背景：恶性肿瘤是严重危害人类生命健康的疾病之一。近十年来我国癌症的发病和死亡数持续上升，给人民的健康生活带来了极大的负担。寻找新的有效的癌症治疗手段是当前医学研究领域的关键问题。近年来，免疫疗法发展迅速并取得了前所未有的临床疗效。程序性死亡配体1（Programmed cell death 1 ligand 1, PD-L1）和程序性死亡受体1（Programmed cell death protein 1, PD-1）是一对T细胞活化共抑制分子。肿瘤细胞高表达PD-L1并抑制T细胞激活是抑制性肿瘤免疫微环境产生的主要原因。肿瘤细胞PD-L1的表达调控涉及基因组水平、转录水平、转录后水平、翻译及翻译后水平等多个层面，系统研究PD-L1的表达调控机制有助于改善针对PD-1/PD-L1疗法的临床疗效。PD-L1的翻译后修饰，例如糖基化、泛素化和棕榈酰化等会影响PD-L1在肿瘤细胞的表达水平和在细胞中的定位。泛素-蛋白酶体系统是真核生物主要的蛋白降解系统之一，PD-L1在翻译后水平受到去泛素化酶的调控并影响其发生内质网相关蛋白降解。去泛素化酶是一个庞大的蛋白酶家族，目前关于去泛素化酶对PD-L1蛋白水平的调控机制缺乏系统性研究。我们对去泛素化酶家族蛋白进行筛选，发现去泛素化酶USP2能够显著影响肿瘤细胞PD-L1蛋白的表达。USP2能够调控MDM2的泛素化，并间接促进p53/TP53的降解抑制其活性发挥促癌作用，但其作为PD-L1的正调控因子的研究还未见报道，因此我们对USP2调控PD-L1蛋白的机制以及对肿瘤免疫微环境的作用及机制进行了研究。

在免疫检查点疗法（Immune checkpoint therapy, ICTs）中，用抗体阻断PD-1或PD-L1，取得了显著的治疗效果，并提高了多种实体瘤患者的总体生存率，但也存在临床反应率低、免疫相关不良反应严重等问题。小分子肿瘤免疫治疗药物的研发是目前免疫治疗的热点问题，相比抗体药物，小分子药物具有给药途径多、生产成本低以及有更好的肿瘤微环境渗透性等优势。丹酚酸B是从唇形科植物丹参的干燥根和根茎中提取分离出的一种天然产物来源小分子化合物。此前的研究表明，丹酚酸B具有抗氧化、对心肌缺血再灌注损伤的保护以及对动脉粥样硬化的防治等作用，但其通过影响肿瘤微环境发挥抗肿瘤作用的研究还未见报道。

方法：以人类结肠癌细胞RKO为研究对象，使用基于CRISPR-CAS9的筛选方法，通过流式细胞术检测不同去泛素化酶对RKO细胞PD-L1蛋白表达的影响；利用western blot、流式细胞术检测USP2敲除对不同肿瘤细胞PD-L1和其他免疫检查点蛋白水平的影响；放线菌酮（CHX）实验检测USP2对PD-L1蛋白稳定性的影响；通过qRT-PCR的方法检测USP2的敲除对PD-L1 mRNA表达水平的影响；通过免疫共沉淀（Co-IP）和DuoLink原位荧光实验检测USP2与PD-L1蛋白的相互作用；用泛素化实验、抑制剂实验和免疫荧光分析USP2调控PD-L1泛素化的机制；通过流式细胞术、激光共聚焦检测USP2敲除后肿瘤细胞结合PD-1蛋白的变化；细胞阻抗分析、结晶紫染色以及流式细胞术检测肿瘤细胞USP2敲除后共培养的T细胞对肿瘤细胞的杀伤活性；在C57BL/6小鼠前列腺癌RM-1和结肠癌MC38移植瘤模型中检测USP2敲除、USP2敲除后过表达小鼠PD-L1以及过表达小鼠PD-L1的移植瘤的生长情况；同时使用多色流式细胞仪检测移植瘤中CD8⁺T细胞功能的变化，以及免疫抑制性细胞Tregs和MDSCs的活性；组织芯片实验研究结肠癌临床样本中USP2蛋白和PD-L1蛋白表达的关联性；通过TCGA数据分析USP2表达水平对肿瘤免疫浸润和患者生存期的影响；以结肠癌和前列腺癌细胞为研究对象，通过MTT实验检测丹酚酸B对RKO细胞、PC3细胞细胞增殖的影响；利用western blot检测丹酚酸B对细胞内PD-L1蛋白水平的影响；通过Discovery Studio 4.5软件分析丹酚酸B在USP2的结合区域；利用表面等离子共振技术测定丹酚酸B对USP2重组蛋白的结合常数；在结肠癌MC38小鼠移植瘤模型中检测丹酚酸B的体内抑瘤活性。

