Examining the role of let-7 microRNA transfer in the metastatic niche

Abstract

Metastasis is a hallmark of malignant disease and still accounts for the majority of cancer related deaths. The term metastasis describes a multistep process. One of the most important steps is the formation of micrometastases in the distant soil. The intercellular transfer of cellular bioactive compounds, like mRNAs and miRNAs, has emerged as an important factor within this process. Tumor suppressor microRNA let-7 serves as a potential player in cancer physiology, but could potentially be used for treatment of cancer. However, the role of let-7 microRNA transfer in the metastatic niche remains unknown. In this dissertation, I examined the roles of let-7 transfer by establishing simplified cell co-culture systems to simulate the metastatic niche in vitro. Dual-luciferase reporter-based contact and non-contact co-culture systems were generated to investigate let-7 level during co-culture of fibroblasts and tumor cells. Interestingly, let-7 level in recipient cells (NIH 3T3, BE2C and Kelly) was increased after co-culturing with low let-7 expressing tumor cells (BE2C and Kelly) compared to high let-7 expressing embryonic fibroblasts (NIH 3T3). In addition, let-7 level in recipient cells (LIN28B KO Kelly) was diminished after co-culturing with donor malignant neuroblastoma cell lines in the absence of LIN28B. The alterations of let-7s activities were independent of direct cell contact. Furthermore, let-7 levels in recipient cells (LIN28B KO BE2C and LIN28B KO PANC1) were downregulated after incubation with cell culture-conditioned media (CCM) and exosome-rich CCM derived from LIN28B KO BE2C compared to parental BE2C. The expression of precursor let-7s was significantly reduced in cell-free exosomes in the absence of LIN28B pointing towards an important role of LIN28B in this process. These results indicate an active let-7 transfer in the micrometastatic niche with LIN28B as a potential mediator.