Preview

PDF, English Download (19MB) | Terms of use

Abstract

Cancer is one of the most common causes of death in the modern world and almost every second person will experience a cancer disease during their lifetime. In general, cancer cells exhibit a degenerated regulation of their gene- and protein expression, which enables them to grow and proliferate unrestrainedly. Furthermore, cancer cells gain the ability to avoid normal control mechanisms like apoptosis or elimination by immune cells. In this study we wanted to analyse the miRNA mediated regulation of immune checkpoint molecules in cancer cells. Expression of immune checkpoint molecules by cancer cells can counter-act tumour reactive immune responses, for example by reducing the susceptibility of tumour cells to cytotoxic T cell (CTL) mediated cytolysis, thereby promoting tumour immune evasion. miRNAs are small non-coding RNAs involved in post-transcriptional regulation. Thus, binding of miRNAs to the 3‘-UTR of target mRNAs can block translation or lead to degradation of the targeted mRNAs. Cancer cells often exhibit aberrant miRNA expression profiles, thus tumour derived miRNAs can be utilized as biomarkers for early tumour detection. Within this study we conducted a FACS-based human miRNA library screen and determined miRNAs affecting surface expression of the immune checkpoint molecules NT5E (CD73), ENTPD1 (CD39) and PD-L1 (CD274) on the human tumour cell lines SK-Mel-28 (melanoma) and MDA-MB-231 (breast cancer). We could identify a set of potential tumour-suppressor miRNAs that decreased expression of the immune checkpoints molecules in these cell lines, as well as a group of potential oncomiRs that induced increased immune checkpoint molecule expression. The results of the high-throughput screen for NT5E were verified in a validation process including up to 12 distinct cancer cell lines. Thus, miR-1285-5p, miR-3134, miR-22-3p and miR-193a-3p were determined as potent inhibitors of NT5E expression. Luciferase-based reporter assays proved that these NT5E inhibitory miRNAs act through direct binding to the NT5E 3’-UTR. Using functional malachite-green assays the net effect of reduced NT5E expression on adenosine production caused by miR-1285-5p and miR-3134 was assessed. As adenosine is known to inhibit effector function of cytotoxic T cells, miRNA mediated alterations in NT5E expression might affect the susceptibility of cancer cells to T cell mediated cytolysis. Moreover, we determined miR-134-3p, miR-6859-3p and miR-224-3p as miRNAs that could enhance NT5E expression most effectively. Notably, the observed increase of NT5E was very consistent across a panel of melanoma and breast cancer cell lines. Different from the NT5E inhibiting miRNAs above, we suspect indirect mechanisms for the miRNA-mediated enhancement of NT5E expression. In summary, microarray expression profiling was performed on miRNA transfected tumour cells to unravel the mechanisms responsible for the up-regulated NT5E expression observed. By applying different bioinformatic analyses we could identify several promising candidates for the ”missing link” like ARNT2 or SOX9. Future experiments will have to clarify, whether these transcription factors really inhibit NT5E expression. As a next step we want to measure the direct impact of our identified miRNAs on killing of human cancer cells by T cells. Furthermore, we want to review whether these miRNAs might be suitable biomarkers or could be even used for therapeutical intervention.