The influence of the WWOX gene on the regulation of biological processes during endometrial carcinogenesis

Abstract

The purpose of the present study was to investigate the role of WW domain containing oxidoreductase (WWOX) downregulation in biological cancer-related processes in normal (non-malignant) and cancer endometrial cell lines. We created an in vitro model using the normal endometrial cell line, THESC, and 2 endometrial cancer cell lines with varying degrees of differentiation, the Ishikawa (well-differentiated) and the MFE296 (moderately differentiated) cells, in which the WWOX tumor suppressor gene was silenced using Gipz lentiviral shRNA. In this model, we examined the changes in invasiveness via biological assays, such as zymography, migration through a basement membrane, the adhesion of cells to extracellular matrix proteins, anchorage-independent growth and colony formation assay. We also evaluated the correlation between the mRNA expression of the WWOX gene and genes involved in the processes of carcinogenesis, namely catenin beta-1 (CTNNB1) and zinc finger E-box binding homeobox 1 (ZEB1) (gene transcription), cadherin 1 (CDH1) and ezrin (EZR) (cell adhesion), vimentin (VIM) (structural proteins), as well as phosphatase and tensin homolog (PTEN) (tumor suppression) and secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) (SPARC) (cell growth regulation) by RT-qPCR. Downregulation of the WWOX gene in the moderately differentiated MFE296 cell line caused decreased migratory capacity, and a reduction of matrix metalloproteinase-2 (MMP-2) activity. However, these cells grew in semisolid medium and exhibited higher expression of CDH1 and EZR (cell adhesion) and secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) (cell growth regulation). Moreover, in the well-differentiated endometrial cancer (Ishikawa) cell line, WWOX gene silencing resulted in an increased ability of the cells to proliferate indefinitely. Additionally, WWOX regulated changes in adhesion potential in both the normal and cancer cell lines. Our results suggest that the WWOX tumor suppressor gene modulated the processes of cell motility, cell adhesion, gene expression and remodeling in endometrial cell lines.

WWOX Loses the Ability to Regulate Oncogenic AP-2γ and Synergizes with Tumor Suppressor AP-2α in High-Grade Bladder Cancer

“…In few cases, a synergistic effect was required between WWOX and AP-2α to demonstrate the adhesiveness reduction. The example of endometrial carcinoma revealed that integrin β4 and α2 are upregulated after WWOX silencing [62], suggesting that WWOX may regulate adhesion to laminin and collagens, respectively [63]. This is in line with another study on BLCA, where WWOX silencing increased adhesion to collagen IV [47].…”

“…Many pathways appeared in at least three comparisons; MAPK, TGFβ, WNT, NOTCH or ERBB signaling were found to be dependent on WWOX, AP-2α and AP-2γ, i.e., MAPK [78][79][80], TGFβ [80][81][82], WNT [83,84], NOTCH [85][86][87] and ERBB [88,89]. Since WWOX is considered a global gene expression modulator [62] and both AP-2 transcription factors regulate thousands of genes and biological processes via specific pathways [79], it was foreseeable that changes in their expression affect cellular events at the broad scale. CAGE data were filtered to indicate the most frequent DEGs; these genes were also screened as putative AP-2 targets.…”

Fragile Gene WWOX Guides TFAP2A/TFAP2C-Dependent Actions Against Tumor Progression in Grade II Bladder Cancer

“…The remaining comparisons indicated that adhesion to collagen IV and laminin I was reduced during overexpression of WWOX and AP-2α or WWOX and AP-2γ (W/A or W/C, respectively). A previous study from our department identified that after WWOX silencing, an increase in the expression of integrin α2 and β4 in MFE-296 endometrial cancer cell line was observed ( 19 ), thus indicating the possibility of regulating adhesion to collagen and laminin, respectively ( 58 ). To the best of our knowledge, there are no scientific reports analyzing changes in the integrin profile regarding AP-2α/γ, while with the use of ELIXIR-approved ( 59 ) Integrated System for Motif Activity Response Analysis (ISMARA) tool ( 60 ), it was possible to confirm that the α2 integrin-encoding gene is in the top 200 AP-2α targets (based on the deposited project “ Inflammatory response time course, HUVEC (Inoue, 2006) ”; dataset3_v2; ZNF711_TFAP2A_TFAP2D), and both α2 and β4 integrin-encoding genes in the top 200 AP-2γ targets (based on the deposited project “ ENCODE cell lines, expression (Ernst 2011) ”; dataset6.1_ENCODE_expression_v2; TFAP2C).…”

“…Assays of colony formation, wound healing and 3D culture growth were performed to systematize function of each cellular variant in terms of self-renewal capacity, cell motility or ability to form tumorspheres, respectively. For instance, WWOX has become involved in the regulation of colony formation ( 19 ), as has AP-2α ( 30 ). In turn, AP-2γ has been associated with cell motility ( 25 ) as well as stemness and chemoresistance ( 31 ).…”

“…The cell starvation medium was collected after 48 h incubation (37°C, 5% CO 2 ) and centrifuged. After measuring the protein concentration using Qubit Protein Assay on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Netherlands), 3µg of protein was used for SDS-PAGE analysis as described previously ( 19 ). The assay was performed in biological triplicate, technical duplicate for each cell variant.…”