Ecto-5'-nucleotidase (Nt5e/CD73)-mediated adenosine signaling attenuates TGFß-2 induced elastin and cellular contraction.

Arterial calcification due to deficiency of CD73 (ACDC) is a rare genetic disease caused by a loss-of-function mutation in the NT5E gene encoding the ecto-5'-nucleotidase (cluster of differentiation 73, CD73) enzyme. Patients with ACDC develop vessel arteriomegaly, tortuosity, and vascular calcification in their lower extremity arteries. Histological analysis shows that patients with ACDC vessels exhibit fragmented elastin fibers similar to that seen in aneurysmal-like pathologies. It is known that alterations in transforming growth factor ß (TGFß) pathway signaling contribute to this elastin phenotype in several connective tissue diseases, as TGFß regulates extracellular matrix (ECM) remodeling. Our study investigates whether CD73-derived adenosine modifies TGFß signaling in vascular smooth muscle cells (SMCs). We show that Nt5e-/- SMCs have elevated contractile markers and elastin gene expression compared with Nt5e+/+ SMCs. Ecto-5'-nucleotidase (Nt5e)-deficient SMCs exhibit increased TGFß-2 and activation of small mothers against decapentaplegic (SMAD) signaling, elevated elastin transcript and protein, and potentiate SMC contraction. These effects were diminished when the A2b adenosine receptor was activated. Our results identify a novel link between adenosine and TGFß signaling, where adenosine signaling via the A2b adenosine receptor attenuates TGFß signaling to regulate SMC homeostasis. We discuss how disruption in adenosine signaling is implicated in ACDC vessel tortuosity and could potentially contribute to other aneurysmal pathogenesis.

NT5E DNA methylation in papillary thyroid cancer: Novel opportunities for precision oncology.

The ectoenzyme CD73, encoded by the NT5E gene, has emerged as a potential prognostic and therapeutic marker for papillary thyroid carcinoma (PTC), which has increased in incidence in recent decades. Here, from The Cancer Genome Atlas Thyroid Cancer (TCGA-THCA) database, we extracted and combined clinical features, levels of NT5E mRNA, and DNA methylation of PTC samples and performed multivariate and random forest analyses to evaluate the prognostic relevance and the potential of discriminating between adjacent non-malignant and thyroid tumor samples. As a result, we revealed that lower levels of methylation at the cg23172664 site were independently associated with BRAF-like phenotype (p = 0.002), age over 55 years (p = 0.012), presence of capsule invasion (p = 0.007) and presence of positive lymph node metastasis (LNM) (p = 0.04). The methylation levels of cg27297263 and cg23172664 sites showed significant and inversely correlations with levels of NT5E mRNA expression (r = -0.528 and r = -0.660, respectively), and their combination was able to discriminate between adjacent non-malignant and tumor samples with a precision of 96%-97% and 84%-85%, respectively. These data suggest that combining cg23172664 and cg27297263 sites may bring new insights to reveal new subsets of patients with papillary thyroid carcinoma.

Novel microRNAs modulating ecto-5'-nucleotidase expression.

Introduction: The 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. Methods: In 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. Results: As 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. Discussion: The miRNAs identified might have clinical relevance as potential therapeutic agents and biomarkers or therapeutic targets, respectively.