Micrornas Control Signaling Pathways and the Biologic Relevance in Multiple Myeloma Side Population Cells

Abstract 721

Novel agents and stem cell transplantation have dramatically improved the remission and survival rates of multiple myeloma (MM). However, almost all patients would experience a relapse, which indicates the existence of tumor-initiating population and/or cancer stem cells (CSCs). Side population (SP) cells are an enriched source of cancer-initiating cells with stem cell properties, which has be acted as a unique hallmark for no specific marker of MM CSC study. The emerging role of microRNAs (miRNAs) in numerous types of human cancer led us to hypothesize that this group of non-coding genes might serve as key regulators of the cell signaling pathways in myeloma SP cells.

Firstly, we successfully isolated and identified SP cells in MM cell lines and primary cells by a MoFlo cytometer, which represented a small fraction of the total cell population ranged from 0.04% to 2.0%. SP cells showed trend significantly high proliferation ability in formation 1392 ± 257 colonies in medium compared with 550 ±15 colonies for the MP cells after 14 days culture. We also have observed that SP cells contain majority percentage of quiescent population (G0/G1 stage) with 95.47% compared to major population (MP) cells with 49.36% in cell cycle assay, which may be one of the reasons for the drug resistance in SP cells besides to the overexpression of the ABC transporters. Indeed, ABCG2 were significantly enhanced in SP cells in comparison with MP cells both in mRNA and protein levels in our study.

We next aimed to investigate the contribution of miRNA expression profiling on myeloma SP cells. Therefore, miRCURY™ LNA array analysis of sorted SP and MP cells from two primary myeloma patients was performed. Among the miRNAs exhibiting at least 2-fold and statistically significant difference in expression, 43 miRNAs were identified in total, with 12 upregulated and 31 down-regulated in SP cells compared with MP cells. Five interesting miRNAs, which may be involved in cancer stem cell pathology, were confirmed by qRT-PCR. We subsequently identified the high-probability interesting predicted target genes and signaling pathways of these dysregulated miRNAs by bioinformatic analysis. The majority of the affected pathways are preferentially related to SP cell presented in PI3K/AKT/mTOR, MAPK/ERK, ErbB/EGF, Wnt/b-catenin, Ubiquitin mediated proteolysis, TGF-β pathways and so on.

Previous recently studies have addressed that mTOR signaling in the maintenance of CSCs have played a vital role. We further identified several activated signaling pathway proteins of PI3K/AKT/mTOR pathway in MM SP cells using phosphor-specific antibodies by immunofluorescence staining and western blot, including the PI3K/AKT/mTOR protein CAB39, TSC1, p-S6 and p-P70S6K. And then, a specific inhibitor of mTOR, rapamycin, was found to reduce remarkably the SP fraction within NCI-H929 cells, which contained 1.3% 3.57% SP cells and 0.05% SP after treatment. We next focus on to assess the miR-451 function analysis, a PI3K/mTOR signaling target, which was overexpressed in MM SP cells. Inhibition expression of miR-451 did not change the proliferation, apoptosis, and cell cycle in NCI-H929 cells. However, after MM cells treatment with As₂O₃, bortezomib, and lenalidomide, knockdown of miR-451 showed a dramatically increases in apoptosis (24%, 15%, and 8% elevation of apoptosis compared to control, respectively), S-phase cell cycle (10%, 8%, 5% elevation compared to control cells, respectively), and CAB39 expression.

In summary, we have identified the MM SP cell and its microRNAs differentially expressed pattern. We further identify that mTOR signaling in the maintenance of CSCs have played an important role, and inhibition of miR-451 contributes to enhance anti-myeloma novel agents' effectiveness, through increased cells apoptosis, differentiation, and CAB39 expression. Our findings might give more insights into the mechanisms regulating MM SP cells and provide a novel strategy to overcome resistance to existing therapies against myeloma.