LY75 Ablation Mediates Mesenchymal-Epithelial Transition (MET) in Epithelial Ovarian Cancer (EOC) Cells Associated with DNA Methylation Alterations and Suppression of the Wnt/β-Catenin Pathway

Abstract

Growing evidence demonstrates that epithelial–mesenchymal transition (EMT) plays an important role in epithelial ovarian cancer (EOC) progression and spreading; however, its molecular mechanisms remain poorly defined. We have previously shown that the antigen receptor LY75 can modulate EOC cell phenotype and metastatic potential, as LY75 depletion directed mesenchymal–epithelial transition (MET) in EOC cell lines with mesenchymal phenotype. We used the LY75-mediated modulation of EMT as a model to investigate for DNA methylation changes during EMT in EOC cells, by applying the reduced representation bisulfite sequencing (RRBS) methodology. Numerous genes have displayed EMT-related DNA methylation patterns alterations in their promoter/exon regions. Ten selected genes, whose DNA methylation alterations were further confirmed by alternative methods, were further identified, some of which could represent new EOC biomarkers/therapeutic targets. Moreover, our methylation data were strongly indicative for the predominant implication of the Wnt/β-catenin pathway in the EMT-induced DNA methylation variations in EOC cells. Consecutive experiments, including alterations in the Wnt/β-catenin pathway activity in EOC cells with a specific inhibitor and the identification of LY75-interacting partners by a proteomic approach, were strongly indicative for the direct implication of the LY75 receptor in modulating the Wnt/β-catenin signaling in EOC cells.

LY75 Suppression in Mesenchymal Epithelial Ovarian Cancer Cells Generates a Stable Hybrid EOC Cellular Phenotype, Associated with Enhanced Tumor Initiation, Spreading and Resistance to Treatment in Orthotopic Xenograft Mouse Model

“…Finally, our in vivo data have also confirmed our previous findings [ 27 ] for the direct implication of the LY75 receptor in modulating the Wnt/β-catenin signaling in EOC cells. Here, we show that Ly75 gene ablation also directed the suppression of the Wnt/β-catenin pathway in tumor samples from mice IB-injected with SKOV3-E and SKOV3-E+M cells.…”

“…We now show that SKOV3-E cells display hybrid phenotype and stemness characteristics, which could explain their aggressive behavior in our experimental EOC animal model. As reported before [ 26 , 27 ], SKOV3-M cells exhibited a typical EMT mesenchymal markers-expression profile, while SKOV3-E (Ly75KD) cells have displayed EMT-related epithelial features; however, we have now found that SKOV3-E cells also strongly express the mesenchymal marker vimentin ( Figure 3 ), as a strong vimentin expression was also retained in tumors, extracted from mice, IB-injected with SKOV3-E and SKOV3-E+M cells ( Figure 4 ). Vimentin is highly expressed in most epithelial cancers including EOC, and its expression correlates with disease progression and poor prognosis [ 42 , 43 ].…”

“…For our in vivo experiments, we used the orthotopic intrabursal (IB) mice model to investigate the tumor-initiating and metastatic potential of EOC cells with mesenchymal (M), epithelial (E) and mixed E+M phenotype. The previously generated cell clones sh-control-SKOV3 (SKOV3-M) and sh-LY75KD-SKOV3 (SKOV3-E) [ 27 ] were initially transfected with firefly luciferase plasmid in order to facilitate further monitoring by bioluminescent imaging for tumor formation and spreading. In our initial set (phase 1) of experiments, stable luciferase-expressing SKOV3-M and SKOV3-E clones, as well as a mixed population of SKOV3-E+M cells (1:1 ratio), were directly injected under the bursal membrane (between the bursa and the ovary) of female SCID mice (n = 5 for each experimental group; see Materials and Methods for details).…”

“…We have previously shown that LY75 supports the active status of the Wnt/β-catenin pathway in EOC cells, and the LY75 depletion results in the suppression of Wnt/β-catenin activity, associated with decreased β-catenin expression and the strong induction of Axin1 and APC2, representing members of the Wnt/β-catenin suppressor complex [ 27 ]. Analysis of primary ovarian tumor samples obtained from our phase 1 experimental animals confirmed these data, as tumor tissues derived from SKOV3-E- and SKOV3-E+M-injected mice displayed considerably diminished β-catenin expression and enhanced Axin1 and APC2, following both Western blot and immunohistochemistry (IHC) analyses, as compared to SKOV3-M-injected mice ( Figure 2 A,B).…”

“…Moreover, re-expression of a shRNA-resistant LY75 gene variant in the LY75 knockdown (KD) SKOV3 clones completely restored the initial mesenchymal phenotype and re-established the SKOV3 parental pattern of mesenchymal markers’ expression [ 26 ]. We have also demonstrated that the LY75 kD-mediated MET in EOC cells is associated with DNA methylation alterations and suppression of the Wnt/β-catenin pathway [ 27 ].…”