Nucleic acid nanoagents utilizing the intracellular microRNA-200c machinery for switching functions in breast cancer cells

microRNAs (miRs) are small non-coding RNAs which are able to regulate the expression of the genome by RNA interference. Our main focus has been on miR-200c, which plays a crucial role in chemosensitivity, epithelial to mesenchymal transition (EMT) and cell migration of breast cancer. In the previous funding period, we optimized synthetic RNAi nanoagents for therapeutic delivery of siRNA or miR-200c into various tumor types. EGF receptor-targeted miR-200c lipopolyplexes delivered into aggressive MDA-MB-231 breast carcinoma cells triggered inhibition of tumor cell migration, reduced proliferation, and increased chemosensitivity. Genetic miR-200c knock-out (KO-200c) breast cancer cells (generated by TALENs lipoplexes) followed by proteomic analysis highlighted relevant proteins differentially expressed between miRNA-200c wild type and knock-out cells. Further, an inducible miR-200c expression construct (Tripz-200c) stably transfected into KO-200c cells or 200c-null cell lines enables us to regulate miR-200c expression in a time-controlled fashion. Switching on miRNA-200c results in changed morphology and reduced migration of breast cancer cells in a time-resolved 1D-migration assay (collaboration B01).

The established technologies for artificial manipulation of miR-200c plus the information from the proteomics study enable the refined cell biological analysis in the new funding period. This will include in-depth analysis of miR-200c modulation on migration both in time, space and expression of relevant target genes. 1D and 2D cell migration assays (collaborations B01, B08) and novel miR-200c sensors based on fluorescent light-up aptamers (FLAPs, to be generated by partner A12) will be applied. Furthermore, the nanoagent technology allows analysis of miR-200c function in more complex cellular 3D-systems like spheroids or organoids (collaboration A10) and in human tumor bearing mice. For this purpose, miRNA-200c will be expressed in tumors either by miR-200c nanoagent delivery or using tumor cells with inducible Tripz-miR-200c. The previous studies including the new proteomics study indicate a role of miR-200c on chemosensitivity, which will be analyzed in more detail. Combination of miR-200c expression with chemotherapy, optionally co-delivered by novel nanoagents, should result in decreased tumor growth and reduced metastases.