Abstract 2883: Do Wnt3A-mediated canonical Wnt/Beta-catenin signals regulate stem cell phenotypes in brain metastasis-specific TNBC

Abstract

Abstract Introduction: Triple negative (TN) and basal-like subtypes portend one of the worst prognoses in BC and have the most challenging diagnosis among patients due to the aggressive nature of the disease. BC stem cells (BCSCs) have a distinct role in breast oncogenesis and CD44(+)/CD24(-) BCSCs play an important role in the clinical behavior of TNBC. Unique growth abilities of BCSCs including self-renewal, differentiation potential and resistance to anti-cancer agents contribute to the development and overall aggressiveness of recurrent metastatic lesions. Patients with TNBC as compared to non-TNBC have significantly higher incidences of recurrence or distant metastasis and the poorer outcome has been associated with increased frequency of BCSCs phenotypes. Aim: We previously reported that Wnt pathway (WP) is associated with metastasis (Dey et al., 2013) and controls metastasis-associated (MA) tumor cell phenotypes in TNBC (De et al., 2016) and here we tested the role of WP in the regulation of BCSCs and MA tumor cell phenotypes using brain-metastasis specific TNBC model (MDA-MB231BR). Methods: MDA-MB231BR cells were stimulated with WP specific ligand or LWnt3A conditioned media (CM) and MA tumor cell phenotypes were tested following WP inhibition. To model distant metastasis in vitro, we have used a long-term 3D-matrigel assay in the presence of LWnt3ACM. Four BCSCs markers, CD44, CD44V6, CD24 and CD133 were selected for the flow cytometric analyses of cells following LWnt3ACM stimulation and downregulation of WP by sulindac sulfide. Results: Specific stimulation of WP by LWnt3ACM led to the clonogenic growth from a single cell. Inhibition of WP blocked micro-metastatic growth of these colonies. Although WP stimulation alone did not alter % of CD44(+)/CD24(-) population in 2D format, inhibition of WP following treatment with sulindac sulfide caused a significant decrease in the % of CD44(+)/CD24(-) population (66%) as compared to the control (79%). To further understand the mechanistic long-term role of BCSCs during the micro-metastatic 3D growth of colonies, we have developed a novel method to identify the % of BCSCs in the micro-metastatic 3D colonies. After 15 days of 3D colony growth, cells are recovered from matrigel and the live cell fraction is analyzed for CD24, CD44, CD44v6 and CD133 by flow cytometric analysis. We previously presented data demonstrating the CD44+/CD24- fraction is enriched in 3D colonies as compared to correspoding 2D cells. Here we’ve extended our analysis and examined 3D colonies following either stimulation or inhibition of WP. Conclusion: Our data demonstrates that WP mediates the BCSCs phenotype(s) of TNBC in a context-dependent manner. Studies are on-going to delineate the mechanistic role of different BCSCs markers particularily CD44 and CD44v6 in signaling the formation of WP-mediated micro-metastatic 3D colonies, the results of which will be presented at the meeting. Citation Format: Jennifer H. Carlson, Pradip De, Casey Williams, Nandini Dey, Brian Leyland-Jones. Do Wnt3A-mediated canonical Wnt/Beta-catenin signals regulate stem cell phenotypes in brain metastasis-specific TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2883. doi:10.1158/1538-7445.AM2017-2883