结果：肿瘤细胞上程序性死亡配体-1（PD-L1）的异常上调通过与PD-1结合阻碍了细胞毒性T细胞对肿瘤细胞的杀伤，因此进一步探索PD-L1在肿瘤中的调节机制将有机会提高PD-L1阻断疗法的临床疗效。我们使用（sgRNAs）筛选系统，鉴定出泛素羧基末端水解酶2（USP2）是调控PD-L1稳定性促进肿瘤免疫逃逸的新调节因子。USP2通过直接与PD-L1相互作用从PD-L1蛋白上去除K48连接的多聚泛素链，增加PD-L1在肿瘤细胞的表达丰度，而USP2的敲除将导致PD-L1的内质网（ER）相关降解，从而减弱PD-L1/PD-1相互作用并使肿瘤细胞对T细胞介导的杀伤敏感。同时，USP2敲除诱导的PD-L1降低能够通过增强CD8+T细胞功能和减少骨髓来源的抑制性细胞（Myeloid-derived suppressor cells，MDSCs）和调节性T细胞（Regulatory cells，Tregs）的免疫抑制从而增强小鼠的抗肿瘤免疫，而PD-L1过表达逆转了通过USP2的敲除导致的肿瘤生长抑制。此外，对临床组织样本的分析也表明USP2存在在肿瘤中上调PD-L1表达的潜在作用。综上，我们的数据揭示了USP2存在调控肿瘤细胞PD-L1稳定性方面的关键作用，并揭示了USP2蛋白是肿瘤免疫治疗的潜在治疗靶点。随后，本研究对丹酚酸B (salvianolic acid B, SAB) 下调肿瘤细胞中PD-L1表达发挥抗肿瘤作用及机制进行了研究。结果表明, SAB能分别以浓度依赖性和时间依赖性方式下调 RKO、PC3细胞中以及细胞膜表面 PD-L1的水平, 这与 SAB抑制肿瘤细胞中去泛素化酶 USP2的活性有关。机制研究发现, SAB 可与 USP2 发生直接相互作用并抑制其去泛素化酶活性, 从而促进 PD-L1 发生泛素-蛋白酶体途径降解。SAB与USP2重组蛋白的结合常数为42.6 µmol·L^-1。此外, SAB可促进共培养的PBMC对RKO细胞的杀伤作用。小鼠荷瘤实验证实, SAB可显著抑制C57BL/6小鼠中MC38移植瘤的生长。20 mg·kg-1 SAB处理荷瘤小鼠后, 可使瘤体积减少63.2%。

结论：在本研究中，我们鉴定出DUBs中泛素特异性蛋白酶（USPs）亚家族成员的USP2，是调控PD-L1蛋白稳定性并促进肿瘤细胞免疫逃逸的新调节因子。我们发现USP2与PD-L1特异性相互作用并去除PD-L1的K48连接的多聚泛素链，从而阻止其发生蛋白酶体降解。进一步研究发现USP2与PD-L1的ICD区域特异性相互作用，并通过其去泛素化活性调节PD-L1的稳定性。USP2的敲除导致PD-L1的ERAD依赖性降解，从而在功能上阻断与PD-1的结合，增强T细胞毒性，并重新编程肿瘤微环境。我们的发现表明，靶向USP2可能潜在地改善针对PD-L1的肿瘤免疫治疗效果。此外，我们的研究结果揭示了SAB通过直接结合USP2并抑制其活性, 促进 PD-L1发生泛素-蛋白酶体途径降解, 从而发挥抗肿瘤作用。本研究为将 SAB研发成靶向USP2-PD-L1轴的小分子肿瘤免疫治疗药物奠定了基础。

