ASCL2 is a key regulator of the proliferation-differentiation equilibrium in the esophageal epithelium.

The esophagus is protected from the hostile environment by a stratified epithelium, which renews rapidly. Homeostasis of this epithelium is ensured by a rare population of stem cells in the basal layer: Keratin 15+ (Krt15+) cells. However, little is known about the molecular mechanisms regulating their distinct features, namely self-renewal, potency and epithelial regeneration. Achaete-scute family BHLH transcription factor 2 (ASCL2) is strongly upregulated in Krt15+ stem cells and is known to contribute to stem cell maintenance in other tissues. Herein, we investigated the role of ASCL2 in maintaining homeostasis under normal and stress conditions in the esophageal epithelium. ASCL2 overexpression severely dysregulated cell differentiation and cell fate. Proliferation was also reduced due potentially to a blockage in the G1 phase of the cell cycle or an induction of quiescence. Mass spectrometry analysis confirmed alterations in several proteins associated with differentiation and the cell cycle. In addition, overexpression of ASCL2 enhanced resistance to radiation and chemotherapeutic drugs. Overall, these results denote the role of ASCL2 as a key regulator of the proliferation-differentiation equilibrium in the esophageal epithelium.

Psychosocial determinants of healthy sleep habits in adults with type 1 and type 2 diabetes.

Background: Many adults with type 1 (T1D) and type 2 diabetes (T2D) have inadequate sleep increasing their risk of hyperglycemia and developing complications. The objective was to identify psychosocial determinants of healthy sleep habits (HSH) among adults with T1D and T2D. Methods: The two HSH were: avoiding screen use in bed and having sleep regularity. Adults (≥18 years) with T1D and T2D were invited to complete an anonymous online survey. The questionnaires were based on the Reasoned Action Approach and formative qualitative research previously conducted in 56 adults with T1D and T2D. Habit was included as an additional variable for screen use in bed. Results: In total, 320 adults with diabetes (T1D: 39%; T2D: 61%) completed the questionnaires (screen use in bed: 174; sleep timing: 146). Close to 75% of participants reported screen use in bed and close to 90% reported sleep timing variability in the last month. Perceived behavioral control (PBC) to avoid screen use in bed (ß = -0.4486, p < 0.0001), habit of using screens in bed (ß = 0.4002; p < 0.0001), and age (ß = -0.0202; p = 0.0086) were determinants of screen use in bed, and this model explained 71% of the variance. PBC for sleep regularity (ß = -0.2909; p = 0.0004) and being female (ß = 0.5057; p = 0.0069) were determinants of sleep timing variability, and this model explained 28% of the variance. The most important beliefs associated with each HSH were identified to obtain information to design targeted interventions. Conclusions: Few adults with diabetes have HSH. Screen use in bed was strongly influenced by habit and the results suggest that both HSH are not easy to adopt among adults with diabetes. Younger adults with diabetes should be prioritized for screen use in bed, while females with diabetes should be prioritized for sleep timing variability. Adults with diabetes should have access to behavior change interventions to encourage them to adopt HSH.

Hepatocyte nuclear factor 4α multiple isoforms, their functions, and their interactomes.

Hepatocyte nuclear factor 4-alpha (HNF4α) is a master regulator gene belonging to the nuclear receptor superfamily and is involved in regulating a wide range of critical biological processes in different organs. Structurally, the HNF4A locus is organized into two independent promoters and is subjected to alternative splicing to produce twelve distinct isoforms. However, little is known about the biological impact of each isoform and the mechanisms by which they regulate transcription. Proteomic analyses have led to the identification of proteins that interact with specific HNF4α isoforms. The identification and validation of these interactions and their roles in the co-regulation of targeted gene expression are essential to better understand the role of this transcription factor in different biological processes and pathologies. This review addresses the discoveries of different HNF4α isoforms and the main functions of the P1 and P2 isoform subgroups. It also provides information on the most recent focus areas in research on the nature and function of proteins associated with each of the isoforms in some biological contexts.

HNF4α Acts as Upstream Functional Regulator of Intestinal Wnt3 and Paneth Cell Fate.

