Bortezomib Reduces the Tumorigenicity of Multiple Myeloma via Downregulation of Upregulated Targets in Clonogenic Side Population Cells

Abstract

Side population (SP) cells in cancers, including multiple myeloma, exhibit tumor-initiating characteristics. In the present study, we isolated SP cells from human myeloma cell lines and primary tumors to detect potential therapeutic targets specifically expressed in SP cells. We found that SP cells from myeloma cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11) express CD138 and that non-SP cells include a CD138-negative population. Serial transplantation of SP and non-SP cells into NOD/Shi-scid IL-2γnul mice revealed that clonogenic myeloma SP cells are highly tumorigenic and possess a capacity for self-renewal. Gene expression analysis showed that SP cells from five MM cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11, JJN3) express genes involved in the cell cycle and mitosis (e.g., CCNB1, CDC25C, CDC2, BIRC5, CENPE, SKA1, AURKB, KIFs, TOP2A, ASPM), polycomb (e.g., EZH2, EPC1) and ubiquitin-proteasome (e.g., UBE2D3, UBE3C, PSMA5) more strongly than do non-SP cells. Moreover, CCNB1, AURKB, EZH2 and PSMA5 were also upregulated in the SPs from eight primary myeloma samples. On that basis, we used an aurora kinase inhibitor (VX-680) and a proteasome inhibitor (bortezomib) with RPMI 8226 and AMO1 cells to determine whether these agents could be used to selectively target the myeloma SP. We found that both these drugs reduced the SP fraction, though bortezomib did so more effectively than VX-680 due to its ability to reduce levels of both phospho-histone H3 (p-hist. H3) and EZH2; VX-680 reduced only p-hist. H3. This is the first report to show that certain oncogenes are specifically expressed in the myeloma SP, and that bortezomib effectively downregulates expression of their products. Our approach may be useful for screening new agents with which to target a cell population possessing strong tumor initiating potential in multiple myeloma.

Dual Inhibition of EZH2 and EZH1 Sensitizes PRC2-Dependent Tumors to Proteasome Inhibition

“…A recent study showed that bortezomib reduces EZH2 protein (26), which prompted us to thoroughly investigate the impact of proteasome inhibitors on EZH2. Interestingly, bortezomib downregulated not only EZH2 protein but also its mRNA in multiple myeloma cells in dose- and time-dependent manners (Fig.…”

Mitochondrial-Targeted Decyl-Triphenylphosphonium Enhances 2-Deoxy-D-Glucose Mediated Oxidative Stress and Clonogenic Killing of Multiple Myeloma Cells

“…In MM.1S, OPM-2, H929, and U266 cells, a distinct sub-population of CD138 low cells (approximately 1–4.2%) was detected by flow cytometry (Fig 1A and 1B). As the robustness of CD138 to correctly identify stem-like MM cells remains debatable [59], side population (SP) staining was also done which an established functional marker of adult stem cells and many types of CSCs including MM [6, 60–62]. The SP fractions in MM.1S and OPM-2 cells were ~ 0.4% ± 0.1% and 2.3% ± 0.5%, respectively and verapamil treatment reduced SP fraction by approximately 80% confirming correct SP gating (Fig 1C and 1D).…”

Extramedullary relapse of multiple myeloma defined as the highest risk group based on deregulated gene expression data

“…20 ). This paper showed that bortezomib treatment can reduce MM tumorigenicity by reduction of target gene expression, including ASPM, in this cell population.…”