Introduction & Objectives:
The WW Domain Containing Oxidoreductase (WWOX) tumor suppressor gene is located in direct proximity to chromosomal locus 16q24 that is related to tumor progression of 20-45% bladder cancers. It encodes protein containing two WW domains, the first of which recognizes proline-rich motifs of proteins such as Activating Enhancer-Binding Protein 2 Alpha (AP-2α) transcription factor (encoded by TFAP2A gene). The literature data indicate that WWOX sequestrates AP-2α outside nucleus, which suppresses its transcriptional transactivation functions.
The purpose of this research was to examine diverse properties of bladder cancer with various WWOX and TFAP2A expression levels in CAL-29 cell line (grade IV) and determine their effect on useful clinical features in patients.

Materials & Methods:
Using two lentiviral systems we created in vitro models with various WWOX and TFAP2A expression on which the cell viability, adhesion, proliferation and 3D culture growth assays were performed. 
Bioinformatic analyzes were conducted on bladder cancer data acquired from The Cancer Genome Atlas (TCGA) using Weighted Gene Correlation Network Analysis (WGCNA) and NETwork-based Gene Enrichment (NET-GE) tool with Molecular Signatures Database (MSigDB) used to assign the role of a particular gene to the biological process.

Results:
In variant with ↑WWOX/↑TFAP2A expression we observed reduced mitochondrial redox potential, decrease in the number and size of spatial colonies and lowered adhesion to fibronectin, collagen I and collagen IV in comparison to cells with ↑WWOX expression and native TFAP2A expression. Similar tendencies were noticed in variant with ↑TFAP2A overexpression and native WWOX expression compared to control cells although with additional demonstration of lower proliferative potential. Decreased size of 3D colonies was also observed in cells with ↑WWOX expression and native TFAP2A, compared to its control. 
To further investigate WWOX role, in silico analyzes (WGCNA, NET-GE) have been conducted on TCGA data of patients having ↑TFAP2A but various WWOX expression. Using WGCNA, we extracted cluster of 530 genes that noticeably differentiated samples in terms of bladder cancer papillary and non-papillary subtypes. Subsequently, ontological analysis of these genes indicated that these genes are primarily implicated in adhesion and proliferation but also in apoptosis, cell cycle or several signaling pathways (e.g. JAK-STAT, MAPK, Wnt, NFkB).

Conclusions:
Taken together, our study indicates that WWOX interplays with AP-2α in regulation of bladder cancer spatial colonies growth. However, the effect on tumor cell viability, proliferation and adhesion appears to be dependent on the AP-2α level. Thus, subsequent in silico analyzes were focused on characterizing the dissimilarities of groups differing in WWOX gene expression level. Ultimately, these tools revealed valuable data concerning correlation of clinical traits with expression profile that once examined through gene ontology, disclosed influence on crucial biological processes or signaling pathways.

This work was supported by NCN Poland grant number 2016/21/B/NZ2/01823.

WWOX–AP-2α interaction affects biology of bladder cancer and demonstrates usefulness in its clinical features’ discrimination.