Abstract PO1-24-12: Investigating the immunological function of alpha-2-glycoprotein 1, zinc-binding in regulating tumor response in the breast cancer microenvironment
Authors: Hanamura Toru , Yokoyama Kozue , Kitano Shigehisa , Kagamu Hiroshi , Yamashita Makiko , Terao Mayako , Okamura Takuho , Kumaki Nobue , Hozumi Katsuto , Iwamoto Takayuki , Honda Chikako , Kurozumi Sasagu , Richer Jennifer , Niikura Naoki 
Published: May 2024

Abstract
Purpose: Tumor immunology has attracted considerable attention as an innovative therapeutic strategy for various types of cancer. Elucidating the unique immunoregulatory mechanisms in the breast cancer microenvironment will assist in the development of novel treatment strategies. Recently, we and other researchers have found that androgen receptor (AR) expression is associated with the immunosuppressive phenotype in the breast cancer microenvironment, suggesting some immunoregulatory function in breast cancer; however, the mechanism remains unclear. Here, we focused on AR-dependent secreted protein, alpha-2-glycoprotein 1, zinc-binding (ZAG) encoded by the AZGP1 gene in breast cancer, which is structurally similar to HLA class I and is implicated in immune regulation. In this study, we investigated the immunological function of AZGP1/ZAG in the breast cancer microenvironment. Methods: We performed a gene set enrichment analysis (GSEA) to screen the biological processes associated with AZGP1 expression using a gene expression profile dataset of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). Subsequently, we analyzed the correlation between AZGP1 expression and the immune cell composition in breast cancer tissues estimated with the CIBERSORTx using METABRIC and another dataset of The Sweden Cancerome Analysis Network-Breast. In our previous study of 45 breast cancer tissue samples, we evaluated the infiltration of 11 types of immune cells using flow cytometry (FCM). ZAG expression was further evaluated by immunohistochemistry, and the relationship between ZAG expression and the immune cell composition in breast cancer tissue was analyzed. Based on the results of this analysis (shown in the results section) we hypothesized that ZAG has some effect on the macrophage (Mφ). The action of ZAG in M1/M2 polarization models constructed using primary culture of human peripheral blood mononuclear cells (PBMC)-derived Mφ was assessed based on the expression of M1/M2 differentiation markers (CD86, CD80/CD163, MRC1) and HLA class I/II expression evaluated by FCM (n=15 each). Results: GSEA demonstrated that AZGP1 expression was negatively correlated with multiple gene sets representing immunological processes, including inflammatory response, allograft rejection, interferon gamma response, IL6/JAK/STAT3 signaling, complement, and IL2/STAT5 signaling. Analysis by CIBERSORTx showed that AZGP1 expression was negatively correlated with the absolute score (the absolute abundance of total immune cell infiltration), Mφ M1, NK cells activated, CD4+ T memory activated, and CD8+ T (r&lt;-3 and p&lt; 0.05). Analyses of our in-house dataset using breast cancer tissue showed that ZAG expression was associated with decreased infiltration of monocytes/macrophages, non-classical monocytes, and myeloid-derived suppressor cells in breast cancer tissues assessed by FCM. In the in vitro analyses, ZAG decreased the expression of CD80, CD163, MRC1, and HLA classes I and II in the M1 polarization model and the expression of CD163 and MRC1 in the M2 polarization model. Conclusion: AZGP1/ZAG was associated with an immunosuppressive phenotype and reduced infiltration of specific immune cell subsets, particularly Mφ, into breast cancer tissues. In the in vitro analysis, ZAG demonstrated some regulatory effects on the phenotypic change of Mφ. Our findings strongly suggest ZAG is a novel mediator of AR-dependent immunomodulation in the breast cancer microenvironment, laying the foundation for future studies to elucidate the immunological role of ZAG in breast cancer.
Citation Format: Toru Hanamura, Kozue Yokoyama, Shigehisa Kitano, Hiroshi Kagamu, Makiko Yamashita, Mayako Terao, Takuho Okamura, Nobue Kumaki, Katsuto Hozumi, Takayuki Iwamoto, Chikako Honda, Sasagu Kurozumi, Jennifer Richer, Naoki Niikura. Investigating the immunological function of alpha-2-glycoprotein 1, zinc-binding in regulating tumor response in the breast cancer microenvironment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-12.