析ssGSEA被用来探究免疫功能，区分免疫细胞亚群，并使用R package ggplot2(版本3.2.1)绘制小提琴图。TIMER17、CIBERSORT-ABS18、QUANTISEQ19、MCPCOUNTER20、XCELL21 5种分析免疫细胞浸润的方法被用来评估2组间免疫细胞浸润的差异。1.7 探索SLC7A11和对免疫检查点阻断的反应使用肿瘤免疫功能障碍和排斥(Tumor Immune Dysfunction and Exclusion，TIDE)评分22(http:/tide.dfci.harvard.edu/)来预测对免疫检查点抑制剂的疗效反应。TIDE评分低的患者倾向于对免疫治疗效果好，评分高的患者被认为免疫治疗效果差22。1.8 生存分析使用survminer包中的“surv_cutpoint”函数确定连续变量的最佳截断点23。根据最佳截断值将患者分为SLC7A11high组和SLC7A11low组，比较2组的生存差异。1.9 统计学处理所有的数据分析均在R软件(版本4.1.0)中进行。计量资料符合正态分布且方差齐，用均数标准差(x s)表示，2组比较采用独立样本t检验；计量资料不符合正态性和方差齐性时，2组比较采用Wilcoxon检验。计数资料用构成比表示，2组比较采用2检验。Kaplan-Meier 生存分析和时序检验被用来评估SLC7A11high组和SLC7A11low组患者的生存差异。使用survival(版本2.41-3)和survminer(版本0.4.9)的R软件包进行所有的分析。P0.05)，肿瘤的病理类型差异有统计学意义(P0.001，表1)。2.2 SLC7A11的表达与HPV阳性宫颈癌的免疫状态相关与SLC7A11low组相比，SLC7A11high组中的铁死亡抑制评分更高，差异有统计学意义(P=0.004，图1A)；SLC7A11high组的免疫评分较低，差异有统计学意义(P=0.046，图1B)。同时，2组的Kaplan-Meier生存分析也提示SLC7A11high组相对于SLC7A11low组预后 更 差(P=0.025，HR=4.45，95%CI 1.9510.15；图1C)。2.3 SLC7A11high组和 SLC7A11low组免疫相关通路活性的差异2组差异表达的基因的火山图结果提示SLC7A11high组低表达免疫相关基因，如IL9R和CNTN2(图2A)。进 一 步 的 GSEA 提 示 与 SLC7A11low组 相 比，SLC7A11high组下调了抗原处理和呈递的调节、巨噬细胞迁移、肿瘤坏死因子超家族细胞因子产生的负调控和T细胞分化的调节等通路(图2B)。除此以外，1325临床与病理杂志,2023,43(7)http:/进一步的特征信号通路的GSVA表明SLC7A11high组富集更多的细胞增殖通路，如 E2F targets 和 G2/M checkpoint(图2C)；而在SLC7A11low组富集到了炎症反应通路(图2C)。图1 铁死亡相关基因SLC7A11在HPV阳性宫颈癌中的作用Figure 1 Role of ferroptosis-related gene SLC7A11 in HPV-positive cervical cancerA:Ferroptosis suppressor score for patients with HPV+cervical cancer,stratified by high(green)and low(blue)SLC7A11 expression.Wilcoxon-test:P=0.004.B:Immune score for patients with HPV+cervical cancer,stratified by high(green)and low(blue)SLC7A11 expression.Wilcoxon-test:P=0.046.C:Kaplan-Meier analysis showing overall survival of patients with HPV+cervical cancer in the TCGA dataset,stratified by high(red)and low(blue)expression of SLC7A11.HPV:Human papillomavirus;HPV+:HPV-positive;TCGA:The Cancer Genome Atlas.表1 SLC7A11high组和SLC7A11low组一般基线资料比较Table 1 Comparison of baseline data between the SLC7A11high and SLC7A11low groups组别SLC7A11high组SLC7A11low组Pn12445年龄/岁(x s)59.529.8161.039.440.819HPV感染状态/例1680230.16218207其他2415淋巴结状态/例阳性23100.407阴性5724其他4411种族/例白种人93270.177亚洲人126黑种人87其他115组别SLC7A11high组SLC7A11low组P临床分期/例I77280.320II219IIIIV256其他12T分期/例T161300.163T22310T330T410Tx81其他284组别SLC7A11high组SLC7A11low组PN分期/例N058230.195N12712Nx116其他284M分期/例M050180.337M110Mx4321其他306病理分型/例鳞癌9842腺癌2611);red dots representing upregulated genes while blue dots representing downregulated genes in the SLC7A11high group.B:GSEA analysis displaying immune response pathways downregulated in the SLC7A11high group.C:GSVA analysis displaying enrichment pathways in SLC7A11high and SLC7A11low groups,respectively.DEGs:Differently expressed genes;GSEA:Gene Set Enrichment Analysis;GSVA:Gene Set Variation Analysis;HPV:Human papillomavirus;HPV+:HPV-positive.1327临床与病理杂志,2023,43(7)http:/2.4 SLC7A11high组和SLC7A11low组肿瘤微环境中浸润的免疫细胞的差异对2组免疫细胞群体进行分析，结果表明：与SLC7A11low组相比，SLC7A11high组免疫细胞浸润更少，包括树突状细胞、B细胞、CD8T细胞、辅助性T细胞、滤泡辅助性T细胞和Th2细胞，同时T细胞共刺激分子和T细胞共抑制分子的表达水平也更低(图 3A)。