Abstract 865: Inhibition of Wnt co-receptor LRP6 sensitizes basal-like breast cancer stem cells to TRA-8 anti-DR5 monoclonal antibody.
Authors: Londono-Joshi Angelina I. , Arend Rebecca C. , Aristizabal Laura , Li Yonghe , Lu Wenyan , Samant Rajeev S. , Metge Brandon J. , Forero Andres , Zhou Tong , LoBuglio Albert F. , Buchsbaum Donald J. 
Published: August 2014

Abstract
Introduction: Basal-like breast cancers (BLBC) are poorly differentiated and display aggressive clinical behavior. These tumors generally become resistant to cytotoxic agents and resistance has been attributed to the presence of tumor initiating cancer stem cells (CSC). Recent studies have shown that the Wnt co-receptor LRP6 is an indispensable element of the Wnt/β-catenin signaling pathway known to regulate CSC proliferation. Furthermore, LRP6/Wnt appears to play a crucial role in BLBC, and may represent an excellent target for this type of cancer. We have previously described that TRA-8, a monoclonal antibody specific to TRAIL death receptor 5, kills both the CSC and non-CSC population of BLBC. This study examined whether niclosamide (an FDA approved antihelminthic) which inhibits Wnt/β-catenin signaling by degrading LRP6 is cytotoxic to BLBC and its stem cell population, and whether niclosamide in combination with TRA-8 produced synergistic killing.
Methods: BLBC cell lines HCC1187, HCC1143, 2LMP and SUM159 were examined for cytotoxicity using the ATPlite assay, inhibition of tumorsphere formation, TOPflash reporter assay, and Western blot analysis for LRP6, free and total β-catenin and Axin 2. 2LMP tumorspheres were treated for 3h ex vivo with TRA-8 and/or niclosamide (10 nM TRA-8, 1 uM niclosamide), and orthotopically implanted into NOD/SCID mice to assess inhibition of tumorigenicity. 2LMP cells were also orthotopically implanted into athymic nude mice to test for in vivo therapeutic efficacy with TRA-8 and niclosamide.
Results: All four BLBC cell lines, maintained as either attached parental cells or as non-adherent tumorspheres showed a dose-dependent cytotoxic response to niclosamide alone and additive or synergistic cytotoxicity with TRA-8. However, tumorspheres exhibited enhanced sensitivity at a lower dose range and earlier time points. There was also a dose-dependent reduction of free β-catenin, phosphorylated LRP6 and Axin 2 after treatment with niclosamide. The combination of niclosamide and TRA-8 further reduced TOPflash activity in the 2LMP and SUM159 cell lines (p=0.05). Interestingly, TRA-8 inhibited Wnt/β-catenin signaling in all four BLBC cell lines. Ex vivo treatments with niclosamide suppressed 2LMP tumorigenicity (p=0.05), which was further suppressed by combination treatment with TRA-8 (p=0.01). Preliminary in vivo studies also showed that niclosamide in combination with TRA-8 suppressed growth of established 2LMP orthotopic tumor xenografts (p=0.009).
Conclusions: Niclosamide inhibited Wnt/β-catenin signaling in BLBCs by degrading LRP6 and β-catenin. Niclosamide in combination with TRA-8 further inhibited Wnt signaling which resulted in synergistic killing of both the CSC and non-CSC populations. Treatment with niclosamide in combination with TRA-8 may be an effective therapy against BLBC.
Citation Format: Angelina I. Londono-Joshi, Rebecca C. Arend, Laura Aristizabal, Yonghe Li, Wenyan Lu, Rajeev S. Samant, Brandon J. Metge, Andres Forero, Tong Zhou, Albert F. LoBuglio, Donald J. Buchsbaum. Inhibition of Wnt co-receptor LRP6 sensitizes basal-like breast cancer stem cells to TRA-8 anti-DR5 monoclonal antibody. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 865. doi:10.1158/1538-7445.AM2013-865