Abstract 2232: Down-regulation of dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A mediated by miR-1246 and miR-1290 in multiple myeloma cancer stem-like cells
Authors: Wong Terry Hei Yan , Cheung Coty HY , Chan Natalie Pui Ha , Ip Rosalina KL , Cheng Chi-Keung , Ng Kitty Ka Yan , Ng Margaret H.L. 
Published: August 2015

Multiple Myeloma (MM) is a clonal plasma cell derived malignancy characterized with high relapse rate and fatality despite recent therapeutic advances. The existence of the rare population of CD138neg cancer stem-like cells had been identified and demonstrated to bear higher clonogenicity and self-renewal capacity when compared to the CD138pos major population. Besides, CD138neg MM cells are more resistant to common therapeutic agents treating MM including dexamethasone, lenalidomide and bortezomib. Therefore, targeting MM cancer-stem like cells could be critical to achieve a cure in MM. Previously, our lab had identified 2 microRNAs, miR-1246 and miR-1290 that are overexpressed in CD138neg MM cells in both cell lines and patient samples. Their target genes are being studied in the present study. CD138pos and CD138neg MM cells were isolated using MACS-immunomagnetic system and the purity of both CD138neg and CD138pos fraction were accessed by flow cytometry using PE anti-human CD138 antibody. The purity of each fraction was over 95% in 4 cell lines (U266, RPMI8226, OPM2 and KMS-12-PE) used. mRNA level of 7 putative targets (DYRK1A, KDM5A, NKRF, STK17A, PHLDA1, FOXA1, SCAI) chosen from TargetScan 6.2, miRanda or previous studies, which are related to tumor development in other cancers, of miR-1246 or miR-1290 were examined using RT-qPCR. Among the 7 putative targets, DYRK1A, KDM5A and NKRF were down-regulated in all four cell lines used. 3′UTR of the 3 down-regulated targets were cloned into pmirGLO Dual-Luciferase miRNA Target Expression Vector, which were co-transfected with miRNA-mimic into 293FT cells. Down-regulation of luciferase signal was observed in DYRK1A (p&lt;0.05) and KDM5A (p&lt;0.05). Mutagenesis of miR-1246 and miR-1290 binding sites of DYRK1A (but not in KDM5A) led to restoration of luciferase signal. Besides, mRNA and protein level of DYRK1A were also down-regulated 48 hours post-transfection of miRNA-mimics. The mRNA level of DYRK1A were also found to be significantly down-regulated in CD138neg cells when compared to CD138pos cells in 30 pairs of MM patient samples. Therefore, we conclude that DYRK1A should be a target of miR-1246 and miR-1290 in MM cells. Next, DYRK1A was knocked down by shRNA in U266. Preliminary results showed that down-regulation of DYRK1A reduced the percentage of apoptotic cells induced by treatment with bortezomib for 24 hours (p&lt;0.05). Taken together, we had identified DYRK1A, which is down-regulated in MM cancer stem-like cells and regulated by miR-1246 and miR-1290. It may play a role in drug resistance in MM cancer stem-like cells. Our findings may provide insight on how MM cancer stem-like cells would escape from chemotherapy and facilitate the development of a novel therapeutic target in effective MM therapy.

Citation Format: Terry Hei Yan Wong, Coty HY Cheung, Natalie Pui Ha Chan, Rosalina KL Ip, Chi-Keung Cheng, Kitty Ka Yan Ng, Margaret H.L. Ng. Down-regulation of dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A mediated by miR-1246 and miR-1290 in multiple myeloma cancer stem-like cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2232. doi:10.1158/1538-7445.AM2015-2232