Abstract
目的
肺癌免疫治疗疗效与免疫细胞浸润密切相关。花生四烯酸5-脂氧合酶(arachidonic acid 5-lipoxygenase，ALOX5)可激活炎症反应并触发各种细胞死亡模式；然而，ALOX5与肺癌免疫细胞浸润的相关性尚不清楚。本研究利用在线数据库分析ALOX5在非小细胞肺癌(non-small cell lung cancer，NSCLC)中的表达，探讨其与NSCLC免疫细胞浸润的相关性及其与预后的关系。
方法
分析TIMER、GEPIA和UALCAN等在线数据库中NSCLC和正常组织ALOX5 mRNA和蛋白表达的差异；应用Kaplan-Meier数据库探讨ALOX5的预后价值，GeneMANIA和String网站探索与ALOX5基因表达相关联的基因及蛋白质；对TCGA数据库挖掘出的差异基因进行基因本体(Gene Ontology，GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)富集分析；应用基因集富集分析(gene set enrichment analysis，GSEA)软件预测ALOX5可能参与的信号通路，TIMER数据库分析ALOX5对免疫细胞浸润水平的影响。
结果
与正常肺组织相比，ALOX5在NSCLC组织中呈低表达(P<0.05)，且影响NSCLC患者预后。基因互作网络分析发现：与ALOX5基因相互作用的基因主要有毛状样蛋白(coactosin like protein 1，COTL1)、白三烯C4合酶(leukotriene C4 synthase，LTC4S)和环加氧酶2(prostaglandin endoperoxide synthase 2，PTGS2)等脂质氧化和促炎介质生成的相关基因，蛋白质-蛋白质相互作用(protein-protein interaction，PPI)网络分析结果与之一致。GO、KEGG富集分析发现：ALOX5基因参与多种免疫细胞功能活化的生物过程，并参与免疫反应功能通路。GSEA结果显示ALOX5可能通过影响细胞因子与细胞因子受体的相互作用、自然杀伤细胞介导的细胞毒性和T细胞受体等信号通路，从而激活免疫反应，介导与免疫相关的预后。肺腺癌及肺鳞状细胞癌中ALOX5 mRNA表达与肿瘤浸润性免疫细胞(B细胞、CD8+T细胞、CD4+T细胞等)浸润水平均呈正相关(均P<0.05)，并且与经典的T细胞免疫检查点抑制剂基因标志物呈正相关(P<0.001)。
结论
ALOX5基因在NSCLC中表达显著下调，可影响NSCLC预后和肿瘤免疫细胞浸润水平。ALOX5基因可能是潜在的与免疫细胞浸润相关的NSCLC预后生物标志物。
Keywords: 花生四烯酸5-脂氧合酶, 非小细胞肺癌, 预后生物标志物, 免疫细胞浸润水平
Abstract
Objective
The efficacy of immunotherapy for lung cancer is closely related to immune cell infiltration. Arachidonic acid 5-lipoxygenase (ALOX5) can activate inflammatory responses and trigger various cell death patterns; however, the relevance of ALOX5 to immune cell infiltration in lung cancer is unclear. The expression of ALOX5 in non-small cell lung cancer (NSCLC) is analyzed using an online database to explore the correlation between ALOX5 and immune cell infiltration in NSCLC and its relationship with prognosis.
Methods
Differences in ALOX5 expression in NSCLC and normal lung tissues were analyzed by online databases such as TIMER, GEPIA and HPA; the UALCAN database was used to reveal the relationship between ALOX5 and clinical features; Kaplan-Meier database was applied to explore the prognostic value of ALOX5; GeneMANIA and String Website was used to explore genes and proteins associated with ALOX5 expression, respectively; the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to analyze ALOX5 differential genes which were picked up through the TCGA database; GSEA software was applied to predict the signal pathways that ALOX5 may be involved in; and the TIMER database was used to analyze the effect of ALOX5 expression on the level of immune cell infiltration.
