ER stress and the unfolded protein response in gastrointestinal stem cells and carcinogenesis.

Endoplasmic reticulum (ER) stress and the adaptive response that follows, termed the unfolded protein response (UPR), are crucial molecular mechanisms to maintain cellular integrity by safeguarding proper protein synthesis. Next to being important in protein homeostasis, the UPR is intricate in cell fate decisions such as proliferation, differentiation, and stemness. In the intestine, stem cells are critical in governing epithelial homeostasis and they are the cell of origin of gastrointestinal malignancies. In this review, we will discuss the role of ER stress and the UPR in the gastrointestinal tract, focusing on stem cells and carcinogenesis. Insights in mechanisms that connect ER stress and UPR with stemness and carcinogenesis may broaden our understanding in the development of cancer throughout the gastrointestinal tract and how we can exploit these mechanisms to target these malignancies.

Grp78 is required for intestinal Kras-dependent glycolysis proliferation and adenomagenesis.

In development of colorectal cancer, mutations in APC are often followed by mutations in oncogene KRAS The latter changes cellular metabolism and is associated with the Warburg phenomenon. Glucose-regulated protein 78 (Grp78) is an important regulator of the protein-folding machinery, involved in processing and localization of transmembrane proteins. We hypothesize that targeting Grp78 in Apc and Kras (AK)-mutant intestines interferes with the metabolic phenotype imposed by Kras mutations. In mice with intestinal epithelial mutations in Apc, Kras G12D and heterozygosity for Grp78 (AK-Grp78 HET ) adenoma number and size is decreased compared with AK-Grp78 WT mice. Organoids from AK-Grp78 WT mice exhibited a glycolysis metabolism which was completely rescued by Grp78 heterozygosity. Expression and correct localization of glucose transporter GLUT1 was diminished in AK-Grp78 HET cells. GLUT1 inhibition restrained the increased growth observed in AK-mutant organoids, whereas AK-Grp78 HET organoids were unaffected. We identify Grp78 as a critical factor in Kras-mutated adenomagenesis. This can be attributed to a critical role for Grp78 in GLUT1 expression and localization, targeting glycolysis and the Warburg effect.

Epithelial argininosuccinate synthetase is dispensable for intestinal regeneration and tumorigenesis.

The epithelial signaling pathways involved in damage and regeneration, and neoplastic transformation are known to be similar. We noted upregulation of argininosuccinate synthetase (ASS1) in hyperproliferative intestinal epithelium. Since ASS1 leads to de novo synthesis of arginine, an important amino acid for the growth of intestinal epithelial cells, its upregulation can contribute to epithelial proliferation necessary to be sustained during oncogenic transformation and regeneration. Here we investigated the function of ASS1 in the gut epithelium during tissue regeneration and tumorigenesis, using intestinal epithelial conditional Ass1 knockout mice and organoids, and tissue specimens from colorectal cancer patients. We demonstrate that ASS1 is strongly expressed in the regenerating and Apc-mutated intestinal epithelium. Furthermore, we observe an arrest in amino acid flux of the urea cycle, which leads to an accumulation of intracellular arginine. However, loss of epithelial Ass1 does not lead to a reduction in proliferation or increase in apoptosis in vivo, also in mice fed an arginine-free diet. Epithelial loss of Ass1 seems to be compensated by altered arginine metabolism in other cell types and the liver.

Endoplasmic reticulum stress regulates the intestinal stem cell state through CtBP2.

Enforcing differentiation of cancer stem cells is considered as a potential strategy to sensitize colorectal cancer cells to irradiation and chemotherapy. Activation of the unfolded protein response, due to endoplasmic reticulum (ER) stress, causes rapid stem cell differentiation in normal intestinal and colon cancer cells. We previously found that stem cell differentiation was mediated by a Protein kinase R-like ER kinase (PERK) dependent arrest of mRNA translation, resulting in rapid protein depletion of WNT-dependent transcription factor c-MYC. We hypothesize that ER stress dependent stem cell differentiation may rely on the depletion of additional transcriptional regulators with a short protein half-life that are rapidly depleted due to a PERK-dependent translational pause. Using a novel screening method, we identify novel transcription factors that regulate the intestinal stem cell fate upon ER stress. ER stress was induced in LS174T cells with thapsigargin or subtilase cytotoxin (SubAB) and immediate alterations in nuclear transcription factor activity were assessed by the CatTFRE assay in which transcription factors present in nuclear lysate are bound to plasmid DNA, co-extracted and quantified using mass-spectrometry. The role of altered activity of transcription factor CtBP2 was further examined by modification of its expression levels using CAG-rtTA3-CtBP2 overexpression in small intestinal organoids, shCtBP2 knockdown in LS174T cells, and familial adenomatous polyposis patient-derived organoids. CtBP2 overexpression organoids were challenged by ER stress and ionizing irradiation. We identified a unique set of transcription factors with altered activation upon ER stress. Gene ontology analysis showed that transcription factors with diminished binding were involved in cellular differentiation processes. ER stress decreased CtBP2 protein expression in mouse small intestine. ER stress induced loss of CtBP2 expression which was rescued by inhibition of PERK signaling. CtBP2 was overexpressed in mouse and human colorectal adenomas. Inducible CtBP2 overexpression in organoids conferred higher clonogenic potential, resilience to irradiation-induced damage and a partial rescue of ER stress-induced loss of stemness. Using an unbiased proteomics approach, we identified a unique set of transcription factors for which DNA-binding activity is lost directly upon ER stress. We continued investigating the function of co-regulator CtBP2, and show that CtBP2 mediates ER stress-induced loss of stemness which supports the intestinal stem cell state in homeostatic stem cells and colorectal cancer cells.

