The ELK3-CXCL16 axis regulates NK cell cytotoxicity via the chemotactic activity of CXCL16 in TNBC

Background Triple-negative breast cancer (TNBC) is the most challenging subtype of breast cancer because of its aggressive behavior and the limited therapeutic strategies available. In the last decade, immunotherapy has become a promising treatment to prolong survival in advanced solid cancers including TNBC. However, the efficacy of immunotherapy in solid cancers remains limited because solid tumors contain few tumor-infiltrating lymphocytes. Methods A proteome profiler array was performed to identify secreted CXCL16 protein, the expression of which is regulated by ELK3 to control natural killer (NK) cell-mediated cytotoxicity. The correlation between ELK3 and CXCL16 was investigated by microarray and TCGA data analysis. NK cell cytotoxicity and migration assays were performed to examine the role of ELK3-CXCL16 in regulating the anticancer effect in TNBC. CXCL16-mediated NK cell recruitment and NK cell cytotoxicity were examined in an experimental metastasis mouse model and in an MDA-MB231 orthotopic mouse model. Results We show that targeting the ETS transcription factor ELK3 recruits immune cells including NK cells into tumors via the chemotactic activity of the chemokine. ELK3 depletion upregulated CXCL16 expression, thereby inducing NK cell recruitment to tumors and increasing NK cell cytotoxicity in TNBC . In silico analysis showed that ELK3 is negatively correlated with CXCL16 expression in breast cancer patient samples. Low expression of ELK3 and high expression of CXCL16 were associated with a better prognosis. Low expression of ELK3 and high expression of CXCL16 were associated with increased expression of NK cell-related genes. Conclusions The ELK3-CXCL16 axis regulates NK cell recruitment and increases NK cell cytotoxicity, suggesting that targeting the ELK3 gene could be an adjuvant strategy for increasing the efficacy of immunotherapy in TNBC. Affymetrix expression microarray analysis of the Hs578T cell line was performed by BioCore. Briefly, a library was constructed using the standard Affymetrix protocol. Labeled DNA was hybridized to the Affymetrix GeneChip Array (Affymetrix, Santa Clara, CA, USA) and scanned on a GCS3000 scanner (Affymetrix). Data processing and analysis were performed using Affymetrix GeneChip Command Console Software (Affymetrix). Genes with fold change >1.50 and p <0.05 were first selected. Gene lists were sorted using the KEGG mapper tool (https://www.genome.jp/kegg/mapper/). Gene clustering and heatmaps were analyzed using the MeV tool (version 4.9.0). The Hs578T microarray data are deposited in the Gene Expression Omnibus data repository under the code GSEXXXXX. Gene expression data from breast cancer cell lines were downloaded from Cancer Dependency Map (DepMap). Gene expression data were obtained from 24 breast cancer cell lines.

背景 三阴性乳腺癌（triple-negative breast cancer, TNBC）是乳腺癌中最具挑战性的亚型，因其侵袭性强且可用治疗策略有限。近十年来，免疫治疗已成为包括三阴性乳腺癌在内的晚期实体瘤延长患者生存的极具前景的治疗手段。然而，由于实体瘤中浸润的肿瘤淋巴细胞稀少，免疫治疗在实体瘤中的疗效仍较为有限。
方法 本研究采用蛋白质组谱分析芯片（proteome profiler array）鉴定分泌型CXCL16蛋白，该蛋白的表达受ELK3调控，进而参与调控自然杀伤（natural killer, NK）细胞介导的细胞毒性。通过芯片测序及癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据分析，探究ELK3与CXCL16的表达相关性。采用NK细胞毒性与迁移实验，验证ELK3-CXCL16轴在三阴性乳腺癌中调控抗肿瘤效应的作用。在实验性转移小鼠模型与MDA-MB231原位小鼠模型中，检测CXCL16介导的NK细胞招募及NK细胞毒性效应。
结果 本研究证实，靶向ETS转录因子ELK3可通过趋化因子的趋化活性，招募包括NK细胞在内的免疫细胞进入肿瘤微环境。ELK3敲低可上调CXCL16的表达，进而诱导NK细胞向三阴性乳腺癌肿瘤组织招募，并增强NK细胞的细胞毒性。生物信息学分析显示，在乳腺癌患者样本中，ELK3的表达与CXCL16呈负相关。ELK3低表达联合CXCL16高表达与更好的预后相关。同时，ELK3低表达联合CXCL16高表达可上调NK细胞相关基因的表达水平。
结论 ELK3-CXCL16轴可调控NK细胞招募并增强NK细胞毒性，提示靶向ELK3基因可作为增强三阴性乳腺癌免疫治疗疗效的辅助策略。
本研究中Hs578T细胞系的Affymetrix表达芯片分析由BioCore完成。简要流程如下：采用标准Affymetrix实验流程构建文库；将标记后的DNA与Affymetrix GeneChip芯片（美国加利福尼亚州圣克拉拉市Affymetrix公司）进行杂交，并通过GCS3000扫描仪（Affymetrix公司）进行扫描；使用Affymetrix GeneChip Command Console软件（Affymetrix公司）进行数据处理与分析。首先筛选出折叠变化>1.50且P值<0.05的差异基因；通过KEGG Mapper工具（https://www.genome.jp/kegg/mapper/）对基因列表进行注释分类；使用MeV工具（版本4.9.0）进行基因聚类与热图分析。本研究中Hs578T细胞的芯片数据已提交至基因表达综合（Gene Expression Omnibus, GEO）数据库，登录号为GSEXXXXX。乳腺癌细胞系的基因表达数据下载自癌症依赖性图谱（Cancer Dependency Map, DepMap），共涵盖24株乳腺癌细胞系的基因表达信息。