TY - JOUR
T1 - Endoplasmic reticulum stress regulates the intestinal stem cell state through CtBP2
AU - Meijer, Bartolomeus J.
AU - Smit, Wouter L.
AU - Koelink, Pim J.
AU - Westendorp, Barbara F.
AU - de Boer, Ruben J.
AU - van der Meer, Jonathan H. M.
AU - Vermeulen, Jacqueline L. M.
AU - Paton, James C.
AU - Paton, Adrienne W.
AU - Qin, Jun
AU - Dekker, Evelien
AU - Muncan, Vanesa
AU - van den Brink, Gijs R.
AU - Heijmans, Jarom
N1 - Funding Information: We would like to thank professor Dr. Jeremy Blaydes BSc, PhD, University of Southampton, UK for providing the sequence for CtBP2 overexpression. And the Jun Qin lab, Baylor College of Medicine, Houston, for performing the CatTFRE assay. We would also like to thank Prof. Dr. J.P. Medema for kindly providing patient-derived APC and APC-KRAS mutant colon organoids. This work was supported in part by Grants of the Dutch cancer foundation (KWF/UVA 2013-6135 and KWF/Alpe 11053/2017-1) and by a Grant of the Netherlands organisation for Scientific Research (NWO-Veni 91615032). Publisher Copyright: © 2021, The Author(s).
PY - 2021/12/1
Y1 - 2021/12/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85105583312&partnerID=8YFLogxK
U2 - https://doi.org/10.1038/s41598-021-89326-w
DO - https://doi.org/10.1038/s41598-021-89326-w
M3 - Article
C2 - 33972635
SN - 2045-2322
VL - 11
JO - Scientific reports
JF - Scientific reports
IS - 1
M1 - 9892
ER -