Oxidative Stress Induces WNT Canonical/Non-Canonical Pathway Modulation in Colon Cancer Cells with APC or beta-Catenin Mutation

09-P28. Oxidative Stress Induces WNT Canonical/Non-Canonical Pathway Modulation in Colon Cancer Cells with APC or β-Catenin Mutation Introduction: It has long been recognised that the aberrant regulation of WNT/β-catenin signaling is involved in the pathogenesis of colorectal cancer. Chronic inflammation predisposes to colon carcinogenesis by increased reactive oxygen species (ROS) levels and can impair the Wingless/It (WNT)/β-catenin. This pathway is essential for gut morphogenesis, tissue homeostasis, and self-renewal, and its aberrant activation may drive the colorectal cancer (CRC), but the molecular mechanisms involved in CRC progression are still undefined. To evaluate the molecular relationship between oxidative stress and canonical/non-canonical WNT pathways, we analyzed the response to ROS exposure in CRC cell lines with different WNT signaling behaviour. Methods: HCT116 (β-catenin mutated) and SW480 (APC mutated) cells were exposed to hydrogen peroxide (H2O2) as oxidizing agent for different times and concentrations (from 50 μM to 10 mM). We assayed cell viability/mitochondria activity by MTS and cell cycle by FACS. Gene expression was evaluated by SYBR Green qRT-PCR in cells under acute stress conditions [2 mM and 10 mM] for 15' and 30'. Protein expression was analysed by IHC. Statistical analysis was performed by t-test (p value <0.05). Results: MTS revealed rates of cell inhibition and growth at different H2O2 concentrations. FACSafter H2O2 [2mM] treatment at 15', SW480 increased in G1 and G2 and decreased in S, whereas HCT116 increased in G1 and slightly reduced in G2; after 30', SW480 enhanced in G1 and S, and reduced in G2, whereas HCT116 diminished in G1 and increased in S/G2. In SW480 cells, acute stress induced by lower H2O2 concentration [2 mM] upregulated gene expression of canonical LRP6 and LEF1, and non-canonical ROR2 and JUN/AP1 molecules. In HCT116, the same H2O2 concentration [2 mM] reduced ROR2 and LRP6 expression of WNT co-receptor and differently affected the WNT transcription factors, upregulating LEF1 (canonical) and downregulating JUN/AP1 (noncanonical). Regarding APC, it showed a behaviour dependent on time and concentration of H2O2 treatment. IHC protein expression analysis showed that H2O2 treatment induced FZD6 in HCT116 cytoplasm and Ecadherin in SW480 cytoplasm, whereas β-catenin increased in both cell lines. Intriguingly, oxidative stress induced a de novo APC expression in cytoplasm of both cell lines. Conclusions: In CRC cells harbouring APC or β-catenin mutations, oxidative stress differently affects the WNT pathways at gene and protein expression levels. Our results could unravel an APC central role in driving and maintaining tumorigenesis and a novel scenario for innovative CRC therapeutic approaches.