TIMER、CIBERSORT、QUANTISEQ、MCPCOUNTER和XCELL 5种免疫细胞浸润分析的热图结果均提示SLC7A11high组有更少的免疫细胞浸润(图3B)。2.5 SLC7A11high组和SLC7A11low组对免疫治疗反应的差异对2组的免疫检查点的表达水平进行分析，结果表明：与SLC7A11low组相比，SLC7A11high组免疫检查 点 表 达 水 平 较 低，如 LAG3、CD40、CD86、TIGIT、PDCD1等(图4A)。进一步的功能分析提示图3 HPV阳性宫颈癌中SLC7A11high组和SLC7A11low组免疫谱上的差异Figure 3 Differences in immune profile between SLC7A11high and SLC7A11low groups in HPV+cervical cancerA:Box plot by Wilcoxon-test showing immune cell infiltration in patients with HPV+cervical cancer,stratified by high(orange)and low(blue)SLC7A11 expression.B:The heat map showing the differences of immune cell infiltration between the 2 groups based on different methods of assessing immune cell infiltration.*P0.05,*P0.01,*P0.001.HPV:Human papillomavirus;HPV+:HPV-positive.1328SLC7A11对HPV阳性宫颈癌患者免疫应答和生存的影响 雷田雨，等SLC7A11的表达水平与T细胞耗竭、肿瘤相关成纤维细胞、髓系来源的抑制细胞和肿瘤相关巨噬细胞M2型呈正相关，与干扰素-、Merck18、CD274和CD8呈负相关(图4B)。应用TIDE算法来预测2组对免疫检查点抑制剂的反应，发现SLC7A11high组TIDE评分更高(图4B)。3 讨 论 铁死亡相关基因SLC7A11在多种肿瘤中高表达，如肺癌、肾上腺皮质癌及胰腺癌，与肿瘤微环境中浸润的免疫细胞密切相关，且与较差预后相关24-25。本研究发现SLC7A11的表达可以作为HPV阳性宫颈癌中潜在的预后标志物。SLC7A11的高表达能够抑制铁死亡，减少肿瘤微环境中免疫细胞的浸润，降低免疫检查点的表达。通过TIDE分析，SLC7A11表达水平高的患者可能对免疫检查点抑制剂反应更差。在高危HPV感染情况下，由宫颈鳞状上皮内病变向宫颈鳞状细胞癌转化的过程中，细胞完成了从发生铁死亡到出现抗铁死亡的转变，提示铁死亡在宫颈癌发展中的重要作用26。不仅如此，越来越多的研究27-29表明铁死亡相关基因在预测肿瘤的预后和重塑肿瘤微环境方面起重要作用。其中，SLC7A11是调节铁死亡的一个重要基因。SLC7A11的表达可以在各种应激条件下被诱导，包括氧化应激，氨基酸饥饿，代谢应激和遗传毒性应激，这可能是一种适应性反应，使细胞能够恢复氧化还原稳态，并在应激条件图4 HPV阳性宫颈癌中SLC7A11high组和SLC7A11low组免疫治疗反应标志物的比较Figure 4 Comparison of the markers of immunotherapy response between SLC7A11high and SLC7A11low groups in HPV+cervical cancerA:Immune checkpoint gene expression levels in the SLC7A11high and SLC7A11low groups.Yellow represents the SLC7A11low group and blue represents the SLC7A11high group.Wilcoxon-test:*P0.05,*P0.01.B:Correlation analysis showing the relationship between SLC7A11 expression and the markers of immunotherapy response.Solid lines represent positive correlations,and dashed lines represent negative correlations.The color of the line represents the P value.HPV:Human papillomavirus;HPV+:HPV-positive;MDSC:Myeloid-derived suppressor cells;TAM:Tumor-associated macrophage.1329临床与病理杂志,2023,43(7)http:/下维持生存30。而癌细胞中SLC7A11过表达促进谷胱甘肽生物合成和铁死亡抵抗，提示SLC7A11介导的胱氨酸摄取在抑制铁死亡和维持氧化应激条件下细胞生存中起关键作用31-32。p53 缺失部分通过SLC7A11抑制铁死亡来促进癌细胞或肿瘤组织的放射治疗抵抗33。放射治疗和免疫治疗可以通过抑制SLC7A11促进铁死亡而达到杀伤肿瘤细胞的作用34，此外，也有研究35表明顺铂可以通过增加宫颈癌的铁死亡水平激活巨噬细胞的抗肿瘤免疫反应，抑制肿瘤细胞的耐药性。本研究发现：SLC7A11高表达会减少肿瘤微环境中免疫细胞浸润(如B细胞、CD8细胞、辅助 T 细胞)以及细胞表面免疫检查点的表达(如TIGIT、CTLA4和PD1)，从而削弱抗肿瘤免疫反应。尽管SCL7A11调节T细胞免疫和肿瘤免疫治疗的机制尚不清楚，但已有研究表明CD8+T细胞分泌的干扰素可以下调SLC7A11的表达从而促进肿瘤细胞发生铁死亡36。因此，靶向SLC7A11联合免疫检查点抑制剂是潜在的治疗方案36。此外，SLC7A11基因的表达也与肿瘤相关成纤维细胞、髓系来源的抑制细胞和肿瘤相关巨噬细胞M2型呈正相关，而这些细胞能重塑免疫微环境37-39，从而促使肿瘤细胞发生免疫逃逸。因此，SLC7A11可以作为进一步研究和治疗的靶点。综 上，本 研 究 探 讨 了 HPV 阳 性 宫 颈 癌 中SLC7A11的高表达与肿瘤免疫浸润及患者预后的关系，提示其在HPV相关宫颈癌治疗中的潜在机制和可能发挥的作用，为HPV相关宫颈癌患者的风险分层和治疗提供了一定的参考。