Stromal p53 Regulates Breast Cancer Development, the Immune Landscape, and Survival in an Oncogene-Specific Manner

Abstract

Coevolution of tumor cells and adjacent stromal elements is a key feature during tumor progression; however, the precise regulatory mechanisms during this process remain unknown. Here, we show stromal p53 loss enhances oncogenic KrasG12D, but not ErbB2, driven tumorigenesis in murine mammary epithelia. Stroma-specific p53 deletion increases both epithelial and fibroblast proliferation in mammary glands bearing the KrasG12D oncogene in epithelia, while concurrently increasing DNA damage and/or DNA replication stress and decreasing apoptosis in the tumor cells proper. Normal epithelia was not affected by stromal p53 deletion. Tumors with p53-null stroma had a significant decrease in total, cytotoxic, and regulatory T cells; however, there was a significant increase in myeloid-derived suppressor cells, total macrophages, and M2-polarized tumor-associated macrophages, with no impact on angiogenesis or connective tissue deposition. Stroma-specific p53 deletion reprogrammed gene expression in both fibroblasts and adjacent epithelium, with p53 targets and chemokine receptors/chemokine signaling pathways in fibroblasts and DNA replication, DNA damage repair, and apoptosis in epithelia being the most significantly impacted biological processes. A gene cluster in p53-deficient mouse fibroblasts was negatively associated with patient survival when compared with two independent datasets. In summary, stroma-specific p53 loss promotes mammary tumorigenesis in an oncogene-specific manner, influences the tumor immune landscape, and ultimately impacts patient survival.

Implications: Expression of the p53 tumor suppressor in breast cancer tumor stroma regulates tumorigenesis in an oncogene-specific manner, influences the tumor immune landscape, and ultimately impacts patient survival.

Figure 1.. Stroma-specific deletion of p53 cooperates robustly with the Kras^G12D, but not ErbB2, epithelial oncogene to accelerate tumorigenesis in mammary transplants.

A) Mammary glands collected 26 weeks after syngeneic transplantation (top panels) and matched H&E staining (bottom panels) from FspCre;p53^fl/fl, MMTV-ErbB2;p53^fl/fl, MMTV-ErbB2;FspCre;p53^fl/fl, MMTV-rtTa;Tet-o-Kras^G12D;p53^fl/fl, and MMTV-rtTa;Tet-o-Kras^G12D;FspCre;p53^fl/fl mice. Bars represent 100 μM; and B) Histological classification of the most severe lesion per mouse observed in mammary gland transplants in each genetic group. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

Figure 2.. Stroma-specific p53 deletion cooperates only with the Kras^G12D epithelial oncogene to accelerate mammary tumorigenesis in a non-transplant model.

This figure involves a more direct analysis using a non-transplant model where mammary glands were evaluated in situ in MMTV-ErbB2;p53^fl/fl, MMTV-ErbB2;FspCre;p53^fl/fl, MMTV-rtTa;Tet-o-Kras^G12D;p53^fl/fl, and MMTV-rtTa;Tet-o-Kras^G12D;FspCre;p53^fl/fl mice. Tumor-free Kaplan-Meier plot and histological classification of the most severe mammary gland lesion per mouse from the two genetic groups in A) MMTV-ErbB2;p53^fl/fl and MMTV-ErbB2;FspCre;p53^fl/fl; and B) MMTV-rtTA;Tet-o-Kras^G12D;p53^fl/fl and MMTV-rtTA;Tet-o-Kras^G12D;FspCre;p53^fl/fl mice. In B), most tumor-bearing mice died or met early removal criteria within a week of first palpation of a tumor. Representative tumor histology from both genetic groups for C) MMTV-ErbB2 (100X); and D) MMTV-rtTa;Tet-o-Kras^G12D (200X). Bars represent 100 μM. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

Figure 3.. Ablation of stromal p53 increases tumor cell proliferation, DNA damage and/or DNA replication stress, and survival.

Immunofluorescent co-staining for A) Ki67, K8, and DAPI and quantification in hyperplastic, MIN, and carcinoma lesions from MMTV-rtTA;Tet-o-Kras^G12D;p53^fl/fl and MMTV-rtTA;Tet-o-Kras^G12D;FspCre;p53^fl/fl mice with quantification of the percentage of Ki67⁺ epithelial cells; B) p-H2AX and DAPI with quantification of the percentage of p-H2AX⁺ cells in mammary carcinomas; and C) Caspase3 and DAPI with quantitation of the percentage of Caspase3⁺ cells in mammary carcinomas. Bars represent 200 μM. Graphs are presented as mean ± standard deviation with n=3–12. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

Figure 4.. p53 deletion in tumor stroma alters the immune landscape.

Immunohistochemistry and immunofluorescence staining and quantification in mammary carcinomas from MMTV-rtTA;Tet-o-Kras^G12D;p53^fl/fl and MMTV-rtTA;Tet-o-Kras^G12D;FspCre;p53^fl/fl mice for A) CD3; B) GR1; C) CD3, CD8, and DAPI; and D) F4/80, CD163⁺, and DAPI. Bars represent 400 μM in A) and 200 μM in B) – D); Graphs represent the percentage of positive staining cells or the absolute number of cells per field of view and are presented as mean ± standard deviation with n=3. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

Figure 5.. A stromal fibroblast p53 deletion gene signature distinguished normal from neoplastic stroma and correlates with survival.

A) Heatmap identifies Fibroblast Gene Cluster 1 (116 differentially expressed genes identified only in MMTV-rtTA;Tet-o-Kras^G12D;FspCre;p53^fl/fl fibroblasts) and Fibroblast Gene Cluster 2 (236 differentially expressed genes in p53-deficient fibroblasts with and without epithelial Kras^G12D; B) Of the 236 genes in Fibroblast Gene Cluster 2, 129 genes (Supplemental Table 2) matched the stromal heatmap and separated normal from tumor stroma in BrCa patients; C) GSEA analysis revealed that targets of p53 and chemokine receptors that bind chemokines were significantly affected biological processes in fibroblasts following p53 deletion; D) KM plot with Fibroblast Gene Cluster 2 against McGill’s survival data and METABRIC’s survival data considering all BrCa subtypes combined.

Figure 6.. Stromal deletion of p53 reprograms gene expression in adjacent Kras^G12D-expressing epithelium, but did not correlate with survival when compared to clinical isolated stromal samples.

A) p53 loss in fibroblasts led to a noticeable reprogramming of gene expression in epithelial cells, independent of epithelial Kras^G12D oncogene expression. Gene clusters were observed in epithelia associated with p53 loss in fibroblasts either 1) in the presence of epithelial Kras^G12D (Epithelial Gene Cluster 1) and 2) both in the presence or absence of epithelial Kras^G12D (Epithelial Gene Cluster 2); B) GSEA of differentially expressed genes in Kras^G12D-expressing epithelia revealed genes involved in DNA replication, DNA damage repair, and apoptosis were significantly affected biological processes in adjacent epithelium following p53 deletion in fibroblasts; and C) Epithelial Gene Clusters 1 and 2 could not distinguish human tumor stroma from normal tissue and did not correlate with survival in the McGill cohort considering all BrCa subtypes combined.

Figure 7.. Epithelial gene clusters correlate with METABRIC survival data.

Epithelial Gene Clusters A) 1 and B) 2 correlate with survival in the METABRIC cohort considering all BrCa subtypes combined.