Abstract 3506: Carfilzomib demonstrates antiproliferative and proapoptotic activities in preclinical triple-negative breast cancer models
Authors: Larson Mathew , Carlson Jennifer H. , Sun Yuliang , Bucholz Tonia , Williams Casey , Dey Nandini , Leyland-Jones Brian , De Pradip 
Published: July 2016

Abstract
BACKGROUND: The proteosome is clinically validated as a target for cancer therapeutics. Carfilzomib (CFZ) is a proteasome inhibitor that selectively and irreversibly binds to its target and has been approved in the US for treatment of relapsed and refractory multiple myeloma. Phase 1/2 studies of CFZ reported signals of clinical activity in solid tumors. The aim of this study was to investigate the activity of CFZ in triple negative breast cancer models. METHODS: A diverse panel of human triple negative breast cancer cell lines was used to investigate the anti-tumor activity of CFZ. Cell viability following CFZ treatment was assessed using MTT assay. Proliferation was monitored both with IncuCyte Zoom real-time imaging system and 3D-ON-TOP clonogenic assay. Annexin-V flow cytometry was used to measure apoptosis. Apoptotic protein levels were measured with Western blot and proteome profiler array. MCL1 and Ki67 expression were also assessed using immunocytochemistry. RESULTS: CFZ had marked anti-proliferative activity in MDA-MD231, MDA-MB468, BT20, and SUM149 TNBC cell lines, with IC50 values following 96-hour exposure from 8 nM to 25 nM by MTT assay. The anti-proliferative activity of CFZ was observed by 3D-ON-TOP clonogenic assay. The anti-proliferative activity of CFZ was also demonstrated with IncuCyte proliferation assay. Proliferation was inhibited by low nanomolar concentrations in both MDA-MB468 and BT20 cells. Western blot analysis of CFZ-treated BT20, MDA-MB231 and MDA-MB468 cells showed cleavage of poly ADP ribose polymerase (PARP) and CASPASE-3, indicative of apoptosis, and induction of microtubule-associated protein-1 light chain-3B (LC3B), indicative of autophagy. However, expression level of anti-apoptotic protein MCL1 was increased in RAS-RAF mutated cells (MDA-MB231) but not in PTEN-null MDA-MD468 cells following the treatment of CFZ. Double immune-fluorescence staining of MDA-MB231 cells following the treatment of CFZ showed higher expression of MCL1 but no change in Ki67 expression. Interestingly, a combination of CFZ and TOR kinase inhibitor (MLN0128) enhanced cleaved CASPASE3 and BIM expression with a concomitant increased induction of apoptosis (annexinV expression) in MDA-MB231 cells. CONCLUSIONS: CFZ demonstrated anti-proliferative activity in TNBC cell lines in vitro. Flow cytometric analysis of annexin V-positive cells and Western blot expression of cleaved CASPASE 3, cleaved PARP and BIM indicated that CRZ -induced TNBC cell death through an apoptosis-dependent manner.
Citation Format: Mathew Larson, Jennifer H. Carlson, Yuliang Sun, Tonia Bucholz, Casey Williams, Nandini Dey, Brian Leyland-Jones, Pradip De. Carfilzomib demonstrates antiproliferative and proapoptotic activities in preclinical triple-negative breast cancer models. [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 3506.