MicroRNA-15a/16-mediated cytokine/chemokine reprogramming in Kupffer cells prevents the development of hepatocellular carcinoma

摘要 由于治疗效果不佳，肝细胞癌在癌症相关死亡率中排名第三。 已发现抑制抗肿瘤免疫的调节性T细胞(Tregs)在肿瘤内聚集 在肝细胞癌。尽管对肝癌中的microRNAs进行了广泛的研究，但它们在调节免疫抑制方面的作用是 描述得很差。AKT(v-AKT小鼠胸腺瘤病毒癌基因同源1)和NRAS的异常激活 在Kupffer细胞(KCs)和肝细胞(Hcs)中观察到神经母细胞瘤ras病毒癌基因同源物(Ras)。 在74%的肝癌患者中。小鼠AKT和NRAS(AKT/RAS)活化形式的流体动力注射(HDI) 在6-8周内引发了致命性肝癌的发展。MiRNA图谱显示miR-15a和miR-16- AKT/RAS小鼠和患者肝癌KCs中有1个簇(miR-15a/16)减少。KC-特定 MiR-15a/16的表达完全抑制了AKT/RAS小鼠侵袭性肝癌的生长，而AKT/RAS的100% 小鼠在注射后6-8周内死于致死的肿瘤负担。MiR-15a/16的KC特异性表达也 导致AKT/RAS荷瘤小鼠肝细胞癌显著消退。机械上，AKT/RAS 将KCs的转录组重新编程为KCs的M2极化，并驱动血清显着增加 C-C基序趋化因子22(CCL22)及其在AKT/RAS小鼠KC中的表达。相比之下，MIR-15A/16 通过直接靶向激活转录的NF-κB逆转这一过程并抑制CCL22的过度生产 CCL22。CCL22通过CCR4(C-C趋化因子受体4型)招募Tregs。事实上，CCL22的额外治疗 在AKT/RAS小鼠体内，miR-15a/16完全阻止了对肝癌的免疫抑制和生长。 我们假设，受过AKT/RAS教育的KCs通过过度生产CCL22来启动Tregs的肝脏募集， 从而促进肝癌的发展；而KC特异性表达miR-15a/16抑制肝脏募集 通过减弱CCL22的产生来抑制Tregs和肝癌的发展。这个项目的目标是阐明 AKT/RAS感染的KCs在促进免疫抑制中的作用促进肝癌的生长，而miR-15a/16 通过抑制KCs中的NF-κB信号来逆转这一过程。长期目标是发展KCs作为一种治疗方法 靶向和miR-15A/16作为一种新的抗肝癌免疫疗法。我们设计了三个具体目标来测试我们的 假设。具体地说，我们将(1)揭示受过AKT/RAS教育的KC促进(S)的机制 免疫抑制与肝癌的发生发展。(2)建立MIR-15A/16驱动机制(S) 抗肿瘤免疫；以及(3)评价miR-15a/16对散发性肝癌的治疗潜力。 背景。强大的初步数据和基于逻辑和理性的实验设计的结合 使这个项目具有高度的可行性。我们期望这项研究将为免疫抑制提供一个新的视角。 肝癌的机制和促进KCs作为新的治疗靶点和miR-15a/16作为合理的治疗靶点的设计 针对肝癌的免疫治疗。

Abstract Hepatocellular carcinoma (HCC) ranks the third in cancer-related mortality because of ineffective therapy. Intratumoral accumulation of regulatory T-cells (Tregs), which suppresses antitumor immunity, has been identified in HCC. Despite the extensive research of microRNAs in HCC, their roles in regulating immunosuppression are poorly described. Aberrant activation of AKT (v-akt murine thymoma viral oncogene homolog 1) and Nras (neuroblastoma ras viral oncogene homolog) (Ras) was observed in Kupffer cells (KCs) and hepatocytes (HCs) in 74% of HCC patients. Hydrodynamic injection (HDI) of activated forms of AKT and Nras (AKT/Ras) into mice triggered the development of lethal HCC within 6-8 weeks. MiRNA profiling revealed that miR-15a and miR-16- 1 cluster (miR-15a/16) was reduced in KCs from HCC tumors of AKT/Ras mice and patients. KC-specific expression of miR-15a/16 fully prevented growth of aggressive HCC in AKT/Ras mice, while 100% of AKT/Ras mice died from lethal tumor burden within 6-8 weeks post-injection. KC-specific expression of miR-15a/16 also led to a significant regression of HCC tumors in AKT/Ras mice bearing tumors. Mechanistically, AKT/Ras reprogrammed the transcriptome of KCs toward M2 polarization of KCs and drove a significant increase in serum C-C motif chemokine 22 (CCL22) and mRNA levels of Ccl22 in KCs of AKT/Ras mice. In contrast, miR-15a/16 reversed this process and inhibited CCL22 overproduction by directly targeting NF-κB that activates transcription of Ccl22. CCL22 recruits Tregs via CCR4 (C-C chemokine receptor type 4). Indeed, additional treatment of CCL22 recovered immunosuppression and growth of HCC that was fully prevented by miR-15a/16 in AKT/Ras mice. We hypothesize that AKT/Ras-educated KCs initiate hepatic recruitment of Tregs by overproducing CCL22, thereby promoting HCC development; while KC-specific expression of miR-15a/16 suppresses hepatic recruitment of Tregs and HCC development by attenuating CCL22 production. The objective of this project is to elucidate the role of AKT/Ras-educated KCs in promoting immune suppression that promotes growth of HCC, while miR-15a/16 reverses this process by inhibiting NF-κB signaling in KCs. The long-time goal is to develop KCs as a therapeutic target and miR-15a/16 as a novel immunotherapy against HCC. Three specific aims are designed to test our hypothesis. Specifically, we will (1) unravel the mechanism(s) by which AKT/Ras-educated KCs promote immunosuppression and HCC development. (2) establish the mechanism(s) by which miR-15a/16 drives antitumor immunity; and (3) evaluate the therapeutic potential of miR-15a/16 against HCC with divergent backgrounds. The combination of strong preliminary data and a logical and rationally based experimental design make this project highly feasible. We expect that this study will provide a novel insight into the immunosuppression mechanisms in HCC and facilitate the design of KCs as a novel therapeutic target and miR-15a/16 as a rational immunotherapy against HCC.