J. A. Campbell1, I. Huffnagle1, G. P. Robertson2, C. R. Pameijer1 1Penn State Hershey Medical Center,Department Of Surgery,Hershey, PA, USA 2Penn State Hershey Medical Center,Department Of Pharmacology,Hershey, PA, USA
Introduction: WEE1 is a protein kinase in the BRAF pathway and is over-expressed in melanoma. It is involved in regulating cell cycle progression and is involved in tumor progression in several malignant tumors. In melanoma, experimentally induced down-regulation of WEE1 has been shown to inhibit cell growth in vitro and in vivo. Like many other proteins, WEE1 expression is regulated by microRNA. MicroRNAs are small, non-coding segments of RNA that are involved in post transcriptional regulation of gene expression and have been implicated in oncogenesis. Melanoma tumors have been shown to have very low levels of microRNA-155 (miR-155), although the downstream effect of this low expression is unknown. We evaluated a group of melanoma patient tumor samples for miR-155 expression, clinical outcome and possible mechanism of action of miR-155.
Methods: RNA was extracted from melanoma patient tumor samples. miRNA microarray analysis was performed, with confirmation by qRT-PCR. Melanoma cell lines transfected with miR-155 were used for a mouse experimental metastasis model. Luciferase reporter assay was used to show interaction between miR-155 and the 3’UTR of WEE1. Western blotting was used to show the effect of miR-155 transfection on the protein levels of WEE1 in melanoma cells.
Results: In patient tumor samples miR-155 was lost in patients who develop metastatic disease. In the mouse model, transfection of cells from two metastatic melanoma cell lines with miR-155 mimic decreased the metastatic potential of melanoma cells, with a significant difference in the number of lung metastases between miR-155 and control mice (figure 1a). Transfection of melanoma cells with miR-155 reduced WEE1 protein levels (figure 1b) while inhibition of endogenous miR-155 up-regulated WEE1 expression. Luciferase reporter assay demonstrated that miR-155 interacts with the WEE1 3’UTR and impaired gene expression in melanoma cells.
Conclusion: miR-155 is lost in patients who develop metastatic melanoma. Loss of miR-155 allows for upregulation of WEE1 in melanoma. Our in vivo studies showed significant decrease in metastasis with cells over expressing miR-155 suggesting that in melanoma miR-155 affects metastasis through WEE1 kinase.