BACKGROUND & AIMS: The intestinal epithelium intrinsically renews itself ex vivo via the proliferation of Lgr5+ intestinal stem cells, which is sustained by the establishment of an epithelial stem cell niche. Differentiated Paneth cells are the main source of epithelial-derived niche factor supplies and produce Wnt3 as an essential factor in supporting Lgr5+ stem cell activity in the absence of redundant mesenchymal Wnts. Maturation of Paneth cells depends on canonical Wnt signaling, but few transcriptional regulators have been identified to this end. The role of HNF4α in intestinal epithelial cell differentiation is considered redundant with its paralog HNF4γ. However, it is unclear whether HNF4α alone controls intrinsic intestinal epithelial cell growth and fate in the absence of a mesenchymal niche. METHODS: We used transcriptomic analyses to dissect the role of HNF4α in the maintenance of jejunal epithelial culture when cultured ex vivo as enteroids in the presence or absence of compensatory mesenchymal cells. RESULTS: HNF4α plays a crucial role in supporting the growth and survival of jejunal enteroids. Transcriptomic analyses revealed an autonomous function of HNF4α in Wnt3 transcriptional regulation and Paneth cell differentiation. We showed that Wnt3a supplementation or co-culture with intestinal subepithelial mesenchymal cells reversed cell death and transcriptional changes caused by the deletion of Hnf4a in jejunal enteroids. CONCLUSIONS: Our results support the intrinsic epithelial role of HNF4α in regulating Paneth cell homeostasis and intestinal epithelium renewal in the absence of compensatory Wnt signaling.

The transcription factor hepatocyte nuclear factor 4A acts in the intestine to promote white adipose tissue energy storage.

The transcription factor hepatocyte nuclear factor 4 A (HNF4A) controls the metabolic features of several endodermal epithelia. Both HNF4A and HNF4G are redundant in the intestine and it remains unclear whether HNF4A alone controls intestinal lipid metabolism. Here we show that intestinal HNF4A is not required for intestinal lipid metabolism per se, but unexpectedly influences whole-body energy expenditure in diet-induced obesity (DIO). Deletion of intestinal HNF4A caused mice to become DIO-resistant with a preference for fat as an energy substrate and energetic changes in association with white adipose tissue (WAT) beiging. Intestinal HNF4A is crucial for the fat-induced release of glucose-dependent insulinotropic polypeptide (GIP), while the reintroduction of a stabilized GIP analog rescues the DIO resistance phenotype of the mutant mice. Our study provides evidence that intestinal HNF4A plays a non-redundant role in whole-body lipid homeostasis and points to a non-cell-autonomous regulatory circuit for body-fat management.

Altered Mucus Barrier Integrity and Increased Susceptibility to Colitis in Mice upon Loss of Telocyte Bone Morphogenetic Protein Signalling.

FoxL1+-Telocytes (TCFoxL1+) are subepithelial cells that form a network underneath the epithelium. We have shown that without inflammatory stress, mice with loss of function in the BMP signalling pathway in TCFoxL1+ (BmpR1aΔFoxL1+) initiated colonic neoplasia. Although TCFoxL1+ are modulated in IBD patients, their specific role in this pathogenesis remains unclear. Thus, we investigated how the loss of BMP signalling in TCFoxL1+ influences the severity of inflammation and fosters epithelial recovery after inflammatory stress. BmpR1a was genetically ablated in mouse colonic TCFoxL1+. Experimental colitis was performed using a DSS challenge followed by recovery steps to assess wound healing. Physical barrier properties, including mucus composition and glycosylation, were assessed by alcian blue staining, immunofluorescences and RT-qPCR. We found that BmpR1aΔFoxL1+ mice had impaired mucus quality, and upon exposure to inflammatory challenges, they had increased susceptibility to experimental colitis and delayed healing. In addition, defective BMP signalling in TCFoxL1+ altered the functionality of goblet cells, thereby affecting mucosal structure and promoting bacterial invasion. Following inflammatory stress, TCFoxL1+ with impaired BMP signalling lose their homing signal for optimal distribution along the epithelium, which is critical in tissue regeneration after injury. Overall, our findings revealed key roles of BMP signalling in TCFoxL1+ in IBD pathogenesis.

Organoids and Their Use in Modeling Gut Epithelial Cell Lineage Differentiation and Barrier Properties During Intestinal Diseases.

