Loss of WWOX induces ANGPTL4 and ROS production in breast cells

FERGUSON BW; GAO X; ZELAZOWSKI MJ; LANGE S; ABBA MC; WOOD RD; ALDAZ CM

Washington, DC

Congreso; 104th Annual Meeting of the American Association for Cancer Research; 2013

AACR

Expression of the WW-domain containing oxidoreductase (WWOX) gene is lost in many human malignancies including breast cancer. It has been suggested that WWOX is potentially acting as a tumor suppressor in breast cancer. In order to understand the effects of loss of WWOX expression we used an shRNA-mediated approach to silence expressionof this gene in normal human breast cells (MCF10F). Microarray analysis identified 671 commonly deregulated probes between two WWOX-silenced lines generated from shRNAs targeting two independent regions of the WWOX transcript. We found that genes involved in cell cycle/proliferation and DNA damage were significantly enriched in the list of up-regulated genes while genes involved in oxidation-reduction and wound healing were enriched in the list of down-regulated genes. ANGPTL4 was found to be one of the most significantly up-regulated genes in WWOX-silenced cells. ANGPTL4 is known to be an important player in lipid metabolism and energy balance. Importantly recent findings have demonstrated ANGPTL4 to also play key roles in breast cancer progression andmetastasis. We validated the upregulation of ANGPTL4 observed in the microarray at them RNA level by qPCR and the protein level by ELISA. We also show that this upregulation can be reversed by reestablishing WWOX expression in previously silenced cells suggesting an inverse correlation between WWOX and ANGPTL4. Supporting these findings, meta-analysis of data from three independent breast cancer gene expression studies (n= 819 cases) demonstrated a significant inverse correlation between WWOX and ANGPTL4 expression. WWOXlow/ANGPTL4high tumors were enriched in triple-negative breast cancer and basal-like tumors. ANGPTL4 signaling is known to upregulate production of reactive oxygen species (ROS) in metastatic breast cancer cells. Interestingly, we found that silencing WWOX in human breast cells or MEFs results in a significant increase in NADPH oxidase-dependent ROS production. Since ROS is a well-known inducer of DNA damage we hypothesized that WWOX silenced cells could have higher levels of DNA damage. In tune with this hypothesis, further analysis of our gene expression data set revealed significant upregulation of multiple genes involved in DNA repair including BRCA1, RAD51 and FANCA in WWOX-silenced MCF10 cells. In additional studies, we determined that WWOX KO MEFs display significantly higher levels of DNA double-strand breaks when compared with WT counterparts as determined by colabeling with 53BP1 and phospho-H2AX antibodies.In summary, we show that loss of the putative tumor suppressor WWOX results in increased levels of the breast metastasis associated gene ANGPTL4 in breast cells and is associated with increased levels of ROS.