Abstract 2221: Necroptosis and apoptosis in human colorectal tissue
Authors: Timothy Su, Yong Li, Chang Yu, Martha J. Shrubsole, Xiangzhu Zhu, Xinqing Deng, Eugene Shubin, Wei Zheng, Harvey J. Murff, Douglas L. Seidner, Reid M. Ness, Qi Dai
Journal: Cancer Research (2018)

Abstract Background: The molecular mechanisms for colorectal carcinogenesis still remain elusive. In recent years, necroptosis has emerged as a programmed cell death regulated by RIP kinases /mixed lineage kinase domain-like (MLKL) pathway, involving the release of intracellular "danger signals" which results in inflammation. On the other hand, classic apoptosis is regulated by Bax-induced caspase pathway and does not lead to inflammation. Transient receptor potential melastatin related 7 (TRPM7), a MLKL downstream target for the mediation of Ca2+ influx, was found to be involved in TNF-induced necroptosis (Nat Cell Bio 2014;16:55). Both necroptosis and apoptosis are important barriers to prevent normal cells from developing cancer. No study has examined how necroptosis biomarkers (MLKL, TRPM7) and pro-apoptosis biomarker (Bax) are related to inflammation (COX-2) and cell proliferation (Ki-67) and how MLKL relates to TRPM7 in human colorectal tissue. Methods: In our randomized trial (R01CA149633, NCT01105169) “Personalized Prevention of Colorectal Cancer Trial” conducted among colorectal polyp patients or those at high risk of colorectal cancer, colorectal biopsies from 250 participants were collected prior to intervention in the trial. Formalin-fixed paraffin-embedded tissues were serially sectioned, and three levels of serial sections spaced 50 µm apart were mounted on one slide for each tissue block. The protein expressions of COX-2, Bax, pMLKL, TRPM7, and Ki-67 were detected immunohistochemically following the EnVision+ System-HRP kit. Positive cells in the upper and bottom zones were evaluated separately by a zonal quantitative analysis procedure using BioQuant imaging software. Correlation analysis was used to compute Pearson product-moment correlation coefficient between necroptosis, apoptosis and inflammation biomarkers. Results: The expression of selected biomarkers showed stronger positive signals in the upper zone than in the bottom zone, with the exception of Ki-67 which showed stronger staining in the bottom zone of crypts. Epithelial Bax had strong positive correlation with COX-2 in the same zones (r=0.25 - 0.36, p&amp;lt; 0.001), and overall Bax expression showed weaker association with stromal COX-2 (r=0.13, p=0.04). pMLKL and TRPM7 were not significantly correlated with COX-2 except a weak correlation between stromal COX-2 and overall TRPM7 (r=-0.174, p=0.008). pMLKL expression was positively correlated with TRPM7 in the upper zone of crypts (r=0.19, p=0.004), and both pMLKL and TRPM7 were not significantly correlated with Bax expression. Conclusions: Our study confirmed a positive correlation of pMLKL and TRPM7, as suggested by previous cell studies, in human non-tumoral colorectal tissue. The positive correlation between Bax and COX-2 suggests that elevated inflammation may result in increased apoptosis. On the other hand, pMLKL is not related to COX-2, although stromal COX-2 is inversely associated with overall TRPM7. Citation Format: Timothy Su, Yong Li, Chang Yu, Martha J. Shrubsole, Xiangzhu Zhu, Xinqing Deng, Eugene Shubin, Wei Zheng, Harvey J. Murff, Douglas L. Seidner, Reid M. Ness, Qi Dai. Necroptosis and apoptosis in human colorectal tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2221.