Abstract
BackgroundWW Domain Containing Oxidoreductase (WWOX) belongs to the unusual tumor suppressors, whose molecular function is not fully understood in bladder cancer, especially regarding interaction with Activator Protein 2 (AP-2) α/γ transcription factors. Thus, using lentiviral systems we created an in vitro model overexpressing or downregulating WWOX in CAL-29 cell line to assess invasiveness pathways. Surprisingly, while WWOX overexpression was accompanied with increased expression of both AP-2 factors, its downregulation only affected AP-2α level but not AP-2γ which remained high.MethodsUsing cellular models and unpaired t-test or Wilcoxon test, we investigated significant changes in biological processes: clonogenicity, extracellular matrix adhesion, metalloproteinases activity, 3D culture growth, proliferation, mitochondrial redox potential and invasiveness. Relative gene expression acquired through Real-Time qPCR has been analyzed by Welch's t-test. Additionally, using oncoprint analysis we distinguished groups for bioinformatics analyzes in order to perform a follow-up of in vitro experiments.ResultsDownregulation of WWOX in bladder cancer cell line intensified ability of single cell to grow into colony, mitochondrial redox potential and proliferation rate. Moreover, these cells shown elevated pro-MMP-2/9 activity but reduced adhesion to collagen I or laminin I, as well as distinct 3D culture growth. Through global in silico profiling we determined that WWOX alters disease-free survival of bladder cancer patients and modulates vital processes through AP-2 downstream effectors.ConclusionsOur research indicates that WWOX possesses tumor suppressor properties in bladder cancer but consecutive examination is required to entirely understand the contribution of AP-2γ or AP-2α.
Highlights
WW Domain Containing Oxidoreductase (WWOX) belongs to the unusual tumor suppressors, whose molecular function is not fully understood in bladder cancer, especially regarding interaction with Activator Protein 2 (AP-2) α/γ transcription factors
Construction of an in vitro CAL‐29 cell line model To investigate the relationship between WWOX, AP-2α and AP-2γ in bladder cancer, WWOX gene expression was downregulated or upregulated in the CAL-29 cell line
As the WWOX interacts with many proteins including transcription factors AP-2 α and γ [26], the present study examined relationship between these proteins in bladder cancer we pursued this issue in further bioinformatics analyzes
Summary
WW Domain Containing Oxidoreductase (WWOX) belongs to the unusual tumor suppressors, whose molecular function is not fully understood in bladder cancer, especially regarding interaction with Activator Protein 2 (AP-2) α/γ transcription factors. Using lentiviral systems we created an in vitro model overexpressing or downregulating WWOX in CAL-29 cell line to assess invasiveness pathways. While WWOX overexpression was accompanied with increased expression of both AP-2 factors, its downregulation only affected AP-2α level but not AP-2γ which remained high. The incidence of bladder cancer (BLCA) has been decreasing over recent years in the US yet in some European countries it is steadily rising [1]. It remains the sixth most common cancer among men [1]. Several subtypes of BLCA are recognized, and these are distinguished on the basis of their molecular profile. BLCA is a heterogeneous disease in terms of its molecular and clinicopathological status; it is associated with disruptions of pathways, with the best known being FGFR3/ RAS, PI3K/AKT/mTOR or TP53/RB1 [4]
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