Results
Compared with the normal lung tissues, the ALOX5 expression was low in NSCLC tissues (P<0.05), and which affected the prognosis of lung cancer patients. The expression level of ALOX5 was related to clinical features such as sex, age, metastasis, and pathological staging in NSCLC patients (all P<0.05). The gene interaction network analysis found that the genes interacting with ALOX5 mainly included the genes related to lipid oxidation and pro-inflammatory mediators such as coactosin like protein 1 (COTL1), leukotriene C4 synthase (LTC4S), and prostaglandin endoperoxide synthase 2 (PTGS2), and the protein-protein interaction analysis results were consistent. GO and KEGG analysis found that ALOX5 was involved in the biological process of activation of immune cell function and was involved in immune response function pathways. The GSEA analysis showed that ALOX5 may activate immune responses and mediate immune-related prognosis by affecting the cytokine-cytokine receptor interactions, natural killer-mediated cytotoxicity, and T cell receptor signaling pathways. The ALOX5 mRNA expressions in lung adenocarcinoma and lung squamous cell carcinoma were positively correlated with the tumor infiltration immune cells (B cells, CD8+ T cells, CD4+ T cells, etc.) (all P<0.05), and the ALOX5 mRNA expression was positively correlated with the expression of classic T cell immune checkpoint inhibitor genes (P<0.001).
Conclusion
The ALOX5 gene expression in NSCLC is significantly downregulated, and which can affect NSCLC prognosis and immune cell infiltration levels. ALOX5 gene may be a potential biomarker of NSCLC prognosis associated with immune cell infiltration.
Keywords: arachidonic acid 5-lipoxygenase, non-small cell lung cancer, prognostic biomarkers, immune cell infiltration level
肺癌是全球癌症相关死亡的主要原因，全球癌症统计数据显示2020年肺癌新发病例达220.7万，仅次于乳腺癌；死亡例数为179.6万，位居各种癌症之首[1-2]。肺癌分为非小细胞肺癌(non-small cell lung cancer，NSCLC)和小细胞肺癌(small cell lung cancer，SCLC)，其中以NSCLC为主，约占85%。NSCLC主要病理类型有肺腺癌(lung adenocarcinoma，LUAD)和肺鳞状细胞癌(lung squamous carcinoma，LUSC)[3]。近年来，靶向癌细胞的药物研发取得了一定的进展，但这些治疗方法仍然不能使晚期NSCLC患者获得理想的生存期[4]。肿瘤微环境(tumor microenvironment，TME)是指肿瘤中出现的非癌变细胞及其组分，包括它们产生和释放的分子[5]。肿瘤细胞与TME之间不断的相互作用在肿瘤的发生、发展、转移和对治疗的反应中起决定性的作用。研究[6]表明TME在患者特定治疗干预的反应方面也起关键作用，TME的免疫学分析可能可以预测预后和免疫治疗反应。