Maintenance of intestinal epithelium homeostasis is a complex process because of the multicellular and molecular composition of the gastrointestinal wall and the involvement of surrounding interactive signals. The complex nature of this intestinal barrier system poses challenges in the detailed mechanistic understanding of intestinal morphogenesis and the onset of several gut pathologies, including intestinal inflammatory disorders, food allergies, and cancer. For several years, the gut scientific community has explored different alternatives in research involving animals and in vitro models consisting of cultured monolayers derived from the immortalized or cancerous origin cell lines. The recent ability to recapitulate intestinal epithelial dynamics from mini-gut cultures has proven to be a promising step in the field of scientific research and biomedicine. The organoids can be grown as two- or three-dimensional structures, and are derived from adult or pluripotent stem cells that ultimately establish an intestinal epithelium that is composed of all differentiated cell types present in the normal epithelium. In this review, we summarize the different origins and recent use of organoids in modeling intestinal epithelial differentiation and barrier properties.

NCOR1 Sustains Colorectal Cancer Cell Growth and Protects against Cellular Senescence.

NCOR1 is a corepressor that mediates transcriptional repression through its association with nuclear receptors and specific transcription factors. Some evidence supports a role for NCOR1 in neonatal intestinal epithelium maturation and the maintenance of epithelial integrity during experimental colitis in mice. We hypothesized that NCOR1 could control colorectal cancer cell proliferation and tumorigenicity. Conditional intestinal epithelial deletion of Ncor1 in ApcMin/+ mice resulted in a significant reduction in polyposis. RNAi targeting of NCOR1 in Caco-2/15 and HT-29 cell lines led to a reduction in cell growth, characterized by cellular senescence associated with a secretory phenotype. Tumor growth of HT-29 cells was reduced in the absence of NCOR1 in the mouse xenografts. RNA-seq transcriptome profiling of colon cancer cells confirmed the senescence phenotype in the absence of NCOR1 and predicted the occurrence of a pro-migration cellular signature in this context. SOX2, a transcription factor essential for pluripotency of embryonic stem cells, was induced under these conditions. In conclusion, depletion of NCOR1 reduced intestinal polyposis in mice and caused growth arrest, leading to senescence in human colorectal cell lines. The acquisition of a pro-metastasis signature in the absence of NCOR1 could indicate long-term potential adverse consequences of colon-cancer-induced senescence.

A novel class of inhibitors that target SRSF10 and promote p53-mediated cytotoxicity on human colorectal cancer cells.

The elevated expression of the splicing regulator SRSF10 in metastatic colorectal cancer (CRC) stimulates the production of the pro-tumorigenic BCLAF1-L splice variant. We discovered a group of small molecules with an aminothiazole carboxamide core (GPS167, GPS192 and others) that decrease production of BCLAF1-L. While additional alternative splicing events regulated by SRSF10 are affected by GPS167/192 in HCT116 cells (e.g. in MDM4, WTAP, SLK1 and CLK1), other events are shifted in a SRSF10-independent manner (e.g. in MDM2, NAB2 and TRA2A). GPS167/192 increased the interaction of SRSF10 with the CLK1 and CLK4 kinases, leading us to show that GPS167/192 can inhibit CLK kinases preferentially impacting the activity of SRSF10. Notably, GPS167 impairs the growth of CRC cell lines and organoids, inhibits anchorage-independent colony formation, cell migration, and promotes cytoxicity in a manner that requires SRSF10 and p53. In contrast, GPS167 only minimally affects normal colonocytes and normal colorectal organoids. Thus, GPS167 reprograms the tumorigenic activity of SRSF10 in CRC cells to elicit p53-dependent apoptosis.

JAK-STAT Pathway Inhibition Partially Restores Intestinal Homeostasis in Hdac1- and Hdac2-Intestinal Epithelial Cell-Deficient Mice.

