FAT4（FAT非典型粘附蛋白4）是粘附蛋白相关蛋白家族的成员，在抑制增殖和转移方面已被证明具有肿瘤抑制作用。Wnt/β-连环蛋白途径的激活与PD-L1相关的肿瘤免疫逃逸密切相关。在这里，我们报告了FAT4过表达通过β-连环蛋白依赖方式抑制PD-L1 N-糖基化和细胞膜定位，调控子宫颈癌抗肿瘤免疫的机制。首先检测了子宫颈癌组织和细胞系中的FAT4表达。通过细胞增殖、克隆形成和免疫荧光方法，确定了FAT4过表达在体外具有肿瘤抑制作用，并在免疫缺陷和完全免疫小鼠异种移植模型中得到了验证。通过体内和体外的功能和机制实验，我们研究了FAT4过表达如何通过β-连环蛋白/STT3/PD-L1轴调节抗肿瘤免疫。FAT4在子宫颈癌组织和细胞系中被下调。我们确定FAT4与β-连环蛋白结合，并对其核定位起到拮抗作用，通过降解复合物（AXIN1、APC、GSK3β、CK1）促进其磷酸化和降解。FAT4过表达降低了程序性死亡配体1（PD-L1）的mRNA表达，导致PD-L1在翻译后修饰（PTM）水平上通过STT3A异常糖基化，进而在内质网（ER）积累和多泛素化依赖性降解。我们发现FAT4过表达通过β-连环蛋白依赖方式促进异常PD-L1糖基化和降解，从而增加了免疫反应性小鼠模型中的细胞毒性T淋巴细胞（CTL）活性。这些发现揭示了子宫颈癌中Wnt/β-连环蛋白途径激活的基础，为针对FAT4/β-连环蛋白/STT3/PD-L1轴的联合免疫治疗提供了选择。方案示意图显示了FAT4的抗肿瘤免疫机制。（左）当Wnt与其受体结合时，受体由Frizzled蛋白和LRP5/6蛋白组成，激活胞浆蛋白DVL，诱导降解复合物（AXIN、GSK3β、CK1、APC）聚集到受体上。随后，稳定的β-连环蛋白转运到细胞核中，并与TCF/LEF和TCF7L2转录因子结合，引起靶基因转录。寡糖基转移酶的催化活性亚单位STT3A增强PD-L1的糖基化，N-糖基化的PD-L1通过ER到高尔基体路径转运到细胞膜上，导致免疫逃逸。（右）FAT4主要通过以下机制发挥抗肿瘤免疫作用：（i）FAT4与β-连环蛋白结合，拮抗其核定位，通过降解复合物（AXIN1、APC、GSK3β、CK1）促进β-连环蛋白的磷酸化和降解；（ii）FAT4以β-连环蛋白依赖方式抑制PD-L1和STT3A的转录，并诱导异常PD-L1糖基化和泛素化依赖降解；（iii）促进细胞毒性T淋巴细胞（CTL）的激活并浸润到肿瘤微环境。© 2023年 意大利国家癌症研究所'Regina Elena'。

FAT4 (FAT Atypical Cadherin 4) is a member of the cadherin-associated protein family, which has been shown to function as a tumor suppressor by inhibiting proliferation and metastasis. The Wnt/β-catenin pathway activation is highly associated with PD-L1-associated tumor immune escape. Here, we report the mechanism by which FAT4 overexpression regulates anti-tumor immunity in cervical cancer by inhibiting PD-L1 N-glycosylation and cell membrane localization in a β-catenin-dependent manner.FAT4 expression was first detected in cervical cancer tissues and cell lines. Cell proliferation, clone formation, and immunofluorescence were used to determine the tumor suppressive impact of FAT4 overexpression in vitro, and the findings were confirmed in immunodeficient and immunocomplete mice xenografts. Through functional and mechanistic experiments in vivo and in vitro, we investigated how FAT4 overexpression affects the antitumor immunity via the β-catenin/STT3/PD-L1 axis.FAT4 is downregulated in cervical cancer tissues and cell lines. We determined that FAT4 binds to β-catenin and antagonizes its nuclear localization, promotes phosphorylation and degradation of β-catenin by the degradation complexes (AXIN1, APC, GSK3β, CK1). FAT4 overexpression decreases programmed death-ligand 1 (PD-L1) mRNA expression at the transcriptional level, and causes aberrant glycosylation of PD-L1 via STT3A at the post-translational modifications (PTMs) level, leading to its endoplasmic reticulum (ER) accumulation and polyubiquitination-dependent degradation. We found that FAT4 overexpression promotes aberrant PD-L1 glycosylation and degradation in a β-catenin-dependent manner, thereby increasing cytotoxic T lymphocyte (CTL) activity in immunoreactive mouse models.These findings address the basis of Wnt/β-catenin pathway activation in cervical cancer and provide combination immunotherapy options for targeting the FAT4/β-catenin/STT3/PD-L1 axis. Schematic cartoons showing the antitumor immunity mechanism of FAT4. (left) when Wnts bind to their receptors, which are made up of Frizzled proteins and LRP5/6, the cytoplasmic protein DVL is activated, inducing the aggregation of degradation complexes (AXIN, GSK3β, CK1, APC) to the receptor. Subsequently, stable β-catenin translocates into the nucleus and binds to TCF/LEF and TCF7L2 transcription factors, leading to target genes transcription. The catalytically active subunit of oligosaccharyltransferase, STT3A, enhances PD-L1 glycosylation, and N-glycosylated PD-L1 translocates to the cell membrane via the ER-to-Golgi pathway, resulting in immune evasion. (Right) FAT4 exerts antitumor immunity mainly through following mechanisms: (i) FAT4 binds to β-catenin and antagonizes its nuclear localization, promotes phosphorylation and degradation of β-catenin by the degradation complexes (AXIN1, APC, GSK3β, CK1); (ii) FAT4 inhibits PD-L1 and STT3A transcription in a β-catenin-dependent manner and induces aberrant PD-L1 glycosylation and ubiquitination-dependent degradation; (iii) Promotes activation of cytotoxic T lymphocytes (CTL) and infiltration into the tumor microenvironment.© 2023. Italian National Cancer Institute ‘Regina Elena’.