Lin28B/Let-7 Axis Regulates Multiple Myeloma Proliferation By Enhancing c-Myc and Ras Survival Pathways

• Published in: Blood 2013-11, Vol.122 (21), p.273-273
• Main Authors: Manier, Salomon, Powers, John T, Sacco, Antonio, Reagan, Michaela R, Moschetta, Michele, Glavey, Siobhan, Maiso, Patricia, Sahin, Ilyas, Mishima, Yuji, Aljaway, Yosra, Leleu, Xavier, Roccaro, Aldo M, Daley, George Q., Ghobrial, Irene M.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V122.21.273.273

MicroRNAs (miRNAs) play a pivotal role in tumorigenesis, due to their ability to target mRNAs involved in the regulation of cell proliferation, survival and differentiation. Lin28B is an RNA binding protein that regulates Let-7 miRNA maturation. Lin28B and Let-7 have been described to act as oncogenes or tumor suppressor genes, respectively, as demonstrated both in solid cancer and hematologic malignancies. However, the role of the Lin28B/Let-7 axis in Multiple Myeloma (MM) has not been studied. Lin28B level expression in MM patients was studied using previously published gene expression profiling (GEP) datasets. Let-7 expression levels were assessed in CD138+ primary MM cells and bone marrow stromal cells (BMSCs) by using PCR, as well as in circulating exosomes using miRNA array (Nanostring® Technology). Exosomes were collected from both normal and MM peripheral blood, using ultracentrifugation; and further studied by using electron microscopy and immunogold labeling for the detection of CD63 and CD81. The knockdown of Lin28B was performed on MM cell lines (U266, MM.1S, MOLP-8) by using a lentiviral Lin28B shRNA. Gain- and loss-of function studies for Let-7 were performed using Let-7 mimic and anti-Let-7 transfection in MM cell lines (MM1S, U266) and primary BMSCs. Cell proliferation has been evaluated by using thymidine assays. Effects of Let-7 and Lin28B on signaling cascades have been evaluated by western blot. Two independent GEP datasets (GSE16558; GSE2658) were analyzed for Lin28B expression, showing a significantly higher level in MM patients compared to healthy controls. In addition, high Lin28B levels correlated with a shorter overall survival (p = 0.0226). We next found that the Let-7 family members are significantly down-regulated in MM primary cells, particularly Let-7a and b (5 fold change, p < 0.05), as demonstrated by using qRT-PCR. Similarly, miRNA arrays showed a lower expression of Let-7-related miRNAs in circulating exosomes obtained from MM patients compared to healthy individuals. We further dissected the functional relevance of Lin28B in MM cells, by performing Lin28 knockdown (KD) in MM cell lines (U266, MOLP-8). This led to a significant decrease in MM cell proliferation associated with G1 phase cell cycle arrest. This was supported by up-regulation of Let-7 and down-regulation of c-Myc, Ras and Cyclin D1 in Lin28 KD MM cells. To further prove that Lin28B-dependent effects on MM cells are mediated by Let7, we next showed that let-7