We have previously reported that histone deacetylase epigenetic regulator Hdac1 and Hdac2 deletion in intestinal epithelial cells (IEC) disrupts mucosal tissue architecture and barrier, causing chronic inflammation. In this study, proteome and transcriptome analysis revealed the importance of signaling pathways induced upon genetic IEC-Hdac1 and Hdac2 deletion. Indeed, Gene Ontology biological process analysis of enriched deficient IEC RNA and proteins identified common pathways, including lipid metabolic and oxidation-reduction process, cell adhesion, and antigen processing and presentation, related to immune responses, correlating with dysregulation of major histocompatibility complex (MHC) class II genes. Top upstream regulators included regulators associated with environmental sensing pathways to xenobiotics, microbial and diet-derived ligands, and endogenous metabolites. Proteome analysis revealed mTOR signaling IEC-specific defects. In addition to mTOR, the STAT and Notch pathways were dysregulated specifically in jejunal IEC. To determine the impact of pathway dysregulation on mutant jejunum alterations, we treated mutant mice with Tofacitinib, a JAK inhibitor. Treatment with the inhibitor partially corrected proliferation and tight junction defects, as well as niche stabilization by increasing Paneth cell numbers. Thus, IEC-specific histone deacetylases 1 (HDAC1) and 2 (HDAC2) support intestinal homeostasis by regulating survival and translation processes, as well as differentiation and metabolic pathways. HDAC1 and HDAC2 may play an important role in the regulation of IEC-specific inflammatory responses by controlling, directly or indirectly, the JAK/STAT pathway. IEC-specific JAK/STAT pathway deregulation may be, at least in part, responsible for intestinal homeostasis disruption in mutant mice.

Human Hepatocyte Nuclear Factor 4-α Encodes Isoforms with Distinct Transcriptional Functions.

HNF4α is a nuclear receptor produced as 12 isoforms from two promoters by alternative splicing. To characterize the transcriptional capacities of all 12 HNF4α isoforms, stable lines expressing each isoform were generated. The entire transcriptome associated with each isoform was analyzed as well as their respective interacting proteome. Major differences were noted in the transcriptional function of these isoforms. The α1 and α2 isoforms were the strongest regulators of gene expression whereas the α3 isoform exhibited significantly reduced activity. The α4, α5, and α6 isoforms, which use an alternative first exon, were characterized for the first time, and showed a greatly reduced transcriptional potential with an inability to recognize the consensus response element of HNF4α. Several transcription factors and coregulators were identified as potential specific partners for certain HNF4α isoforms. An analysis integrating the vast amount of omics data enabled the identification of transcriptional regulatory mechanisms specific to certain HNF4α isoforms, hence demonstrating the importance of considering all isoforms given their seemingly diverse functions.

A Novel Organoid Model of Damage and Repair Identifies HNF4α as a Critical Regulator of Intestinal Epithelial Regeneration.

BACKGROUND & AIMS: 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. METHODS: We established and characterized an in vitro model of intestinal damage and repair by applying γ-radiation on small-intestinal organoids. We then used this model to identify novel regulators of intestinal regeneration. RESULTS: We identified hepatocyte nuclear factor 4α (HNF4α) as a pivotal upstream regulator of the intestinal regenerative response. Organoids lacking Hnf4a were not able to propagate in vitro. Importantly, intestinal Hnf4a knock-out mice showed impaired regeneration after whole-body irradiation, confirming intestinal organoids as a valuable alternative to in vivo studies. CONCLUSIONS: In conclusion, we established and validated an in vitro damage-repair model and identified HNF4α as a crucial regulator of intestinal regeneration. Transcript profiling: GSE141515 and GSE141518.

Acceptability of a computer-tailored and pedometer-based socio-cognitive intervention in a secondary coronary heart disease prevention program: A qualitative study.

When developing an innovative intervention, its acceptability to patients, health care professionals and managers must be considered to ensure the implementation into practice. This study aims to identify factors influencing the acceptability of a computer-tailored and pedometer-based socio-cognitive intervention for patients with heart disease. Focus group interviews were conducted in two outlying regions of the province of Quebec (Canada). The Theory of Planned Behavior formed the theoretical basis of the interview guide. Two researchers performed verbatim analysis independently until consensus was achieved. The sample included 44 participants divided into six groups (patients n = 7 + 8, health care professionals n = 8 + 8, managers n = 6 + 7). Health care professionals and managers mentioned benefits concerning partners' opportunity to improve assessment and monitoring. Patients believed the intervention could be useful to improve adherence to physical activity. Additional benefits indicated were self-monitoring behavior and improved health-related outcomes. However, patients expressed concern about the online security, fearing possible data breach. Some clinicians felt the pedometer may not be able to evaluate physical activities other than walking. With regard to behavioral control, a web application and pedometer must be easy to use and compatible with services already in place. Further barriers include level of literacy, cost and the various difficulties associated with wearing a pedometer. Findings suggest that, to improve the acceptability of a computer-tailored and pedometer-based socio-cognitive intervention, users must be assured of a secure website, validated, affordable and easy-to-use pedometers, and an intervention adapted to their level of literacy.

