The role of WT1 isoforms in vasculogenic mimicry and metastatic potential of human triple negative breast cancer cells

Abstract

Triple negative breast cancer (TNBC) is highly aggressive and has a few therapeutic treatments, so new targeted therapy and biomarkers are required to provide alternative choices for treating TNBC patients. Recent studies showed that vasculogenic mimicry (VM), the formation of blood channels by aggressive cancer cells that mimic endothelial cells, is a factor contributing to poor prognosis in TNBC. Wilms' tumor 1 (WT1) gene has been found to be highly expressed in TNBC, and has 4 major distinct isoforms; isoform A (-17AA/-KTS; -/-), isoform B (+17AA/-KTS; +/-), isoform C (-17AA/+KTS; -/+) and isoform D (+17AA/+KTS; +/+). The involvement of each WT1 isoform in TNBC progression remains largely unclear. In this study, WT1 isoform-overexpressing cell sublines were established from a TNBC cell line, MDA-MB-231, by stable transfection, and the aggressive behavior of the cell sublines were evaluated. Only the WT1 isoform B- and isoform C-overexpressing cell sublines showed the significant increase in VM forming capability compared to the parental cell line and other isoform cell sublines. qRT-PCR was used to explore the change in expression level of two VM-related genes, EphA2 and VE-cadherin. All WT1 isoform cell sublines showed up-regulation of EphA2 but the levels detected in the isoform B- and isoform C-cell sublines were higher than those observed in other cell sublines. In contrast, significant up-regulation of VE-cadherin was found only in isoform A- and isoform D-cell sublines. Isoform B- and isoform C-cell sublines showed higher rates of cell migration compared to those of other cell sublines, as determined by both wound healing and Transwell assays. Gelatin zymography revealed increased MMP-9 enzyme production in isoform D-cell subline compared to the parental cell line, but this change was not observed in other cell sublines. Western blot analysis showed significantly increased expression of β-catenin in isoform B- and isoform C-cell sublines, compared to parental cell line and other isoform cell sublines. In conclusion, our findings demonstrate that WT1 isoforms play different roles in modulating the VM-forming capacity and metastatic potential of TNBC cells.

Keywords: Cell migration; EphA2; Triple negative breast cancer; Vasculogenic mimicry; WT1 isoforms; β-catenin.