Tumor immune microenvironment permissive to metastatic progression of ING4-deficient breast cancer

Abstract

Deficiencies in the ING4 tumor suppressor are associated with advanced stage tumors and poor patient survival in cancer. ING4 was shown to inhibit NF-{kappa}B in several cancers. As NF-{kappa}B is a key mediator of immune response, the ING4/NF-{kappa}B axis is likely to manifest in tumor-immune modulation but has not been investigated. To characterize the tumor immune microenvironment associated with ING4-deficient tumors, three approaches were employed in this study: First, tissue microarrays composed of 246 primary breast tumors including 97 ING4-deficient tumors were evaluated for the presence of selective immune markers, CD68, CD4, CD8, and PD-1, using immunohistochemical staining. Second, an immune-competent mouse model of ING4-deficient breast cancer was devised utilizing CRISPR-mediated deletion of Ing4 in a Tp53 deletion-derived mammary tumor cell line; mammary tumors were evaluated for immune markers using flow cytometry. Lastly, the METABRIC gene expression dataset was evaluated for patient survival related to the immune markers associated with Ing4-deleted tumors. The results showed that CD68, CD4, CD8, or PD-1, was not significantly associated with ING4-deficient breast tumors, indicating no enrichment of macrophages, T cells, or exhausted T cell types. In mice, Ing4-deleted mammary tumors had a growth rate comparable to Ing4-intact tumors but showed increased tumor penetrance and metastasis. Immune marker analyses of Ing4-deleted tumors revealed a significant increase in tumor-associated macrophages (Gr-1loCD11b+F4/80+) and a decrease in granzyme B-positive (GzmB+) CD4+ T cells, indicating a suppressive and/or less tumoricidal immune microenvironment. The METABRIC data analyses showed that low expression of GZMB was significantly associated with poor patient survival, as was ING4-low expression, in the basal subtype of breast cancer. Patients with GZMB-low/ING4-low tumors had the worst survival outcomes (HR=2.80, 95% CI 1.36-5.75, p=0.0004), supportive of the idea that the GZMB-low immune environment contributes to ING4-deficient tumor progression. Collectively, the study results demonstrate that ING4-deficient tumors harbor a microenvironment that contributes to immune evasion and metastasis.