Department of Radiotherapy Center, Hubei Cancer Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Hubei Provincial Clinical Research Center for Breast Cancer, Wuhan, Hubei, China; Wuhan Clinical Research Center for Breast Cancer, Wuhan, Hubei, China.
Department of Radiotherapy Center, Hubei Cancer Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Hubei Provincial Clinical Research Center for Breast Cancer, Wuhan, Hubei, China.
Int Immunopharmacol. 2024 Oct 25;140:112875. doi: 10.1016/j.intimp.2024.112875. Epub 2024 Aug 9.
The aim of this study was to assess the prognostic significance of α-1,3-mannitrotransferase (ALG3) in triple-negative breast cancer (TNBC) and investigate its impact and potential mechanism on the efficacy of anti-PD-1 therapy.
Bioinformatics analysis was used to examine the expression of ALG3 in cancer patients using UACLAN and other databases. The associations of the ALG3 gene and the clinicopathological features of breast cancer were examined with bc-GenExMiner database. Correlation between ALG3 expression and survival was further established utilizing the Kaplan-Meier Plotter database. Immunohistochemistry (IHC) was used to analyze the expression of ALG3 in cohort of breast cancer patients from Hubei cancer hospital to confirmed the prognostic value of ALG3 in TNBC. The effect of ALG3 on the levels of infiltrating immune cells was also analyzed. And the mutation module within cBioPortal was utilized to visualize ALG3 mutations in BRCA. The CRISPR/Cas9 technique was used to establish ALG3 low-expression TNBC cell lines. Influence of ALG3 expression on cancer cell proliferation and chemotherapeutic responsiveness was scrutinized in vitro. Animal models were constructed to evaluate the alteration of tumor sensitivity to anti-PD-1 therapy with decreased ALG3 expression. And flow cytometry and IHC were used to investigate the tumor immune microenvironment. Association of PD-L1 Glycosylation and ALG3 expression were also investigated by western blot.
ALG3 expression was elevated in TNBC and was strikingly linked to unfavorable clinical features such as lymphatic node metastasis, high NPI, advanced stage and age, etc. Furthermore, high ALG3 expression was associated with shorter OS in TNBC patients. Mechanistically, ALG3 expression was negatively correlated with the infiltration of CD8 T cells, CD4 T cells, and NK cells. ALG3-KO cells had increased sensitivity to chemotherapeutic agents. In animal models, the volume of ALG3-KO tumors was lower than the control group with immunotherapy. ALG3-KO tumors showed an increased proportion of CD8 T cells, while a decreased proportion of regulatory T cells and M2-type macrophages. The expression level of PD-L1 protein was not affected by ALG3 level, but the glycosylation level was significantly decreased in tumor. Similarly, the glycosylation level of PD-L1 is reduced in ALG3-KO cell in vitro. Additionally, ALG3 knockout lead to reduced tolerance of tumor cells to IFN-γ, thereby enhancing the efficacy of immunotherapy.
ALG3 is a potential biomarker for poor prognosis of TNBC and may reduce the efficacy of immunotherapy by modulating the tumor microenvironment and glycosylation of PD-L1.
本研究旨在评估 α-1,3-甘露糖基转移酶（ALG3）在三阴性乳腺癌（TNBC）中的预后意义，并探讨其对抗 PD-1 治疗疗效的影响及其潜在机制。
使用 UACLAN 和其他数据库进行生物信息学分析，检测癌症患者中 ALG3 的表达。利用 bc-GenExMiner 数据库检测 ALG3 基因与乳腺癌临床病理特征的相关性。利用 Kaplan-Meier Plotter 数据库进一步建立 ALG3 表达与生存的相关性。免疫组化（IHC）分析湖北肿瘤医院乳腺癌患者队列中 ALG3 的表达，以证实 ALG3 在 TNBC 中的预后价值。还分析了 ALG3 对浸润免疫细胞水平的影响。并利用 cBioPortal 中的突变模块可视化 BRCA 中的 ALG3 突变。利用 CRISPR/Cas9 技术构建 ALG3 低表达 TNBC 细胞系。体外研究 ALG3 表达对癌细胞增殖和化疗反应性的影响。构建动物模型，评估降低 ALG3 表达对肿瘤对抗 PD-1 治疗敏感性的改变。并利用流式细胞术和 IHC 检测肿瘤免疫微环境。利用 Western blot 研究 PD-L1 糖基化与 ALG3 表达的相关性。
ALG3 在 TNBC 中表达上调，与不良临床特征如淋巴结转移、高 NPI、晚期和年龄等显著相关。此外，高 ALG3 表达与 TNBC 患者的总生存期缩短相关。机制上，ALG3 表达与 CD8 T 细胞、CD4 T 细胞和 NK 细胞的浸润呈负相关。ALG3-KO 细胞对化疗药物的敏感性增加。在动物模型中，与对照组相比，ALG3-KO 肿瘤的体积较低，免疫治疗后。ALG3-KO 肿瘤中 CD8 T 细胞的比例增加，而调节性 T 细胞和 M2 型巨噬细胞的比例降低。PD-L1 蛋白的表达水平不受 ALG3 水平的影响，但肿瘤中的糖基化水平显著降低。同样，体外 ALG3-KO 细胞中 PD-L1 的糖基化水平降低。此外，ALG3 敲除导致肿瘤细胞对 IFN-γ的耐受性降低，从而增强了免疫治疗的疗效。
ALG3 是 TNBC 预后不良的潜在生物标志物，通过调节肿瘤微环境和 PD-L1 的糖基化可能降低免疫治疗的疗效。