Effectiveness of a web-based computer-tailored intervention promoting physical activity for adults from Quebec City: a randomized controlled trial.

BACKGROUND: The primary objective of this study was to determine the effectiveness of a 3-month web-based computer-tailored intervention on moderate-to-vigorous physical activity (MVPA) in adults. METHODS: A total of 242 Canadian adults aged between 35 and 70 years were randomized to an experimental group receiving the intervention or a waiting list control group. The fully automated web-based computer-tailored physical activity intervention consists of seven 10- to 15-min sessions over an 8-week period. The theoretical underpinning of the intervention is based on the I-Change Model. RESULTS: A repeated-measures ANOVA using a linear mixed model showed a significant 'group-by-time' interaction favoring the intervention group in self-reported MVPA (p = .02). The MVPA was similar in both groups at baseline (mean ± SD; 176 ± 13 vs. 172 ± 15 min/week, p = .72) and higher in the intervention than in the control group at a 3-month follow-up (259 ± 21 vs. 201 ± 22 min/week, p = .04). This finding was comparable across women and men (group-by-sex, p = .57) and across participants meeting or not physical activity guidelines at baseline (group-by-baseline physical activity, p = .43). Although engagement to the web-based sessions declined over time, participants completing more web sessions achieved higher self-reported MVPA (p < .05). CONCLUSION: These findings suggest that this intervention is effective in enhancing self-reported MVPA in this adult population in the short term; however, this needs to be confirmed in a larger trial with better engagement to the web-based sessions.

Quantitative Proteomics Identifies DNA Repair as a Novel Biological Function for Hepatocyte Nuclear Factor 4α in Colorectal Cancer Cells.

Hepatocyte nuclear factor 4α (HNF4α) is a transcription factor that acts as a master regulator of genes for several endoderm-derived tissues, including the intestine, in which it plays a central role during development and tumorigenesis. To better define the mechanisms by which HNF4α can influence these processes, we identified proteins interacting with HNF4α using stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics with either immunoprecipitation of green fluorescent protein (GFP) or with proximity-dependent purification by the biotin ligase BirA (BioID), both fused to HNF4α. Surprisingly, these analyses identified a significant enrichment of proteins characterized with a role in DNA repair, a so far unidentified biological feature of this transcription factor. Several of these proteins including PARP1, RAD50, and DNA-PKcs were confirmed to interact with HNF4α in colorectal cancer cell lines. Following DNA damage, HNF4α was able to increase cell viability in colorectal cancer cells. Overall, these observations identify a potential role for this transcription factor during the DNA damage response.

HDAC1 and HDAC2 independently regulate common and specific intrinsic responses in murine enteroids.

Both HDAC1 and HDAC2 are class I deacetylases acting as erasers of lysine-acetyl marks on histones and non-histone proteins. Several histone deacetylase inhibitors, either endogenous to the cell, such as the ketogenic ß-hydroxybutyrate metabolite, or exogenous, such as butyrate, a microbial-derived metabolite, regulate HDAC activity. Different combinations of intestinal epithelial cell (IEC)-specific Hdac1 and/or Hdac2 deletion differentially alter mucosal homeostasis in mice. Thus, HDAC1 and HDAC2 could act as sensors and transmitters of environmental signals to the mucosa. In this study, enteroid culture models deleted for Hdac1 or Hdac2 were established to determine IEC-specific function as assessed by global transcriptomic and proteomic approaches. Results show that Hdac1 or Hdac2 deficiency altered differentiation of Paneth and goblet secretory cells, which sustain physical and chemical protection barriers, and increased intermediate secretory cell precursor numbers. Furthermore, IEC Hdac1- and Hdac2-dependent common and specific biological processes were identified, including oxidation-reduction, inflammatory responses, and lipid-related metabolic processes, as well as canonical pathways and upstream regulators related to environment-dependent signaling through steroid receptor pathways, among others. These findings uncover unrecognized regulatory similarities and differences between Hdac1 and Hdac2 in IEC, and demonstrate how HDAC1 and HDAC2 may complement each other to regulate the intrinsic IEC phenotype.

HNF4α is a novel regulator of intestinal glucose-dependent insulinotropic polypeptide.

