Abstract
Deregulated expression of microRNAs (miR) is a hallmark of cancer. Tumor suppressor miRNAs are generally down-regulated in cancer cells compared to their normal counterpart, and their enforced expression indeed represents a promising strategy for cancer treatment. We have found miR-23b to be downregulated in CD138+ myeloma cells from 38 multiple myeloma (MM) patients and 18 plasma cell leukemia (PCL) patients compared to normal PCs. Decreased expression of miR-23b was further confirmed in an independent dataset of 66 MM patients by TaqMan miRNA assays. The downregulation of miR-23b expression was also observed in several myeloma cells lines when compared with PBMC and BMSC.
Interestingly, interaction of BMSC with MM cells resulted in further decrease in miR-23b expression in both cell types. Moreover, Interleukin-6 (IL-6) also suppressed the expression of miR-23b in a time- and dose- dependent pattern, indicating that the human bone marrow microenvironment (huBMM) modulates miR-23b levels. miR-23b is commonly repressed in autoimmune conditions by IL-17, a cytokine shown to promote myeloma cell growth and inhibit its immune function. We have indeed observed further decrease in miR-23b expression in MM cells after IL-17 treatment for 24 hours. We have also observed downregulation of miR-23b in CD19+ Waldenstrom’s Macroglobulinemia (WM) cells compared to CD19+ B cells from healthy donors, which was further decreased in the presence of components of the WM bone marrow milieu.
We further assessed the functional significance of miR-23b by both gain- and loss-of-function studies. A significant decrease in cell proliferation and survival, along with induction of caspase 3/7 activity was observed over time in miR-23b mimic–transfected myeloma (H929, KMS11) and WM cell lines (MWCL1) with low miR-23b expression.
At the molecular level, we have identified Sp1, a transcription factor endowed with oncogenic activity in MM and WM, as a target of miR-23b. Expression of miR-23b decreased Sp1 mRNA levels via 3’UTR binding, as assessed in luciferase reporter assays. On the other hand, genetic and/or pharmacological inhibition of Sp1 led to miR-23b upregulation, thus highlighting the occurrence of a feedback loop between miR-23b and its target. Of note, miR-23b transfection significantly reduced Sp1-driven NF-kB activity in MM and WM cells.
Finally, c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, has been shown to transcriptionally repress miR-23b. Moreover, treatment with the demethylating agent 5-aza-deoxycitidine significantly increase the expression of miR-23b in MM1S and KMS-11 cells suggesting that promoter methylation may be an additional mechanism of miR-23b suppression in myeloma. Thus MYC-dependent miR-23b repression in myeloma cells may allow activation of oncogenic transcription factors Sp1 and NF-κB, representing the first feed forward loop with critical growth and survival role in myeloma.
Taken together, these data support a model in which the humoral environment reduces miR-23b expression in tumor cells, suggesting a tumor suppressor role in MM and WM and highlighting the potential of a miR-23b-based replacement therapy to treat these hematologic malignancies.
Disclosures: Anderson:
gilead: Consultancy; onyx: Consultancy; celgene: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.
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