Abstract #5530: Downregulation of Topoisomerase II\#945; expression in drug resistant cells is associated with altered NF-YB expression that is mediated by miR-485-3p

Downregulation of Topoisomerase II \#945; expression in drug resistant cells is associated with altered NF-YB expression that is mediated by miR-485-3p Cheng Fen Chen 1 , Xiaolong He 1 , Yin-Yuan Mo 2 and William T. Beck 1 1 Department of Biopharmaceutical Sciences, University of Illinois at Chicago, Chicago, Illinois; 2 Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University, Springfield, Illinois DNA topoisomerase II\#945; (TopoII\#945;) is a nuclear enzyme essential for DNA metabolism and cell cycle progression. Many TopoII inhibitors are used clinically as anti-tumor agents. However, tumors are frequently refractory to treatment due to drug resistance. Our teniposide-resistant (and etoposide cross-resistant) human leukemic lymphoblastic cells (CEM/VM-1-5) express reduced TopoII\#945; protein, as measured by western blot. To determine the role of TopII\#945; in drug resistance, we knocked down TopoII\#945; in parental CCRF-CEM cells by introducing a shTopoII\#945; lentiviral vector. Compared to control cells, CEM-shTopoII\#945; cells are more resistant to etoposide, revealing the essential role of TopoII\#945; in etoposide resistance. Our previous work suggested that the transcription factor NF-YB is a negative regulator of TopoII\#945;, working through the TopoII\#945; promoter ( Morgan et al . Mol Pharmacol , 2001). We have found by western blot that NF-YB protein expression is increased in CEM/VM-1-5 cells compared to CEM cells. This suggests that increased NF-YB may be the cause of reduced TopoII\#945; in CEM/VM-1-5 cells. Accordingly, we then asked what causes the upregulation of NF-YB in CEM/VM-1-5 cells. MicroRNAs are a recently identified group of non-protein coding RNAs that regulate posttranscriptional gene expression by targeting mRNAs\#8217; 3\#8217; untranslated region (UTR) for degradation or translational inhibition. We found by microRNA profiling that tens of microRNAs are either up- or down-regulated in CEM/VM-1-5 cells, compared to CEM cells. One of the microRNAs, hsa-miR-485-3p, is consistently substantially lower in CEM/VM-1-5 cells compared to CEM cells. Using miRanda TM and Target Scan TM servers, we determined that the 3\#8217; UTR of NF-YB harbors a putative miR-485-3p binding site. We thus hypothesized that hsa-miR-485-3p regulates drug resistance by increasing NF-YB expression, which in turn negatively regulates TopoII\#945; expression. To test this hypothesis, we overexpressed miR-485-3p in CEM/VM-1-5 cells. This led to reduced expression of NF-YB and a corresponding upregulation of TopoII\#945;. Moreover, preliminary results suggested that these CEM/VM-1-5/miR-485-3p cells express increased sensitivity to etoposide compared to control cells. In conclusion, we have demonstrated that miR-485-3p is associated with NF-YB expression that mediates etoposide resistance by altering TopoII\#945; expression. (Supported in part by CA40570 (to WTB) and in part by UIC.) Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5530.