花生四烯酸5-脂氧合酶(arachidonic acid 5-lipoxygenase，ALOX5)是一种含铁的非血红素双加氧酶，可催化花生四烯酸(arachidonic acid，AA)等多不饱和脂肪酸的过氧化作用[7]，在炎症反应、细胞死亡及肿瘤发生、发展过程中具有重要作用。既往研究[8-12]发现：ALOX5可影响肿瘤的侵袭和迁移，在胃癌、结直肠癌、乳腺癌、慢性髓细胞白血病等癌症的发生、发展过程及临床预后中发挥重要的作用。另外，ALOX5已被证明是免疫相关基因，其基因多态性与肺癌患病风险增加相关[13]。然而，ALOX5基因在NSCLC发生、发展中的作用及其潜在的分子机制尚不清楚。本研究拟整合多种生物信息学方法，分析ALOX5基因在NSCLC中的表达及其对NSCLC预后的影响，并探讨其可能存在的分子机制，为NSCLC的诊治及预后判断提供新的依据。
1. 资料与方法
1.1. 使用的网站及数据库
GEPIA网站(http://gepia.cancer-pku.cn/index.html)：在GEPIA数据库中研究LUAD/LUSC与正常相邻肺组织样本ALOX5基因的表达情况。采用Spearman相关系数法进行相关分析，确定ALOX5基因与程序性细胞死亡蛋白-1(programmed death-1，PD-1)及其配体(PD-L1)、细胞毒T淋巴细胞相关抗原4(cytotoxic T lymphocyte-associated antigen-4，CTLA-4)的关系。
Tumor Immune Estimation Resource (TIMER)网站(https://cistrome.shinyapps.io/timer/)：利用TIMER数据库评估ALOX5基因在多种类型癌症中的表达。分析ALOX5基因与LUAD、LUSC中免疫细胞浸润的相关性。
基因本体(Gene Ontology，GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)富集分析、基因集富集分析(gene set enrichment analysis，GSEA)：通过GO和KEGG分析探讨ALOX5基因在肺癌中的生物学功能。利用GSEA研究ALOX5基因的潜在作用机制。GO、KEGG由R包ClusterProfiler执行。GSEA富集分析应用GSEA软件进行。
CIBERSORT(https://cibersort.stanford.edu/)网站：采用1个已验证的包含547个基因和22个人类免疫细胞亚群的白细胞基因签名矩阵来描述免疫细胞组成。通过CIBERSORT测量肿瘤浸润免疫细胞(tumor infiltrating immune cells，TIICs)的比例，并检查ALOX5基因表达和免疫细胞亚群之间的相关性。
Kaplan-Meier Plotter (http://kmplot.com)网站：包含3 452例临床肺癌患者基因表达数据和生存信息的在线数据库，可分析ALOX5基因在肺癌中的预后价值。
PrognoScan (http://www.abren.net/PrognoScan/)网站：通过PrognoScan数据库分析ALOX5基因表达与NSCLC生存率之间的相关性。
采用GeneMANIA(http://www.genemania.org)数据库构建ALOX5的基因-基因相互作用网络。利用STRING在线数据库(https://string-db.org/)构建ALOX5的蛋白质-蛋白质相互作用(protein-protein interaction，PPI)网络。
1.2. 统计学处理
Kaplan-Meier、PrognoScan和GEPIA网站的结果显示为HR和P或Cox P值(来自log-rank检验)。基因表达差异采用独立样本t检验。基因表达的相关性采用Spearman相关分析。ALOX5基因与铁死亡相关基因(ferroptosis-related genes，FRGs)的相关热图由R软件包与Spearman相关的pheatmap生成。P<0.05为差异有统计学意义。
2. 结 果
2.1. NSCLC患者 ALOX5 表达降低
通过TIMER数据库分析ALOX5基因在泛癌中的表达情况(图1A)。在GEPIA和UALCAN数据库中，ALOX5 mRNA在LUAD和LUSC组织中的表达均低于正常肺组织(图1B、1C)。直接用TCGA数据库获得的数据显示：相较于邻近正常组织，ALOX5基因在LUAD和LUSC组织中表达均明显降低(均P<0.001，图1D)。此外，在58对LUAD和50对LUSC肿瘤标本中，与相邻的正常组织相比，ALOX5基因在和LUSC中的表达均明显降低(均P<0.001，图1E)。
图1.
