Exploring GZMK as a Prognostic Marker and Predictor of Immunotherapy Response in Breast Cancer: Unveiling Novel Insights into Treatment Outcomes
Authors: Li Zitao , Xie Qiqi , Zhao Fuxing , Huo Xinfa , Ren Dengfeng , Liu Zhilin , Zhou Xiaofeng , Shen Guoshuang , Zhao Jiuda 
Published: March 2024

Abstract

Background
Granzyme K (GZMK) is an important mediator released by immune cells to eliminate tumor cells and plays crucial roles in inflammation and tumorigenesis. However, the precise role of GZMK in breast cancer and its underlying mechanisms remain poorly understood.
Methods
Utilizing data from TCGA and GEO databases, along with various analytical approaches including GO, KEGG, GSEA, ssGSEA, and PPI, we investigated the impact of GZMK on breast cancer. RT-qPCR, CCK-8 assay, cell cycle experiments, apoptosis assays, Celigo scratch assays, Transwell assays, and immunohistochemical methods were employed to validate the in vitro effects of GZMK on breast cancer. Furthermore, Cox regression analysis based on TCGA data and our clinical data was conducted to establish an overall survival (OS) prediction model for breast cancer.
Results
We observed elevated expression of GZMK in breast cancer tissues, particularly in subanalyses showing higher expression in hormone receptor-negative and HER2-positive patients. Clinical pathological feature analysis revealed significant correlations between GZMK expression and lymph node staging, differentiation grade, and molecular subtypes of breast cancer. High expression of GZMK was associated with improved OS, progression-free survival (PFS), and recurrence free survival (RFS) in patients, as confirmed by multifactor Cox regression analysis indicating that GZMK_High improves patient OS. Functional and pathway enrichment analysis of genes positively correlated with GZMK revealed involvement in lymphocyte differentiation, T cell differentiation, and T cell receptor signaling pathways. Analysis of immune cell infiltration in the breast cancer tumor microenvironment (TME) demonstrated a strong association between GZMK expression and T cells, as well as favorable correlations with ESTIMATEScore_estimate (Cor = 0.743, P < 0.001), ImmuneScore_estimate (Cor = 0.802, P < 0.001), and StromalScore_estimate (Cor = 0.516, P < 0.001). Moreover, GZMK exhibited significant correlations with immune checkpoint molecules including CTLA4 (Cor = 0.856, P < 0.001), PD-1 (Cor = 0.82, P < 0.001), PD-L1 (Cor = 0.56, P < 0.001), CD48 (Cor = 0.75, P < 0.001), and CCR7 (Cor = 0.856, P < 0.001). Three immune-related studies suggested that high expression of GZMK enhances patients' responsiveness to immunotherapy, with higher levels observed in immunotherapy-responsive patients compared to non-responsive ones. In vitro experiments validated that GZMK enhances cell proliferation, promotes cell division, facilitates apoptosis, increases cell migration capacity, and enhances cell invasiveness.
Conclusion
Our study provides insights into the differential expression of GZMK in breast cancer and offers preliminary insights into its potential mechanisms in breast cancer pathogenesis. Elevated GZMK expression improves OS and RFS in breast cancer patients, suggesting its potential as a prognostic marker for breast cancer survival and as a predictor of immunotherapy efficacy in tumor patients.