P0051 Is NKCC1, a marker of dysplasia associated with inflammation and CAC, playing a role in colorectal carcinogenesis?
Authors: Adam P , Salée C , Stepniak M , Loly J P , Vieujean S , Reenaers C , Coimbra C , Decker E , Massot C , Delvenne P , Louis E , Meuwis M A 
Published: January 2025

Background

Colitis-associated cancer (CAC) is a distinct entity from sporadic colorectal cancer (CRC)1. Previous studies have demonstrated that Solute Carrier Family 12 Member 2 (SLC12A2/NKCC1), a Na-K-Cl cotransporter, is a tissue marker able to discriminate dysplasia from inflammatory and normal tissues2. NKCC1 is overexpressed in both pre-tumoral and tumoral lesions of CAC and CRC. It is located at the cell membrane and in the cytoplasm and is mainly expressed in fluid-secreting cells. In IBD, NKCC1 mutations cause defects in mucus secretion and chronic inflammation3. Our objective is to study its specific role in colorectal carcinogenesis.

Methods

Oxidative stress was induced in 3 CRC cell lines using H2O2 concomitantly with bumetanide (BMT), a chemical inhibitor of NKCC1, or by silencing its expression via shRNA. Cell viability, the mTORC1 pathway, and markers of stemness, proliferation, and differentiation were assessed by Western blot and qPCR. Human intestinal organoids deriving form patient tumours and normal margins, grown under the proliferative or differentiated states were used to characterize NKCC1 at the transcriptional and protein levels. Finally, NKCC1 expression in CRC was investigated using public single-cell datasets (GEO: GSE178341) to determine its specific expression within epithelial and tumoral cell-clusters4.

Results

Exposure of Caco-2 cells to 1 mM H2O2 for 24h resulted in 68% cell viability. However, when co-treated with 200 µM bumetanide cell viability dropped to 18%. Similarly, HT-29 cell viability dropped from 62% to 36%, while HCT116 cells showed a reduction from 12% to 5% with BMT and was shown to be BMT concentration-dependent. BMT-treated organoids/tumoroids revealed an activation of the mTORC1 pathway. Silencing NKCC1 expression via shRNA significantly increased cancer stem cell markers (LGR5, CD44, ALDH1A1). Upon differentiation, human intestinal organoids showed a marked decrease in NKCC1 expression mirroring the reduction of stem cell markers. This was confirmed by the single-cell data exploration, revealing that NKCC1 is highly expressed in both intestinal normal and cancer stem cells.

Conclusion

We confirmed that NKCC1 is expressed in both intestinal and cancer stem cells of colorectal tumours. Inhibition of NKCC1 transporter activity reduced cell viability in CRC cell lines only under oxidative stress exposure. NKCC1 silencing enhanced cancer stem cell marker expression, while its specific chemical inhibition activated the mTORC1 pathway. These results suggest that NKCC1 could be an important player in carcinogenesis at least by its ability to regulate oxidative stress in tumoral cells. Investigation of its role in ulcerative colitis and CAC remains to be clarified.

References

1. Fantini MC, Guadagni I. From inflammation to colitis-associated colorectal cancer in inflammatory bowel disease: Pathogenesis and impact of current therapies. Dig Liver Dis. 2021;53(5):558-565. doi:10.1016/j.dld.2021.01.012

2. Merli AM, Vieujean S, Massot C, et al. Solute Carrier Family 12 Member 2 as a Proteomic and Histological Biomarker of Dysplasia and Neoplasia in Ulcerative Colitis. J Crohn’s Colitis. 2021;15(2):287-298. doi:10.1093/ecco-jcc/jjaa168

3. Koumangoye R, Omer S, Kabeer MH, Delpire E. Novel Human NKCC1 Mutations Cause Defects in Goblet Cell Mucus Secretion and Chronic Inflammation. Cmgh. 2020;9(2):239-255. doi:10.1016/j.jcmgh.2019.10.006

4. Pelka K, Hofree M, Chen JH, et al. Spatially organized multicellular immune hubs in human colorectal cancer. Cell. 2021;184(18):4734-4752.e20. doi:10.1016/j.cell.2021.08.003