肺癌中 ALOX5 的表达
Figure 1 Expression of ALOX5 in lung cancer
A: Expression of ALOX5 in different types of cancers is investigated in the TIMER database; B: Compared to the normal tissues, ALOX5 expression is reduced in the lung cancer tissues in the GEPIA database; C: Expression of ALOX5 in lung cancer is investigated in the UALCAN database; D: Expression analysis of ALOX5 is analyzed in the lung cancer tissues and the normal tissues adjacent to the cancer in TCGA database; E: TCGA database and statistical analysis of ALOX5 expression in 58 pairs of LUAD tissues and paraneoplastic normal tissues and 50 pairs of LUSC tissues and paraneoplastic normal tissues. *P<0.05, **P<0.01, ***P<0.001. ALOX5: Arachidonic acid 5-lipoxygenase gene; TPM: Transcripts per million; LUAD: Lung adenocarcinoma; LUSC: Lung squamous carcinoma; TCGA: The Cancer Genome Atlas; num(T): Number of tumor tissues; num(N): Number of normal tissues.
2.2. 肺癌患者 ALOX5 表达降低与预后相关
KaplanMeier plotter数据库显示：ALOX5基因表达降低与LUAD患者较好的总生存期(overall survival，OS)和无进展生存期(progression-free survival，PFS)相关，而在LUSC患者中不相关(图2A)。另外，在LUAD和LUSC中均显示ALOX5基因表达高低与进展后生存期(post progression survival，PPS)无显著相关性(图2A)。此外，PrognoScan数据库显示：在GSE31210队列中，LUAD中ALOX5基因表达升高与较差的OS、无复发生存时间(recurrence free survival，RFS)、疾病特异性生存期(disease-specific survival，DSS)显著相关，而在GSE4573队列中也显示NSCLC中ALOX5基因表达升高与较差的DSS显著相关(图2B)。为了更好地了解ALOX5基因表达在肺癌中的预后价值和潜在机制，利用Kaplan-Meier数据库探讨ALOX5 mRNA表达与临床特征之间的关系。结果显示：在LUAD中，ALOX5高表达与较差的OS相关，而不论性别和吸烟与否，ALOX5高表达是OS和FPS的危险因素。
图2.
评价 ALOX5 预后价值的生存曲线
Figure 2 Survival curves to evaluate the prognostic value of ALOX5
A: Survival curves of OS, PFS, and PPS for LUAD and LUSC by Kaplan-Meier plotter; B: Survival curves for DSS, OS, and RFS in the PrognoScan database; C: Correlation between ALOX5 expression and the clinicopathological parameters in the LUAD and LUSC patients by forest plots. ALOX5: Arachidonic acid 5-lipoxygenase gene; LUSC: Lung squamous carcinoma; LUAD: Lung adenocarcinoma; OS: Overall survival; PFS: Progression-free survival; PPS: Post progression survival; RFS: Recurrence free survival; DSS: Disease-specific survival.
2.3. ALOX5 相互作用基因和蛋白质的鉴定及遗传改变
利用GeneMANIA构建ALOX5与改变的邻近基因的基因-基因相互作用网络。结果显示：与ALOX5密切相关的前20个基因包括COTL1、ALOX5AP、ALOX15B和LTC4S等(图3A)。利用STRING数据库生成ALOX5的PPI网络(图3B)共有21个节点，包括COTL1、LTC4S和ALOX5AP等(图3B)。基于TCGA数据库，探讨ALOX5与FRGs的关系。ALOX5在LUAD中与SLC40A1、TFRC、FTL、FTH1、CP、ACO1呈显著正相关(图3C)。此外，在LUSC中ALOX5与TFRC、TFR2、IREB2呈显著负相关，而与SLC40A1、FTL、CP和ACO1呈正相关(图3D)。
图3.
ALOX5 基因相互作用的基因和蛋白质
Figure 3 Genes and proteins interacted with ALOX5 gene
A: Construction of the gene-gene interaction network of ALOX5 by GeneMANIA; B: PPI network of ALOX5 generated by STRING; C: Heatmap showing the correlation of ALOX5 with FRGs in LUAD; D: Heatmap showing the correlation of ALOX5 with FRGs in LUSC. *P<0.05, **P<0.01; ***P<0.001. ALOX5: Arachidonic acid 5-lipoxygenase gene; LUSC: Lung squamous carcinoma; LUAD: Lung adenocarcinoma; TCGA: The Cancer Genome Atlas; PPI: Protein-protein interaction; FRGs: Ferroptosis-related genes.
