Background A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. Better understanding of the molecular mechanisms leading to platelet activation is of importance for the development of improved therapies. Recently, protein tyrosine phosphatases (PTPs) have emerged as critical regulators of platelet function. Methods and Results This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated through the collagen receptor glycoprotein VI (GPVI) and the C-type lectin-like receptor 2 (CLEC-2). DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism, compared to wild-type mice, and showed severely impaired thrombus formation upon ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of PLCγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen and CLEC-2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. Conclusions DUSP3 plays a selective and essential role in collagen- and CLEC-2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a PTP, implicated in platelet signaling, has been targeted with a small-molecule drug.

WNT5A has been identified as an important ligand in the malignant progression of a number of tumours. Although WNT5A signalling is often altered in cancer, the ligand’s role as either a tumour suppressor or oncogene varies between tumour types and is a contemporary issue for investigators of β-catenin-independent WNT signalling in oncology. Here, we report that one of the initial effects of active WNT5A signalling in malignant melanoma cells is an alteration in cellular energy metabolism and specifically an increase in aerobic glycolysis. This was found to be at least in part due to an increase in active Akt signalling and lactate dehydrogenase (LDH) activity. The clinical relevance of these findings was strengthened by a strong correlation (P < 0.001) between the expression of WNT5A and LDH isoform V in a cohort of melanocytic neoplasms. We also found effects of WNT5A on energy metabolism in breast cancer cells, but rather than promoting aerobic glycolysis as it does in melanoma, WNT5A signalling increased oxidative phosphorylation rates in breast cancer cells. These findings support a new role for WNT5A in the metabolic reprogramming of cancer cells that is a context- dependent event.

BackgroundWNT5A (-/-) mammary tissue has been shown to exhibit increased ductal elongation, suggesting elevated mammary cell migration. Increased epithelial cell migration/invasion has often but not always been linked to the epithelial-mesenchymal transition (EMT). In the current study, we investigated the loss of WNT5A in HB2 human mammary epithelial cells and hypothesized that this loss increased their invasion via the EMT. Based on these results, we postulated that suppression of breast cancer cell migration and invasion by WNT5A is due to EMT reversal.MethodsWNT5A was transiently knocked down using specific siRNAs, whereas WNT5A signaling was induced in MDA-MB468 and MDA-MB231 breast cancer cells by stably transfecting cells with WNT5A or treating them with recombinant WNT5A (rWNT5A). Changes in EMT markers, CD44, pAKT and AKT expression were assessed using Western blotting and immunofluorescence. The physiological relevance of altered WNT5A signaling was assessed using migration and invasion assays.ResultsWNT5A knockdown in HB2 mammary epithelial cells resulted in EMT-like changes and increased invasiveness, and these changes were partially reversed by the addition of rWNT5A. These data suggest that WNT5A might inhibit breast cancer cell migration and invasion by a similar EMT reversal. Contrary to our expectations, we did not observe any changes in the EMT status of breast cancer cells, either after treatment with rWNT5A or stable transfection with a WNT5A plasmid, despite the parallel WNT5A-induced inhibition of migration and invasion. Instead, we found that WNT5A signaling impaired CD44 expression and its downstream signaling via AKT. Moreover, knocking down CD44 in breast cancer cells using siRNA impaired cell migration and invasion.ConclusionsWNT5A bi-directionally regulates EMT in mammary epithelial cells, thereby affecting their migration and invasion. However, the ability of WNT5A to inhibit breast cancer cell migration and invasion is an EMT-independent mechanism that, at least in part, can be explained by decreased CD44 expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0421-0) contains supplementary material, which is available to authorized users.

No abstract