Targeting CXCR4 impaired T regulatory function through PTEN in renal cancer patients

Abstract Background: Tregs trafficking is controlled by CXCR4. In Renal Cell Carcinoma (RCC), the new CXCR4 antagonist R54 was explored in peripheral blood Tregs isolated from primary RCC patients. Methods: PB-Tregs were isolated from 77 RCC patients and 38 healthy donors-(HD) and CFSE-Tregs suppression assay, IL-35 secretion and Nrp-1+Tregs frequency was conducted. PB-Tregs were analyzed for PTEN, CD25, TGF-β1, FOXP3, DNMT1 expression. PTEN-pAKT signaling was evaluated in the presence of R54 and/or triciribine (TCB), Akt inhibitor. TSDR (Treg-specific Demethylated Region) methylation analysis was conducted. Results: Ex vivo R54 impaired PB-RCC-Tregs function, reduced IL-35 release and Nrp-1+Tregs. As PTEN and CD25 expression significantly decreased in R54-PB-RCC-Tregs, PTEN signaling was evaluated. While CXCL12 recruited PTEN+CD25+Tregs cells, R54 significantly reduced it in PB-RCC-Tregs. Tregs activation through IL-2/PMA impaired pAKT+Tregs while R54 increased it. The Akt inhibitor, triciribine, prevented the pAKT+Tregs-R54 induction. As active Tregs present demethylated Treg-specific region (TSDR), a significant decrease in demethylation rate (DMR) of Foxp3-TSDR was observed in R54 treated PB-RCC-Tregs with significant reduction in DNMT1 and FOXP3 expression. Conclusion: R54 affects Tregs function in primary RCC patients targeting PTEN/PI3K/AKT pathway, reducing TSDR demethylation and thus Foxp3 and DNMT1 expression. CXCR4 Tregs targeting may improve the efficacy on RCC tumor microenvironment.

摘要
背景：调节性T细胞（Tregs）的迁移受趋化因子受体4（CXCR4）调控。本研究针对肾细胞癌（RCC），从原发性RCC患者的外周血中分离得到Tregs，对新型CXCR4拮抗剂R54展开探索。
方法：本研究从77例RCC患者及38名健康供者（HD）体内分离外周血Tregs（PB-Tregs），并开展CFSE标记Tregs抑制实验、白细胞介素35（IL-35）分泌检测及神经纤毛蛋白1阳性Tregs（Nrp-1⁺Tregs）频率分析。对PB-Tregs的张力蛋白同源缺失的磷酸酶（PTEN）、CD25、转化生长因子β1（TGF-β1）、叉头框P3（FOXP3）及DNA甲基转移酶1（DNMT1）的表达水平进行检测。在CXCR4拮抗剂R54和/或Akt抑制剂曲西立滨（TCB）的存在条件下，对PTEN-磷酸化Akt（pAKT）信号通路进行评估。同时开展Treg特异性去甲基化区域（TSDR）甲基化分析。
结果：体外实验中，R54可损害PB-RCC-Tregs的功能，降低IL-35的分泌量及Nrp-1⁺Tregs的比例。鉴于R54处理后的PB-RCC-Tregs中PTEN与CD25的表达水平显著下调，本研究对PTEN信号通路进行了进一步评估。趋化因子配体12（CXCL12）可募集PTEN⁺CD25⁺Tregs，而R54可显著降低PB-RCC-Tregs中此类细胞的数量。经白细胞介素2（IL-2）/佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）激活的Tregs中pAKT⁺Tregs比例降低，但R54可提升该比例。Akt抑制剂曲西立滨可阻断R54诱导的pAKT⁺Tregs升高现象。由于活化的Tregs呈现TSDR去甲基化状态，经R54处理的PB-RCC-Tregs中，叉头框P3特异性去甲基化区域（Foxp3-TSDR）的去甲基化率（DMR）显著降低，同时DNMT1与FOXP3的表达水平也明显下调。
结论：R54可通过靶向PTEN/PI3K/AKT信号通路，影响原发性RCC患者体内Tregs的功能，降低TSDR的去甲基化水平，进而下调FOXP3与DNMT1的表达。靶向CXCR4介导的Tregs调控，或可改善RCC肿瘤微环境的治疗效果。