2.4. GO、KEGG富集分析
LUAD和LUSC中与ALOX5正相关的前30个基因见图4A、4B。然后，选择前300个与ALOX5基因显著正相关的基因，通过clusterProfiler包进行GO和KEGG分析。GO术语可分为3类：分子功能(molecular function，MF)、生物过程(biological process，BP)和细胞成分(cell component，CC)。构建了表征GO分析中最富集的前15个BP、MF和CC项的柱状图(图4B)。结果显示：在LUAD中与ALOX5共表达的基因介导的BP主要包括中性粒细胞活化、免疫反应中的中性粒细胞活化和中性粒细胞脱颗粒等；在LUSC中主要为T细胞活化、淋巴细胞活化的调节、白细胞活化的阳性调节等(图4C、4D)。KEGG通路分析还发现：在LUAD和LUSC中，ALOX5参与细胞因子及其受体相互作用、细胞因子信号通路、Th1和Th2细胞分化等免疫相关通路(图4E、4F)。
图4.
ALOX5 的GO和KEGG富集分析
Figure 4 GO and KEGG enrichment analysis of ALOX5
A: Heat map of the top 30 genes positively associated with ALOX5 in LUAD; B: Heat map of the top 30 genes positively associated with ALOX5 in LUSC; C: Top 15 BP class enrichment in LUAD; D: Top 15 BP class enrichment in LUSC; E: Top 15 KEGG-enriched pathways in LUAD; F: Top 15 KEGG enrichment pathways in LUSC. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; ALOX5: Arachidonic acid 5-lipoxygenase gene; LUSC: Lung squamous carcinoma; LUAD: Lung adenocarcinoma; BP: Biological process.
2.5. GSEA鉴定 ALOX5 相关信号通路
在KEGG中，GSEA揭示了多个在肺癌中富集的免疫功能基因集。在LUAD中，包括细胞因子和细胞因子受体的相互作用、自然杀伤细胞介导的细胞毒性、B细胞受体信号通路、T细胞受体信号通路等相关基因集(图5A~5F)。在LUSC中，ALOX5主要富集于细胞因子和细胞因子受体的相互作用、自然杀伤细胞介导的细胞毒性、T细胞受体信号通路(图5G~5L)。
图5.
GSEA分析显示的肺腺癌及肺鳞状细胞癌中 ALOX5 的基因富集通路
Figure 5 GSEA analysis showing gene enrichment pathway of ALOX5 in lung adenocarcinoma and lung squamous carcinoma
A-F: Enriched plot in LUAD; G-L: Enriched plot in LUSC. ALOX5: Arachidonic acid 5-lipoxygenase gene; LUSC: Lung squamous carcinoma; LUAD: Lung adenocarcinoma; GSEA: Gene set enrichment analysis.
2.6. ALOX5 表达与多种免疫标志物的相关性研究
为了加深对ALOX5与免疫应答之间相互作用的理解，使用TCGA数据库和TIMER数据库验证ALOX5表达与LUAD和LUSC中不同的免疫特征之间的相关性。用于表征各种免疫细胞的基因见表1。调整肿瘤纯度后，ALOX5表达与LUSC和LUAD中不同类型的免疫细胞中的大多数免疫标志物显著相关。
表1.
