Abstract 5330: Differential expression and roles of miR-1246 and miR-1290 in multiple myeloma cancer stem cell-like subpopulation.
Authors: Cheung Coty HY , Cheng Suk-Hang , Li Libby , Chan Natalie Pui Ha , Ip Rosalina KL , Cheng Chi-Keung , Ng Margaret H.L. 
Published: August 2014

Multiple myeloma (MM) remains an incurable clonal plasma cell malignancy. The existence of cancer stem cell-like subpopulation in MM may explain for its unfavourable prognosis and high relapse rate. Studies have shown that CD138neg MM cells were clonogenic and could be serially transplanted, while CD138+ cells could not. We characterized CD138neg cells by checking the presence of such subpopulation and their tumorigenic properties. In concordance with literature, CD138neg cells were present in MM cell lines, and possessed higher clonogenic potential than its non-tumorigenic counterpart as shown in colony formation assay. They were more quiescent, with less than 1% in G2/M phase compared to 5-10% in CD138+ cells. They also demonstrated higher resistance to chemotherapeutic agents, including thalidomide and bortezomib. Since studies in solid tumors have demonstrated that microRNA (miRNA) plays a role in self-renewal, tumorigenicity and chemoresistance in cancer stem cells, we hypothesized that specific miRNAs are the key regulators of the cancer stem cell-like functions in MM clonogenic cells. To search for these candidates, we first identified miRNAs that are differentially expressed among the two subpopulations, CD138+ and CD138neg cells, which were isolated from MM cell lines (NCI-H929, U266 and MM17, our in-house MM cell line) using MACS-immunomagnetic separation system and subjected to Exiqon miRCURY LNA miRNA microarray analysis. Ten candidate miRNAs were identified and selected for further validation by Taqman miRNA assay. By further independent investigation using fifteen MM patient samples, concordantly we found frequent up-regulation of miR-483-5p, miR-1246, miR-1275, miR-1290 and miR-3196 in CD138neg compared to CD138+ cells. MiR-1246 (93.4%, p = 0.006) and miR-1290 (93.4%, p = 0.0015) were chosen for further examination of the potential roles in MM pathogenesis and prognosis because they showed significantly higher expression levels in CD138neg cells than the CD138+ counterparts. Preliminary results showed that inhibiting miR-1246 (p = 0.0478) and miR-1290 (p = 0.0386) reduced cell proliferation in CD138neg cells at 24h or 48h of transfection in unsorted U266 cells, while CD138+ cells were not affected. As predicted using computational algorithms (miRANDA and TargetScan), the downstream targets of the two miRNAs include SEMA6A, STK17A, PHLDA1 and KLF9, which bear functions related to MM pathogenesis or known to be tumor suppressors. Moreover, we also confirmed that their mRNA expressions were down-regulated in U266 CD138neg cells compared with CD138+ cells.Taken together, we suggest that miR-1246 and miR-1290 may play a regulatory role in MM clonogenic cell proliferation. Our findings may provide a novel insight for the microRNA-based therapeutics that specifically targets the clonogenic progenitors, which may prove to be more effective for cure of MM.

Citation Format: Coty HY Cheung, Suk-Hang Cheng, Libby Li, Natalie Pui Ha Chan, Rosalina KL Ip, Chi-Keung Cheng, Margaret H.L. Ng. Differential expression and roles of miR-1246 and miR-1290 in multiple myeloma cancer stem cell-like subpopulation. [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 5330. doi:10.1158/1538-7445.AM2013-5330