利益冲突声明：作者声称无任何利益冲突。参考文献1Sung H,Ferlay J,Siegel RL,et al.Global cancer statistics 2020:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countriesJ.CA Cancer J Clin,2021,71(3):209-249.https:/doi.org/10.3322/caac.21660.2de Sanjose S,Quint WG,Alemany L,et al.Human papillomavirus genotype attribution in invasive cervical cancer:a retrospective cross-sectional worldwide studyJ.Lancet Oncol,2010,11(11):1048-1056.https:/doi.org/10.1016/S1470-2045(10)70230-8.3Handler MZ,Handler NS,Majewski S,et al.Human papillomavirus vaccine trials and tribulations:clinical perspectivesJ.J Am Acad Dermatol,2015,73(5):743-756;quiz757-758.https:/doi.org/10.1016/j.jaad.2015.05.040.4赵琛,宋淑芳.我国宫颈癌流行病学及病因学的研究J.医学信 息,2021,34(5):6-8.https:/doi.org/10.3969/j.issn.1006-1959.2021.05.002ZHAO Chen,SONG Shufang.The epidemiology and etiology of cervical cancer in ChinaJ.Medical Information,2021,34(5):6-8.https:/doi.org/10.3969/j.issn.1006-1959.2021.05.002.5Zheng R,Zhang S,Zeng H,et al.Cancer incidence and mortality in China,2016J.Journal of the National Cancer Center,2022,2(1):1-9.https:/doi.org/10.1016/j.jncc.2022.02.002.6Varn FS,Schaafsma E,Wang Y,et al.Genomic characterization of six virus-associated cancers identifies changes in the tumor immune microenvironment and altered genetic programsJ.Cancer Res,2018,78(22):6413-6423.https:/doi.org/10.1158/0008-5472.CAN-18-1342.7Lei T,Li X,Wang F,et al.Immune landscape of viral cancers:insights from single-cell sequencingJ/OL.J Med Virol,2023,95(1):e283482023-02-05.https:/doi.org/10.1002/jmv.28348.8Liu C,Zhang M,Yan X,et al.Single-cell dissection of cellular and molecular features underlying human cervical squamous cell carcinoma initiation and progressionJ.Sci Adv,2023,9(4):eadd8977.https:/doi.org/10.1126/sciadv.add8977.9艾海兵,陈亚楠,谷春会,等.人乳头瘤病毒(HPV)相关性宫颈癌的研究进展J.吉林医学,2017,38(1):169-171.https:/doi.org/10.3969/j.issn.1004-0412.2017.01.069.AI Haibing,CHEN Yanan,GU Chunhui,et al.Research progress of human papillomavirus(HPV)-associated cervical cancerJ.Jilin Medical Journal,2017,38(1):169-171.https:/doi.org/10.3969/j.issn.1004-0412.2017.01.069.10Mou Y,Wang J,Wu J,et al.Ferroptosis,a new form of cell death:opportunities and challenges in cancerJ.J Hematol Oncol,2019,12(1):34.https:/doi.org/10.1186/s13045-019-0720-y.11Jiang X,Stockwell B,Conrad M.Ferroptosis:mechanisms,biology and role in diseaseJ.Nat Rev Mol Cell Biol,2021,22:266-282.https:/doi.org/10.1038/s41580-020-00324-8.12Cruz-Gregorio A,Aranda-Rivera AK,Ortega-Lozano AJ,et al.Lipid metabolism and oxidative stress in HPV-related cancersJ.Free Radic Biol Med,2021,172:226-236.https:/doi.org/10.1016/j.freeradbiomed.2021.06.009.13Stockwell BR,Friedmann Angeli JP,Bayir H,et al.Ferroptosis:a regulated cell death nexus linking metabolism,redox biology,and diseaseJ.Cell,2017,171(2):273-285.https:/doi.org/10.1016/j.cell.2017.09.021.14Long Y,Tao H,Karachi A,et al.Dysregulation of glutamate transport