ALOX5 与免疫细胞基因标志物的相关性分析
Table 1 Correlation analysis of ALOX5 with immune cell gene markers
| Description | Gene markers | LUAD | LUSC | ||||||
|---|---|---|---|---|---|---|---|---|---|
| None | Purity | None | Purity | ||||||
| cor | P | cor | P | cor | P | cor | P | ||
| B cell | CD19 | 0.23 | *** | 0.09 | 0.054 | 0.48 | *** | 0.34 | *** |
| CD79A | 0.20 | *** | 0.06 | 0.172 | 0.49 | *** | 0.34 | *** | |
| T cell (general) | CD3D | 0.40 | *** | 0.26 | *** | 0.59 | *** | 0.49 | *** |
| CD3E | 0.45 | *** | 0.33 | *** | 0.67 | *** | 0.58 | *** | |
| CD2 | 0.48 | *** | 0.37 | *** | 0.65 | *** | 0.56 | *** | |
| CD8+ T cell | CD8A | 0.31 | *** | 0.19 | *** | 0.56 | *** | 0.48 | *** |
| CD8B | 0.24 | *** | 0.14 | ** | 0.45 | *** | 0.42 | *** | |
| Monocyte | CD86 | 0.67 | *** | 0.62 | *** | 0.73 | *** | 0.66 | *** |
| CSF1R | 0.75 | *** | 0.71 | *** | 0.79 | *** | 0.73 | *** | |
| TAM | CCL2 | 0.40 | *** | 0.32 | *** | 0.51 | *** | 0.44 | *** |
| CD68 | 0.69 | *** | 0.65 | *** | 0.62 | *** | 0.54 | *** | |
| IL10 | 0.46 | *** | 0.36 | *** | 0.54 | *** | 0.47 | *** | |
| M1 macrophage | IRF5 | 0.60 | *** | 0.55 | *** | 0.27 | *** | 0.27 | *** |
| PTGS2 | -0.10 | * | -0.10 | * | 0.12 | ** | 0.05 | 0.278 | |
| M2 macrophage | CD163 | 0.62 | *** | 0.56 | *** | 0.71 | *** | 0.65 | *** |
| VSIG4 | 0.70 | *** | 0.67 | *** | 0.68 | *** | 0.61 | *** | |
| Neutrophils | CEACAM8 | 0.39 | *** | 0.39 | *** | 0.16 | *** | 0.14 | ** |
| ITGAM | 0.80 | *** | 0.77 | *** | 0.75 | *** | 0.69 | *** | |
| CCR7 | 0.47 | *** | 0.36 | *** | 0.64 | *** | 0.56 | *** | |
| Natural killer cell | KIR2DL1 | 0.08 | 0.054 | 0.03 | 0.556 | 0.26 | *** | 0.21 | *** |
| KIR2DL3 | 0.14 | ** | 0.06 | 0.176 | 0.31 | *** | 0.27 | *** | |
| KIR3DL1 | 0.10 | ** | 0.03 | 0.572 | 0.40 | *** | 0.36 | *** | |
| KIR3DL2 | 0.16 | *** | 0.08 | 0.063 | 0.34 | *** | 0.28 | *** | |
| KIR2DS4 | 0.13 | ** | 0.06 | 0.159 | 0.27 | *** | 0.23 | *** | |
| Dendritic cell | HLA-DPB1 | 0.68 | *** | 0.63 | *** | 0.80 | *** | 0.74 | *** |
| HLA-DRA | 0.66 | *** | 0.61 | *** | 0.75 | *** | 0.69 | *** | |
| HLA-DPA1 | 0.66 | *** | 0.61 | *** | 0.79 | *** | 0.73 | *** | |
| CD1C | 0.56 | *** | 0.51 | *** | 0.58 | *** | 0.45 | *** | |
| ITGAX | 0.70 | *** | 0.66 | *** | 0.77 | *** | 0.70 | *** | |
*P<0.05, **P<0.01, ***P<0.001. cor: Correlation coefficient; LUSC: Lung squamous carcinoma; LUAD: Lung adenocarcinoma; TAM: Tumor-associated macrophage; None: Unadjusted tumor purity; Purity: Adjustment of tumor purity.
