Hu Changpeng, Qin Ming, Lai Wenjing, Zhou Huyue, Yuan Chengsha, Liu Yafeng, Dai Yue, Yang Mengmeng, Hu Min, Wang Xuan, Luo Menglin, Zhang Rong, Li Guobing
Department of Pharmacy, The Second Affiliated Hospital of Army Medical University, Chongqing, China.
Department of Pharmacy, The Second Affiliated Hospital of Army Medical University, Chongqing, China
J Immunother Cancer. 2025 Nov 28;13(11):e012586. doi: 10.1136/jitc-2025-012586.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a newly identified immunosuppressive regulator, but its mechanism of suppressing antitumor immunity remains ambiguous. This study aims to uncover the underlying mechanism by which PCSK9 promotes hepatocellular carcinoma (HCC) immune escape and to explore potential intervention strategies.
Co-culture assay assessed the cytotoxicity of CD8 T cells against PCSK9-knockout HCC cells. Hepa1-6, H22, and HepG2 cells were used to establish HCC mouse models. Tumor microenvironment changes were evaluated using flow cytometry and single-cell RNA sequencing. Additionally, we developed a CRISPR adenine base editing (ABE) base editor and screened small molecule inhibitors for PCSK9 inhibition in HCC treatment.
We found that PCSK9 was highly expressed and correlated with poor survival in patients with HCC. While PCSK9 deficiency did not affect HCC growth in vitro, it significantly enhanced CD8 T cell-mediated selective killing in vitro and in vivo. This selective killing of PCSK9-deficient HCC cells could not be explained by existing theories related to major histocompatibility complex-I and T-cell receptor (TCR) degradation. Instead, our study revealed that PCSK9 knockout inhibited the expression of secreted phosphoprotein 1 (SPP1) and programmed death-ligand 1 (PD-L1) in HCC cells, and identified friend leukemia virus integration 1 (FLI1) as their co-transcription factor. Overexpression of FLI1 reversed the PCSK9 knockout-induced downregulation of SPP1 and PD-L1, thereby promoting HCC immune escape. Furthermore, PCSK9 upregulated FLI1 expression through the neurogenic locus notch homolog protein 3 (NOTCH3) pathway. Additionally, we designed an all-in-one ABE base editor with thyroxine-binding globulin promoter (ABE-TBG-PCSK9) to knock down PCSK9 and identified parecoxib as a small molecule inhibitor. We confirmed both approaches enhanced CD8 T-cell antitumor activity, significantly inhibiting HCC tumor growth and prolonging mouse survival.
PCSK9 promoted HCC immune escape by upregulating SPP1 and PD-L1 via NOTCH3/FLI1 signaling. CRISPR ABE-mediated PCSK9 deficiency and PCSK9 inhibitor parecoxib may serve as effective strategies to inhibit HCC.
前蛋白转化酶枯草溶菌素/kexin 9型（PCSK9）是一种新发现的免疫抑制调节因子，但其抑制抗肿瘤免疫的机制仍不明确。本研究旨在揭示PCSK9促进肝细胞癌（HCC）免疫逃逸的潜在机制，并探索潜在的干预策略。
共培养试验评估CD8 T细胞对PCSK9基因敲除的HCC细胞的细胞毒性。使用Hepa1-6、H22和HepG2细胞建立HCC小鼠模型。采用流式细胞术和单细胞RNA测序评估肿瘤微环境变化。此外，我们开发了一种CRISPR腺嘌呤碱基编辑（ABE）碱基编辑器，并筛选用于HCC治疗中抑制PCSK9的小分子抑制剂。
我们发现PCSK9在HCC患者中高表达且与较差的生存率相关。虽然PCSK9缺陷在体外不影响HCC生长，但在体外和体内均显著增强了CD8 T细胞介导的选择性杀伤作用。现有与主要组织相容性复合体-I和T细胞受体（TCR）降解相关的理论无法解释这种对PCSK9缺陷的HCC细胞的选择性杀伤作用。相反，我们的研究表明，PCSK9基因敲除抑制了HCC细胞中分泌性磷蛋白1（SPP1）和程序性死亡配体1（PD-L1）的表达，并确定Friend白血病病毒整合1（FLI1）为它们的共转录因子。FLI1的过表达逆转了PCSK9基因敲除诱导的SPP1和PD-L1的下调，从而促进HCC免疫逃逸。此外，PCSK9通过神经源性位点Notch同源蛋白3（NOTCH3）途径上调FLI1表达。另外，我们设计了一种带有甲状腺素结合球蛋白启动子的一体化ABE碱基编辑器（ABE-TBG-PCSK9）来敲低PCSK9，并确定帕罗西汀为小分子抑制剂。我们证实这两种方法均增强了CD8 T细胞的抗肿瘤活性，显著抑制HCC肿瘤生长并延长小鼠生存期。
PCSK9通过NOTCH3/FLI1信号上调SPP1和PD-L1促进HCC免疫逃逸。CRISPR ABE介导的PCSK9缺陷和PCSK9抑制剂帕罗西汀可能是抑制HCC的有效策略。