enhances treg function that promotes VEGF blockade resistance in glioblastomaJ.Cancer Res,2020,80(3):499-509.https:/doi.org/10.1158/0008-5472.CAN-19-1577.15Hmon A,Louandre C,Lailler C,et al.SLC7A11 as a biomarker and therapeutic target in HPV-positive head and neck squamous cell carcinomaJ.Biochem Biophys Res Commun,2020,533(4):1083-1087.https:/doi.org/10.1016/j.1330SLC7A11对HPV阳性宫颈癌患者免疫应答和生存的影响 雷田雨，等bbrc.2020.09.134.16Subramanian A,Tamayo P,Mootha VK,et al.Gene set enrichment analysis:a knowledge-based approach for interpreting genome-wide expression profilesJ.Proc Natl Acad Sci USA,2005,102(43):15545-15550.https:/doi.org/10.1073/pnas.0506580102.17Li B,Severson E,Pignon JC,et al.Comprehensive analyses of tumor immunity:implications for cancer immunotherapyJ.Genome Biol,2016,17(1):174.https:/doi.org/10.1186/s13059-016-1028-7.18Newman AM,Liu CL,Green MR,et al.Robust enumeration of cell subsets from tissue expression profilesJ.Nat Methods,2015,12(5):453-457.https:/doi.org/10.1038/nmeth.3337.19Finotello F,Mayer C,Plattner C,et al.Molecular and pharmacological modulators of the tumor immune contexture revealed by deconvolution of RNA-seq dataJ.Genome Med,2019,11(1):34.https:/doi.org/10.1186/s13073-019-0638-6.20Becht E,Giraldo NA,Lacroix L,et al.Erratum to:estimating the population abundance of tissue-infiltrating immune and stromal cell populations using gene expressionJ.Genome Biol,2016,17(1):249.https:/doi.org/10.1186/s13059-016-1113-y.21Aran D,Hu Z,Butte AJ.xCell:digitally portraying the tissue cellular heterogeneity landscapeJ.Genome Biol,2017,18(1):220.https:/doi.org/10.1186/s13059-017-1349-1.22Jiang P,Gu S,Pan D,et al.Signatures of T cell dysfunction and exclusion predict cancer immunotherapy responseJ.Nat Med,2018,24(10):1550-1558.https:/doi.org/10.1038/s41591-018-0136-1.23Wang B,Mou H,Liu MM,et al.Multiomics characteristics of neurogenesis-related gene are dysregulated in tumor immune microenvironmentJ.NPJ Genom Med,2021,6(1):37.https:/doi.org/10.1038/s41525-021-00202-y.24Shi ZZ,Tao H,Fan ZW,et al.Prognostic and immunological role of key genes of ferroptosis in pan-cancerJ.Front Cell Dev Biol,2021,9:748925.https:/doi.org/10.3389/fcell.2021.748925.25李青芸,刘晓雯,向凤怡,等.SLC7A11在胰腺癌中的研究进展 J.生 命 科 学,2022,34(7):815-820.https:/doi.org/10.13376/j.cbls/2022089.LI Qingyun,LIU Xiaowen,XIANG Fengyi,et al.Research progress of SLC7A11 in pancreatic cancerJ.Chinese Bulletin of Life Sciences,2022,34(7):815-820.https:/doi.org/10.13376/j.cbls/2022089.26Wang T,Gong M,Cao Y,et al.Persistent ferroptosis promotes cervical squamous intraepithelial lesion development and oncogenesis by regulating KRAS expression in patients with high risk-HPV infectionJ.Cell Death Discov,2022,8(1):201.https:/doi.org/10.1038/s41420-022-01013-5.27You Y,Fan Q,Huang J,et al.Ferroptosis-related gene signature promotes ovarian cancer by influencing immune infiltration and invasionJ.J Oncol,2021,2021:9915312.h