分析ALOX5表达与6种TIICs(包括B细胞、CD8+ T细胞、CD4+ T细胞、巨噬细胞、中性粒细胞和树突状细胞)之间的相关性，结果显示ALOX5表达水平与LUAD和LUSC中TIICs均呈显著正相关(图6A)。ssGSEA分析结果显示：ALOX5表达与肺癌免疫细胞浸润相关性的棒棒糖图见图6，ALOX5与LUAD和LUSC中巨噬细胞、活化树突状细胞、中性粒细胞等的浸润水平呈正相关(图6B)。进一步研究发现ALOX5表达与T细胞检查点PD-1、PD-L1和CTLA-4的表达均有显著相关性(均P<0.001，图6C、6D)。
图6.
ALOX5 表达与免疫细胞浸润水平的相关性
Figure 6 Correlation of ALOX5 expression with the levels of immune cell infiltration
A: ALOX5 is significantly correlated with tumor purity, and positively correlated with different immune cell infiltration in TIMER database; B: Lollipop plot shows the correlation between ALOX5 expression and immune cell infiltration in lung cancer using ssGSEA analysis; C and D: Scatter plot shows ALOX5 expression correlating with PD-1, PD-L1 and CTLA-4 in LUAD (C) and LUSC (D) in the TCGA database. ALOX5: Arachidonic acid 5-lipoxygenase gene; LUSC: Lung squamous carcinoma; LUAD: Lung adenocarcinoma; GSEA: Gene set enrichment analysis; TCGA: The Cancer Genome Atlas; TPM: Transcripts per million; PD-1: Programmed death-1; PD-L1: Programmed cell death-ligand 1; CTLA-4: Cytotoxic T lymphocyte antigen 4.
3. 讨 论
ALOX5是触发脂质过氧化的关键酶，可激发炎症反应，并引发各种细胞死亡模式，包括凋亡、焦亡和铁死亡[7]。研究[14]发现：ALOX5是介导细胞死亡的重要的酶，依赖于ERK-1磷酸化，介导核膜脂质过氧化，导致细胞溶解分子的核易位，诱导DNA损伤和细胞死亡。既往研究[8]发现：在多种肿瘤类型中，ALOX5基因表达上调并介导转移或化学治疗(以下简称“化疗”)耐药，导致不良预后。ALOX5基因在胃癌患者中表达上调，并且通过其代谢物5-羟基二十碳四烯酸(5-HETE)激活胃癌细胞，降低化疗疗效。ALOX5基因可能通过抑制STAT/K-RAS信号通路，提高急性髓系白血病的化疗敏感性，在急性髓系白血病中表现出抗肿瘤作用[15]。ALOX5基因通过调节PI3K/AKT通路，促进膀胱癌细胞的增殖和转移[16]。本研究发现：ALOX5基因在NSCLC中表达明显下调，ALOX5基因表达下调只在LUAD中与较好的OS和PFS显著相关，而在LUSC中预后差异不显著，提示ALOX5基因可以作为LUAD预后生物标志物。
