Colon cancer stem cells (colon-CSCs) are more resistant to conventional chemotherapy than differentiated cancer cells. This subset of therapy refractory cells is therefore believed to play an important role in post-therapeutic tumor relapse. In order to improve the rate of sustained response to conventional chemotherapy, development of approaches is warranted that specifically sensitize colon-CSCs to treatment. Here, we report that ER-stress-induced activation of the unfolded protein response (UPR) forces colon-CSCs to differentiate, resulting in their enhanced sensitivity to chemotherapy in vitro and in vivo. Our data suggest that agents that induce activation of the UPR may be used to specifically increase sensitivity of colon-CSCs to the effects of conventional chemotherapy.

Cell-type plasticity within a tumor has recently been suggested to cause a bidirectional conversion between tumor-initiating stem cells and nonstem cells triggered by an inflammatory stroma. NF-κB represents a key transcription factor within the inflammatory tumor microenvironment. However, NF-κB's function in tumor-initiating cells has not been examined yet. Using a genetic model of intestinal epithelial cell (IEC)-restricted constitutive Wnt-activation, which comprises the most common event in the initiation of colon cancer, we demonstrate that NF-κB modulates Wnt signaling and show that IEC-specific ablation of RelA/p65 retards crypt stem cell expansion. In contrast, elevated NF-κB signaling enhances Wnt activation and induces dedifferentiation of nonstem cells that acquire tumor-initiating capacity. Thus, our data support the concept of bidirectional conversion and highlight the importance of inflammatory signaling for dedifferentiation and generation of tumor-initiating cells in vivo.

In gastric cancer, a new epigenetic mechanism of tumour suppressor loss has been suggested where the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is responsible for loss of expression of RUNX3. This is consistent with EZH2 upregulation in multiple cancer types being associated with poor prognosis. We investigated whether EZH2 influences the expression of RUNX3 in colorectal cancer (CRC) and whether this is independent of methylation. We determined protein and messenger RNA (mRNA) levels of EZH2 and RUNX3 and assessed RUNX3 methylation with methylation-specific polymerase chain reaction using 72 human CRCs and 8 CRC cell lines. We assessed the effect of efficient RNA interference-mediated knockdown of EZH2 on RUNX3 levels, cell viability and H3K27 trimethylation of the RUNX3 promoter using chromatin immunoprecipitation. Despite higher levels of EZH2 and lower levels of RUNX3 in CRC specimens in general, no inverse correlation between EZH2 and RUNX3 in paired samples was found arguing against a major role for histone methylation in silencing RUNX3 in CRC. Conversely, downregulation of RUNX3 mRNA in the same tumours was associated with RUNX3 DNA methylation (P < 0.05). In cell lines, knockdown of EZH2 removed the repressive chromatin marks from RUNX3 but did not result in RUNX3 re-expression. However, it prevented the re-silencing of RUNX3 after the removal of demethylating agents. In conclusion, DNA methylation is primarily responsible for the transcriptional silencing of RUNX3 in CRC, but EZH2 and histone methylation are necessary for its methylation-dependent re-silencing after the removal of demethylating agents. These results would predict that inhibitors of EZH2 and histone methylation would enhance the effects of demethylating agents in cancer therapy.

Coronavirus disease 2019 (COVID-19) can lead to systemic coagulation activation and thrombotic complications.

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.

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.

It recently has been recognized that men develop colonic adenomas and carcinomas at an earlier age and at a higher rate than women. In the Apc(Pirc/+) (Pirc) rat model of early colonic cancer, this sex susceptibility was recapitulated, with male Pirc rats developing twice as many adenomas as females. Analysis of large datasets revealed that the Apc(Min/+) mouse also shows enhanced male susceptibility to adenomagenesis, but only in the colon. In addition, WT mice treated with injections of the carcinogen azoxymethane (AOM) showed increased numbers of colonic adenomas in males. The mechanism underlying these observations was investigated by manipulation of hormonal status. The preponderance of colonic adenomas in the Pirc rat model allowed a statistically significant investigation in vivo of the mechanism of sex hormone action on the development of colonic adenomas. Females depleted of endogenous hormones by ovariectomy did not exhibit a change in prevalence of adenomas, nor was any effect observed with replacement of one or a combination of female hormones. In contrast, depletion of male hormones by orchidectomy (castration) markedly protected the Pirc rat from adenoma development, whereas supplementation with testosterone reversed that effect. These observations were recapitulated in the AOM mouse model. Androgen receptor was undetectable in the colon or adenomas, making it likely that testosterone acts indirectly on the tumor lineage. Our findings suggest that indirect tumor-promoting effects of testosterone likely explain the disparity between the sexes in the development of colonic adenomas.

Stem cells generate rapidly dividing transit-amplifying cells that have lost the capacity for self-renewal but cycle for a number of times until they exit the cell cycle and undergo terminal differentiation. We know very little of the type of signals that trigger the earliest steps of stem cell differentiation and mediate a stem cell to transit-amplifying cell transition. We show that in normal intestinal epithelium, endoplasmic reticulum (ER) stress and activity of the unfolded protein response (UPR) are induced at the transition from stem cell to transit-amplifying cell. Induction of ER stress causes loss of stemness in a Perk-eIF2α-dependent manner. Inhibition of Perk-eIF2α signaling results in stem cell accumulation in organoid culture of primary intestinal epithelium. Our findings show that the UPR plays an important role in the regulation of intestinal epithelial stem cell differentiation.

