Abstract
Wnt5a is a non-canonical signaling Wnt. Low expression of WNT5A is correlated with poor prognosis in breast cancer patients. The highly invasive breast cancer cell lines, MDA-MB-231 and 4T1, express very low levels of WNT5A. To determine if enhanced expression of WNT5A would affect metastatic behavior, we generated WNT5A expressing cells from the 4T1 and MDA-MB-231 parental cell lines. WNT5A expressing cells demonstrated cobblestone morphology and reduced in vitro migration relative to controls. Cell growth was not altered. Metastasis to the lung via tail vein injection was reduced in the 4T1-WNT5A expressing cells relative to 4T1-vector controls. To determine the mechanism of WNT5A action on metastasis, we performed microarray and whole-transcriptome sequence analysis (RNA-seq) to compare gene expression in 4T1-WNT5A and 4T1-vector cells. Analysis indicated highly significant alterations in expression of genes associated with cellular movement. Down-regulation of a subset of these genes, Mmp13, Nos2, Il1a, Cxcl2, and Lamb3, in WNT5A expressing cells was verified by semi-quantitative RT-PCR. Significant differences in transcript splicing were also detected in cell movement associated genes including Cd44. Cd44 is an adhesion molecule with a complex genome structure. Variable exon usage is associated with metastatic phenotype. Alternative spicing of Cd44 in WNT5A expressing cells was confirmed using RT-PCR. We conclude that WNT5A inhibits metastasis through down-regulation of multiple cell movement pathways by regulating transcript levels and splicing of key genes like Cd44.
Highlights
The Wnt family of proteins consists of at least 19 members, that can be broadly divided into two general categories: 1) the canonical, ß-catenin pathway; and (2) the non-canonical, ßcatenin independent pathway [1,2,3]
Previous studies have shown that loss of WNT5A is associated with early relapse of invasive breast cancer and, in a retrospective study, immunohistochemical detection of WNT5A in tumors was inversely correlated with metastasis and survival [8,9,10]
We propose that WNT5A acts at least in part to inhibit metastasis by regulating movement of tumor cells through these changes in gene expression and transcript splicing
Summary
The Wnt family of proteins consists of at least 19 members, that can be broadly divided into two general categories: 1) the canonical, ß-catenin pathway; and (2) the non-canonical, ßcatenin independent pathway [1,2,3]. Many canonical signaling Wnts have a clear role in breast cancer progression [2,6]. A screen of Wnt expression in various established tumor cell lines showed that, in general, canonical Wnts were up-regulated in cancer cell lines relative to normal human mammary epithelial cells while the expression of non-canonical Wnts, including WNT5A, WNT5B and WNT16, was down-regulated [7,8]. It was shown that WNT5A is critical for macrophage-induced invasion of breast cancer cell lines [11,12]. This suggests WNT5A may play different roles, which may be stage dependent or involve cues from the microenvironment An in-depth understanding of the mechanism of WNT5A action in breast cancer progression and metastasis is required
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