p53R172H and p53R245W Hotspot Mutations Drive Distinct Transcriptomes in Mouse Mammary Tumors Through a Convergent Transcriptional Mediator

Abstract

Aggressive breast cancers harbor TP53 missense mutations. Tumor cells with TP53 missense mutations exhibit enhanced growth and survival through transcriptional rewiring. To delineate how TP53 mutations in breast cancer contribute to tumorigenesis and progression in vivo, we created a somatic mouse model driven by mammary epithelial cell-specific expression of Trp53 mutations. Mice developed primary mammary tumors reflecting the human molecular subtypes of luminal A, luminal B, HER2-enriched, and triple-negative breast cancer with metastases. Transcriptomic analyses comparing MaPR172H/- or MaPR245W/- mammary tumors to MaP-/- tumors revealed (1) differences in cancer-associated pathways activated in both p53 mutants and (2) Nr5a2 as a novel transcriptional mediator of distinct pathways in p53 mutants. Meta-analyses of human breast tumors corroborated these results. In vitro assays demonstrate mutant p53 upregulates specific target genes that are enriched for Nr5a2 response elements in their promoters. Co-immunoprecipitation studies revealed p53R172H and p53R245W interact with Nr5a2. These findings implicate NR5A2 as a novel mediator of mutant p53 transcriptional activity in breast cancer.

Significance: Our findings implicate NR5A2 as a novel mediator of mutant p53 transcriptional activity in breast cancer. NR5A2 may be an important therapeutic target in hard-to-treat breast cancers such as endocrine-resistant tumors and metastatic triple-negative breast cancers harboring TP53 missense mutations.

Figure 1

Molecular characterization of mammary tumors from MaP mice. A, Incidence of primary mammary tumor formation across MaP^R172H/− (n = 7), MaP^R245W/− (n = 5), and MaP^−/− (n = 6) mice. B, Tumor-free survival of mammary tumor-bearing mice. C, Histological subtypes across MaP mice (*Indicates four mice had two primary tumors). D, Histology of mammary tumors in MaP mice. Yellow scale bar, 100 µm. E, Breast cancer molecular subtypes defined by qPCR for Esr1 (Estrogen Receptor), Pgr (progesterone receptor), and Erbb2 (HER2).

Figure 2

Metastasis in MaP mice. A, Metastasis incidence in MaP mice. B, Histology and immunofluorescence staining of TdTomato in a primary breast tumor and matched lung metastasis from a MaP^R172H/− mouse, YZ2. C, Histology and immunofluorescence staining of TdTomato in a primary breast tumor and matched lung metastasis from a MaP^R245W/− mouse, JM1233. H&E, hematoxylin and eosin; IF, immunofluorescence.

Figure 3

Transcriptomes of MaP^R172H/− and MaP^R245W/− mice disrupt nonoverlapping cancer pathways. A, Heatmap showing hierarchical clustering of MaP^R172H/− and MaP^−/− mammary tumors using the Pearson distance and Ward linkage. Order of samples for MaP^R172H/−: YZ17, YZ11, YZ4, YZ6, YZ10, YZ2, and YZ3. Order of samples for MaP^−/−: JM663-T2, JM779-T2, JM836-T2, JM663-T1, JM833-T1, and JM833-T2. B, Pie chart representing significantly DEGs for MaP^R172H/− mammary tumors compared with MaP^−/− mammary tumors (DESeq2, significance criteria were FDR <5% and log₂ fold change > 2). C, Enrichment plot for Wnt Beta Catenin signaling. D, Heatmap showing hierarchical clustering of MaP^R245W/− and MaP^−/− mammary tumors. Order of samples for MaP^R245W/−: JM1233-T1, JM1126, JM1133, JM1564, and RM0014. Order of samples for MaP^−/−: JM836-T2, JM663-T1, JM663-T2, JM779-T2, JM833-T1, and JM833-T2. E, Pie chart representing significantly DEGs for MaP^R245W/− mammary tumors compared with MaP^−/− mammary tumors. F, Representation of significantly enriched Hallmarks pathways as defined by gene set enrichment analysis. G, Enrichment plots for pathways involved in oxidative phosphorylation signaling, mTORC1 signaling (H), myogenesis signaling (I), Myc targets V1 (J), cholesterol homeostasis (K), peroxisome (L), epithelial–mesenchymal transition (M), Myc targets V2 (N), DNA repair (O), unfolded protein response (P), and glycolysis (Q) in MaP^R245W/− compared with MaP^−l− mammary tumors.

