Background: The canonical Wnt/β-catenin signaling pathway directs self-renewal cues for stem cells in multiple tissues. Through alternative receptors, noncanonical Wnt/β-catenin-independent pathways exist, mediating cellular processes that include planar cell polarity, convergent extension, actin/cytoskeletal rearrangements, and in some contexts, antagonism of canonical Wnt signaling. How different Wnt pathways cooperate within heterogeneous cellular landscapes in breast cancers remains an outstanding question. Within the current study, we investigated the role of the alternative Wnt receptor, Ror2, in mediating Wnt/β-catenin-independent functions during tumor progression.
Experimental design and methods: A TP53 null transplantable GEM model was used in the current study, which exhibits subtype-specific molecular signatures characteristic of human breast cancers. A lentiviral-based shRNA strategy was developed for gene silencing of noncanonical Wnt pathway components in vivo, with emphasis on the alternative Wnt receptor, Ror2. This strategy was combined with pathway-specific Wnt reporters to survey the interplay of canonical and noncanonical Wnt pathways in vivo.
Results: TP53 null mammary tumors differentially expressed the noncanonical Wnt receptor Ror2, with the highest levels of expression evident in basal-like tumors relative to luminal and claudin-low subtypes analyzed. Basal-like tumors also displayed Wnt/β-catenin active populations, which were inversely correlated with Ror2 expression. Lentiviral shRNA silencing of Ror2 within 3 independent basal-like models (T1, T2, 2225L) resulted in elevated Wnt/β-catenin activity along with various phenotypic outcomes, including squamous differentiation. Ror2, therefore, functions to restrain Wnt/β-catenin-dependent signaling in vivo. Despite the similarity in Wnt/β-catenin-dependent signaling outcome upon Ror2 depletion, gene expression profiling of Ror2-intact (shLUC) vs. Ror2-depleted (shRor2) tumors unveiled diverse gene expression changes within basal-like tumor models. Intriguingly, Ror2 silencing within 2225L basal-like tumors resulted in a change in the composition of tumor cell subpopulations. Additionally, gene expression analysis of shLUC vs. shRor2 tumors revealed that genes upregulated in shRor2 tumors were associated with claudin-low features, while genes downregulated were associated with basal-like features, reflecting changes in the cellular landscape upon Ror2 loss. Gene ontology analysis also revealed alterations in actin/cytoskeletal- and cell adhesion-associated genes upon Ror2 depletion.
Conclusion: The current study highlights the differential effects of Ror2 loss within TP53 null basal-like models. In particular, Ror2 can regulate the plasticity between basal-like and claudin-low states, impacting cytoskeletal and cell adhesion dynamics. Current efforts are focused on how these changes impact tumor progression and metastasis.
Citation Format: Kevin Roarty, Adam D. Pfefferle, Chad J. Creighton, Charles M. Perou, Jeffrey M. Rosen. Wnt pathway dynamics in breast cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3306.