Patients with hematological cancers (HC) are at high risk of infections, in particular community-acquired pneumonia (CAP). Recent data on incidence and predictors of CAP among patients with HC are scarce.

Recent evidence has suggested that the intact intestinal epithelial barrier protects our body from a range of immune-mediated diseases. The epithelial layer has an impressive ability to reconstitute and repair upon damage and this process of repair increasingly is seen as a therapeutic target. In vitro models to study this process in primary intestinal cells are lacking.

The unfolded protein response (UPR) acts through its downstream branches, PERK-eIF2α signaling, IRE1α-XBP1 signaling and ATF6 signaling. In the intestine, activation of the UPR through the kinase PERK results in differentiation of intestinal epithelial stem cells and colon cancer stem cells, whereas deletion of XBP1 results in increased stemness and adenomagenesis. How downstream activation of XBP1 and ATF6 influences intestinal stemness and proliferation remains largely unknown. We generated colorectal cancer cells (LS174T) that harbor doxycycline inducible expression of the active forms of either XBP1(s) or ATF61-373. Activation of either XBP1 or ATF6 resulted in reduced cellular proliferation and reduced expression of markers of intestinal epithelial stemness. Moreover, XBP1 and ATF6 activation reduced global protein synthesis and lowered the threshold for UPR activation. XBP1-mediated loss of stemness and proliferation resulted from crossactivation of PERK-eIF2α signaling and could be rescued by constitutive expression of eIF2α phosphatase GADD34. We thus find that enforced activation of XBP1 and ATF6 results in reduction of stemness and proliferation. We expose a novel interaction between XBP1 and PERK-eIF2α signaling.

Cholestasis-induced accumulation of bile acids in the liver leads to farnesoid X receptor (FXR)-mediated transcriptional down-regulation of the bile acid importer Na+-taurocholate cotransporting protein (NTCP) and to induction of endoplasmic reticulum (ER) stress. However, whether ER stress affects bile acid uptake is largely unknown. Here, we investigated the role of ER stress on the regulation and function of the bile acid transporter NTCP. ER stress was induced using thapsigargin or subtilase cytotoxin in human osteosarcoma (U2OS) and human hepatocellular carcinoma (HepG2) cells stably expressing NTCP. Cellular bile acid uptake was determined using radiolabeled taurocholate (TCA). NTCP plasma membrane expression was determined by cell surface biotinylation. Mice received a single injection of thapsigargin, and effects of ER stress on NTCP messenger RNA (mRNA) and protein were measured by reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis. Effects of cholestasis on NTCP and ER stress were assessed in response to 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) feeding or bile duct ligation in FXR-/- mice after 7 or 3 days, respectively. Novel NTCP-interacting proteins were identified by mass spectrometry (MS), interaction verified, and assessed by co-immunoprecipitation and TCA uptake for functional relevance in relation to ER stress. ER stress induction strongly reduced NTCP protein expression, plasma membrane abundance, and NTCP-mediated bile acid uptake. This was not controlled by FXR or through a single unfolded protein response (UPR) pathway but mainly depended on the interaction of NTCP with calnexin, an ER chaperone. In mice, expression of both NTCP and calnexin was reduced by thapsigargin or cholestasis-induced ER stress. Calnexin down-regulation in vitro recapitulated the effect of ER stress on NTCP. Conclusion: ER stress-induced down-regulation of calnexin provides an additional mechanism to dampen NTCP-mediated bile acid uptake and protect hepatocytes against bile acid overload during cholestasis.

In many mammalian species, the intestinal epithelium undergoes major changes that allow a dietary transition from mother's milk to the adult diet at the end of the suckling period. These complex developmental changes are the result of a genetic programme intrinsic to the gut tube, but its regulators have not been identified. Here we show that transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1) is highly expressed in the developing and postnatal intestinal epithelium until the suckling to weaning transition. Intestine-specific deletion of Blimp1 results in growth retardation and excessive neonatal mortality. Mutant mice lack all of the typical epithelial features of the suckling period and are born with features of an adult-like intestine. We conclude that the suckling to weaning transition is regulated by a single transcriptional repressor that delays epithelial maturation.

Upon intestinal epithelial damage a complex wound healing response is initiated to restore epithelial integrity and defend against pathogenic invasion. Epithelium-derived Indian Hedgehog (Ihh) functions as a critical sensor in this process. Signaling occurs in a paracrine manner because the receptor for Ihh is expressed only in the mesenchyme, but the exact Hedgehog target cell has remained elusive. The aim of this study was to elucidate further the nature of this target cell in the context of intestinal inflammation.

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.

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.

The majority of colorectal cancers (CRCs) present with early mutations in tumor suppressor gene APC. APC mutations result in oncogenic activation of the Wnt pathway, which is associated with hyperproliferation, cytoskeletal remodeling, and a global increase in mRNA translation. To compensate for the increased biosynthetic demand, cancer cells critically depend on protein chaperones to maintain proteostasis, although their function in CRC remains largely unexplored. In order to investigate the role of molecular chaperones in driving CRC initiation, we captured the transcriptomic profiles of murine wild type and Apc-mutant organoids during active transformation. We discovered a strong transcriptional upregulation of Hspb1, which encodes small heat shock protein 25 (HSP25). We reveal an indispensable role for HSP25 in facilitating Apc-driven transformation, using both in vitro organoid cultures and mouse models, and demonstrate that chemical inhibition of HSP25 using brivudine reduces the development of premalignant adenomas. These findings uncover a hitherto unknown vulnerability in intestinal transformation that could be exploited for the development of chemopreventive strategies in high-risk individuals.