Mutations in the HNF4A gene cause MODY1 and are associated with an increased risk of Type 2 diabetes mellitus. On the other hand, incretins are hormones that potentiate reductions in blood glucose levels. Given the established role of incretin-based therapy to treat diabetes and metabolic disorders, we investigated a possible regulatory link between intestinal epithelial HNF4α and glucose-dependent insulinotropic polypeptide (GIP), an incretin that is specifically produced by gut enteroendocrine cells. Conditional deletion of HNF4α in the whole intestinal epithelium was achieved by crossing Villin-Cre and Hnf4αloxP/loxP C57BL/6 mouse models. GIP expression was measured by qPCR, immunofluorescence and ELISA. Gene transcription was assessed by luciferase and electrophoretic mobility shift assays. Metabolic parameters were analyzed by indirect calorimetry and dual-energy X-ray absorptiometry. HNF4α specific deletion in the intestine led to a reduction in GIP. HNF4α was able to positively control Gip transcriptional activity in collaboration with GATA-4 transcription factor. Glucose homeostasis and glucose-stimulated insulin secretion remained unchanged in HNF4α deficient mice. Changes in GIP production in these mice did not impact nutrition or energy metabolism under normal physiology but led to a reduction of bone area and mineral content, a well described physiological consequence of GIP deficiency. Our findings point to a novel regulatory role between intestinal HNF4α and GIP with possible functional impact on bone density.

P1 promoter-driven HNF4α isoforms are specifically repressed by ß-catenin signaling in colorectal cancer cells.

HNF4α is a key nuclear receptor for regulating gene expression in the gut. Although both P1 and P2 isoform classes of HNF4α are expressed in colonic epithelium, specific inhibition of P1 isoforms is commonly found in colorectal cancer. Previous studies have suggested that P1 and P2 isoforms might regulate different cellular functions. Despite these advances, it remains unclear whether these isoform classes are functionally divergent in the context of human biology. Here, the consequences of specific inhibition of P1 or P2 isoform expression was measured in a human colorectal cancer cell transcriptome. Results indicate that P1 isoforms were specifically associated with the control of cell metabolism, whereas P2 isoforms globally supported aberrant oncogenic signalization, promoting cancer cell survival and progression. P1 promoter-driven isoform expression was found to be repressed by ß-catenin, one of the earliest oncogenic pathways to be activated during colon tumorigenesis. These findings identify a novel cascade by which the expression of P1 isoforms is rapidly shut down in the early stages of colon tumorigenesis, allowing a change in HNF4α-dependent transcriptome, thereby promoting colorectal cancer progression.This article has an associated First Person interview with the first author of the paper.

The G protein-coupled P2Y6 receptor promotes colorectal cancer tumorigenesis by inhibiting apoptosis.

Colorectal tumors are immersed in an array of tumor-promoting factors including extracellular nucleotides such as uridine 5'­diphosphate (UDP). UDP is the endogenous agonist of the G protein-coupled P2Y6 receptor (P2Y6R), which may contribute to the formation of a tumor-promoting microenvironment by coordinating resistance to apoptosis. Colorectal cancer (CRC) was chemically induced in P2ry6 knockout (P2ry6-/-) mice using azoxymethane and dextran sulfate sodium challenges. Mice were euthanatized and their tumor load determined. Fixed tissues were stained for histological and immunohistochemistry analysis. Tumoroids were also prepared from CRC tumors resected from P2ry6+/+ mice to determine the role of P2Y6R in resistance to apoptosis, whereas HT29 carcinoma cells were used to elucidate the signaling mechanism involved in P2Y6R anti-apoptotic effect. P2ry6-/- mice developed a reduced number of colorectal tumors with apparent tumors having smaller volumes. Overall dysplastic score was significantly lower in P2ry6-/- animals. Stimulation of P2Y6R with the selective agonist MRS2693 protected HT-29 cells from TNFα-induced apoptosis. This protective effect was mediated by the stabilizing phosphorylation of the X-linked inhibitor of apoptosis protein (XIAP) by AKT. Using CRC-derived tumoroids, P2Y6R activation was found to contribute to chemoresistance since addition of the P2Y6R agonist MRS2693 significantly prevented the cytotoxic effect of 5-fluorouracil. The present study shows that sustained activation of P2Y6R may contribute to intestinal tumorigenesis by blocking the apoptotic process and by contributing to chemoresistance, a substantial concern in the treatment of patients with CRC. These results suggest that P2Y6R may represent a prime target for reducing colorectal carcinogenesis.