Abstract
Expression of the Wnt ligand Wnt5a is frequently elevated in melanoma and is thought to be a critical regulator of cell movement during metastasis. However, the mechanisms regulating its expression are unknown. We find that the level of secreted Wnt5a varies by as much as 10-fold between cell lines and correlates more strongly with invasion than total cellular levels. Our results indicate that the RNA helicase Mov10 plays a role in Wnt5a synthesis and secretion. Inhibition of Mov10 increases secreted Wnt5a levels in melanoma cells by increasing Wnt5a synthesis and acylation. This is achieved by increasing fatty acid synthase (FASN) and stearoyl-CoA desaturase expression, leading to elevated levels of palmitoleoyl-CoA, required for Wnt ligand lipid modification and secretion. Melanoma tumors exhibit reduced expression of Mov10 compared with benign nevi and Mov10 levels inversely correlate with FASN levels in primary tumors. These results reveal a previously unappreciated role for aberrant lipid metabolism in regulating Wnt5a signaling that may be a critical step in melanoma progression.
Highlights
We expected to see greater invasion in the higher Wnt5a-expressing lines if Wnt5a is a significant factor in determining invasiveness; we found little correlation between the invasiveness of the cell line and the Levels of Wnt5a secretion correlates with cell invasion in melanoma cells level of Wnt5a measured in the cell lysates in a three-dimensional collagen invasion assay (Figures 1a and b)
A similar response was seen in cells ectopically expressing Wnt3a. These results indicate the increase in Wnt5a secretion was not caused by Mov[10] binding to Wnt5a mRNA as the Mov10-binding sites are in the 5′ and 3′-untranslated region (UTR)
Our studies indicate that Mov[10] short hairpin RNA (shRNA) increases expression of fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) in cultured melanoma cell lines promoting Wnt5a secretion
Summary
Wnt5a is a member of the Wnt family of secreted ligands that signals independently of β-catenin-mediated transcription.[1,2] Wnt5a is strongly implicated in promoting metastatic behavior in melanoma and other types of cancer and is thought to function as an autocrine signaling factor to promote cell motility.[3,4,5,6,7,8,9,10,11] It has been demonstrated that Wnt5a protein levels are elevated in latestage melanoma patient samples, and increased Wnt5a expression increases cell motility, invasion and polarization of the cytoskeleton in melanoma cell lines.[9,10,11] Wnt5a interacts with the receptor tyrosine kinase Ror[2] and downstream signaling requires the cytosolic adaptor protein Disheveled.[12,13] Inhibition of the Wnt5a receptor Ror[2] in melanoma blocks lung colonization in mice, demonstrating a requirement for the Wnt5a pathway in extravasation and colonization during metastasis.[14]. The low Wnt5a secreting WM239A cells expressing Mov[10] shRNA displayed significantly higher collagen invasion, and the effect was observed with two short hairpins (Figures 2e and f). To address whether the increase in invasion is caused by Wnt5a signaling activity the Wnt5a receptor Ror[2] was inhibited by two separate short hairpins in control and Mov[10] shRNA-expressing UACC903 cells (Supplementary Figure S2). FASN is expressed at low levels in most cells but is elevated in many types of cancer including melanoma.[33,34,35,36,37,38] Inhibition of Mov[10] by shRNA caused an increase in the levels of FASN protein that was suppressed by expression of a bypass Mov[10] containing shRNA-resistant mutations (Figure 4d). We found inhibiting FASN with the a Palmitic acid b shCtrl shMov[10]
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