BRCA Status Dictates Wnt Responsiveness in Epithelial Ovarian Cancer

Abstract

The association of BRCA1 and BRCA2 mutations with increased risk for developing epithelial ovarian cancer is well established. However, the observed clinical differences, particularly the improved therapy response and patient survival in BRCA2-mutant patients, are unexplained. Our objective is to identify molecular pathways that are differentially regulated upon the loss of BRCA1 and BRCA2 functions in ovarian cancer. Transcriptomic and pathway analyses comparing BRCA1-mutant, BRCA2-mutant, and homologous recombination wild-type ovarian tumors showed differential regulation of the Wnt/β-catenin pathway. Using Wnt3A-treated BRCA1/2 wild-type, BRCA1-null, and BRCA2-null mouse ovarian cancer cells, we observed preferential activation of canonical Wnt/β-catenin signaling in BRCA1/2 wild-type ovarian cancer cells, whereas noncanonical Wnt/β-catenin signaling was preferentially activated in the BRCA1-null ovarian cancer cells. Interestingly, BRCA2-null mouse ovarian cancer cells demonstrated a unique response to Wnt3A with the preferential upregulation of the Wnt signaling inhibitor Axin2. In addition, decreased phosphorylation and enhanced stability of β-catenin were observed in BRCA2-null mouse ovarian cancer cells, which correlated with increased inhibitory phosphorylation on GSK3β. These findings open venues for the translation of these molecular observations into modalities that can impact patient survival.

Significance: We show that BRCA1 and BRCA2 mutation statuses differentially impact the regulation of the Wnt/β-catenin signaling pathway, a major effector of cancer initiation and progression. Our findings provide a better understanding of molecular mechanisms that promote the known differential clinical profile in these patient populations.

Figure 1

Transcriptomic differences between patients with BRCA1mt, BRCA2mt, and HRwt ovarian cancer. RNA-seq was performed on patients with HRwt (n = 375), BRCA1mt (n = 16), and BRCA2mt (n = 15) ovarian cancer. A, Volcano plots showing the distribution of DEGs (P = 0.05; FC > 1.5) in the compared groups as indicated. B, Venn diagram of DEGs in each of the groups compared showing 163 genes unique to BRCA2mt tumors. C, Venn diagram of differentially regulated pathways in each of the groups compared showing six pathways unique to BRCA2mt tumors compared with BRCA1mt and HRwt; these six pathways are shown in D.

Figure 2

Wnt signaling is predicted to be inhibited in BRCA2mt ovarian tumors. A, DEGs comparing BRCA2mt vs. BRCA1mt and (B) BRCA2mt vs. HRwt reported as logFC; red bars represent upregulated, and blue bars represent downregulated; note that several Wnt inhibitors are upregulated in BRCA2mt tumors (red asterisk). C, Perturbation analysis of the Wnt signaling pathway comparing BRCA2mt and BRCA1mt tumors; red genes are predicted to be upregulated, and blue genes are predicted to be downregulated; the dashed circle shows β-catenin is predicted to be downregulated. Red arrows indicate upregulation of several Wnt signaling inhibitors at the level of the receptor.

Figure 3

Characterization of the Wnt signaling pathway in isogenic mouse ovarian cancer cells upon loss of Brca1 or Brca2. Cellular fractionation was performed to separate cytoplasmic (A) and nuclear (B) fractions in control no-treatment cells or cells treated with 100 ng/mL Wnt3A for 8 hours; ILF3 and Cox-IV were used as integrity controls for nuclear and cytoplasmic fractions, respectively. C, Levels and cellular location of β-catenin were determined by immunofluorescence for β-catenin (green). DAPI nuclear staining is shown in blue. Images show both green and DAPI channels; white arrows show increased green staining in the nucleus after Wnt3A treatment in ID8^Trp53−/−; the red arrow shows increased green staining in the cytoplasm after Wnt3A treatment in ID8^{Trp53−/−;Brca1−/−}; yellow arrows show membranal staining in ID8^{Trp53−/−;Brca2−/−}; images shown are specific foci in the cultures. D, Levels of canonical Wnt/β-catenin gene targets were determined by Western blot analysis using whole-cell lysates. The red box shows truncated forms of Tcf1/7. DAPI, 4′,6′-diamidino-2-phenylindole.

Figure 4

β-Catenin is stabilized upon loss of BRCA2. A, ID8 mouse ovarian cancer cells were treated with 10 µg/mL cycloheximide at designated time points, and levels of β-catenin were determined by Western blot analysis. B, Quantification of A. C, ID8 mouse ovarian cancer cells were treated with 50 µmol/L MG132, and levels of phosphorylated and total β-catenin were determined by Western blot analysis. D, Figures depicting components of the β-catenin destruction complex. E, Basal levels of phosphorylated and total GSK3β were determined by Western blot analysis. NT, no treatment. (Created with Biorender.com.)

Figure 5

The noncanonical Wnt pathway is activated upon loss of BRCA1. A, Model of the noncanonical Wnt signaling pathway. B, ID8 mouse ovarian cancer cells were treated with 100 ng/mL Wnt3A followed by immunofluorescence for F-actin (green). DAPI staining is shown in blue. Red arrows point to F-actin filaments. Images show both green and DAPI channels. C, Biological processes related to filament and microtubule polymerization are differentially regulated in BRCA1mt vs. BRCA2mt and HRwt ovarian tumors. DAPI, 4′,6′-diamidino-2-phenylindole.

Figure 6

Decreased tumor growth kinetics and improved survival in mice bearing ID8^{Trp53−/−;Brca2−/−} cells. ID8 mouse ovarian cancer cells were injected intraperitoneally in C57BL/6 mice (n = 6). A, Abdominal width was used as surrogate for intraperitoneal tumor growth; note the significant decrease in tumor growth in mice bearing ID8^{Trp53−/−;Brca2−/−} cells. Data are presented as mean ± SEM. Two-way ANOVA with mixed-effect analysis was used to calculate statistical significance. B, Kaplan–Meier survival curve showing improved overall survival (defined as the day abdominal width reached 3.4 cm) in mice bearing ID8^{Trp53−/−;Brca2−/−} cells. The log-rank test was used to calculate statistical significance. C, Formalin-fixed, paraffin-embedded sections of intraperitoneal tumors were immunostained for β-catenin. D, Quantification of C as detailed in “Materials and Methods” (n = 3); ordinary one-way ANOVA with multiple comparisons was used to calculate statistical significance. n.s., not significant.

Figure 7

Proposed model showing differential response to Wnt3A in ovarian cancer cells in the context of BRCA mutation. Wnt3A activates the canonical Wnt/β-catenin pathway in ovarian cancer cells with WT BRCA1 and BRCA2. Loss of BRCA1 function leads to preferential activation of the noncanonical signaling pathway, leading to actin polymerization. Loss of BRCA2 stabilizes β-catenin and leads to preferential upregulation of negative regulators in response to Wnt3A. (Created with Biorender.com.)