Background: Malignant tumor is one of the diseases that seriously endanger human life and health. There are 4 million new cancer cases in China every year, accounting for about a quarter of the new cases worldwide. About 3 million residents in China are killed by cancer every year, and the number of cancer incidence and death in China has continued to rise in the past decade, which has brought a great burden to the healthy life of people. Therefore, finding new and effective cancer treatments is a key issue in the current medical research field. In recent years, immunotherapy has been developing rapidly and has achieved unprecedented clinical efficacy. Programmed death ligand 1 (PD-L1) and programmed death receptor 1 (PD-1) are a pair of T-cell activation co-suppressor molecules. High expression of PD-L1 in tumor cells and inhibition of T-cell activation are the main reasons for the generation of suppressive tumor immune microenvironment. The regulation of PD-L1 expression in tumor cells involves multiple levels including genomic level, transcriptional level, post-transcriptional level, translational and post-translational levels, and systematic study of PD-L1 expression regulation mechanisms can help improve the clinical efficacy of therapies targeting PD-1/PD-L1. Post-translational modifications of PD-L1, such as glycosylation, ubiquitination and palmitoylation, have been reported to affect the expression level of PD-L1 in tumor cells and its localization in the cells. The ubiquitin-proteasome system is one of the major protein degradation systems in eukaryotes, and it has now been shown that PD-L1 is regulated by deubiquitinating enzymes at the post-translational level and influences its occurrence of endoplasmic reticulum-associated protein degradation. Deubiquitinating enzymes are a large family of proteases, and there is a lack of systematic studies on the mechanism of regulation of PD-L1 protein levels by deubiquitinating enzymes. We screened the deubiquitinating enzyme family proteins and found that the deubiquitinating enzyme USP2 could significantly affect the expression of PD-L1 protein in tumor cells. Previous studies have shown that USP2 can regulate the ubiquitination of MDM2 and indirectly promote the degradation of p53/TP53 to inhibit its activity to play a pro-cancer role, but its role as a positive regulator of PD-L1 has not been reported, so we investigated the mechanism of USP2 regulation of PD-L1 protein and its role and mechanism on tumor immune microenvironment.

  In Immune checkpoint therapy (ICTs), blocking PD-1 or PD-L1 with antibodies has achieved remarkable therapeutic effects and improved the overall survival of patients with a variety of solid tumors, but there are also problems such as low clinical response rates and serious immune-related adverse effects. The development of small molecule tumor immunotherapy drugs is currently a hot issue in immunotherapy. Compared with antibody drugs, small molecule drugs have the advantages of multiple routes of administration, low production cost and better permeability to the tumor microenvironment. Salvianolic acid B is a small molecule compound of natural product origin isolated from the dried roots and rhizomes of Salvia miltiorrhiza, a plant of the family Labiatae. Previous studies have shown that salvianolic acid B has antioxidant effects, protection against myocardial ischemia-reperfusion injury and prevention of atherosclerosis, but its antitumor effects by affecting the tumor microenvironment have not been reported.

Methods: Using human colon cancer cells RKO as the target, the effect of different deubiquitinating enzymes on PD-L1 protein expression in RKO cells was detected by flow cytometry using a CRISPR-CAS9-based screening method; the effect of USP2 knockdown on PD-L1 and other immune checkpoint protein levels in different tumor cells was detected by western blot and flow cytometry. The effect of USP2 on PD-L1 protein stability was detected by actinomycin (CHX) assay; the effect of USP2 knockdown on PD-L1 mRNA expression level was detected by qRT-PCR; the interaction of USP2 with PD-L1 protein was detected by immunoprecipitation (Co-IP) and DuoLink in situ fluorescence assay; the ubiquitination assay, inhibitor assay and immunofluorescence to analyze the mechanism of USP2-regulated PD-L1 ubiquitination; detecting changes in PD-1 protein binding by tumor cells after USP2 knockdown by flow cytometry and laser confocal; cell impedance analysis, crystalline violet staining, and flow cytometry to detect the killing activity of co-cultured T cells against tumor cells after USP2 knockdown in tumor cells; in C57BL/6 mice with prostate cancer RM-1 and MC38 transplantation tumor models of colon cancer, the growth of transplantation tumors with USP2 knockdown, PD-L1 overexpressing mice after USP2 knockdown, and PD-L1 overexpressing mice was examined; changes in CD8+ T cell function and the activity of immunosuppressive cells Tregs and MDSCs in transplantation tumors were also examined using multicolor flow cytometry; tissue microarray experiments were performed to study The correlation between USP2 protein and PD-L1 protein expression in colon cancer clinical samples; the effect of USP2 expression level on tumor immune infiltration and patient survival was analyzed by TCGA data; the effect of tannic acid B on cell proliferation of RKO cells and PC3 cells was examined by MTT assay using colon cancer and prostate cancer cells; the effect of tannic acid B on cell proliferation of RKO cells and PC3 cells was examined by western blot using The effect of salvianolic acid B on intracellular PD-L1 protein level was examined by western blot; the binding region of salvianolic acid B in USP2 was analyzed by Discovery Studio 4.5 software; the binding constant of salvianolic acid B on USP2 recombinant protein was determined by surface plasmon resonance technique; the in vivo tumor suppressive activity of salvianolic acid B was examined in MC38 mouse transplantation tumor model of colon cancer.

