O025 Triggering mRNA stability switch by a miRNA inhibitor: Normalization of aberrant chemokine response in invasive breast cancer

Introduction Aberrant mRNA stability for a number of genes coding for AU-rich (ARE) mRNAs, including chemokines and growth factors, occurs in many cancer types. The CXCR4-CXCL12 (SDF-1 α ) axis is among the most deregulated chemokine responses that promote the progression of invasive breast cancer. Normalization of the aberrant CXCR4-CXCL12 response by triggering CXCR4ARE-mRNA destabilization mediated by the zinc finger mRNA binding protein, tristetraprolin(TTP, ZFP36), is a potential and intriguing approach. Methods Several breast cancer cell lines were used for comparison with the MDA-MB-231 cell line as an invasive model of breast cancer. We treated the cells with a cell-permeable miR-29a inhibitor which triggers TTP expression, and monitored mRNA stability changes for key RNA binding proteins using RT-QPCR and reporter assays. RNA-immunoprecipitation was also employed to study the mRNA–protein interactions. Subsequent alterations in protein levels and associated functional attributes, including invasion and migration assays were investigated. Further, clinical data from a public database were used to understand these relationships. Results We found that CXCR-4 over-expression in invasive breast cancer is causally linked to deficient TTP expression. TTP deficiency was partly due to the high miR-29a levels that target TTP 3’UTR- this is observed in the highly-invasive MDA-MB-231 cells when compared to other non-invasive or non-tumorigenic cell lines. We also show that CXCR-4 is an mRNA target for TTP but not its zinc finger mutant, C124R, as the wild type was able to bind to CXCR-4 mRNA. TTP deficiency in MDA-MB-231 cells led to increased stability of mRNA coding for the RNA stabilizing protein, HuR, and subsequently HuR protein levels were increased. We demonstrated that HuR is able to bind to and increase CXCR-4 mRNA stability. Thus, the aberrant TTP-HuR balance results in CXCR-4 mRNA stabilization in invasive breast cancer. To overcome this aberration, we employed a cell-permeable peptide-linked miR-29a inhibitor to promote TTP mRNA upregulation and HuR mRNA down-regulation. Indeed this was the case, which subsequently led to CXCR-4 mRNA destabilization and reduction of CXCR-4 levels. This paralleled a reduction in the invasiveness of the cells and migration towards CXCR4ligand, CXCL12. Clinical data showed that TTP-HuR mRNA ratios which reflect ARE-mRNA stability changes are perturbed in invasive breast cancer samples when compared to breast tissues ( p p Conclusion We establish that the chemokine receptor CXCR-4 is a novel mRNA target for TTP and HuR, and that its over-expression and breast cancer invasiveness is normalized by the miR29a inhibitor. The study shows that aberrant ARE-mediated mRNA stability in invasive breast cancer can be corrected by switching TTP-HuR balance towards mRNA destabilization in the CXCR4/CXCRL12 axis, indicating a potential therapeutic approach.