Inhibition of Percupine has a differential impact on the gene expression signature of breast cancer cell lines of different levels of agrressiveness

Abstract

Wnt pathway is involved in cellular processes which are dysregulated in cancer as cell renewal, proliferation and EMT. In particular, in breast cancer (BC) it has a role in both tumor initiation and progression. Porcupine’s palmitoylation of Wnt ligands is required for their proper signaling and release and its inhibition showed anti-tumoral effects on different BC models. In order to study if the inhibition of Porcupine (PORCN) had different effects regarding to the aggressiveness of the BC cell lines, we analyzed the impact of IWP-2, a PORCN inhibitor, in the TNBC cell line MDA-MB231 and in the less aggressive MCF-7 cell line. Cells were treated with 5 µM IWP-2 or DMSO as control. IWP-2 reduced the synthesis of Wnt ligands and target genes in MCF-7 but not in MDA-MB-23 1 cells (qPCR, 24 h, *p<0.05: WNT3A*, AXIN2*, CCND1*, C-MYC*, CCNB1*). We also found a decrement in CYCLIN B1 protein levels in MCF-7 cells. However, it reduced the expression of ß-CATENIN in both cell lines (WB, 48 h). Furthermore, cell number was also reduced in both cases (TB, 72 h). When analyzing stem cell markers, SOX2 mRNA was decreased and KLF-4 increased in MCF-7 and MDA-MB-231 suggesting that PORCN inhibition has a similar effect on the stem-cell phenotype in both cell lines. However, the number of colonies was reduced in MDA-MB-231 but not in MCF-7 cells (CFA, 8 d). In hanging drop assays (6 d), both cell lines formed bigger but less compact mammospheres than controls, with a stronger effect in MCF-7 cells, suggesting a loss of cell adhesion in this cell line. This was also consistent with reduced E-CADHERIN expression in MCF-7 cells. Regarding EMT, no differences were found in SNAIL1, ZEB-1 and E-CADHERIN expression at the mRNA level with IWP-2 treatment although TGF-ß synthesis was decreased in MCF-7 cells. Our results suggest that the differences in the genetic background and phenotype of breast cancer cells can modulate the effect of PORCN inhibition over tumoral properties in vitro.