Sa1686 Characteristics of and Associations Among the HER2 Signal-Related Factors Thought to Be Predictive Factors for the Effectiveness of Trastuzumab Therapy in Gastric Cancer

Abstract

Background: Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that is highly up-regulated in inflammatory bowel disease and reduces the expression of the intestinal specific homeodomain transcription factor, CDX2. CDX2 is a master regulator of the intestinal homeostasis and is considered to be a tumor suppressor. WNT/ β-catenin signaling is critical for intestinal cell proliferation, but decreased CDX2 expression contributes to an abnormal increased activation of WNT signaling and progression of cancer. Furthermore, low CDX2 expression is associated with enhanced epithelial-to-mesenchymal transition (EMT) in colorectal cancer (CRC). Inflammation is often observed at the invasive front of the colon cancer, and it has been suggested that the inflammatory microenvironment around a tumor also induces EMT. Hence, it is likely that down-regulation of CDX2 expression in the invasive front in CRC contributes to enhanced tumorigenicity by regulating genes associated with the WNT/β-catenin pathway. Aim: To determine the role of TNFα in the regulation of CDX2 expression in CRC and its influence onWNT/β-catenin signaling. Methods: Carcinoma specimens were obtained from patients undergoing surgical resection of the rectum. Ten cases with tumor cell buddings were selected, and the expression patterns of TNFα and CDX2 at the invasive front were evaluated on immunostained slides. The mechanisms behind TNF-α-mediated down-regulation of CDX2 were investigated in vitro by selective inhibitors, and the impact of TNF-α on APC, AXIN2 and GSK3β expression were analyzed by quantitative real-time PCR (qPCR) in Caco-2 cells. Subsequently, in vivo CDX2-DNA interactions to APC, AXIN2 and GSK3β gene regulatory elements were investigated by chromatin immunoprecipitation in relation to the TNF-α status (presence vs. absence). Results: Immunohistochemical staining showed reduced CDX2 positive-cells in tumor buddings in areas with TNF-α expression in the surrounding inflammatory cells. TNF-α treatment showed a dosedependent decrease of CDX2 mRNA and protein expression dependent on p38 and NFκB, but not on JNK and ERK signaling pathways. Down-regulation of CDX2 leads to significantly decreased levels of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05) mRNA. In line with these results, TNF-α treatment impaired the ability of CDX2 to interact and activate the expression of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05). Conclusions: This study suggests that TNF-α has a tumor-promoting effect on CRC by influencing the activity of WNT signaling through down-regulation of CDX2 expression. These findings provide a novel insight into the molecular regulation of genes in the β-catenin degradation complex in response to the pro-inflammatory cytokine TNFα in cancer cells.