Abstract
We have shown that Wnt5A increases the motility of melanoma cells. To explore cellular pathways involving Wnt5A, we compared gain-of-function (WNT5A stable transfectants) versus loss-of-function (siRNA knockdown) of WNT5A by microarray analysis. Increasing WNT5A suppressed the expression of several genes, which were re-expressed after small interference RNA-mediated knockdown of WNT5A. Genes affected by WNT5A include KISS-1, a metastasis suppressor, and CD44, involved in tumor cell homing during metastasis. This could be validated at the protein level using both small interference RNA and recombinant Wnt5A (rWnt5A). Among the genes up-regulated by WNT5A was the gene vimentin, associated with an epithelial to mesenchymal transition (EMT), which involves decreases in E-cadherin, due to up-regulation of the transcriptional repressor, Snail. rWnt5A treatment increases Snail and vimentin expression, and decreases E-cadherin, even in the presence of dominant-negativeTCF4, suggesting that this activation is independent of Wnt/beta-catenin signaling. Because Wnt5A can signal via protein kinase C (PKC), the role of PKC in Wnt5A-mediated motility and EMT was also assessed using PKC inhibition and activation studies. Treating cells expressing low levels of Wnt5A with phorbol ester increased Snail expression inhibiting PKC in cells expressing high levels of Wnt5A decreased Snail. Furthermore, inhibition of PKC before Wnt5A treatment blocked Snail expression, implying that Wnt5A can potentiate melanoma metastasis via the induction of EMT in a PKC-dependent manner.
Highlights
From the ‡Laboratory of Immunology, Gerontology Research Center, and the ʈResearch Resources Branch, Gerontology Research Center, NIA, National Institutes of Health (NIH), Baltimore, Maryland 21224, the §Cancer Genetics Branch, NHGRI, NIH, Bethesda, Maryland 20892, the ¶Department of Pharmacology, Howard Hughes Medical Institute, and Institute for
The most efficient sequence, A2, was used to decrease Wnt5A in a melanoma cell line that had been stably transfected with WNT5A (UACC1273-4-7), and both M93-047 cells, and the UACC1273-4-7 cells were stained for expression of the Wnt5A protein, using immunofluorescent detection (Fig. 1c)
Cells treated with Wnt5A-A2 siRNA had not yet closed a scratch wound in this time, confirming that WNT5A knockdown resulted in a decrease in melanoma cell motility (Fig. 1D)
Summary
We have previously demonstrated the ability of Wnt5A to increase PKC activation, and melanoma cell motility, and have shown that inhibition of Wnt signaling results in a decrease in melanoma cell migration [9]. To assess this in another manner and to visually determine the effects of phorbol ester on PKC activation, we transfected our Wnt5Alow cells with the PKC-GFPII vector and subjected them to PMA treatment (200 nM) at different time points, fixed the slides, and imaged the results using confocal microscopy (Fig. 3D).
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