High Expression of Wee1 Is Associated with Poor Disease-Free Survival in Malignant Melanoma: Potential for Targeted Therapy

Notoriously resistant malignant melanoma is one of the most increasing forms of cancer worldwide; there is thus a precarious need for new treatment options. The Wee1 kinase is a major regulator of the G2/M checkpoint, and halts the cell cycle by adding a negative phosphorylation on CDK1 (Tyr15). Additionally, Wee1 has a function in safeguarding the genome integrity during DNA synthesis. To assess the role of Wee1 in development and progression of malignant melanoma we examined its expression in a panel of paraffin-embedded patient derived tissue of benign nevi and primary- and metastatic melanomas, as well as in agarose-embedded cultured melanocytes. We found that Wee1 expression increased in the direction of malignancy, and showed a strong, positive correlation with known biomarkers involved in cell cycle regulation: Cyclin A (p<0.0001), Ki67 (p<0.0001), Cyclin D3 (p = 0.001), p21Cip1/WAF1 (p = 0.003), p53 (p = 0.025). Furthermore, high Wee1 expression was associated with thicker primary tumors (p = 0.001), ulceration (p = 0.005) and poor disease-free survival (p = 0.008). Transfections using siWee1 in metastatic melanoma cell lines; WM239WTp53, WM45.1MUTp53 and LOXWTp53, further support our hypothesis of a tumor promoting role of Wee1 in melanomas. Whereas no effect was observed in LOX cells, transfection with siWee1 led to accumulation of cells in G1/S and S phase of the cell cycle in WM239 and WM45.1 cells, respectively. Both latter cell lines displayed DNA damage and induction of apoptosis, in the absence of Wee1, indicating that the effect of silencing Wee1 may not be solely dependent of the p53 status of the cells. Together these results reveal the importance of Wee1 as a prognostic biomarker in melanomas, and indicate a potential role for targeted therapy, alone or in combination with other agents

PPARβ activation inhibits melanoma cell proliferation involving repression of the Wilms’ tumour suppressor WT1

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that strongly influence molecular signalling in normal and cancer cells. Although increasing evidence suggests a role of PPARs in skin carcinogenesis, only expression of PPARγ has been investigated in human melanoma tissues. Activation of PPARα has been shown to inhibit the metastatic potential, whereas stimulation of PPARγ decreased melanoma cell proliferation. We show here that the third member of the PPAR family, PPARβ/δ is expressed in human melanoma samples. Specific pharmacological activation of PPARβ using GW0742 or GW501516 in low concentrations inhibits proliferation of human and murine melanoma cells. Inhibition of proliferation is accompanied by decreased expression of the Wilms’ tumour suppressor 1 (WT1), which is implicated in melanoma proliferation. We demonstrate that PPARβ directly represses WT1 as (1) PPARβ activation represses WT1 promoter activity; (2) in chromatin immunoprecipitation and electrophoretic mobility shift assays, we identified a binding element for PPARβ in the WT1 promoter; (3) deletion of this binding element abolishes repression by PPARβ and (4) the WT1 downstream molecules nestin and zyxin are down-regulated upon PPARβ activation. Our findings elucidate a novel mechanism of signalling by ligands of PPARβ, which leads to suppression of melanoma cell growth through direct repression of WT1

Novel transcripts reveal a complex structure of the human TRKA gene and imply the presence of multiple protein isoforms

Publisher Copyright: © 2015 Luberg et al.Background: Tropomyosin-related kinase A (TRKA) is a nerve growth factor (NGF) receptor that belongs to the tyrosine kinase receptor family. It is critical for the correct development of many types of neurons including pain-mediating sensory neurons and also controls proliferation, differentiation and survival of many neuronal and non-neuronal cells. TRKA (also known as NTRK1) gene is a target of alternative splicing which can result in several different protein isoforms. Presently, three human isoforms (TRKAI, TRKAII and TRKAIII) and two rat isoforms (TRKA L0 and TRKA L1) have been described. Results: We show here that human TRKA gene is overlapped by two genes and spans 67 kb-almost three times the size that has been previously described. Numerous transcription initiation sites from eight different 5' exons and a sophisticated splicing pattern among exons encoding the extracellular part of TRKA receptor indicate that there might be a large variety of alternative protein isoforms. TrkA genes in rat and mouse appear to be considerably shorter, are not overlapped by other genes and display more straightforward splicing patterns. We describe the expression profile of alternatively spliced TRKA transcripts in different tissues of human, rat and mouse, as well as analyze putative endogenous TRKA protein isoforms in human SH-SY5Y and rat PC12 cells. We also characterize a selection of novel putative protein isoforms by portraying their phosphorylation, glycosylation and intracellular localization patterns. Our findings show that an isoform comprising mainly of TRKA kinase domain is capable of entering the nucleus. Conclusions: Results obtained in this study refer to the existence of a multitude of TRKA mRNA and protein isoforms, with some putative proteins possessing very distinct properties.publishersversionPeer reviewe

International Update on Screening for Abdominal Aortic Aneurysms:Issues and Opportunities

Screening for abdominal aortic aneurysm (AAA) has been shown to be effective at reducing AAA-related mortality and is cost-effective. There are, however, significant variations in AAA screening protocols between healthcare systems and numerous challenges to be overcome. Further evidence of cost- and clinical effectiveness is likely to be required before there is widespread international adoption of AAA screening