Novel microRNAs modulating ecto-5′-nucleotidase expression

IntroductionThe expression of immune checkpoint molecules (ICMs) by cancer cells is known to counteract tumor-reactive immune responses, thereby promoting tumor immune escape. For example, upregulated expression of ecto-5′-nucleotidase (NT5E), also designated as CD73, increases extracellular levels of immunosuppressive adenosine, which inhibits tumor attack by activated T cells. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Thus, the binding of miRNAs to the 3′-untranslated region of target mRNAs either blocks translation or induces degradation of the targeted mRNA. Cancer cells often exhibit aberrant miRNA expression profiles; hence, tumor-derived miRNAs have been used as biomarkers for early tumor detection.MethodsIn this study, we screened a human miRNA library and identified miRNAs affecting the expression of ICMs NT5E, ENTPD1, and CD274 in the human tumor cell lines SK-Mel-28 (melanoma) and MDA-MB-231 (breast cancer). Thereby, a set of potential tumor-suppressor miRNAs that decreased ICM expression in these cell lines was defined. Notably, this study also introduces a group of potential oncogenic miRNAs that cause increased ICM expression and presents the possible underlying mechanisms. The results of high-throughput screening of miRNAs affecting NT5E expression were validated in vitro in 12 cell lines of various tumor entities.ResultsAs result, miR-1285-5p, miR-155-5p, and miR-3134 were found to be the most potent inhibitors of NT5E expression, while miR-134-3p, miR-6859-3p, miR-6514-3p, and miR-224-3p were identified as miRNAs that strongly enhanced NT5E expression levels.DiscussionThe miRNAs identified might have clinical relevance as potential therapeutic agents and biomarkers or therapeutic targets, respectively

SOX5 is involved in balanced MITF regulation in human melanoma cells

Background: Melanoma is a cancer with rising incidence and new therapeutics are needed. For this, it is necessary to understand the molecular mechanisms of melanoma development and progression. Melanoma differs from other cancers by its ability to produce the pigment melanin via melanogenesis; this biosynthesis is essentially regulated by microphthalmia-associated transcription factor (MITF). MITF regulates various processes such as cell cycling and differentiation. MITF shows an ambivalent role, since high levels inhibit cell proliferation and low levels promote invasion. Hence, well-balanced MITF homeostasis is important for the progression and spread of melanoma. Therefore, it is difficult to use MITF itself for targeted therapy, but elucidating its complex regulation may lead to a promising melanoma-cell specific therapy. Method: We systematically analyzed the regulation of MITF with a novel established transcription factor based gene regulatory network model. Starting from comparative transcriptomics analysis using data from cells originating from nine different tumors and a melanoma cell dataset, we predicted the transcriptional regulators of MITF employing ChIP binding information from a comprehensive set of databases. The most striking regulators were experimentally validated by functional assays and an MITF-promoter reporter assay. Finally, we analyzed the impact of the expression of the identified regulators on clinically relevant parameters of melanoma, i.e. the thickness of primary tumors and patient overall survival. Results: Our model predictions identified SOX10 and SOX5 as regulators of MITF. We experimentally confirmed the role of the already well-known regulator SOX10. Additionally, we found that SOX5 knockdown led to MITF up-regulation in melanoma cells, while double knockdown with SOX10 showed a rescue effect; both effects were validated by reporter assays. Regarding clinical samples, SOX5 expression was distinctively up-regulated in metastatic compared to primary melanoma. In contrast, survival analysis of melanoma patients with predominantly metastatic disease revealed that low SOX5 levels were associated with a poor prognosis. Conclusion: MITF regulation by SOX5 has been shown only in murine cells, but not yet in human melanoma cells. SOX5 has a strong inhibitory effect on MITF expression and seems to have a decisive clinical impact on melanoma during tumor progression

Living in the Post

30 Jahre nach dem ersten Aufkommen des Begriffes Postsozialismus widmet sich dieser Band den vielfältigen (Re)Konfigurationen, die das Konzept seither durchlaufen hat und fragt nach seinem gegenwärtigen analytischen Gehalt. Anhand unterschiedlicher Kontexte untersuchen die Autor*innen die multiplen Verwendungen von und Perspektiven auf das Konzept. Sie wenden sich verschiedenen Formen von (materiellem) Erbe, künstlerisch-aktivistischen Projekten des Erinnerns oder Ost/West-Machtdynamiken zu und diskutieren das spannungsvolle Verhältnis zu post- und dekolonialen Theorien. Den Rahmen bildet dabei ein Fokus auf Praktiken des Erinnerns, der Vergegenwärtigung von Vergangenem und seinem Wirken in die Zukunft.Thirty years after the emergence of the term postsocialism, this volume is dedicated to the multiple (re)configurations the concept has undergone since then and inquires its current analytical value. Looking at different contexts, the authors examine the multiple uses of and perspectives on the concept. They turn to various forms of (material) heritage, artistic-activist projects of remembering, or East-West power dynamics and discuss the ambivalent relationship to post- and decolonial theories. The articles are framed by a focus on practices of remembering, the presencing of the past and its impact on the future.Peer Reviewe

Controlling the Immune Suppressor: Transcription Factors and MicroRNAs Regulating CD73/NT5E

The NT5E (CD73) molecule represents an ecto-5′-nucleotidase expressed on the cell surface of various cell types. Hydrolyzing extracellular adenosine monophosphate into adenosine and inorganic phosphate, NT5E performs numerous homeostatic functions in healthy organs and tissues. Importantly, NT5E can act as inhibitory immune checkpoint molecule, since free adenosine generated by NT5E inhibits cellular immune responses, thereby promoting immune escape of tumor cells. MicroRNAs (miRNAs) are small non-coding RNA molecules regulating gene expression on posttranscriptional level through binding to mRNAs, resulting in translational repression or degradation of the targeted mRNA molecule. In tumor cells, miRNA expression patterns are often altered which in turn might affect NT5E surface expression and eventually influence the efficacy of antitumor immune responses. This review describes the diverse roles of NT5E, summarizes current knowledge about transcription factors controlling NT5E expression, and highlights the significance of miRNAs involved in the posttranscriptional regulation of NT5E expression

Controlling the immune suppressors: miRNAs regulating NT5E, ENTPD1 and CD274

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