Multiple myeloma cells modify VEGF/IL-6 levels and osteogenic potential of bone marrow stromal cells via Notch/miR-223.

Bone marrow mesenchymal stromal cells (BMMSCs) represent a crucial component of multiple myeloma (MM) microenvironment supporting its progression and proliferation. Recently, microRNAs have become an important point of interest for research on micro-environmental interactions in MM with some evidence of tumor supportive roles in MM. In this study, we examined the role of miR-223 for MM support in BMMSCs of 56 patients with MM (MM-BMMSCs). miR-223 expression in MM-BMMSCs was reduced by the presence of MM cells in vitro in a cell-contact dependent manner compared to mono-cultured MM-BMMSCs. Co-cultivation of MM cells and MM-BMMSCs induced activation of notch amongst others via jagged-2/notch-2 leading to increased expression of Hes1, Hey2, or Hes5 in both cell types. Cultivation of MM-BMMSCs with increasing levels of recombinant jagged-2 reduced miR-223 and increased Hes1 levels in a concentration-dependent manner. Transient reduction of miR-223 levels increased VEGF and IL-6 expression and secretion by MM-BMMSCs. In addition, reduction of miR-223 degraded the osteogenic differentiation potential of MM-BMMSCs. Inhibition of notch signaling induced apoptosis in both MM cells and MM-BMMSCs. Furthermore, it increased miR-223 levels and reduced expression of VEGF and IL-6 by both cell types. These data provide first evidence that miR-223 participates in different MM supporting pathways in MM-BMMSCs inlcuding regulation of cytokine secretion and expression as well as osteogenic differentiation of MM-BMMSCs. More insights on the role of miR-223 in MM-BMMSCs and in cellular interactions between MM cells and MM-BMMSCs could provide starting points for a more efficient anti-myeloma treatment by targeting of notch signaling. © 2015 Wiley Periodicals, Inc.

Multiple myeloma cells alter the senescence phenotype of bone marrow mesenchymal stromal cells under participation of the DLK1-DIO3 genomic region.

BACKGROUND: Alterations and senescence in bone marrow mesenchymal stromal cells of multiple myeloma patients (MM-BMMSCs) have become an important research focus. However the role of senescence in the pathophysiology of MM is not clear. METHODS: Correlation between senescence, cell cycle and microRNA expression of MM-BMMSCs (n = 89) was analyzed. Gene expression analysis, copy number analysis and methylation specific PCR were performed by Real-Time PCR. Furthermore, cyclin E1, cyclin D1, p16 and p21 genes were analyzed at the protein level using ELISA. Cell cycle and senescence were analyzed by FACS. MiRNA transfection was performed with miR-485-5p inhibitor and mimic followed by downstream analysis of senescence and cell cycle characteristics of MM-BMMSCs. Results were analyzed by Mann-Whitney U test, Wilcoxon signed-rank test and paired t-test depending on the experimental set up. RESULTS: MM-BMMSCs displayed increased senescence associated ß-galactosidase activity (SA-ßGalA), cell cycle arrest in S phase and overexpression of microRNAs. The overexpressed microRNAs miR-485-5p and miR-519d are located on DLK1-DIO3 and C19MC, respectively. Analyses revealed copy number accumulation and hypomethylation of both clusters. KMS12-PE myeloma cells decreased SA-ßGalA and influenced cell cycle characteristics of MM-BMMSCs. MiR-485-5p was significantly decreased in co-cultured MM-BMMSCs in connection with an increased methylation of DLK1-DIO3. Modification of miR-485-5p levels using microRNA mimic or inhibitor altered senescence and cell cycle characteristics of MM-BMMSCs. CONCLUSIONS: Here, we show for the first time that MM-BMMSCs have aberrant methylation and copy number of the DLK1-DIO3 and C19MC genomic region. Furthermore, this is the first study pointing that multiple myeloma cells in vitro reduce both the senescence phenotype of MM-BMMSCs and the expression of miR-223 and miR-485-5p. Thus, it is questionable whether senescence of MM-BMMSCs plays a pathological role in active multiple myeloma or is more important when cell interaction with myeloma cells is inhibited. Furthermore, we found that MiR-485-5p, which is located on the DLK1-DIO3 cluster, seems to participate in the regulation of senescence status and cell cycle characteristics of MM-BMMSCs. Thus, further exploration of the microRNAs of DLK1-DIO3 could provide further insights into the origin of the senescence state and its reversal in MM-BMMSCs.

Dual PPARalpha/gamma ligand TZD18 either alone or in combination with imatinib inhibits proliferation and induces apoptosis of human CML cell lines.

Despite progress in the treatment of early-stage chronic myeloid leukemia (CML), the accelerated and blastic phases of CML still remain a therapeutic challenge. Persistence of BCR-ABL-positive (bcr-abl(+)) cells or secondary resistance during imatinib therapy frequently occurs. In this study, we investigated the activity of a novel dual ligand specific for peroxisome proliferator-activated receptor alpha and gamma (PPARalpha/gamma) against CML blast crisis cell lines. Exposure of these cell lines (K562, KU812 and KCL22) to TZD18 resulted in a growth inhibition in a dose- and time-dependent manner. This effect may not be mediated through PPARgamma and PPARalpha activation, since antagonists of PPARgamma and/or PPARalpha could not reverse this inhibition. Western blotting analysis showed that expression of the cyclin dependent kinase inhibitor (CDKI) p27(kip1) was enhanced, whereas levels of cyclin E, cyclin D2 and cyclin dependent kinase 2 (CDK-2) were decreased when these cells were treated with TZD18. Most interestingly, TZD18 synergistically enhanced the antiproliferative and pro-apoptotic effect of imatinib. Overall, our findings strongly suggest that either TZD18, either alone or in combination with imatinib may be beneficial for the treatment of CML in myeloid blast crisis.