Result: Aberrant upregulation of programmed death ligand-1 (PD-L1) on tumor cells impedes tumor cell killing by cytotoxic T cells through binding to PD-1, thus further exploration of PD-L1 regulatory mechanisms in tumors has the opportunity to improve the clinical efficacy of PD-L1 blockade therapies. Using the (sgRNAs) screening system, we identified ubiquitin carboxy-terminal hydrolase 2 (USP2) as a novel regulator of PD-L1 stability promoting tumor immune escape.USP2 increases PD-L1 expression abundance in tumor cells by directly interacting with PD-L1 by removing the K48-linked polyubiquitin chain from PD-L1 protein, and knockdown of USP2 would lead to endoplasmic reticulum (ER)-associated degradation of PD-L1, thereby attenuating the PD-L1/PD-1 interaction and sensitizing tumor cells to T-cell-mediated killing. Meanwhile, USP2 knockdown-induced PD-L1 reduction was able to enhance anti-tumor immunity in mice by enhancing CD8⁺ T cell function and reducing immunosuppression of bone marrow-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), while PD-L1 overexpression reversed the tumor growth suppression caused by USP2 knockdown. Furthermore, analysis of clinical tissue samples also suggested a potential role for USP2 in upregulating PD-L1 expression in tumors. Taken together, our data reveal the existence of a key role of USP2 in regulating PD-L1 stability in tumor cells and reveal USP2 protein as a potential therapeutic target for tumor immunotherapy. Subsequently, we investigated the anti-tumor effect of salvianolic acid B (SAB) on PD-L1 expression in tumor cells and its mechanism. The results showed that SAB downregulated PD-L1 levels in RKO and PC3 cells and on the cell membrane surface in a concentration-dependent and time-dependent manner, respectively, which was associated with the inhibition of the activity of deubiquitinating enzyme USP2 in tumor cells by SAB. Mechanistic studies revealed that SAB directly interacted with USP2 and inhibited its deubiquitinating enzyme activity, thereby promoting the degradation of PD-L1 by the ubiquitin-proteasome pathway.The binding constant of SAB to USP2 recombinant protein was 42.6 µmol-L-1. In addition, SAB promoted the killing effect of co-cultured PBMC on RKO cells. SAB significantly inhibited the growth of MC38 transplanted tumors in C57BL/6 mice. 20 mg-kg-1 SAB treatment in tumor-bearing mice resulted in a 63.2% reduction in tumor volume.

Conclusion: In this study, we identified USP2, a member of the ubiquitin-specific proteases (USPs) subfamily of DUBs, as a novel regulator of PD-L1stabilization for cancer cell evasion of immune surveillance. We found USP2 specifically interacts with PD-L1 and removes the K48-linked polyubiquitin chains of PD-L1, thus prevents its proteasomal degradation.USP2 depletion lead to ERAD-dependent degradation of PD-L1, thereby functionally blocking PD-1 binding and enhancing T cell cytotoxicity as well as reprogramming the tumor microenvironment. Our findings suggest targeting USP2 may potentially improve PD-L1-dependent cancer immunotherapy. Our results proved that USP2 specifically interacts with PD-L1 on the ICD region and regulates PD-L1 stability through its deubiquitination activity. Our results demonstrate that SAB exerts its anti-tumor activity by direct binding and inhibiting the activity of USP2 and reducing the PD-L1 level. Our study provides an important material basis and scientific basis for the potential application of SAB in tumor immunotherapy drug targeting USP2-PD-L1 axis.