Figure 4

p53R172H and p53R245W show distinct transcriptomes via Nr5a2. A, Supervised clustering of MaP^−/−, MaP^R172H/− and MaP^R245W/− tumors based on upregulated and downregulated genes. Green box, comparison of MaP^R172H/− and MaP^−/− downregulated genes; Pink box, comparison of MaP^R172H/− and MaP^−/− upregulated genes; Black boxes, comparison of MaP^R245W/− and MaP^−/− downregulated genes; Yellow boxes, comparison of MaP^R245W/− and MaP^−/− upregulated genes. B, Venn diagrams depicting genes significantly upregulated and (C) significantly downregulated in MaP^R172H/− and MaP^R245W/− mammary tumors. D, Pie charts representing the number of significantly upregulated genes in MaP^R172H/− tumors and (E) MaP^R245W/− tumors that have an Nr5a2 motif.

Figure 5

Transcriptomic meta-analysis of human breast tumors with TP53^R175H and TP53^R248W/Q mutations recapitulates NR5A2 as a co-regulator. A, Supervised clustering of human breast tumors from METABRIC dataset harboring a TP53^R175H mutation compared with human breast tumors with a homozygous deletion of TP53. B, Pie chart representing significantly DEGs for TP53^R175H breast tumors compared with TP53-null breast tumors (limma), significance criteria were FDR <5% and fold change >1.5. C, Supervised clustering of human breast tumors from METABRIC dataset harboring a TP53^R248Q or TP53^R248W mutation compared with human breast tumors with a homozygous deletion of TP53. D, Pie chart representing DEGs for TP53^R248Q/W breast tumors compared with TP53-null breast tumors (limma), significance criteria were FDR <5% and fold change >1.5. E, NR5A2 binding motif sequences identified by MEME from JASPAR database. F, Oncoprint analysis in cBioportal to assess co-occurrence of TP53 mutations with NR5A2 amplifications. G, Prevalence of co-occurrence of TP53 missense mutations with NR5A2 amplification across human molecular breast cancer subtypes: luminal A (38%), luminal B (26%), HER2-enriched (15%), and TNBC (21%).

Figure 6

p53R172H and p53R245W interact with Nr5a2 to alter transcription. A, Western blot analyses of p53R172H and p53R245W protein levels in Trp53-null breast tumor cells infected with lentiviral vectors expressing p53R172H or p53R245W, compared with empty vector control. B, qRT-PCR analysis of p53R172H downstream target genes Argef18, Fbxo2, Lime1, Msi1, Nrtn with Nr5a2 motifs after lentivirus infection of p53R172H (n = 6, P value <0.002, 0.01, 0.002, 0.002, 0.03, respectively, Mann–Whitney U test). C, qRT-PCR analysis of p53R245W downstream target genes Add2, Ankrd2, Myh4, and Prss35 with Nr5a2 motifs after lentivirus infection of p53R245W (n = 6, P value < 0.002, 0.4, 0.04, 0.6, 0.2, respectively, Mann–Whitney U test). C, control. D, qRT-PCR analysis of p53R245W downstream target genes Add2, Ankrd2, and Myh4 after siRNA-mediated knockdown of Nr5a2 in MaP^R245W/− cell line, RM0014. C, control. E, Co-immunoprecipitation experiments of MaP^R172H/− cell line YZ10 and MaP^R245W/− cell line JM1564 using Nr5a2 pull-down and probing for mutant p53.