M2型巨噬细胞不仅介导肿瘤细胞转移，还可以释放多种抑制T淋巴细胞的细胞因子影响其功能，促进肿瘤细胞免疫逃逸，导致不良预后[17]。既往研究[18]发现：肺泡巨噬细胞比间质巨噬细胞表达更高水平的ALOX5和产生更多白三烯(leukotriene，LT)B4。ALOX5抑制剂zileuton的使用可使转移灶的数量减少。本研究采用ssGSEA及TIMER分析的结果显示ALOX5表达与巨噬细胞浸润水平相关性最高，主要为与M2巨噬细胞呈显著正相关。Wculek等[11]研究发现：中性粒细胞是小鼠乳腺癌模型(前)转移肺微环境中建立的主要成分和驱动因素。从机制方面看，LTs通过选择性地扩大具有高致瘤潜能的癌细胞亚池，有助于远处组织的定植。抑制介导LTs生成的限速酶ALOX5可消除中性粒细胞前转移活性，从而减少转移。本研究GO分析发现：在LUAD中，与ALOX5相关的基因富集的功能最主要的包括中性粒细胞活化、免疫反应中的中性粒细胞活化、中性粒细胞脱颗粒。另外，本研究发现与ALOX5相互作用及共表达的基因主要有COTL1、ALOX5AP、LTC4S等，相应表达的蛋白质也是COTL1、ALOX5AP、LTC4S等。研究[19-20]发现COTL1基因在乳腺癌细胞中表达上调，其介导的微丝排列导致乳腺癌细胞迁移和肿瘤转移。ALOX5AP基因可通过增强M2巨噬细胞极化和浆液性卵巢癌微环境中的免疫抑制来预测预后不良[21]。肺癌组织中的癌细胞和外泌体通过LTC4S基因介导LTC4生成LTD4，从而刺激癌细胞迁移和生存[22]。以上研究为进一步探索ALOX5基因在NSCLC发生、发展中的分子机制提供了依据，在多个方面解释了NSCLC转移的可能机制，但NSCLC发生、发展及转移的具体机制仍需要分子生物学实验来验证。
近年来，TME的免疫逃逸机制及免疫疗法在肿瘤治疗中发挥越来越重要的作用[23]。有研究[24]报道免疫细胞浸润对NSCLC的临床和预后具有重要作用，将免疫细胞浸润作为特征纳入预后模型，有助于临床医生对患者的预后进行更准确的预测。本研究发现：LUAD和LUSC中ALOX5 mRNA表达水平与B细胞、CD8+ T细胞、CD4+ T细胞、树突状细胞的浸润水平相关。这些结果提示ALOX5有可能参与NSCLC的免疫细胞浸润过程。肿瘤浸润性淋巴细胞(tumor infiltrating lymphocyte，TILs)反映了癌症中的适应性抗肿瘤免疫反应，通常与良好的预后相关[25]。在NSCLC中，CD8+ T细胞是富集于肿瘤细胞巢周围的基质组织区域最丰富的亚群，较高的间质CD8+ T细胞密度，尤其在PD-L1阳性的肿瘤中，与较长的生存期显著相关[26-27]。本研究发现ALOX5与不同类型的T细胞基因标志物呈显著相关。GSEA富集分析也发现：在LUAD和LUSC中均显示出ALOX5的功能富集于免疫功能基因集，包括细胞因子及其受体的相互作用、自然杀伤细胞介导的细胞毒性、T细胞受体信号通路等。此外，本研究还发现ALOX5与经典的T细胞免疫检查点抑制剂基因标志物CD274、PDCD1和CTLA4呈显著正相关。上述结果提示，ALOX5可作为潜在的与免疫细胞浸润相关的可预测免疫治疗预后的生物标志物。
总之，ALOX5可能作为一种潜在的新的肺癌预后生物标志物。此外，ALOX5与肺癌患者TME中免疫细胞浸润及免疫功能存在密切相关关系，这些发现具有潜在的价值，不仅可以促进对ALOX5作用的理解，而且可以促进其在肺癌预后和免疫治疗中的转译使用。这些结果提示ALOX5可能成为肺癌免疫相关治疗的新靶点。为进一步阐明ALOX5在NSCLC中的功能和作用，还需分子生物学实验进行验证和分析。
基金资助
国家自然科学基金(82172672)；山东省自然科学基金(ZR2021MH379)。
This work was supported by the National Natural Science Foundation (82172672) and the Natural Science Foundation of Shandong Province (ZR2021MH379), China.
利益冲突声明
作者声称无任何利益冲突。
作者贡献
赵倩倩 研究设计，数据分析，论文撰写与修改；孙之、潘月眉、荆琪、李伟 数据采集；王传玺 研究设计，论文指导及修改。所有作者阅读并同意最终的文本。
原文网址
http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/202303311.pdf
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