Microenvironment Confers Resistance to TRAIL Mediated Apoptosis.
Authors: Perez Lia E. , Parquet Nancy , Nimmanapalli Ramadevi , Alsina Melissa , Anasetti Claudio , Dalton William 
Published: April 2020

Abstract
TNF-related apoptosis inducing ligand (TRAIL), a member of the TNF superfamily of death ligands, is preferentially cytotoxic against neoplastic cells and suppresses tumor development in vivo. TRAIL plays a role in Natural Killer, TNF-activated T and Dendritic cell-dependent tumor surveillance. The bone marrow microenvironment provides tumor protection from chemotherapeutic agents and Fas-death receptor signaling, a process known as environmental mediated drug resistance (EM-DR). In this study we have investigated whether EM-DR confers resistance to apoptosis mediated by TRAIL in Multiple Myeloma. We hypothesize that EM-DR contributes to evade TRAIL mediated apoptosis resulting in Multiple Myeloma progression. Three drug sensitive Mulitple Myeloma cell lines (RPMI-8266, U266 and MM1s) exhibits a dramatic apoptosis resistance to recombinant human TRAIL (10ng/mL) after twenty four hours of exposure when directly attached to HS-5 stromal cells as determined by annexin/7-AAD staining (Table 1). RPMI-8226 TRAIL-mediated apoptosis was demonstrated dose- and time-dependent. Experiments in transwell assay with RPMI-8226 in the upper well and HS-5 in the lower well induced significantly reduced apoptosis (8.9 ± 5.4 %) compared to RPMI-8226 cells exposed to medium in lower well (37 ± 16 %) after four hour treatment with TRAIL (10ng/mL). Western blot analysis of RPMI-8226 cells exposed to HS-5 for four hours in transwell exhibited attenuated caspase-8, caspase-3 and PARP cleavage. Direct contact of RPMI-8226 cells with bone marrow fibronectin did not confer resistant to TRAIL. These findings suggest that cell:cell contact and soluble factors produced by stromal cells contribute to TRAIL EM-DR. Pretreatment of RPMI-8226 cells with proteosome inhibitor Bortezomib (10nM) partially overcomes TRAIL EM-DR in cells directly attached to HS-5 and in transwell assays with HS-5 in lower well (Table 2). Bortezomib up-regulated TRAIL receptor-2 (DR5) mRNA expression in RPMI-8226 cells, but did not increase DR5 protein expression as determined by Western blot and flow cytometry. We hypothesize that NF-κB inactivation by bortezomib reduces transcription of genes that mediate TRAIL EM-DR. Mechanistic studies elucidating TRAIL EM-DR are under investigation with the purpose to identify novel targets to enhance anti-tumor activity of TRAIL in vivo.
Table 1. Adhesion of RPMI-8226, U266 and MM1s Myeloma Cells to HS-5 Stromal Cells Attenuates TRAIL Mediated Apoptosis RPMI-8226 (% Specific Apoptosis) U266 (% Specific Apoptosis) MM1s (% Specific Apoptosis) Suspension 75±10 % 11±2.7 % 25±1.7 % Direct Adhesion to HS-5 stromal cells 12±1.8 % 3.5±1 % 4±0.7 % Table 2. Bortezomib Partially Overcomes RPMI-8226 Environment Mediated-TRAIL Resistance Direct Adhesion to HS-5 stromal cells (% Specific Apoptosis) Transwell Assay with HS-5 in lower well (% Specific Apoptosis) *TRAIL (10ng/mL) for 4 hours ** Bortezomib (10nM) for 20 hours followed by TRAIL (10ng/mL) + Bortezomib for 4 hours TRAIL * 1.5±0.7 % 8.9±5.4 % Bortezomib + TRAIL** 23.5±13.5 % 28.6±12.7 %