ATF2 and ATF7 Are Critical Mediators of Intestinal Epithelial Repair.

BACKGROUND & AIMS: Activation factor-1 transcription factor family members activating transcription factors 2 and 7 (ATF2 and ATF7) have highly redundant functions owing to highly homologous DNA binding sites. Their role in intestinal epithelial homeostasis and repair is unknown. Here, we assessed the role of these proteins in these conditions in an intestine-specific mouse model. METHODS: We performed in vivo and ex vivo experiments using Villin-CreERT2Atf2fl/flAtf7ko/ko mice. We investigated the effects of intestinal epithelium-specific deletion of the Atf2 DNA binding region in Atf7-/- mice on cellular proliferation, differentiation, apoptosis, and epithelial barrier function under homeostatic conditions. Subsequently, we exposed mice to 2% dextran sulfate sodium (DSS) for 7 days and 12 Gy whole-body irradiation and assessed the response to epithelial damage. RESULTS: Activating phosphorylation of ATF2 and ATF7 was detected mainly in the crypts of the small intestine and the lower crypt region of the colonic epithelium. Under homeostatic conditions, no major phenotypic changes were detectable in the intestine of ATF mutant mice. However, on DSS exposure or whole-body irradiation, the intestinal epithelium showed a clearly impaired regenerative response. Mutant mice developed severe ulceration and inflammation associated with increased epithelial apoptosis on DSS exposure and were less able to regenerate colonic crypts on irradiation. In vitro, organoids derived from double-mutant epithelium had a growth disadvantage compared with wild-type organoids, impaired wound healing capacity in scratch assay, and increased sensitivity to tumor necrosis factor-α-induced damage. CONCLUSIONS: ATF2 and ATF7 are dispensable for epithelial homeostasis, but are required to maintain epithelial regenerative capacity and protect against cell death during intestinal epithelial damage and repair.

Amino Acid Loss during Continuous Venovenous Hemofiltration in Critically Ill Patients.

BACKGROUND/OBJECTIVES: During continuous venovenous hemofiltration (CVVH), there is unwanted loss of amino acids (AA) in the ultrafiltrate (UF). Solutes may also be removed by adsorption to the filter membrane. The aim was to quantify the total loss of AA via the CVVH circuit using a high-flux polysulfone membrane and to differentiate between the loss by ultrafiltration and adsorption. METHODS: Prospective observational study in ten critically ill patients, receiving predilution CVVH with a new filter, blood flow 180 mL/min, and predilution flow 2,400 mL/h. Arterial blood, postfilter blood, and UF samples were taken at baseline, and 1, 8, and 24-h after CVVH initiation, to determine AA concentrations and hematocrit. Mass transfer calculations were used to determine AA loss in the filter and by UF, and the difference between these 2. RESULTS: The median AA loss in the filter was 10.4 g/day, the median AA loss by UF was 13.4 g/day, and the median difference was -2.9 g/day (IQR -5.9 to -1.4 g/day). For the individual AA, the difference ranged from -1 g/day to +0.4 g/day, suggesting that some AA were consumed or adsorbed and others were generated. AA losses did not significantly change over the 24-h study period. CONCLUSION: During CVVH with a modern polysulfone membrane, the estimated AA loss was 13.4 g/day, which corresponds to a loss of about 11.2 g of protein per day. Adsorption did not play a major role. However, individual AA behaved differently, suggesting complex interactions and processes at the filter membrane or peripheral AA production.