Ror2 Signaling and Its Relevance in Breast Cancer Progression

Abstract

Breast cancer is a heterogeneous disease and has been classified into five molecular subtypes based on gene expression profiles. Signaling processes linked to different breast cancer molecular subtypes and different clinical outcomes are still poorly understood. Aberrant regulation of Wnt signaling has been implicated in breast cancer progression. In particular Ror1/2 receptors and several other members of the non-canonical Wnt signaling pathway were associated with aggressive breast cancer behavior. However, Wnt signals are mediated via multiple complex pathways, and it is clinically important to determine which particular Wnt cascades, including their domains and targets, are deregulated in poor prognosis breast cancer. To investigate activation and outcome of the Ror2-dependent non-canonical Wnt signaling pathway, we overexpressed the Ror2 receptor in MCF-7 and MDA-MB231 breast cancer cells, stimulated the cells with its ligand Wnt5a, and we knocked-down Ror1 in MDA-MB231 cells. We measured the invasive capacity of perturbed cells to assess phenotypic changes, and mRNA was profiled to quantify gene expression changes. Differentially expressed genes were integrated into a literature-based non-canonical Wnt signaling network. The results were further used in the analysis of an independent dataset of breast cancer patients with metastasis-free survival annotation. Overexpression of the Ror2 receptor, stimulation with Wnt5a, as well as the combination of both perturbations enhanced invasiveness of MCF-7 cells. The expression-responsive targets of Ror2 overexpression in MCF-7 induced a Ror2/Wnt module of the non-canonical Wnt signaling pathway. These targets alter regulation of other pathways involved in cell remodeling processing and cell metabolism. Furthermore, the genes of the Ror2/Wnt module were assessed as a gene signature in patient gene expression data and showed an association with clinical outcome. In summary, results of this study indicate a role of a newly defined Ror2/Wnt module in breast cancer progression and present a link between Ror2 expression and increased cell invasiveness.

Keywords: Ror2; Wnt signaling; breast cancer; metastasis; module; network integration.

Figure 1

Conceptual workflow of the study. (1.) We explored activation and outcome of the non-canonical Wnt signaling by overexpression of the Ror2 receptor in the ER+ breast cancer cell line MCF-7. (2.) To explore the effect of perturbation on the phenotypic level, the invasive capacity of the cells was measured and (3.) on gene expression level the mRNA of the cell lines was profiled. (4.) The results of differential analysis were integrated with the non-canonical Wnt signaling network to identify an induced subnetwork—the Ror2/Wnt module. (5.) The Ror2/Wnt module genes were further translated into independent gene expression data of breast cancer patients in order to assess the association of non-canonical Ror2/Wnt signaling with metastasis-free survival in breast cancer.

Figure 2

Overexpression of Ror2 in MCF-7 cells activates non-canonical Wnt signaling and enhances tumor invasion. (A) Flow cytometry: Ror2 expression was analyzed in MCF-7 cells transfected with an empty vector (pcDNA, black) or a Ror2 overexpression construct (pRor2, green). The isotype control (ctl) is shown in gray. (B) Quantification of ROR2 gene expression by quantitative real-time PCR (n = 3, mean ± SD, *p < 0.0001). (C) Expression of non-canonical Wnt target proteins in pcDNA and pRor2 cells ± Wnt5a (100 ng/ml, 24 h) was assessed by western blotting. (D) Invasiveness of MCF-7 pcDNA and pRor2 cells with or without Wnt5a stimulation (100 ng/ml) was measured in a modified Boyden chamber (mean ± SD, n = 3, *p < 0.001). (E) Receptor status of MCF-7 pcDNA and pRor2 cells was analyzed by immunohistochemistry. One representative image is shown for each staining and condition.

Figure 3

Venn diagrams of perturbation targets. (A) Wnt5a targets: overlap of significantly differentially expressed targets of Wnt5a stimulation in the MCF-7 cells with and without Ror2 receptor overexpression. (B) Ror2 targets: overlap of three lists of significant differentially expressed genes responsive to the Ror2 overexpression in MCF-7. (C) Gene expression changes of the top five genes regulated in Ror2-overexpressing vs. empty vector MCF-7 cells were validated by quantitative real-time PCR (n = 3, mean ± SD, *p < 0.05, **p < 0.01).

Figure 4

Enrichment analysis of KEGG pathways. Adjusted p-values (q-values) depicting significant enrichment (q-value < 0.05, green) of the gene sets in the three target lists: all targets (All), upregulated targets (Up), and downregulated targets (Down). Non-significant values are displayed in red.

Figure 5

Ror2-expression-responsive module. Non-canonical Wnt subnetwork representing a differentially regulated module of the Wnt pathway. The color coding corresponds to the fold-changes of the targets: blue represents downregulation and red upregulation of the genes in Ror2-overexpressing cells compared to the control MCF-7 cells. The smaller black nodes were introduced by Steiner tree analysis. The directed edges represent controlling interactions.

Figure 6

The Ror2/Wnt module gene signature in the whole patient cohort. (A) Clustering analysis revealed heatmap of expression levels (depicted as row z-scores) and the subsequent dendrogram shape-based analysis yielded four patient clusters. (B) Kaplan–Meier curves showing metastasis-free survival according to the four patient clusters. (C) The pie charts display distribution of the subtypes within each cluster.

Figure 7

The Ror2/Wnt module gene signature applied in (A) luminal A, (B) luminal B, and (C) basal-like subtypes. Subtype-specific heatmaps are displayed at the top, whereas associated Kaplan–Meier curves depicting metastasis-free survival are at the bottom.

Figure 8

Performance of random signatures compared to Ror2/Wnt module gene signature. The boxplots visualize log-transformed p-values from metastasis-free survival analysis of 1,000 random signatures in the whole cohort and lumA and Basal subtypes. Blue bars display p-values of the original Ror2/Wnt module-based gene signature. Red line depicts significance level p = 0.05.

Figure 9

Knockdown of Ror1 in MDA-MB231 cells decreases non-canonical Wnt signaling. (A) Ror2 expression was measured in MDA-MB231 cells expressing an empty vector (pcDNA) or hRor2 overexpression construct (pRor2) by quantitative real-time PCR (n = 3, mean ± SD). (B) Cell invasion assay of MDA-MB231 pcDNA and pRor2 cells ± Wnt5a (100 ng/ml). Invasion rates are shown in relation to invasion of MCF-7 pcDNA cells (mean ± SD, n = 4, *p < 0.0001). (C) Western blot of endogenous Ror2 expression in MDA-MB231 cells. MCF-7 pRor2 cells are shown as positive control. (D) Western blot of Ror1 expression in MDA-MB231 non-sense control (shCTL) and shRor1 cells. Ponceau staining and Tubulin are depicted as loading controls. (E) MDA-MB231 shCTL and shRor1 cells were fractionated and expression of active β-catenin (=ABC) as well as total β-catenin analyzed in cytoplasm (=C) and nucleus (=N) by Western Blotting. HDAC1 expression was analyzed to confirm successful fractionation. (F) Western Blot: MDA-MB231 shCTL and shRor1 cells were characterized for non-canonical Wnt target proteins.