Characterization of the impact of NKCC1 in colorectal carcinogenesis

Introduction
Colorectal cancer associated with inflammatory bowel disease also known as colitis-associated cancer (CAC) is considered as a separate entity from sporadic colorectal cancer (CRC). Previous studies demonstrated that Solute carrier family 12 member 2 (SLC12A2/NKCC1) is a protein overexpressed in pre-tumoral and tumoral lesions of CAC and CRC. This Na-K-Cl cotransporter localized at the cell membrane and in the cytoplasm is mainly expressed in cells involved in fluid secretion and in intestinal stem cells. NKCC1 have been reported to be activated during oxidative stress which is recognized to be a major player in carcinogenesis in CRC and CAC, inducing enhanced cell proliferation and DNA damages. Despite NKCC1 up-regulation and change of distribution within dysplastic and colorectal cancers tissues, little is known about its impact and role in colorectal carcinogenesis in relation to oxidative stress.

Aim
The aim of this study is to investigate the impact of NKCC1 in colorectal carcinogenesis and more precisely its role in the regulation of oxidative stress in tumor cells.

Methods
Oxidative stress was induced on 3 different CRC cell lines (Caco-2, HT-29, HCT116) by adding H202 or by UV irradiation. Concurrently, cells were incubated with NKCC1’s chemical inhibitor, bumetanide (BMT), or by NKCC1 Silencing (siRNA). Viability of cells was measured using Cell titer glo 2.0 kit (Promega). In addition, characterization of protein of Endoplasmic Reticulum (ER) stress (BiP), mTORC1 signaling (p70 S6 kinase and its phosphorylated form), apoptosis (caspase3 cleaved form) and β-catenin were monitored by Western blot.

Results
Caco-2 cells subjected to 24-hour exposure at 1 mM H2O2 and 200 µM bumetanide showed 68% to 18% decreased viability without and with BMT respectively, while HT29 viability decreased from 62% to 36% respectively. HCT116 viability dropped from 12% to 5% with BMT treatment. The loss of viability was demonstrated to be BMT concentration-dependent in Caco-2. This effect was observed exclusively under induced oxidative stress. Western blot analyses performed when using H2O2 and BMT co-treatment revealed the activation of the mTORC1 pathway and an ER stress increase. Upon specific NKCC1 silencing, a significant decrease in mTORC1 pathway activity was observed with a marked increase in the cleaved form of Caspase 3 and a strong reduction of β-catenin signal.

Conclusions
The inhibition of the catalytic site of NKCC1 led to a decrease in the viability of the 3 colorectal cell lines tested when exposed to oxidative stress. At the molecular level, we found an enhanced activation of the mTORC1 pathway, together with an increase in endoplasmic reticulum stress. When NKCC1 expression was silenced, apoptosis event could be detected, as well as the dysregulation of both the mTORC1 pathway and β-catenin. Collectively, these results suggest the importance of NKCC1 expression and activity as a Na-K-Cl cotransporter, in survival and proliferation of CRC tumors cells. The characterization of the impact of NKCC1 in carcinogenesis is currently under investigation using the 3D-organoid model derived from colorectal cancer surgical margins, from ulcerative colitis patients and using tumoroids.